5 15 672 Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 206 Folder The folder containing the original item. 5782 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Letters from Joan M. Maiman to Alvin L. Young and Barclay Shepard, October 5, 1982 ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/8d88717d9aea31be07420f03ddd16cdf.pdf 0156ce9397d4b1fdb24226c39e2774a6 PDF Text Text Item D Number 05731 G fl0t Scanned Author Corporate Author Report/Article TltlB Memorandum to Alvin L. Young from Frederic L Conway, subject: Agent Orange Litigation, dated March 18, 1982 Journal/Book Title Year Month/Day Color [J Number of Images ° Conway is sending Young copies of two reports: Agent Orange Litigation - Report I, January 5, 1982; and Agent Orange Litigation - Report II, March 9, 1982. Tuesday, April 02, 2002 Page 5781 of 5840 �Xl2* Veterans Administration REFERENCE SUP INITIALS-DATE TO (Name or title-Mail routing symbol) 1 Ma^Jor Alvln Young (JLQ2B) 2. 3. 4. 5. REASON FOR REFERENCE [~| AS REQUESTED 1 1 FOR YOUR FILES 1 1 NOTE AND RETURN 1 I COMMENTS 1 | | PER CONVERSATION 1 (CONCURRENCE 1 (NECESSARY ACTION (INFORMATION I [SIGNATURE REMARKS SUBJ: Agent Orange Litigation Attached for your information are copies of Agent Orange Litigation Reports I & II. Att. DATE FROM FREDERIC L. CONWAY (02C) Special Assistant to the General Counsel VA FORM , « , n MAY 1BBO 3230 RECEIVE!) - I/O (102) MAR 131982 EXISTING STOCKS OF VA FORM 3Z3O. AUG I07C, WILL BE USED. 3/18/82 TEL. EXT. x-3723 �AGENT ORANGE LITIGATION - Report I January 5, 1982 This is the first of a series of quarterly reports outlining developments in litigation activities impacting on Agent Orange programs of the VA. This first report will briefly describe the nature of the complaint, the relief sought, and the current status of the litigation. Perhaps the most important case, and one that receives a great deal of media attention, is Chapman, et al. v. Dow Chemical, et al., a case dealing with Agent Orange product liability. A number of veterans and their families have / brought suit in various federal district courts against the manufacturers of the components of a variety of herbicides, Including Agent Orange, that were used by the military during the war in Vietnam. There are four groups of plaintiffs: Vietnam veterans, their spouses, their parents, and their children. They assert numerous theories of liability, Including strict products liability, negligence, breach of warranty^ Intentional tort and nuisance. .Plaintiff veterans seek to recover for personal Injuries caused by their exposure to Agent Orange. The family members seek to recover on various derivative claims; some of the children assert claims in their own right for genetic Injury and birth defects caused by their parent's exposure to Agent Orange; and some of the veterans1 wives seek to recover In their own right for nlscarrlges. �These actions were consolidated under 28 U.S.C. §1^07 for pretrial processing and transferred to the United States District Court for the Eastern Division of New York. The defendants, seeking indemnification or contribution in the event they are held liable to the plaintiffs, served third party complaints against the United States. In their third party complaints they allege negligence, misuse of product, post-discharge failure to warn, implied indemnity, denial of due process and failure to comply with herbicide registration laws. The plaintiff had sought redress of their alleged injuries under Federal common law and Invoked the "federal question" jurisdiction of the district court. (28 U.S.C. §1331(a).) The defendant chemical manufacturers moved to dismiss the complaint for lack of subject matter Jurisdiction, contesting the existence of a federal common law cause of action. The district court, denying respondents1 motion to dismiss the claims for lack of Jurisdiction, held that petitioners' claims were governed by federal common law and hence arose under federal law within the meaning of 28 U.S.C. § 1331(a). (In re 'Agent Orange"Product Liability Litigation, 506 P. Supp. 737 (E.D. N.Y. 1979)). The district court reasoned that there, are significant federal Interests In this litigation (Id. at 749). It noted the " 'distinctively federalffl �3. relationship between the government and members of the armed forces and remarked that "[tjorts committed by war contractors against soldiers in action constitute 'harms inflicted' on the soldiers and 'interference' with the relationship between soldiers and the government. Such harms and interferences implicate federal interests" (id. at 7^6, quoting UnitedStates, v. Standard Oil Co., 332 U.S. 301, 305-306 (19^7;)The court added that "the federal interest in this litigation includes as well the rights of the war contractors since the extent of a contractor's liability undoubtedly will affect future dealings between the contractor and the government" (id. at 7^7). The court explained (id. at n. 5): War contractors might be expected to increase the price of war materials to correspond to any extension in their potential liability. Such adjustments might have a significant effect on the federal treasury. If potential liability increased dramatically, future war contractors might attach conditions to the use of their products, or balk at supplying the military with any products whatsoever. Thus, the government's military capabilities might be affected by this litigation. Applying state law to petitioners' claims, the court stated, "would burden federal interests by creating uncertainty as to the rights of both veterans and war contractors. It would also be unfair in that essentially similar claims, Involving veterans and war contractors identically situated in all �H. relevant respects, would be treated differently under different state laws" (Id. at 784).i/ Finally, the district court remarked that while tort claims are traditionally matters for state law, "state law has not considered the complex question of a war contractor's liability to soldiers injuried by toxic chemicals while engaged in combat and serving abroad" (id. at 7^9). The court concluded that the state interests affected by the application of federal common law to petitioners' claims were slight "(b)ecause state law is no more or less developed as to such claims than federal common law" (ibid.). Respondents appealed under 28 U.S.C. § 1292(b), and a divided panel of the Court of Appeals reversed (In re "Agent Or ange*1 Product Liability Litigation, 635 F.2d 987 (2nd Cir. 1980)), The majority concluded, for two reasons, that there was no "identifiable federal 27 The district court qualified its ruling by noting: "A lone veteran suing the supplier of a single piece of defectivemilitary machinery would implicate only a minimal federal interest. . . . But [In] this litigation . . . [tjhe estimated number of Involved veterans ranges from thousands to millions, and the estimated potential liability . . . ranges from millions to billions of dollars. As the number of veterans and the size of the claims against the war contractors Increase so the federal Interest In this litigation expands" (Id. at 741). In this connection the district court cited three cases — Whltaker v. Harvell-Kllgore Corp. 418 P.2d 1010 (5th Clr. 1969), feoelng Airplane Co. v. Brown, 291 P. 2d 310 (9th Cir. 1961), and Adams v. General Dynamics Corp., 405 P. Supp. 1020 (N.D. Cal. 1975) — in which state law apparently was applied to servicemenfs claims against defense contractors without any discussion of whether federal law should apply. Each of these claims arose from a single event, none of which occurred during armed conflict. See also Day ft Zimmermann, Inc. v. Challoner. 423 U.S. 3 (1975). ~~~~ �5. policy at stake In this litigation that warrants the creation of federal common law. . . . " (id. at 993). First, the majority said, "since this litigation is between private parties and no substantial rights or duties of the government hinge on its outcome, there is no federal interest in uniformity for its own sake . . . [Therefore t]he fact that application of state law may produce a variety of results is of no moment" (id. at 993-99*0. Second, the Court of Appeals distinguished other cases in which federal common law was applied on the ground that in those cases "the government's substantive interest in the litigation (was) essentially mono(lithic), in that it (was) concerned only with preserving the federal fisc" (id. at 99*0. By contrast, the court stated (ibid.): Mere the government has two Interests; and here the two interests have been placed in sharp contrast with one another. Thus, the government has an interest in the welfare of its veterans . . . . But the government also has an Interest in the suppliers of its material; imposition, for example, of strict liability as contended for by plaintiffs would affect the government's ability to procure material without the exaction of significantly higher prices, or the attachment of onerous conditions, or the demand of Indemnification or the like. The Court reasoned that "it Is properly left to Congress in the first Instance to strike the balance between the conflicting Interests of the veterans and the contractors . . . ." (Id. at 944). Accordingly, the Court concluded that "while the federal government has obvious Interests In the welfare of the parties to the litigation, Its Interest In the outcome of the litigation, i.e., in how the parties1 welfares should be balanced, is as yet undetermined. ... In the present litigation the federal policy is not yet identifiable" (id. at 995). �6. For this reason, the majority held that state law governed petitioners' claims and accordingly that the district court lacked federal question jurisdiction. Chief Judge Feinberg dissented. He endorsed the 6 istrict court's view that the government has a substantial interest in the litigation, that the servicemen's claims should be treated uniformly, and that a federal determination of the novel issues Involved in the case would not displace any wellestablished body of state law. A petition for a writ of certiorari was denied by the Supreme Court on December 14, 1981. The United States had submitted an amicus brief taking the position that there is no basis for concluding that the plaintiff's claims arise under federal common law. After the Court of Appeals' ruling, the district court held further proceedings on petitioners' claims. In re "Agent Orange" Product Liability Litigation, 506 F. Supp. 762 (E.D. N.Y. 1980). The court's jurisdiction over these proceedings rested § 1332. on diversity of citizenship, as provided In 28 U.S.C. See 506 P. Supp. at 782-783 4 n. 30, 786. Our understanding Is that a significant number of plaintiffs will not be able to bring diversity suits in federal district court. �7. At these proceedings, respondents asserted an affirmative defense based on federal law. They claimed that the policies underlying the Defense Production Act, 50 U.S.C. App. 2061 et __seq_., and their status as government contractors (see Yearsley v. W.A. Ross Construction Co., 309 U.S. 18 (1940)), barred petitioners1 claims. The district court stated that it was "satisfied that such a ... defense exists and has possible application to the facts at bar." It denied respondents' motion for summary judgment, however, on the ground that respondents would have to establish certain facts about their relationship to the government in order to invoke the defense. at 796. 506 F. Supp. The court scheduled a trial on this issue "at the threshold of this action," before trial on liability. Id. at 796, 785. In addition, the district court dismissed respondents' thirdparty complaint against the United States. The court ruled that the holding in the case of Feres v. United States, 3^0 U.S. 135 (1950), would bar this action against the United States. The Feres case held that under the doctrine of sovereign Immunity, the United States Is not liable for Injuries Incurred by servicemembers which arise out of or are In the course of activity Incident to military service. The court also held that In that case that the Federal Tort Claims Act (28 U.S.C. § 13*»6(b) et. seq.) did not constitute a waiver of sovereign Immunity from liability for these types of Injuries. �8. The district court further ruled that third party claims such as the one brought by the chemical manufacturers were also barred. In reaching this conclusion, the court cited Stgncll__Aer_o__ Engineer ing Corporation v. United States,, 431 U.S. 666 (1977), which held that "the right of a third party to recover In an indemnity action against the United States . . . must be limited by the rationale of Feres where the injured party is a serviceman." ; Regarding derivitive claims of spouses, parents, and children, the district court ruled that the Feres doctrine also bars suits by a servicemember's family for damages resulting from injuries the servicemember suffered incident to and arising out of military service. Concerning the alleged failure to warn, the court ruled that any post-discharge failure to warn would not be sufficiently separate and distinct from the underlying claim for injury occuring Incident to or arising out of military service as to remove it from the Feres doctrine. With regards to the remaining complaints brought by the chemical manufacturers, the court ruled that It lacked Jurisdiction to consider them, the proper forum being the Court of Claims. Judge Pratt did indicate that his holdings would not affect the claims of veterans who allege Improper medical care rendered after military service for a condition allegedly '-;1'" �9. resulting from Agent Orange exposure. Also, In an unusual foot- note, Judge Pratt cautioned that "the views expressed and the rulings made in the decision are not entirely free from doubt." Accordingly, he advised that any plaintiff who believes he or she has a valid claim against the United States should protect his or her interest by filing a claim form (Standard Form 95) with the proper Federal agency . £ ./ The district court also certified the Agent Orange litigation against the chemical manufacturers as a class action and began the implementation of a plan for managing this highly complex case. Ryan et al. v. Cleland et al. takes up where Chapman leaves off. In this case, Vietnam veterans, their spouses, their children, and their parents have brought suit against a number of VA officials. A variety of allegations are made about the knowledge of VA officials and the actions those officials did or did not take. First, It Is alleged that these officials failed to provide information to veterans regarding the hazards associated with exposure to "toxic synthetic chemicals" such as 2,3,7, 8 - TCDD (dloxln). £/ As a result of this comment, the VA received about 10,000 such claims from veterans as well as the spouses, children, and parents of veterans. Only about five percent of these claims allege wrongdoing on the part of the VA. The remainder were forwarded to the Department of Defense for processing. �10. Secondly, it is claimed that the VA failed to provide adequate medical care and treatment, including genetic counseling, to Vietnam veterans and their families, thereby subjecting them to unreasonable risk of sustaining personal Injuries and damages. Third, it is alleged that the officials of the VA engaged in negligent, wanton and reckless conduct. Specifically, it is claimed that these officials knew a variety of "facts" about the harmful effects of exposure to dioxin and the actions taken or recommended by a number of organizations, both government and private at home and abroad, to reduce the risks of exposure. In this regard, it Is also alleged that VA officials conspired to conceal from the plaintiff the deleterious effects of exposure and have actively misrepresented the extent of the risks of exposure. Fourth, unspecified constitutional torts are alleged to have been committed by the named VA officials. Fifth, the "Individual defendant bureaucrats" are claimed to have conspired to disseminate false and misleading Information to persuade individuals not to assert their rights and pursue their legal remedies against the war contractors and the United States. Finally, the defendants are alleged to have conspired to overmedlcate Vietnam combat veterans with psychotropic drugs and to ignore symptoms of serious diseases being experienced by the plaintiffs. �11. Among the reliefs sought is a declaration that the defendants have a fiduciary obligation to notify the plaintiffs of the dangerous properties of the chemicals to which they were' exposed. Also sought are an order compelling the United States to join (as plaintiff) in the lawsuit against the chemical manufacturers or, alternatively, declare that the plaintiffs may proceed as private attorneys general against the chemical manufacturers to seek reimbursement for the cost of providing care and compensation to the afflicted veterans. Other forms of declaratory and injunctive relief are also demanded. Similar complaints have been filed across the country and have been consolidated for pretrial procedures and motion under the multi-district litigation procedure. The United States responded to the complaint by filing a motion to dismiss. Action on the motion is currently pending. In White v. Cleland, the plaintiffs allege certain violations of the Administrative Procedure Act by the VA with regard to the Issuance of an internal instruction document relating to the processing of disability claims involving exposure to herbicides (particularly Agent Orange) during the Vietnam conflict without soliciting public comment. The plaintiffs have argued that the document, Identified Internally as a Program Guide, sets forth rules concerning the handling of Agent Orange �12. compensation claims. Consequently, it is argued, the VA violated the requirements of the Administrative Procedure Act (5 U.S.C. §§552 and 553) regarding rule making, particularly with respect to providing the public with an opportunity to comment on ,the content of the Program Guide. The Veterans Administration has"taken the position that the Program Guide is not regulatory but rather informative and educational. As such, it is argued, it is not required to submit the Program Guide for public comment prior to the distribution to the field for guidance. The Program Guide is characterized as informational, designed to inform agency employees of the existing state of factual knowledge and, to a lesser degree, the state of the law. Cross motions for summary judgment have been filed and action is pending on It. In a related case, Gott v. Cleland, 80-0906, D.D.C., Sept. 30, 1981, a similar Program Guide concerning the handling of radiation-related cases has been found to be in the nature of a rule or regulation, and, as such, subject to the requirements of the APA mandating notice and an opportunity for public comment. In this case, the VA had published the contents of the Guide for public notice, but not for public comment. The district court concluded that the failure to solicit public comment violated the APA and, therefore, Invalidated its use as regulations. This case Is currently under appeal. �Plaintiffs filed a motion to amend the judgment to include an order requiring the VA to readjudicate claims which had been denied while the Program Guide had been in use. was successfully opposed. FREDERIC L. CONWAY Special Assistant to the General Counsel This motion �AGENT ORANGE LITIGATION - Report II March 9, 1982 In re "Agent Orange" Product Liability Litigation, M.D.L. 381 The district court issued a series of pretrial orders and decisions in this litigation between the plaintiff veterans and the defendant chemical manufacturers. The most significant ruling came in response to the defendant's motion for summary judgment based on what has been termed the "Government contract defense." They argued that liability may not be imposed upon them for injuries arising out of the Government's use of "Agent Orange," ("Agent Orange" denotes all herbicides used in Southeast Asia during the relevant period of the Vietnam conflict) because the defendants had manufactured "Agent Orange" under contract with the United States Government and in strict compliance with its specifications. The court, upon consideration of the arguments raised by the parties to the litigation, set forth three elements which, if proved by the defendants, would Insulate them from liability. First, it must be demonstrated that the Govern- ment established the specifications for "Agent Orange". Second, it must be shown that the "Agent Orange" manufactured by the defendants meet the Government's specifications in all material respects. Finally, the defendants must prove that the Government knew as much as or more than the defendants about the hazards of the product being purchased. �2. This last element would not impose a duty on the supplier to perform any testing that was not included in the specifications but it would require the supplier to share with the Government any information it had about the hazards of the herbicides being purchased. If the knowledge level between the defendant suppliers and the Government is at least in balance, the defendants are then shielded by the Government contract defense from liability for damages resulting from use of the herbicides supplied pursuant to and in compliance with Government contract specifications. This case will now enter into a process of "intensive discovery" in preparation for phase one of this litigation, a trial on the Government contract defense. �Ryan v. Cleland, Civ. No. 81-0055 (E.D. N.Y.) The district court ruled favorably on the Government's motion to dismiss this action which was based on a series of allegations against the VA and certain named officials. The court, in granting the Government's motion, stated that the action fell within 38 U.S.C. §211(a) which bars judicial review of the VA's interpretation or application of a particular VA statute in the absence of a valid constitutional claim. The plaintiffs had attempted to plead a variety of constitutional violations which the court did not accept. The court also rejected the Federal Tort Claims Act (PTCA) as a jurisdictional basis because the FTCA provides relief only In the form of monetary damages and the plaintiffs were seeking declaratory and injunctive relief. � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 206 Folder The folder containing the original item. 5781 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Memorandum to Alvin L. Young from Frederic L. Conway, subject: Agent Orange Litigation, dated March 18, 1982 ao_seriesVIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 206 Folder The folder containing the original item. 5780 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource News Clippings, 11/12/81 - 6/84 ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/f9345e8de52ad5f8903d039f40cbe56b.pdf e17e6e44ff6ef709bf6e129601c5f232 PDF Text Text Item D Number °5779 D N0t Scanned Author Corporate Author Report/Article Title Preliminary Materials and Notes for An International Symposium on Chlorinated Dioxins and Related Compounds, October 25-29, 1981, Arlington, Virginia Journal/Book Title Year Month/Day Color Number of Images D ° Descrlpton Notes Thursday, March 28, 2002 Page 5779 of 5780 �AN INTERNATIONAL SYMPOSIUM ON CHLORINATED DIOXINS AND RELATED COMPOUNDS October 25 - 29, 1981 Sheraton National, Arlington, VA About the Symposium: The purpose .of the symposium is to address the risk of chlorinated dioxins and related compounds to human health and the environment. The four day symposium contains both platform and poster sessions and is organized into five major topic areas: The Problem, Human and Environmental Exposure, Toxicology, Risk Assessment, and Courts vs. Science. The Session on The Problem contains overviews on the extent of environmental contamination, fact vs. fiction, information needs, current research, and the available data base. Human and Environmental Exposure is addressed through sessions on sources and releases, environmental chemistry and laboratory safety, and waste management. Toxicology is discussed in three sessions; animal toxicology, environmental toxicology and human observations. The session on Risk Assessment is a principle component of this symposium. This session is designed to" integrate human and environmental exposure with toxicology to estimate potential for risk. Both platform presentations and panel discussion will be used to discuss the assumptions underlying the risk estimate and to identify data gaps and research needs that will help to better understand and estimate risk. A session on Courts vs. Science has been included in this symposium because researchers and their data are being extensively utilized in litigation; and this trend will continue in the future. This session will contain papers on legal implications of scientific research, the legal process vs. the scientific process, data reliability and liability, and estimation of risk and its liability. �f<? iptfc^ PRELIMINARY PROGRAM Sunday, October 25, 1981 5:00 P.M. - 7:00 P.M. Registration 7:00 P.M. Mixer and Reception Monday. October 26. 1981 [ INTRODUCTION AND THE PROBLEM] A.M. Plenary Session • Welcome (purpose and intent of symposium, acknowledgments) • The Problem (overviews from different perspectives)" I HUMAN AND ENVIRONMENTAL EXPOSURES] P.M. SESSION: [ c f l Sources and Releases Tuesday, October 27, 1981 A.M. • • • SESSION: Environmental Chemistry Physical/Chemical Properties Analytical Detection & Monitoring Estimated Environmental Concentration (fate/analysis) Concurrent with: A.M. SESSION: ~~ Laboratory Safety and Waste Management TTOXICOLOGY j P.M. SESSION: ^Animal Toxicity • • • • Pharmacok$netics/metabo1sim Acute, subchronic, chronic toxicity carcinotoxic, embryotoxic,,fetotoxic Teratoxic, mutatoxic Wednesday October 28, 1981 A.M. SESSION: Human Observations P.M. SESSION: Environmental Toxicology • Metabolism • Aquatic effects &^\< • Terrestrial effects Thursday October 29, 1981 A.M. SESSION: Risk Assessment t Platform papers V.tc*J. f«W4. • Panel discussion ,<, , 1 COURTS VS. SCIENCES j P.M. SESSION: Legal Implications of Scientific Data �POSTER SESSIONS Monday: Human and Environmental Exposure Papers on: Sources and Releases Environmental Chemistry Tuesday: Toxicology Wednesday: Papers on: Animal Toxicology Human Observations Environmental Toxicology Thursday: Laboratory Safety and Waste Management �SCHEDULE ITEM COMPLETION DATE 1. Title, location, date, program topics and format, speakers identified February 3rd 2. Call for papers (solidify mailing list) (Deadline for Abstracts) February 20th (May 1st) '3. Mail Symposium announcements to journals February 28th 4. Keynote and wanted speakers committed March 30th 5. Pre-program disseminated (flyer) May 15th 6. Peer review June 15th 7. Notice to authors June 20th 8. Program to printers July 20th 9. Program disseminated August 20th 10. Pre-registrati on closed • October 2nd �^SJCZ^L- .jggr .:3£3^igj _ '6c2Lpmzfcz&/Kfe- �- f, � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5779 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Preliminary Materials and Notes for An International Symposium on Chlorinated Dioxins and Related Compounds, October 25-29, 1981, Arlington, Virginia ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/32f298fc0fdbb99b3850e7f5b14c6161.pdf 2e574275001a87c6de2970e29e0006d7 PDF Text Text Item D Number °5778 D Not Scanned Author Corporate Author JRB Associates, McLean, Virginia RODOrt/ArtiClB TltlB Review °' Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins, Volume II: Annotated Bibliography Journal/Book Title Year 1981 Month/Day October Color Number or Images D ° Descrlpton Notes Thursday, March 28, 2002 Page 5778 of 5780 �Veterans Administration Review of Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins Volume II Annotated Bibliography Department of Medicine and Surgery �VA Contract Number: V101(93)P-823 JRB Project Number: 2-816-03-744-00 Review of Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins Annotated Bibliography Volume II This report was prepared under a cost reimbursement-type contract awarded on a competitive bid basis on December 15, 1980. The contract will run for nine and one-half months and is funded at $111,743. Prepared for Contracting Officer's Technical Representative: Barclay M. Shepard, M.D. Special Assistant to the Chief Medical Director for Environmental Medicine (102) Department of Medicine and Surgery Veterans Administration 810 Vermont Avenue, N.W. Washington, D.C. 20420 Submitted by: JRB Associates 8400 Westpark Drive McLean, Virginia 22102 �FOREWARD Public Law 96-151 enacted December 20, 1979, mandated the Veterans Administration to conduct "...a comprehensive review and scientific analysis" of the worldwide literature on Agent Orange and other phenoxy herbicides. The need to conduct such a review was in response to an increasing awareness among veterans the Congress and the public of the potential long-term health consequences of exposure to these herbicides and the contaminant dioxin. This report was prepared wholly and exclusively by JRB Associates, Inc., and represents an independent assessment of the current state of science relating to the herbicides used in conjunction with the Vietnam conflict. The publication of this document does not signify that the contents necessarily reflect the views and policies of the Veterans Administration. �PREFACE The controversy surrounding the tactical use of herbicides, in Southeast Asia during the Vietnam conflict has now extended into its third decade. Few environmental or occupational health issues have received the sustained national attention that has been focused on "Agent Orange". The controversy centered first on the actual employment and subsequent ecological effects of herbicides in South Vietnam, then on the question of the safe disposal of surplus herbicide following the conflict, and lastly, on whether herbicides were responsible for health problems reported among Vietnam veterans. As each facet of the controversy came to the attention of the public, more government agencies were tasked to deal with the associated issues. Hence, today a great many branches and agencies of the Federal government are involved in seeking resolutions of the scientific, medical, legal and social problems surrounding "Agent Orange." In addition, many state governments have enacted legislation relating to this highly controversial issue. The basis for resolving the Agent Orange controversy must in large measure stem from the results of scientific inquiry. Appropriately, the preparation of extensive reviews and summaries of the scientific literature have appeared in the past, e.g., Midwest Research Institute Report (1967), National Academy of Science Report (1974) and United States Air Force Technical Report (1978). The present report therefore has benefited from the information developed in previous reports as well as from recently published scientific data. The volume of available scientific literature pertaining to the herbicides used in Southeast Asia has increased almost geometrically since the 1967 report. Especially important has been the increase in the scientific data on the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p_-dioxin (TCDD). The present report is published in two volumes. Volume I presents a detailed scientific assessment of the literature, and Volume II contains an annotated bibliography of the relevant literature. Although much is known about the toxicity of the herbicides used in Vietnam, a number of gaps in the scientific knowledge are still present. It is hoped that this two-volume report may serve to focus the continuing public dialogue and future scientific inquiry on those aspects of the problem in which substantial doubt or gaps in information remain. This report should also assist researchers in identifying opportunities for the systematic development of new knowledge based on what is now known and accepted as fact. VETERANS ADMINISTRATION October 1981 �INTRODUCTION This annotated bibliography represents about 1,200 documents identified as relevant to the review of the scientific literature on the environmental fate and toxicity of herbicides, including phenoxy herbicedes, and associated dioxins, used in South Vietnam. Over 900 of these citations, those dealing primarily with health effects, are accompanied by annotations; relevant abstracts, summaries, review articles, editorials, and documents used for background material are included in the bibliography, but are not annotated. The bibliography is arranged alphabetically by author. A subject index is included in the back of the bibliography. Herbicides described in this literature include: Herbicides Orange, Orange II, Purple, Pink, Green, White, Blue (cacodylic acid), Dinoxol, Trinoxol, Bromacil, Diquat, Tandex, Monuron, Diuron, and Dalapon. The key constituent compounds include: 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 2,3,7,8-tetrachlorodibenzo-para-dioxin, cacodylic acid, and picloram. The following abbreviations are used in the annotations: 2,4-D 2,4-dichlorophenoxyacetic acid 2,4,5-T 2,4,5-trichlorophenoxyacetic acid TCDD 2,3,7,8-tetrachlorodibenzo-para-dioxin or 2,3,6,7-tetrachlorodibenzo-para-dioxin ppm parts per million ppb parts per billion ppt parts per trillion kg kilogram (doses expressed per kg always refer to kg of body weight unless otherwise designated) (u)g; ug microgram rag milligram mM raillimolar �ANNOTATED BIBLIOGRAPHY 1. Aberg, B., and Eliasson, L. The herbicidal effects of phenoxy compounds. In Chlorinated Phenoxy Acids and Their Dioxins. ed. C. Rarael (Ecol. Bull. no. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 86-100. [Review article.] 2. Abou-Donia, M. B., Charles, J. M., and Menzel, D. B. (1976) Uptake and metabolism of pesticides by the isolated perfused rat lung. Pharmacologist 18(2):247. [Abstract, only.] 3. Abo-Khatwa N., and Hollingworth, R. M. (1974) Pesticidal chemicals affecting some energy-linked functions of rat liver mitochondria in vitro. Bull. Environ. Contain. Toxicol. 1 ( ) 4 6 4 4 24:4-5. The effects of diuron, 2,4-D, 2,4,5-T and 2-(2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP) on rat liver mitochondrial function in vitro were described. The herbicides were added to mitochondrial suspensions and 4 energy-linked functions were measured. These functions were the efficiency of respiratory chain phosphorylation (measured as the ADP to oxygen ratio), state 4 and state 3 respiration rates (the rates of oxygen uptake in the absence and presence of phosphate acceptor, respectively), and the respiratory control index (the ratio of succinate oxidation with ADP present to the rate after the acceptor is depleted). At 10 M, 2,4,5-TP reduced the respiratory control index, stimulated the state 4 respiration rate, and reduced the ADP to oxygen ratio. Diuron caused these effects at 10~ M, and 2,4-D and 2,4,5-T reduced the respiratory control index and stimulated the state 4 respiration rate at 10~ M. The effects of a total of 47 pesticides were studied on mitochondrial function. The authors concluded that all four compounds were uncouplers of respiratory chain phosphorylation. 4. Adamoli, P., Angeli, E., Bandi, G., Bertolotti, A., and Bianchi, E. Analysis of 2,3,7,8-tetrachlorodibenzo-para-dioxin in the Seveso area. In Chlorinated Phenoxy Acids and Their Dioxins, ed, C. Ramel. (Ecol. Bull. no. 27, Stockholm: Swedish Natural Research Council, 1978) pp. 31-38. The authors present information on the analytical methods used in the analysis of TCDD after its release from a manufacturing plant accident in Meda, Italy on July 10, 1976. Several laboratories participated in the analysis of a large number of samples collected in two phases. During the first phase, which lasted from July 22-August 6, 1976, environmental sampling was performed to determine the distribution and concentration of TCDD in the environment. Levels of 0.1 Mg in lOOg of �soil were measured using a gas chromatograph - mass spectrometer and a Finnigan 3000 instrument. The sensitivity of the instruments was 0.1 ppb. In the second phase, more precise measurements were made to determine the amount of TCDD in the environment in order to plan decontamination procedures. A sampling scheme was developed and soil samples were collected and analyzed using an analytical procedure with sensitivity reaching 1 ppt. The author concludes that samples analyzed simultaneously at several laboratories showed TCDD levels that were closely comparable. 5. "Agent Orange" - Bibliography. (1980) Personal Communication with Dr. J.A. Moore, National Institutes of Environmental Health Sciences, Research Triangle Park, NC. 76 pp. [Bibliography.] 6. Ahling, B., Lindskog, A., Jansson, B., et al. (1977) Formation of polychlorinated dibenzo-p-dioxins and dibenzofurans during composition of a 2,4,5-T formulation. Chemosphere 33:461-468. [Background material.] 7. Ahmed, F. E., Hart, R. W., and Lewis, N.J. (1977) Pesticide induced DNA damage and its repair in cultured human cells. Mutat. Res. 42:161-174. The authors investigated the potential of 2,4-D, diquat and 11 other pesticides to induce unscheduled DNA synthesis in human cells in culture. The cell line VA-4, a human fibroblast cell line transformed by the virus SV-40 grown on cover slips was used as the cell culture system. Diquat and 2,4-D were dissolved in water and added to the cell cultures at 1, 10, 100 and 1000 uM for 1, 3, 5, 8, or 12h. Unscheduled DNA synthesis measured by [ H]-TdR incorporation and autoradiographic analysis was determined in cell cultures with and without rat (S9) metabolic activation. Silver grains in 30 cells per cover slip were counted to determine unscheduled DNA synthesis. The number of replicate slides counted was not reported. Cytotoxicity at experimental herbicide concentrations was not reported. Negative controls were included in the experiment, but no positive controls were mentioned. Results of the assay were analyzed by a t-test comparing the mean number of silver grains in controls versus treated cells. Both diquat and 2,4-D treated cultures had statistically significant increases in mean numbers of silver grains at all concentrations tested. No metabolic activation was necessary for this effect. In addition, the authors also measured 2,4-D-induced 313 nm photolysis of BudR containing repaired regions of DNA to determine the form of DNA repair induced. At 1, 10, and 100 uM, 2,4-D induced 0.7, 1.1 and 1.3 breaks per 10 daltons DNA, respectively. The shape of the curve of induced breaks for 2,4-D resembled the shape of curves obtained in similar experiments using ionizing radiation and alkylating agents such �as ethyl methane sulfonate. This result suggests a similarity between alkylating agents and 2,4-D in the mechanism of induction of unscheduled DNA synthesis. 8. Ahmed, F. E., Lewis, N. J., and Hart, R. W. (1977) Pesticide induced ouabain resistant mutants in Chinese hamster V79 cells. Chem.-Biol. Interactions 19:369-374. The authors studied the mutagenicity of 2,4-D in an assay for detection of forward mutations in Chinese hamster V79 cells. This system measures the induction of ouabain resistant mutants in an ouabain sensitive cell line. 2,4-D was tested in triplicate at a concentration of 10 uM which yielded about 40% survival. A negative control but no positive control was included in the assay. Metabolic activation was also not included in the study. The forward mutation frequency of 2,4-D exposed cells was calculated to be 25.5/10 survivors while mutation frequency of controls was 1.8/10 survivors. The authors concluded that 2,4-D was a weak mutagen in this test system. 9. Air Force will study men who sprayed Agent Orange, [editorial] (1980) JAMA 243(2):102-103. [Editorial.] 10. Aitio A., and Parkki, M. G. (1978) Organ specific induction of drug metabolizing enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Toxicol. Appl. Pharmacol. 44(1):107-114. The effect of TCDD on 6 different enyzme activities from 5 rat tissues is described. Male Wistar rats were administered a single oral dose of 20 ug/kg TCDD in acetone-corn oil (1:90). After 7 days, rats were killed and microsomes were prepared from the liver, kideny, lung, intestine, and testis and assayed for various enzyme activities. Ethoxycorimarin deethylation was induced by TCDD 20 fold in the kidney, and 5 fold in the liver and lung. Benzo(a)pyrene hydroxylation activity was increased 60 fold in the kidney, 7 fold in the lung, and 5 fold in the liver. VDP glucuronosyltransferase activity was induced 7 fold in the liver and 1.5 fold in the lung and kidney by TCDD. Increased liver and kideny VDP Glucuronosyl-transferase activity was elicited in control and TCDD-treated microsomes by addition of digitonin, trypsin or phospholipase A to the incubation mixture. TCDD did not induce cytochrome C reductase activity inthe liver or kidney, epoxide hydratase or glutathione s-transferase activities (with styrene oxide as substrate for these enzymes) in any tissue or any of the assayed enzymes activities in intestine or testis. The authors concluded that TCDD produced a different patterns of induction of enzymes than the patterns reported for phenobarbital and polycycluc hydrocarbon induction. �11. Akamine, E. K. (1951) Persistence of 2,4-D toxicity in Hawaiian soils. Botany 112:312-319. The author studied the persistence of 2,4-D toxicity in soils and the influence of physical and chemical factors on persistence. Indicator crops were planted and harvested every 2 weeks from 2,4-D treated agricultural soils at five locations. Higher soil temperatures resulted in a more rapid rate of 2,4-D dissipation. High pH values also resulted in more rapid dissipation. High aerobic bacterial counts were associated with shorter retention times of 2,4-D in the soils. Organic matter content was not correlated with 2,4-D persistence. The author concluded that there was a considerable variation of 2,4-D toxicity persistence among the soils. 12. Akermark, B, Photochemical reactions of phenoxy acids and dioxins. In Chlorinated Phenoxy Acids and Their Dioxins. ed. C. Ramel (Ecol. Bull, no. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 75-81. [Background material.] 13. Albright, R., Johnson, N., Sanderson, T. W., Farb, R. M., Melton, R., et al. (1974) Pesticide residues in the top soil of five west Alabama counties. Bull. Environ. Contain. Toxicol. 12(3):378-384. The authors reported on the occurrence of 2,4,5-T in five west Alabama counties. In three of the counties, Greene, Sumter, and Marengo, over 60% of the land area was farmland, while in Tuscaloosa County 27% was farmland and in Pickens County 42% was farmland. Amounts and types of pesticides applied to the sampling areas could not be documented. At least 5-100 g soil samples were collected in each county and analyzed by gas-liquid chromatography. 2,4,5-T was found in 24% of the samples analyzed. Concentrations of 2,4,5-T were less than 0.1 ppm. The authors noted that heaviest concentrations of pesticide residues were found in new fields, old farm fields, run-offs and drainage ditches. 14. Aldred, J. E., Belcher, R.S., Christophers, A.J., Clements, A., Danks, D.M., et al. (1978) Report of the consultative council on congenital abnormalities in the Yarram district. Presented to both Houses of the Australian Parliament. The relationship between birth defects that occurred in the Yarram district of Austrialia and use of the herbicides 2,4-D and 2,4,5-T were investigated. Between 1975 and 1978 8 cases of abnormal (human) births and birth effects occurred in the yarram district and were investigated. The incidence of abnormal births in this time period was compared statistically to incidences between 1960 and 1977 for this district. Veteriniarian reports of abnormal births and a review of scientific literature related to the teratogenicity of 2,4-D, 2,4,5-T and TCDD were used to evaluate the significance of the abnormal human �births related to herbicide use. The abnormal births included 2 premature births (at 22 and 27 weeks of gestation), phocomelia of a child whose mother resided outside of Yarram until after the infant was born, cystic hygroma of a child born in 1978, 2 cases of spin bifida, 1 case of anencephalus, and 1 case of renal agenesis. The incidence of 4 neonatal deaths due to birth defects (the last 4 cases listed) among 278 births between 1975 and 1977 exceeded the expected rate of 2.7 per 1000 births. From the case records, no direct herbicide exposure could not be established for any of the 8 mothers. The times of conception also did not correlate with seasons of heavy herbicide usage and to usage was not notably elevated in 1975-1977. From animals studies doses below teratogenic levels for 2,4-D, 2,4,5-T and TCDD were estimated, as well as food residue levels. Consumption of enough contaminated food to reach an effective herbicide level required that a 60 kg pregnant woman ingest unreasonable volume of food (6,000 kg of meat or 60,000 liters of water). No evidence of teratogenic effects in animals was reported in Yarram in 1975-1977. The 3 birth defects related to neural tube closure were considered to be non-specific, with 53 other potential etiotogies, suggested by the authors. The committee concluded that available information did not include any evidence of birth defects that could be attributed to the use of 2,4-D or 2,4,5-T in Yarram. 15. Allebone, J. E., Hamilton, R. J., and Ravenscroft, B. (1975) Environmental organic chemistry of 2,4-dichlorophenoxyacetic acid. Environ. Chem. 1:160-190. [Review article.] 16. Allen, A. M. (1977) Skin Diseases in Vietnam, 1965-72. In Internal Medicine in Vietnam. U.S. Army, Washington, D.C. pp. 1-13, 29-51, 125-129. [Background material.] 17. Allen, J. R., Barsotti, D. A., Lambrecht, L. K., and Van Miller, J. P. (1979) Reproductive effects of halogenated aromatic hydrocarbons on nonhuman primates. Ann. N.Y. Acad. Sci. 320:419-425. [Review article.] 18. Allen, J. R., Barsotti, D. A., Van Miller, J. P., Abrahamson, L. J., and Lalich, J. J. (1977) Morphological changes in monkeys consuming a diet containing low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fd. Cosmet. Toxicol. 15:401-410. The subacute toxicity of TCDD in the monkey is described. Eight young female Rhesus monkeys were fed a diet containing 500 parts per trillion (ppt) TCDD for 9 months. Monkeys were observed daily, food intake was �monitored daily, hematology and blood chemistry were analyzed monthly, and autopsies were performed, including gross and microscopic examinations only on the monkeys that died. Five of the eight animals died; these deaths occurred between months 7 and 12. By 3 months, periorbital edema, loss of facial hair and eyelashes, and dry scaly skin were observed and worsened during the next 3 months. Food intake was unaltered, but all monkeys lost weight. The hematological alterations were decreased hemoglobins and hematocrits by 6 months in all animals and, prior to death, slight increase in albumin and decrease in glutamic-pyruvic transaminase levels. Four monkeys died between 7 and 11 months from the start of TCDD exposure, and all four developed anemia, thrombocytopenia, and leukopenia prior to death. One survivor developed the last two symptoms at 12 months, and all three survivors continued to lose hair and show periorbital edema after exposure ended. Additional observations at necropsy included ascites, edema and numerous hemorrhages in many organs, abnormal nail growth, lymph node atrophy, bile duct distention, degeneration or atrophy of bone marrow and lymphopoietic tissues, and cellular hypertrophy and hyperplasia of epithelia, metaplasia of mucus-secreting cells, including sebaceous glands, and keratinization of hair follicles and adjacent sebaceous glands. 19. Allen, J. R. , and Carstens, L. A. (1967) Light and electron microscopic observations in Macaca mulatta monkeys fed toxic fat. Amer. J. Vet. Res. 28(126):1513-1526^ The effects of toxic fat, which was lethal to exposed chickens, were studied in monkeys. Rhesus monkeys were fed diets containing from 0.125 to 10% toxic fat. Monthly analyses of blood for hematologic and biochemical parameters, liver biopsies and body weights were evaluated. Gross and light and electron microscopic examinations were performed when death occurred. Mean survival times were related to dose and varied for 91 days for groups fed the 5% and 10% toxic fat diets to 445 for the group fed 0.125% toxic fat. The clinical and pathologic findings were the same for all groups. Alopecia and subcutaneous edema developed 1-2 months prior to death. Edema appeared on the lips and eyelids, then included the face and finally the whole body. Anorexia and diarrhea occurred, along with ascites, hydrothorax and hydropericardium. Hematologic changes included decreased erythrocyte and leukocyte counts. Total serum protein levels decreased. Cardiac dilation, myocardial hypertrophy and edema, reduced hematopoiesis, reduced spermatogenesis, blood vessel degeneration, focal necrosis of the liver and gastric ulcers occurred to exposed monkeys. Detailed descriptions of these findings are provided at the light and electron microscopic levels. No evidence of acne was observed and the thymus was not examined. The authors concluded that hepatic parenchymal cells, endothelium and myocardium are the most sensitive tissues to the toxicity of toxic fat. At the time of publication, the authors were unaware of the identity of the toxic components in toxic fat, which have been identified subsequently and include several dioxins, including TCDD. �20. Allen, J. R., and Van Miller, J. P. (1978) Health implications of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in primates. In Pentachlorophenol, K.R. Rao, ed. (New York: Plenum Publishing Co.) pp. 371-379. [Review article. ] 21. Allen, J. R., Van Miller, J. P., and Norback, D. H. .(1975) Tissue distribution, excretion, and biological effects of [ C] tetrachlorodibenzo-p-dioxin in rats. Fd. Cosmet. Toxicol. 13:501-505. The tissue distribution, clearance, and toxicity of TCDD was studied in the rat, Male Sprague-Dawley rats (40 per group) were administered 50 ug/kg [ C}-TCDD in corn oil by gastric intubation or vehicle only. After 1-21 days, 5 rats per group were killed, tissues were weighed, and portions were prepared for light and electron microscopy and analyzed for radioactivity. Hepatic microsomal enyzmes were assayed and liver homogenates were analyzed for RNA, DNA, and protein. Of 10 rats per group that were not scheduled to be killed at specified times after TCDD exposure, half had died within 25 days and the survivors had lost an average of 76 g of 200 g initial body weight. Anorexia did not occur and all hematologic and blood chemistry tests were normal. During the first 3 days, 25% of the dose of radioactivity was excreted in feces and 1-2% per day was excreted thereafter. By day 21, 4.5% of the dose had been excreted in urine. Histological changes included lipid droplets and proliferation of the smooth endoplasmic reticulum of the liver and a paucity of cortical thymocytes. Grossly, liver enlargement and thymic atrophy were observed. The levels of radioactivity were 6-7 times higher in the liver than in other tissues. The levels in the liver remained constant for the first 2 weeks and dropped by 50% in the subsequent week. Most of the hepatic radioactivity was associated with the raicrosomal fraction. The fat had the second highest concentration of radioactivity and the skin accounted for the second highest portion of the body burden. The half-life of TCDD clearance was estimated at 16-21 days. Liver homogenates from exposed animals had decreased protein content and decreased esterase, glucose-6-phosphatase and aromatic hydroxylase activities. The authors concluded that the proliferated endoplasmic reticulum was the site of TCDD localization and that hypoactivity of the associated enzymes occurred. The authors also concluded that TCDD tissue distribution contrasted that of polychlorinated biphenyl, concentration of TCDD in fat remained constant while the fat depots were depleted, and the concentration of TCDD in the liver declined substantially. 22. Allred, P. M., and Strange, J. R. (1977) The effects of 2,4,5trichlorophenoxyacetic acid and 2,3,7,8- tetrachlorodibenzo-p-dioxin developing chicken embryos. Arch. Environ. Contatn. Toxicol. 64:8-8. ()4349 The effects of 2,4,5-T and TCDD on chicken embryo viability and liver weight were evaluated. Test compounds were administered in acetone on �into the airspace of eggs and on day 18 the eggs were opened, embryos were examined for viability and weighed. Livers were weighed and the DNA and RNA contents were analyzed. The LI>en values were 133.1 mg of 2,4,5-T per kg of egg weight 2.4 x 10 rag/kg for TCDD. TCDD contamination of 1.8 ppm in the LD,.n dose of 2,4,5-T would be required to account for the effect of 2,4,5-T. A synergistic effect was observed on embryo viability when the 2 compounds were administered together. The slopes of the probit plots of the data for each compound were substantially different, indicating (along with the synergistic effect) that the compounds probably have different modes of action. Liver weights relative to egg weights were significantly increased in 2,4,5-T treated eggs but not in TCDD-treated eggs. The hepatic nucleic acid levels did not increase in relation to increases in liver size, and did not support the hypothesis that hyperplasia accounted for increased liver size, but rather suggested that hypertrophy from increased lipid content causes liver cell enlargement. The changes in RNA and DNA content were examined at 1 dose of 2,4,5-T and of TCDD, a dose-related response has not been demonstrated and concomitant changes in the vehicle controls complicate the interpretation of these results. 23. Althouse, R., Tomatis, L., Huff, J., and Wilbourn, J. (1979) Chemicals and Industrial Processes Associated with Cancer in Humans. International Agency for Research on Cancer, World Health Organization, Lyon. pp. 46-47. [Review article.] 24. Altom, J. D., and Stritzke, J. F. (1973) Degradation of dicamba, picloram and four phenoxy herbicides in soils. Weed Sci. 21(6):556-560. The authors determined the degradation rates of 2,4-D, 2,4,5-T, picloram, and several other pesticides in three soils. Two of the samples were forest soils and one sample was a grassland soil, but all of similar composition. Collections were made of the top 2.5 cm of soil from the three sampling areas. Each soil sample was mixed and 130 g aliquots were placed in styrofoam cups. The cups were placed in a growth chamber and kept moist to allow for good microbial growth before pesticide application. Herbicides were applied to triplicate soil samples at 4.79 ppm. Soil samples treated with 2,4-D, or 2,4,5-T were removed at days 0, 5, 10, 20, and 40 for gas chromatographic analysis, while soils treated with picloram were sampled on days 0, 20, 40, 80, and 100. The degradation of 2,4-D was the most rapid of the three herbicides with a half-life of 4-5 days. Degradation of 2,4,5-T occurred more slowly, the half life being 14 days in grassland soil and 21-24 days in forest soil. Picloram was the most persistent of the three herbicides. No half-life could be calculated, because degradation was less than 50% by the end of the experiment at 100 days. �25. Aly, O.M., and Faust, S.D. (1964) Studies on the fate of 2,4-D and ester derivatives in natural surface waters. J. Agric. Food Chem. 12:541-546. [Background material.] 26. American Council on Science and Health. March, 1981. 16 pp. The health effects of 2,4,5-T. [Background material.] 27. American Legion (1980) Statement before the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants. Sept. 22, 1980. 4 pp. [Testimony.] 28. Andersen, K. J., Leightty, E. G., and Takahashi, M. T. (1972) Evaluation of herbicides for possible mutagenic properties. J. Agric. Food Chem. 20(3):649-656. The authors evaluated the mutagenicity of cacodylic acid, 2,4-D, dalapon, diquat, diuron, monuron, picloram and 2,4,5-T in four in vitro mutagenicity assays in bacteria and viruses. The test systems used were: 1) eight strains (unspecified) of Salmonella typhimurium that revert from histidine dependence to histidine independence when exposed to a mutagenic chemical; 2) T, bacteriophage in Escherichia coli B cells that form morphologically distinguishable mutant plaques when exposed to a mutagen; 3) and 4) mutants of T, bacteriophage, AP 72 and N17 that revert to the wild type with mutagen treatment. The j^. typhimurium assay was carried out as a "spot test" in which the herbicide to be tested was added as a liquid or as crystals to the surface of the agar plate. No metabolic activation system was used, therefore only direct acting mutagens could be detected. Incubation of the test compound in bacteriophage assays was in liquid suspension followed by plating. All of the herbicides tested were negative in the 4 assays. Several omissions, however, detract from the quality of the experiment: number of replications of experiments and platings were not reported; strains of S. typhimurium used were omitted; concentration of compounds tested Tn S. typhimurium was not included; only 1 test concentration of herbicide in the bacteriophage assays were reported; and no criteria for positive result were defined by the authors. �29. Anderson, D. , McGregor, D. B., and Purchase, I. F. (1976) Dominant lethal studies with paraquat and diquat in male CD-I mice. Mutat. Res. 40:349-358. The authors tested diquat in a dominant lethal test in CD-I mice. Male mice 10-12 weeks old (15 per treatment group) were given 5 daily oral doses of 0.1, 1,00, or 10.00 mg diquat in 0.5% Tweeji 80/kg body weight. The high dose, 10 mg/kg, was determined to be the maximum tolerated dose for these animals by a range finding experiment. Negative controls received 10 ml 0.5% Tween 80 per kg body weight. Positive control groups (15 animals per treatment) received either 5 daily oral doses 100 mg ethyl methane sulfonate in water per kg body weight or one intraperitoneal dose of 200 mg cyclophosphamide in water. Each treated male was placed with 2 virgin 8-10 week old females. After 7 days, males were transferred to cages containing a second batch of females. This process was repeated for a total of 8 weeks to insure that all phases of sperraatogenesis were represented by matings. Fifteen or 16 days after caging females with males, the female mice were killed and each uterus examined for live implantations, early fetal deaths, and late fetal deaths. Data were analyzed according to analysis of variance and chi-square analysis. Number of males which successfully mated was not affected by any of the treatments according to a chisquare test. Similarly, numbers of females that became pregnant showed no statistically significant variations using a chi-square test. This indicates that diquat did not decrease fertility at the doses tested. The total implants per pregnant female and number of early deaths per pregnancy were compared statistically using an analysis of variance and Dunnett's t-test. No statistically significant differences between negative controls and the diquat treated groups were observed. However, the positive control groups had statistically significant decreases in total implants per pregnant female and increases in the number of early deaths per pregnancy. Furthermore, no statistical difference in percentage of total implants recorded as early deaths per pregnancy and percentage induced pre- and post-implantational dominant lethality was observed between negative control and the diquat treated groups. Positive controls did, however, show statistically significant differences. Late deaths were distributed evenly throughout all treatment groups. The authors concluded that there was no evidence of a dominant lethal effect in male CD-I mice orally administered diquat. 30. Appleman, S. M. (1980) Public information aspects of Agent Orange. Presented at the 2d Continuing Education Conference of Agent Orange, Washington, DC, May 28-30. [Background material.] 31. Aquatic Chemistry. An introduction emphasizing chemical equilibria in natural waters, ed. by Stum, W. and Morgan, J.J. (New York: Wiley-Interscience, 583 pp., 1970) [Background material.] 10 �32. Arkhipov, G. N., and Kozlova, I. N. (1974) Research on the carcinogenic properties of the herbicide amino salt 2,4-D. Vop. Pitan. 5:83-84. Forty-five female and 120 male nonparous rats, weighing 80-100 g at the beginning of the study, were administered amino salt 2,4-D at .10 LD,-n (dose not stated) in their feed. A control group received the same diet without 2,4-D. In the test group, two rats developed tumors at 1 year 11 months; one was a fibroadenoma of the mammary gland, and the other was a hemangioma in the abdominal cavity and mesentery. Four months later, one control rat also developed fibroadenoma of the mammary gland. Three groups of approximately 100 female OSVA X S57/VL mice, weighing about 18-20 g at the beginning, were also tested. One group received .10 LD of the herbicide in their food. A second group was administered two arops per week of a 10 percent solution in acetone of the amino salt 2,4-D on a cutoff part of the skin between the shoulder blades. The third group of mice was kept as a control. Testing was continued through the animals' lifetimes, and no tumors developed. Approximately 200 OSVA X S57/VL mice in two groups were also tested throughout their lifetimes (1 year, 8 months). Neither the age nor the weight of the mice at the time testing began is stated. In the first 3 weeks of testing, both groups were pretreated on the skin between the shoulder blades, with 1 drop of a .5 percent benzol solution of 3-methycholanthrene. One group was then treated with a 10 percent solution in acetone of amino salt 2,4-D (purity not stated). Among the test group mice 17.7 percent developed papillomas; no changes were noted in the control group. The authors concluded that these results provided evidence of oncogenic activity in amino salt 2,4-D. However, serious reporting deficiencies in the account of this study make it difficult to assess accurately the conclusions of the authors. 33. Associate Committee on Scientific Criteria for Environmental Quality. (1978) Phenoxy Herbicides—Their effects on environmental quality. (Ottawa, Canada: National Research Council of Canada) 440 p. [Review article.] 34. Aulicino, F., Bignami, M., Carere, A., Conti, G., Morpurgo, G., and Velcich, A. (1976) Mutational studies with some pesticides in Aspergillus nidulans. Mutat. Res. 38(2):138. [Abstract, only.] 35. Axelson, 0., and Sundell, L. (1974) Herbicide exposure, mortality and tumor incidence: An epidemiological investigation on Swedish railroad workers. Work Environ. Health 11(1):21-28. The authors performed an historical prospective study of 348 Swedish railroad workers exposed to combinations of herbicides to determine the effect of amitrol and/or phenoxy acid exposure on mortality or tumor 11 �incidence. The authors compared 4 groups of non-exclusive cohorts exposed to combinations of non-specified herbicides with amitrol and/or phenoxy acid. The authors also performed an age matched case-control retrospective study of phenoxy acid and amitrol to "attempt to evaluate a possible dose response relationship" and to investigate "possible co-carcinogenicity from smoking." The authors calculated the relative risk using age and sex-specific death and cancer incidence for expected rates. Data were analyzed using the Poisson distribution and onetailed P-values. The authors observed an excess tumor incidence in the amitrol and combinations groups and an increased but not significant tumor incidence in the phenoxy acids and combinations groups. Smoking was found to have a co-carcinogenic influence. The authors failed to indicate specifically what herbicides and what combination of each herbicide was used. The authors used a limited population of 348 workers and compared them in non-exclusive groups where an individual could be in more than one group. It is not possible to determine the effects of specific herbicides on the population due to the confounding factor of mixed and multiple exposures to other herbicides. 36. Axelson, 0., and Sundell, L. (1977) Phenoxy acids and cancer. Lakartidningen 74(35):2887-2888. [Review article.] 37. Axelson, 0., Sundell, L., Anderson, K., Edling, C., Hogstedt, C., and Kling, H. Herbicide exposure and tumor mortality: An updated epidemiological investigation on Swedish railroad workers. Unpublished paper. 10 pp. A followup historical prospective study of 348 railroad workers with occupational exposure of greater than 45 days to phenoxy acid and/or assitual was performed. The population was followed from 1957 to 1978. Data were abstracted from death certificates and the National Central Bureau of Statistics. The expected number of total deaths and deaths from different tumors were calculated by multiplying the person years of observations by the cause and age-specific national death rates for males during the respective calendar years. The statistical methodology utilized was the Poisson distribution with one tailed p-values. An over mortality of 6 tumors was observed among persons with combined exposure to phenoxy acid and amitrol where 1.78 were expected. Based upon three and six observed cases, the risk ratio calculated for amitrol was 1.5 and 1.9 for phenoxy acid. Persons exposed to phenoxy acid were found to have a higher rate of tumor incidence than that demonstrated in an earlier study. This increase was associated with the increased latency period which allowed for tumor development. The authors were non-specific in detailing which and what amount of herbicides the workers were exposed to. Therefore, it is not possible to determine a specific causal association between tumor development/mortality and a specific herbicide. 12 �38. Baader, E. W., and Bauer, H. J. (1951) Industrial intoxication due to pentachlorophenol. Ind. Med. Surg. 20(6) .-286-290. Ten cases of chloracne in workers who were exposed to trichlorophenol and tetrachlorophenol are described. The plant produced pentachlorophenol only between August, 1948 and February, 1949; experimentation with trichlorophenol production in this plant was mentioned also, but the dates were not indicated. The first case of chloracne began in December, 1948 and the last case began in May, 1949. Four of the 10 men had severe lesions and 4 others had moderate lesions in the summer of 1950, and only 1 case had cleared. The skin lesions were typical of those reported for other cases of chloracne. Neuralgic pain of the lower extremities was experienced by 8 of the 10 workers, although definite signs of neuritis were not observed during neurological exams. Four workers complained of heart disorders, including palpitation and shortness of breath, 4 complained of libido, and 4 of bursitis of the elbow. Laboratory tests, which were only performed after the maximum clinical symptoms had subsided, revealed nothing significant. Irritation of the eyes and respiratory tract during exposure was attributed to pentachlorophenol. The authors concluded that the symptoms experienced by the workers resulted from their occupational exposure to a noxious substance, and proposed pentachlorophenol to be responsible for the observed toxicity. From other industrial incidents that occurred since this report was published, TCDD produced during the trichlorophenol manufacturing experiments is a likely candidate, at least as the cause of chloracne. 39. Baars, A. J., Jansen M., and Breimer, D. D. (1978) The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-pdioxin on flutathiane s-transferase activity of rat liver cytosol. Biochem. Pharmacol. 27:2487-2494. The enzyme kinetics of TCDD-induced glutathione-S-transferase of rat liver cytosol are described. Male SPF-Wistar rats were administered 10 ug/kg TCDD in dioxane-sesame oil (1:40) intraperitoneally on days 1 and 7. On day 13, the animals were killed and the hepatic microsomal supernatant fraction was prepared and assayed for GSH S-transferase activity using 3 different substrates, 1,2-butylene oxide (BOX), l-chloro-2,4-dinitro-benzene (CDNB), and styrene oxide (STOX). TCDD treated rats and control rats gained weight at the same rate and liver weights were not altered by TCDD treatment. GSH S-transferase activity per gm body weight with each of the 3 substrates were significiantly elevated in TCDD pretreated rats, although enzyme activity per mg hepatic protein were not significantly increased over vehicle controls. TCDD pretreatment resulted in increased apparent V values with the substrates BOX and CDNB, an increased apparent Km value with CDNB and a decreased Km with STOX. The effects of methylcholantrene and phenobarbital pretreatments on GSH S-transferase activity were also described. The authors concluded that TCDD probably induced different types of GSH S-transferases than those induced by the other 2 agents. 13 �40. Backstrotn, J. The phenoxy acid problem in Sweden. In Chlorinated Phenoxy Acids and Their Dioxins, ed., C. Ramel (Ecol. Bull. no. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 108-121. [Review article.] 41. Babbitt, B,, Risch, S., Choffnes, E., Kalis, J., Zupanic, M., et al. (1973) Effects of 2,4,5-T on human chromosomes. Genetics 71(Suppl):53. [Abstract, only.] 42. Bage, G., Cekanova, E., and Larsson, K. S. (1973) Teratogenic and embryotoxic effects of the herbicides di- and trichlorophenoxyacetic acids (2,4-D and 2,4,5-T). Acta Pharmacol. Toxicol. 32:408-416. The teratogenic effects of 2,4,5-T and of 2,4,5-T with 2,4-D were evaluated in mice exposed during days 6 to 14 of gestation. Daily subcutaneous doses of 110 or 50 mg/kg of the butoxy ethyl ester of 2,4,5-T with less than 1 ppm dioxin contaminant or of 2,4-D and 2,4,5-T (2:1; both as acids) were administered in dimethyl sulfoxide. On day 18 of gestation, the numbers of dead, resorbed and viable fetuses were determined. Living fetuses were examined for gross malformations. Skeletal defects were observed in alizarin red-stained fetuses and internal malformations were observed in fetuses fixed in Bouin's fluid. The only adverse effect observed in the pregnant dams was necrosis at the site of injection. All treatment groups had more litters with at least one resorbed fetus (75-100%) than the vehicle control group (58%). Substantial increases in the percentage of resorbed implantation sites and incidence of cleft palate and decreases in fetal weights were produced in the treatment groups. The incidences of rib and vertebral malformations and of hemorrhages increased 2-3 times in the high dose groups, compared to the vehicle controls. Hemorrhages were observed in various locations, but not in the gastrointestinal tract. No cystic kidneys were observed. The data was not analyzed statistically. The authors indicated that the doses of phenoxyacetic acid compounds that were teratogenic in animals were extremely high compared to levels of likely human-exposure. 43. Baker, D. L., Ramsey, F. K., and Sylvester, E. P. (1953) Suspecting poisoning of dogs from eating grasses treated with 2,4-D. North Am. Vet. 34:194. The effects of oral administration of 2,4-D to dogs is reported. Grass was sprayed with 4 Ib per acre of 2,4-D butyl ester and was harvested 2 days later. The grass was then mixed with dog food and administered to 2 dogs for 2 days. No decrease in food consumption or change in behavior was noted in either animal over the next 2 days. The dogs were then administered 500 mg/kg 2,4-D butyl ester in a gelatin capsule. No changes were seen in 1 dog over the next 82 days or in the 14 �other dog over the 4-day period after feeding or at necropsy at this time. The authors concluded that suspected poisoning of dogs admitted to clinics was unlikely to be caused by ingestion of 2,4-D. 44. Baldwin, R. C., Pasi, A., MacGregor, J, T., and Hine, C. H. (1975) The rates of radical formation from the dipyridylium herbicides paraquat, diquat, and morfamquat in homogenates of rat lung, kidney, and liver: an inhibitory effect of carbon monoxide. Toxicol. Appl. Pharmacol. 32(2):298-304. The formation of diquat-derived free radicals was determined in various rat tissues in vitro. The lungs, liver, and kidneys of male SpragueDawley rats were removed, homogenized, centrifuged, and incubated in the presence of 10 M diquat. Formation of diquat free-radicals was monitored by an increase in absorbance (at 435 nm). The inhibitory effects of carbon monoxide, 1.0 mM potassium cyanide, and 1.0 mM SKF 525-A on radical formation were tested. The rates of radical formation were 5.4, 2.1, and 1.1 mol/liter/min/mg protein for lung, lung, and kidney, respectively, and in the presence of carbon monoxide were 0.2 to 0.4 times these rates. No other inhibitors altered the rate of radical formation. The formation of radicals from paraquat and morfamquat were also described. The authors concluded that the organ selectivity observed for the 3 cations studied did not result from differences in radical formation by different tissues and that diquat radicals were the only radicals formed by a carbon-monoxide-sensitive enzyme system. 45. Bamesberger, W. L., and Adams, D. F. aerosol and gaseous 2,4-D compounds. (1966) An atmospheric survey for Advan. Chem. Ser. 60:219-227. [Background material.] 46. Bankowska, J. , and Bojanowska, A. (1973) Induction effect of diuron and linuron in short-term chronic experiments in adult rats. Rocz. Panstu. Zabl. Hyg. 24(4):493-505. [Foreign language.] 47. Barnett, A. P., Hauser, E. W., White, A. W., et al. (1967) Loss of 2,4-D in washoff from cultivated fallow land. Weeds 15:133-137. [Background material.] 48. Barr, M. Jr., Keller, C. A., Rogan, W. J., and Kline, J. (1979) Summary of the workshop on perinatal and postnatal defects and neurologic abnormalities from chemical exposures. Ann. N.Y. Acad. Sci. 320:458-472. [Review article.] 15 �49. Barsotti, D. A., Abrahamson, L. J., and Allen, J. R. (1979) Hormonal alterations in female rhesus monkeys fed a diet containing 2,3,7,8tetrachlorodibenzo-p-dioxin. Bull. Environ. Contam. Toxicol. 21(4-5):463-469. Steroid hromone levels were monitored in female monkeys that were administered TCDD-contaminated diets for 9 months. Female rhesus (Macaca mulatta) monkeys (8 per group) were administered feed containing 500 ppt TCDD or a control diet for 9 months. One treated monkey had an unsuccessful mating history and was eliminated from the study; all other treated animals had previously given birth to normal offspring. Food intake was monitored daily. Hematologic parameters and blood chemistries were assayed monthly. Serum progesterone and estradiol levels were monitored for a complete menstrual cycle, 1, 3, and 6 months after the experimental diets were started. Mating with control males was initiated 6 months after TCDD began and was conducted during the appropriate time of the menstrual cycle. Pregnancy was confirmed by a mouse bioassay for monkey chorionic gonadotropin and by palpation of the uterus (Morphological changes were reported separately; see Allen et al, 1977). The length and duration of the menstrual cycle was not altered in monkeys fed TCDD for 6 months. Of the treated animals that remained in the study, 3 had reduced progesterone levels and 2 of these 3 had reduced estradiol levels as well, compared to pretreatment levels; 2 had anovulatory patterns and 2 had normal patterns. All of the control monkeys and 1 treated monkey with normal steroid patterns conceived and delivered healthy infants. After the treated.infant was weaned, normal menstruations were re-established. The second monkey with normal steroid patterns and the monkey with only decreased progesterones both conceived and then aborted (on days 46 and 62). Both of these monkeys showed prolonged implantation bleeding. The other monkeys failed to conceive. The 2 monkeys with both steroid levels diminished also had intense and sporadic menstruations. Except for 1 anovulatory monkey and the monkey that delivered normally, all other treated monkeys died between the seventh and twelfth months of the experiment. After the anovulatory monkey was returned to a control diet, both steroid patterns returned to normal and this animal was bred and delivered a normal infant. Food consumption of the treated monkey that delivered normally was not reduced. The authors concluded that reproductive toxicity from TCDD was reversible in the monkey that survived TCDD intoxication and suggested that the effects of TCDD on steroid hormones were related to placental and maternal hepatic microsomal enzyme effects of TCDD. 50. Bashirov, A. A. (1969) Health conditions of workers producing herbicides of amine salt and butyl ester of 2,4-D acid. Vrach. Delo. 10:92-95. A summary is given of a survey of the health status of 292 workers from a factory that produces the amine salt and butyl esters of 2,4-D. The group surveyed included 248 men, 264 of the 292 workers were between 21 and 40 years of age, and 194 were exposed for less than 36 years. Frequent headaches that intensified at the end of the work-day were 16 �reported by 63% of the workers, 33% reported dizziness, 27% had respiratory problems during physical stress, 18% reported pain in the region of the heart, 52% complained of digestive problems, and 61% had functional disturbances related to the nervous system. An objective health examination was conducted on 50 workers who were free of illnesses prior to employment in the 2,4-D plant and the results were compared with those of a control group of 20 subjects who were not exposed to toxic substances. Cardiovascular parameters of the experimental group (but not the control group) were reported. Parameters related to the digestive system were examined and revealed compromised function in the exposed group, although the methods used and data were not reported. The author concluded that employment in herbicide plants can lead to diminished health of the digestive and cardiovascular systems. 51. Basrur, S. V. , Fletcher, R. H., and Basrur, P. K. (1976) In vitro effects of 2,4-dichlorophenoxyacetic (2,4-D) on bovine cells. Can. J. Comp. Med. 40(4):403-415. The effects of 2,4-D treatment in vitro on bovine fetal muscle cells were examined histologically. Limb muscles from 2.5-4.5 month bovine fetuses were cultured as cell suspensions for 1 day and then aliquots of cells were cultured in the presence of 2 or 20 mg/liter 2,4-D (99.6% purity). After a 24-96 hour culture period, cell counts were taken and cells were stained for light microscopy. Counts of differentiating cells (multinucleated cells with linear stuations), degenerating cells (interphase cells with pyknotic nuclei or abnormal mutotic cells) and polyploid cells (with over double the nuclear size of the majority of cells in the field) were counted. In control cultures, fibroblast-like cells on day 1 became confluent by day 2-3 and resembled fusing myoblasts by day 3. Cells treated with the higher dose of 2,4-D contained more differentiating cells and were in more advanced stages of differentiation (more nuclei per myotube) than controls, at 72 hours. The numbers of metaphase cells, polyploid cells and degenerating were higher in cells treated with either concentration of 2,4-D than in control cells. Abnormalities of treated cells included unipolar and tripolar spindles of mitotic cells, malorientation of the mitotic apparatus and mitosis of myoblasts undergoing myogenesis. The authors concluded that 2,4-D exerted a mitostatic effect on dividing cells and a mitogenic effect on postmitotic myogenic cells. 52. Bastomsky, C. H. (1977) Enhanced thyroxine metabolism and high uptake goiters in rats after a single dose of 2,3,7,8-tetrachlorodibenzo-pdioxin. Endocrinology 101:292-296. The effect of TCDD pretreatment on thyroid hormone metabolism and excretion were studied in the rat. Rats were administered 25 ug/kg TCDD in acetone-corn oil (1:9) by,stomach tube 9 days prior to measuring biliary excretion. L-[ I]T, was injected intravenously and bile was collected from cannulated bile ducts for 1 hour. Plasma was collected at the end of the hour. All samples were counted for 17 �125 radioactivity [ I] triiodothyronine ( , ) was administered by the same T, procedure and bile was collected for 30 minutes. I was administered to a group of rats and 4 hours later blood was collected and analyzed for T, by competitive protein-binding analysis and T_ by.radioimmunoassay. Thyroids were removed, weighed and analyzed tor I uptake. TCDD,treatment produced a 10.fold stimulation in biliary clearance rate of [ 1]T,. Excretion of [ ^]T, increased 4-fold, with no change in bile flow rate. The proportion of biliary radioactivity as T, glucuronide increased from 50 to 84% with less as T. and I after TCDD treatment. T., excretion in bile was unaltered. Both thyroid weight and I uptake increased and both weight of TCDD treated rats decreased. Serum T, was half of control and T_ and TSH levels were elevated in serum of treated rats. Sephadex uptake of [ llT, from the serum of treated rats was lower than control uptake. The author concluded that TSH-stimulated thyroid secretion produced in treated rats caused elevated T, levels and that T, itself has intrinsic hormone activity rather than serving solely as a precursor. 53. Batten, C. R. (1978) The phenoxy herbicides in California, a history of the conflict. California Forest Protective Assn. 12 pp. [Background material.] 54. Bauer, H., Schulz, K. H., and Spiegelberg, V. Gewerbepath. 18:538-555. (1961) Arch. The clinical states are described for 9 of 31 workers, 5 years after they were involved in the manufacture of 2,4,5-T in Hamburg, Germany and had developed chloracne. Nine workers described were those who remained under medical care, 5 years after exposure ended. The derraatologic symptoms of the workers resembled chloracne in workers exposed to various chlorinated aromatics that were not present in the Hamburg factory. Chloracne was resistent to treatment and left severe scarring in some patients. All patients complained of pronounced tiredness and weakness in the legs, although polyneuropathy was not diagnosed in any of the cases. Six patients had abnormal electroencephalograms, 5 had signs of vegetative over excitability, 5 had gastriontestinal disorders, 3 cases of pathological liver biopsies were reported. All patients exhibited signs of psychological disorders, characterized by depression, fatigue, increased irritability, loss of appetite, loss of potency, and decreased mental efficiency. These symptoms were revealed by the patients and some were confirmed by physicians who conducted interviews and psychological tests of these patients. Some of these tests were briefly described. The authors summarized the dermatologic and psychologic disturbances common to chloracne that appeared in 100 workers in 3 work populations: the group described in this report, a second group of workers manufacturing 2,4,5-T in Middle-Rhein (not described further in this report) and a group of 17 workers exposed to perchlorinated naphthalenes. The authors reviewed previous reports on animal testes that identified TCDD as the probable acnegenic agent in the 2,4,5-T manufacturing process. 18 �The authors concluded that small amounts of manufacturing byproducts were shown to produce severe industrial disease, which upon identification of the etiology, was eliminated by changing manufacturing processes. 55. Baughman, R., and Meselson, M. (1973) An analytical method for detecting TCDD (Dioxin): Levels of TCDD in samples from Vietnam. Environ. Health Perspect. 5:27-35. [Background material.] 56. Baur, J. R., Bovey, R. W., and McCall, H. G. (1973) Thermal and ultraviolet loss of herbicides. Arch. Environ. Contain. Toxicol. 1(4):289-302. The authors studied the thermal and ultraviolet losses of 2,4,5-T and picloram under laboratory conditions. Solutions of 100 (u)g 2,4,5-T and picloram-free acids were prepared in acetone and evaporated in beakers. Aqueous solutions of 100 (u)g K salts of the two herbicides at pH 4.0 and 7.0 were treated in the same manner. All six herbicide preparations were exposed to temperatures of 30°C and 60°C for 7 days in the dark to determine thermal loss. Measurement of thermal loss included both degradation and volatilization of the compounds. The authors made no attempt to separate the two components. Residual herbicide after 7.days was determined by gas-liquid chromatography. Thermal losses of the free acids of 2,4,5-T and picloram at 60°C were 55% and 24%, respectively. No losses of other herbicide were observed at 30°C. Exposure of the potassium salts of 2,4,5-T to 30°C at pH 7.0 resulted in a 31% loss of herbicide. No significant losses were observed at 30°C for picloram at pH 4.0 and 7.0 or 2,4,5-T at pH 7.0. At 60°C, only 2,4,5-T at pH 7.0 was thermally degraded. The herbicides were also exposed to two levels of long wave (356 nm) UV irradiation: low intensity in which the light source was 98 cm from the herbicide and high intensity in which the light source was 3 cm from the herbicide. The wavelength used simulates sunlight. Three concentrations of each herbicide were used: 10, 100, and 1,000 (u)g/dish. The potassium salt herbicides were at pH 7.0. After 7 days of low intensity exposure, the free acid and salt of both 2,4,5-T and picloram were photo-decomposed. Results of high intensity UV exposure were complicated by the fact that at such an intensity the exposure temperature reached 60°C. However, degradation of the herbicides was greater with high intensity UV irradiation than with 60°C alone. The significance of such laboratory studies to a real world situation is not known. However, the authors concluded that picloram and 2,4,5-T are degraded by both temperature and UV light under laboratory conditions. 19 �57. Baur, J. R., and Bovey, R. W. (1974) Ultraviolet and volatility loss of herbicides. Arch. Environ. Contam. Toxicol. 2:275-288. [Background material.] 58. Beale, M. G., Shearer, W. T., Karl, M. M., and Robson, A. M. Long-term effects of dioxin exposure. Lancet 1(8014):748. (1977) The clinical follow-up examinations of three females exposed to TCDD are described. Following the spraying of a farm horse arena with waste-oil contaminated with TCDD, a 6-year-old female, who was in the area daily, developed nosebleeds, headaches, diarrhea, and painful micturition. Her urine was found to contain protein and red and white blood cells, and she had a contracted edematous bladder, a renal caliceal diverticulum, and a systolic murmur. Following identification of TCDD on the arena floor, the patient was removed from exposure, and her symptoms resolved within one week. The child's mother and 10-year old sister also reported symptoms of abdominal pain, diarrhea, and headaches shortly after the TCDD was sprayed. Five years later all three females received thorough examinations, which included neural, renal, hepatic, and thyroid tests, and no health problems were identified except for persistence of the heart murmur in the younger child. No other human exposures during this incident were mentioned. Many animals on the farm, including horses, dogs, cats, and birds, died. 59. Beatty, P. W. (1977) Studies of the metabolism and possible mechanisms of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dissertation Abstracts International Section B; Physical Sciences and Technology 38(8) tSeeSHT [Abstract, only.] 60. Beatty, P. W., Letnbach, K. J., Holscher, M. A., and Neal, R. A. (1975) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on mammalian cells in tissue cultures. Toxicol. Appl. Pharmacol. 31(2):309-312. The effects of TCDD on cell growth and morphology of cultured cells are reported. Cultures of human epithelial carcinoma cells (HeLa), normal mouse (Balb-3T3) fibroblasts, virus-transformed mouse cells (SV101), normal human diploid fibroblast cells (FS), and normal human lymphocytes (NC-37) were cultured in the presence of 10~ M TCDD (98% purity). After 4 days of treatment, cells were trypsinized and counted. Aliquots of lymphocyte suspensions were removed during the treatment period, and counted or grown to confluency and prepared for electron microscopy. ,FS and SV101 cells were cultured in medium that contained 0.32 ug/ml [ C]-TCDD (99% purity), lyzed with sodium hydroxide and the cell lysates were counted for radioactivity. TCDD treatment did not alter cell growth rate for any of the 4 cell lines examined and produced no changes in cell morphology. The TCDD concentration for FS cells was 310 ppb and for SV101 cells was 637 ppb; the corresponding 20 �media concentrations were 2.3 and 5.2 ppb, respectively. The authors concluded that cultured cells took up TCDD, but their growth was unaffected by the compound. 61. Beatty, P., and Neal, R. A. (1978) Factors affecting the induction of DT-diaphorase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Pharmacol. 27(4):505-510. Induction of diaphorase activity in various tissues of the rat and guinea pig by TCDD is examined. Sprague-Dawley rats and Hartley guinea pigs were administered TCDD (98+% purity) in olive oil, intraperitoneally. Tissues were removed 12 hours to 9 days after TCDD treatment. Liver cells were fractionated into mitochondria, microsomes and cytosol. The mitochondrial fraction was assayed for NADH-diaphorase activity and the other 2 fractions for NADPH-diaphorase activity. Brain, heart, lung, kidney, spleen, and thymus tissues were assayed for NADPH-diaphorase activity as well. Diaphorase activity in all rat extraembryonic tissues except the brain was elevated 2 days after 50 ug/kg TCDD was administered compared to vehicle controls, and all levels were higher at 9 days than at 2 (thymic atrophy at 9 days precluded analysis of thymic levels). A dose of 45 ug/kg TCDD induced rat hepatic microsomal and cytosol levels 12-24 hours later and actinomycin D pretreatmen blocked this effect. A dose of 50 ug/kg TCDD to male rats or 25 ug/kg to females produced induction of diaphorase levels in all 3 subcellular fractions up to 17 times the control value by 21 days after treatment. Doses of 0.6-6.0 ug/kg TCDD to guinea pigs failed to produce a dose-related elevation in diaphorase levels of liver tissue over 7 days or of extrahepatic tissues in 48 hours, except for a doubling in lung activity. A dose of 1000 ug/kg of octachlorodibenzo-p-dioxin was required to produce a significant elevation in rat hepatic diaphorase levels 7 and 16 days after treatment. The authors concluded that the role of enzyme induction by TCDD in eliciting toxic effects is unknown, since TCDD did not induce diaphorase activity in the species most sensitive to TCDD toxicity. 62. Beatty, P. W., and Neal, R. A. (1976) Induction of DT-diaphorase activity of rat liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 37(1):189. [Abstract, only.] 63. Beatty, P., and Neal, R. A. (1976) Evidence for a role for DTdiaphorase induction in the toxicity of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD). Pharmacologist 18(2):211. [Abstract, only.] 21 �64. . Beatty, P. W.» Vaughn, W. K., and Neal, R. A. (1978) Effect of alteration of rat hepatic mixed-function oxidase (MFO) activity on the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 45(2)-.513-519. The toxicity of TCDD was compared in male and female rats, in rats of different ages, and in rats given stimulators or inhibitors of mixedfunction oxidase (MFO) activity. TCDD was administered to SpragueDawley rats intraperitoneally in olive oil at 4 doses, including the LDeQ. Adult male rats were given 20-80 ug/kg doses, adult females were given 10-60 ug/kg and weanling males were given 5-50 ug/kg. The L^CQ values of TCDD for each group of rats given TCDD or pretreated with testosterone (100 mg/kg/day for 3 days), phenobarbital (50 mg/kg/day for 3 days), 3 methylcholanthrene (40 mg/kg), TCDD (5 ug/kg), cobaltous chloride (20 mg/kg/day for 3 days), hemin (20 mg/kg/day for 4 days), or piperonyl butoxide before TCDD and for 2 days afterwards) intraperitoneally. Hepatic microsomes were isolated from pretreated and nontreated rats and several MFO activities were measured. Adult male rats had 2-3 fold more MFO activity than adult females or weanling males (cited from another publication) and LD-0 value was 60 ug/kg, compared to 25 ug/kg for both adult females and weanling males. Castrated males showed significantly lower aminopyrine demethylase and aniline hydroxylase activities and a lower TCDD LDcn than non-castrated males, while testosterone treated females had higher enzyme levels and a higher LD,.~ than non-testosterone-treated females. Pretreatment of male weanling rats with phenobarbitai or TCDD increased both aniline hydroxylase and benzpyrene hydroxylase activities as well as the TCDD LD-_ values, while 3-methylcholanthrene pretreatment elevated only the benzopyrene hydroxylase activity and the L^cn. Pretreatment with cobaltous chloride, hemin, or piperonyl butoxide caused decreases in the TCDD L , , which were not statistically significant and the first 2 compounds D.. significantly decreased benzpyrene hydroxylase activity, as well. The authors concluded that the inverse relationship between MFO activity and susceptibility to TCDD toxicity probably indicates that TCDD was metabolized by the MFO system to a less toxic metabolite. Other explanations exist and verification of this conclusion awaits a demonstration of TCDD biotransformat ion by MFO enzymes. 65. Beck, J. Emotional struggle on Agent Orange. Journal, Akron, Ohio. March 24. (1980) Akron Beacon [Editorial.] 66. Beck, S. (1981) Assessment of adult skeletons to detect prenatal exposure to 2,4,5-T or Trifluralin. Teratology 23:33-55. The teratologic effects of 2,4,5-T in the mouse are described and the frequencies of 88 skeletal deviations in exposed offspring at 2 months of age are presented. CD-I mice were administered 20 or 100 mg/kg 2,4,5-T (less than 0.5 ppm dioxin contaminant) in corn oil, by oral gavage on days 6 to 15 of gestation. Litter size, mating efficiency, 22 �frequency of gross malformations, and fetal and neonatal deaths were recorded. At 62+2 days of age, mice were killed and skeletal structures were visualized in cleared, alizarin red stained specimens. Bones were cleared of surrounding tissue, dried, and weighed. Some measurements and observations were made from photographs of the stained articulated skeletons. Each specimen was examined for a total of 88 skeletal variants and the frequencies of each variant were determined for the 2,4,5-T-treated groups and compared to groups of untreated and of vehicle controls. Significant reductions in numbers of live newborns, mean birth weights, mating efficiency, and increased numbers of stillborns and of malformations occurred in the higher dosage 2,4,5-T group, compared to the untreated controls. Nineteen of the 88 skeletal parameters had significantly different incidences in the high dosage group than in the untreated group; (17 had increased frequencies) for the low dosage group and vehicle control group, none and one, respectively, of the variants occurred more frequently than in the untreated controls. The average difference in incidence of each variant for the treated group from control incidence was 23.7%. Types of variants that occurred more frequently in the treated group were presence of an interfrontal bone, parted frontals, fusion of the frontals, variants in cervical vertebrae, reduction in the number of cardal vertebrae, and loss of the prominant dorsal spine of the second thoracic vertebra. The authors concluded that the skeletal variant assay system they used provides a useful method for postnatal detection of prenatal exposure to potentially noxious substances. Considering the small increases in postnatal frequency of variants, the non-specific nature of the variant and the high and maternally toxic doses of 2,4,5-T administered to produce these effects, the potential of this method as a device for predicting exposure is dubious. 67. Becker, D. (1973) The effect of folate overdose and of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on kidney and liver respectively of rat and mouse embryos. Teratology 8:215. [Abstract, only.] 68. Becroft, D. M. 0. (1977) The safety of the herbicide 2,4,5-T. Zealand Medical Journal. 35-36. New [Abstract, only.] 69. Benedetto, A. V., and Taylor, J. S. Cutis 21:483-488. (1978) Porphyria cutanea tarda. [Review article. ] 70. Benes and Scram. (1969) Ind. Med. [Not available.] 23 38:50-62. �71. Benigni, R., Bignami, M., Carere, A., Conti, G., Conti, L., Crebelli, R., Dogliotti, E., Gualandi, G., Novelletto, A., and Ortali, V. A. (1979) Mutational studies with diquat and paraquat in vitro. Mutat. Res. 68:183-193. The authors studied the mutagenicity of diquat in several in vitro mutgencity assays, ranging from prokaryotic bacterial cells to eukaryotic mammalian cells. This battery included: 1) Ames test, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, with and without metabolic activation, measuring reversion from histidine dependence to histidine independence; 2) forward mutation test in S_. typhimurium measuring induction of 8-azaguanine resistance in a sensitive strain; 3) DNA repair test in _S_. typhimurium measuring preferential killing of DNA repair deficient cells compared to DNA repair proficient cells; 4) forward mutation test in Aspergillus nidulans measuring induction of 8-azaguanine resistance and methionine suppresion; 5) induction of recessive lethals in A. nidulans; and 6) induction of unscheduled DNA synthesis in a human cell line. A thorough Ames test was performed. Diquat dissolved in water was tested at 0.25, 0.5, 1, 2.5, and 5 (u)g/plate in triplicate platings. No increase in numbers of revertants was observed at any dose, either with or without metabolic activation. Both positive and negative controls were present. An adequate dose range was selected, i.e., doses ranged over several logs and included a toxic dose. A less complete experimental design was employed in the repair test in J3_. typhimurium. Killing of bacteria was measured in strains TA 1538, a repair deficient strain, and TA1978, a repair proficient strain. Diquat was tested at only one concentration (10 (u)g/plate), with and without metabolic activation. Data for positive controls but not negative controls were reported. Diquat had a relative activity (zone of killing of repair deficient/zone of killing of repair proficient) of 1.07, without metabolic activation, and 1.91, with metabolic activation, which was classified as a weak positive response. A forward mutation test measuring 8-azaguanine resistance was also performed in S_. typhimurium strains hisG46, TA92, and TA1535. Diquat induced dose-dependent forward mutations at 0.1 and 0.25 (u)g/plate, without metabolic activation. Toxicity, evident by decreased numbers of revertants, was present at the highest concentration tested, 0.5 (u)g/plate. Results from both positive and negative controls were included. Diquat also induced forward mutations in the haploid strain 35 of Aspergillus nidulans, a yeast, in both a plate incorporation assay and a liquid suspension test. No exogenous metabolic activation system was included. Results from 5-8 replicate plates were analyzed by a student's + test. Diquat was tested for 8-azaguanine resistance in three separate experiments at 20, 100, 500, and 1,000 (u)g/plate, at 400 and 500 (u)g/plate, and at 600 and 700 (u)g/plate. Statistically significant increases (p less than 0.01) in 8-azaguanine mutants were induced by diquat treatment at concentrations of 400-600 (u)g/plate. However, in the methionine suppression plate assay using A_. nidulans, no increase in mutants was observed at concentrations of diquat from 2-100 (u)g/plate. Concentrations higher than 100 (u)g/plate were toxic. In the liquid suspension test, diquat was tested at 10 mg/ml in both the 8-azaguanine resistance assay and the methionine suppression 24 �assay. Six replicate plates were scored per treatment level. Treatment times for both assays were 0, 1, 2, and 4 hours. At 1, 2, and 4 hour treatment times, diquat significantly increased the induction of 8-azaguanine resistant mutants (p less than 0.001) and at 2 and 4 hours treatment time, significantly increased (p less than 0.001) numbers of mutants in the methionine suppression assay. Data for negative controls, but not positive controls were reported. Another strain of ftidulans, Po, which is diploid, was used as the indicator organism Tn a test for induction of recessive lethals. Diquat (10 mg/ml) was tested at treatment times of 2, 4, and 8 hours. Recessive lethals increased with increasing treatment times from 4 to 24 times that of the negative control. No positive controls were reported. To complete the test battery, induction of unscheduled DNA synthesis by diquat was studied in human cell cultures. The cell line EUE, an epithelial-like cell derived from human skin and muscle explants was used as the indicator cell. Diquat was tested at 20, 100, 1,000 and, 2, 000 (u)g/ml for 1 hour of treatment time. Cells were labeled with [ H] thymidine for 4 hours. Results for both positive and negative controls were reported. Incorporation was detected by autoradiography. The mean number of grains per nucleus was 4-6 times higher in diquat-treated cells than in negative controls. Grains per nucleus increased with increasing diquat concentrations. This study represents a thorough in vitro test battery to study the mutagencity of diquat and revealed much valuable information. Diquat was mutagenic in forward mutation tests in bacteria and yeast, DNA repair tests in bacteria and mammalian cells, and a recessive lethal test in yeast. Reverse mutation tests in bacteria were negative. The authors suggested the reason for these results may be that diquat is unable to induce frameshift or base-pair substitution type mutations, but may be able to cause other damage such as deletions, strand breaks or cross links at the gene level. Diquat was shown to cause damage at the chromosomal level (recessive lethal test) and to damage DNA (unscheduled DNA synthesis). 72. Bennett, P. N. , Davies, D. S., and Hawkesworth, G. M. (1976) In vivo absorption studies with paraquat and diquat in the dog. Br . J . Pharmacol. 58(2):284P. [Abstract, only.] 73. Berkley, M. C. , and Magee, K. R. (1963) Neuropathy following exposure to a dimethylamine salt of 2,4-D. Arch. Int. Med. 111:133-134. The health condition of a 39-year old farmer that had been exposed to 2,4-D is described. Two weeks prior to the clinical examination, the farmer had been spraying weeds with a liquid solution of 2,4-D. The solution contained about 40% 2,4-D that was 98 to 99% pure. He had repeatedly used his hands to unplug the sprayer, so came into direct contact with the solution. Initial symtpoms were constant prickling of the fingers, toes and midabdomen. During the first week, this progressed into a general numbness of the extremities accompanied by muscular aches and stiffness. Despite the numbness, however, the 25 �patient complained of hypersensitivity to touch. By the second week, manual dexterity and coordination had becoma unstable. Clinical examination revealed hypoactive biceps, triceps and ankle reflexes, hand uncoordination, and sensory impairment in the distal extremities. Laboratory tests were conducted, including blood analysis, x-rays, urinalysis, and EEC, but all results were within normal limits. The patient was treated with daily doses of thiamine hydrochloride and vitamin supplementation in the diet. The authors concluded that cutaneous exposure to 2,40D produced a primary sensory neuropathy in this patient. 74. Berlin, A. (ed.) (1976) Proceedings of the expert meeting on the problems raised by TCDD pollution. Commission of the European Communities, Milan, 1976. 178 p. [Background material.] 75. Berndt, W. 0., and Koschier, F. (1973) In vitro uptake of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by renal cortical tissue of rabbits and rats. Toxicol. Appl. Pharmacol. 26(4):559-570. Some characteristics of 2,4-D and 2,4,5-T accumulation by renal cortical slices from the rabbit and rat are described. ..Renal cortical slices were incubated in the presence of C-2,4,-D or C-2,4,5-T and the uptake of radioactivity by the tissue was determined at the end of the incubation period. The time-course for uptake and effects of various inhibitors and nutrients added to the incubation medium were established. Data were expressed as the ratio of slice to medium (S/M) radioactivity. The concentrations of herbicides used were 10 M (except kinetic competition studies). The S/M ratios for 2,4-D uptake were 37 for rabbit slices and 16 for rat slices and for 2,4,5-T were 59 and 30, respectively. Uptake of both compounds by rabbit slices was linear for 5 hours, and for both species was substantially reduced in the presence of 2,4-dinitrophenol, iodoacetamide, probenecid, and nitrogen. Uptake of both substances studied in rabbit slices was increased significantly in the presence of acetate, pyruvate, and lactate was significantly decreased by alpha-ketoglutarate and succinate and was unaltered by glucose and citrate. Lactate and succinate caused the same effects in rat slices as rabbit slices, but acetate was ineffective in altering uptake by rat slices. The only difference in uptake between slices from male and female rats was increased 2,4,5-T uptake by slices from males (S/M ratio = 23) than by those from females (S/M ratio = 16). From Lineweaver-Burke plots of data for both species and compounds in the presence of para-amino hippurate, transport was concluded to be mediated by a carrier, as Michaelis-Menton-type kinetics were involved. Uptake of both herbicides, studied in liver slices, approached a steady state and was inhibited by 2,4-dinitrophenol and, for only 2,4,5-T, by probenecid. The authors concluded that both phenoxy acids were transported by the 26 �classical organic anion transport process but non-specific tissue binding also occurred because a small degree of uptake occurred in the presence of metabolic inhibitors that blocked the transport process. 76. Bernstein, J. Z. (1980a) Statement before the Committee on Veterans' Affairs, United States Senate. [Agent Orange] Feb. 21, 1980. 16 pp. [Testimony.] 77. Bernstein, J. Z. (1980c) Statement at the Public Meeting of the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants, Sept. 22, 1980. 10 pp. [Testimony.] 78. Berry, D. L., DiGiovanni, J., Juchau, M. R., Bracken, W. M., Gleason, G. L., and Slaga, T. J. (1978) Lack of tumor-promoting ability of certain environmental chemicals in a two-stage mouse skin tumorigenesis assay. Res. Coromun. Chem. Pathol. Pharmacol. 20(1):101-108. The authors tested TCDD and several other chemicals for tumor promoting ability in the mouse skin tumorigenesis assay. Female CD-I mice 6-8 weeks old, were shaved *2 days before treatment. Only mice in the resting stage of the hair cycle were used in the experiment. Preshaven mice (30 per group) received 200 nmol dimethyl benzanthracene (DMBA) in 0.2 ml acetone topically. One week after DMBA initiation, the positive control group received TPA in 0.2 ml acetone. Test groups received a topical application 0.1 (u)g TCDD in acetone/mouse. TCDD was one of several compounds tested. The concentration of TCDD was determined by a range-finding histological study. At 2 (u)g/mouse TCDD increased intrafollicular epidermis from 1 to 4 layers, and caused severe gastrointestinal damage, and death in 30% of the animals. At 1 (u)g/mouse TCDD caused only slight increases in intrafollicular epidermis, which the authors did not consider significant. Each compound was applied twice weekly for 30 weeks. Papillomas and carcinomas were observed weekly until the end of the experiment at 30 weeks. The positive control, DMBA, followed by TPA promotion, produced 8.1 papillomas/mouse, while the negative control, TPA promotion without the initiator DMBA, produced 0.03 papillomas/mouse. DMBA followed by TCDD promotion and TCDD alone did not induce any papillomas at the concentration tested. The authors also noted skin rashes on animals (numbers unspecified) treated. From these data, it appears that TCDD at the concentration tested does not act as a tumor promoter. 79. Berry, D. L., Slaga, T. J., DiGiovanni, J., and Juchau, M. R. (1979) Studies with chlorinated dibenzo-p-dioxins, polybrominated biphenyls, and polychlorinated biphenyls in a two-stage system of mouse skin tumorigenesis: Potent anticarcinogenic effects. Ann. N.Y. Acad. Sci. 320:405-414. [Review article.] 27 �80. Berry, D. L., Slaga, T. J., Wilson, N. M., Zachariah, P. K., Namkung, M. J., Bracken, W. M., and Juchau, M. R. (1977) Transplacental induction of mixed function oxygenases in extra-hepatic tissues by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Pharmacol. 26(15):1383-1388. The effects of TCDD on extra-hepatic maternal and fetal enzyme levels are described. Pregnant Sprague-Dawley rats were administered a single dose of 0.2-6.0 ug/kg TCDD in corn oil, intraperitoneally on day 17 of gestation. On day 20 of gestation, maternal and fetal tissues were removed and microsomes were prepared and analyzed for various enzyme activities. Dose-related increases in aryl hydrocarbon hydroxylase activities were demonstrated for fetal tissues, including lung, kidney, and skin, placenta 1, and maternal lung, kidney, adrenal and liver. Formation of some metabolites of benzpyrene, identified by high pressure liquid chromatography were increased in TCDD-treated fetal and maternal tissues. Epoxide hydratase activity in maternal and fetal lung and fetal skin were induced almost 3 fold by TCDD. In vitro covalent binding of [ H]-benzpyrene to DNA was enhanced by tissue horaogenates of TCDD-treated rats. The authors concluded that maternally administered TCDD produced a potent broadspectrum induction of carcinogen-transforming enzymes in extra-hepatic fetal tissues. 81. Berry, D. L., Zacharian, P. K., Namkung, M. J., and Juchau, M. R. (1976) Transplacental induction of carcinogen hydroxylating systems with 2,3,7,8 tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 36(3):569-584. Effects of TCDD administration to pregnant rats on microsomal enzyme activities and histopathology of fetal and maternal tissues is described. Pregnant female rats were administered 0.2-6.0 ug/kg TCDD in corn oil, intraperitoneally on day 17 of gestation. On day 20 of gestation, maternal livers and adrenals, placentas, and fetal livers were prepared for light and electron microscopy and microsomes were isolated and assayed for various enzyme activities. Spectral analyses of microsomal chromosomes were performed on fetal and maternal samples. Maximum induction of fetal aryl hydrocarbon hydroxylase (AHH) was produced by 2.5 ug/kg of TCDD and this dose was used for the remaining experiments. Ring and N-hydroxylation of N-2-fluorenylacetamide (FAA) were elevated 3-20 fold in maternal liver and 30 to 300 fold in fetal liver; Placental levels were also elevated. AHH activity was increased 6-100 fold in fetal livers after TCDD treatment and 15 fold in adult liver; placental levels were also elevated. Adrenal AHH levels were not induced by TCDD and cytochrome P-450 levels increased in hepatic tissues and decreased in placenta. Formation of benzpyrene metabolites, formed by liver homogenates and identified by high-pressure liquid chromatography, was increased by TCDD in fetal and maternal tissue. After TCDD treatment, formation of doils relative to phenols was favored. Cytotoxic effects were evident in fetal hepatic tissue and in placental and maternal liver and included cellular necrosis and placental hemorrhages, the adrenals were not altered ultrastructural changes in fetal liver cells included enlarged mitochondria and rough 28 �endoplasmic reticulura and glycogen deposits. The authors concluded that the fetus contained the genetic potential to respond to hep'atic mixed-function enzyme inducers, although such fetal responses are not usually elicited. 82. Berwick, P. (1970) 2,4-Dichlorophenoxyacetic acid poisoning in man. JAMA 214(6):1114-1117. The clinical symptoms of 2,4-D poisoning are described. A 46-year-old farmer ingested an estimated 30 ml of Knoxweed containing 7200 mg of 2,4-D, 14,700 mg of S-ethyldipropyl thiolcarbamate and 150 mg of epichlorohydrin. The formulation caused oral burning, flushing of the face, gastritis, vomiting, muscular and chest pains, and eventually respiratory, renal, and neurological disorders which subsided after several weeks. Muscular effects included- fibrillary twitching, myotonia and muscle weakness, but no cardiac complications. Skeletal muscle damage was evident from elevated serum enzyme levels, and myoglobinuria. No peripheral neuropathy was ever observed, although the patient complained of insomnia and was moderately depressed. The authors concluded that the toxic effects observed in the patient were produced by 2,4-D because the normal serum cholinesterase level was not consistent with poisoning by S-ethyl-dipropylthiolcarbamate, a cholinesterase inhibitor, and only a small amount of epichlorohydrin was ingested, 83. Bethel, J. S., Turnbull, K. J., Briggs, D., and Flores, J. Military defoliation of Vietnam forests. American Forests 81(1): 26-30, 27-61. [Review article. ] 84. Bezuglyi, V. P., Kokina, K. V., Komarova L. T., Sivitskaia, I. I., Ilina, V. I., Gorskaia, N. Z. (1979) Clinical manifestations of long term sequelae of acute poisoning with 2,4-dichlorophenoxy acetic acid. Gig. Tr. Prof . Zabol. 3:47-48. The clinical symptoms of 11 workers 2 years after they were acutely exposed to 2,4-D is described. All workers were female with a mean of 42.4 years of age and with 17-32 years of field crop working experience. Exposure was described as slight for 8 workers, moderate for 2, and severe for one. Symptoms described by the patients included periodic headaches, general weakness and rapid fatigue, dizziness in 7 patients, the appearance of circles in the visual field, numbness and pain in hands and legs (8 cases) and neck (2 cases), increased irritability (5 cases) and loss of memory (2 cases). In 7 cases, headaches were accompanied by heart palpitations, shortness of breath, cooling of the extremities, profuse sweating, increased urination and sudden weakness. Dysnia occurred in 5 patients and heart irregularities and pain, in 6. Physical examinations revealed a soft grade 1 murmur in all patients and a systolic murmur in 4. Five patients had elevated 29 �arterial blood pressure and 1 had low blood pressure. Various neural parameters were altered in the subjects; the methodology used for the tests and any tests in which no deficiencies were identified were not given by the authors. Gynecological examinations revealed menstrual cycle upsets in 9 of the women, but the parameters measured and types of upsets were not explained. Monocytosis, lymphositosis, hypercholesterolemia lowered blood phospholipid levels and lowered activities of leukocyte oxidizing enzymes were mentioned. Chronic conjunctivitis in 8 subjects and toxic hepatitis in 2 subjects, remained 1 1/2 years after the exposure. The patients were treated with various therapies and all were reported to have been discharged in satisfactory condition. The authors concluded that the principal disorder in these patients was pathology of the nervous system, involving polyneuritis and dystonia. This report the reader with a dim clinical picture of the consequences of an unknown dose of 2,4-D by an unknown route because no description of the conditions of 2,4-D exposure nor an adequate description of the clinical findings, beyond summary statements is provided. 85. Bibliographic References: Dioxins. (1980) Environmental Sciences Laboratory, Mt. Sinai School of Medicine of the City University of New York, Jan. 18. 48 pp. [Bibliography.] 86. Bignami, M., Aulicino, F., Velcich, A., Carere, A., and Mopurgo, G. (1977) Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans. Mutat. Res. 46:395-402. The authors studied the mutagenicity of dalapon, picloram, tordon, and other pesticides in Aspergillus nidulans. Three types of mutagenic events were studied: forward point mutation from 8-azaguanine sensitivity to 8-azaguanine resistance in haploid strain 35; mitotic crossing over in the diploid strain P; and mitotic nondisjunction in the diploid strain P. In all three, a spot test was employed; that is, the chemical to be tested was added to a filter disc which was placed on top of the agar surface of the test plates which contained the indicator organism. 8-Azaguanine resistance is measured by growth of colonies on medium containing the inhibitor. Both mitotic crossingover and mitotic nondisjunction are determined by growth of mutant colonies on medium containing fluorophenylalanine. The colonies derived by crossing-over are pale green, while the nondisfunctional ones are yellow, and the tester strain colonies are white. In the forward mutation test, all three chemicals were negative according to the authors. Doses used, negative and positive controls, were not reported by the authors. No numerical data were presented. Ability of the compounds to induce nondisjunction was also tested by a nonselective test in which fluorophenylalanine was omitted. The endpoint was measured by screening colonies arising on complete medium for colored sectors. The authors reported testing a range of doses for each of the pesticides but only reported on the highest dose tested. 30 �Dalapon, up to 0.8 rag/ml, picloram, up to 0.8 mg/ml, and tordon, up to 1.5 mg/ml, did not induce nondisjunction. Postive controls were not reported. 87. Bignatni, M., and Crebelli, R. (1979) A simplified method for the induction of 8-azaguanine resistance in Salmonella typhitnurium. Toxicol. Lett. 3:169-175. The authors tested diquat and several other chemicals in a forward mutation assay and in the standard reverse mutation assay (Ames test) in Salmonella typhimurium. In the standard Ames test, mutagenicity is detected by induction of histidine independent revertants in a histidine dependent strain. The forward mutation assay measures the induction of 8-azaguanine resistant mutants in a sensitive strain. Diquat dissolved in water was tested at 0.1-1.0 ug/plate in a plate incorporation assay in strains his G46.TA92, TA1535, TA1538, TA100. Data reported were averages of triplicate plates; at least three experiments were performed. The herbicide was not mutagenic in the standard Ames test. In the forward mutation assay, however, diquat induced significant dose dependent increases in 8-aza guanine mutants in strains TA1535 and TA92. , 88. Bimber, D. L., Boening, R. W., and Sharma, M. L. (1976) Respiratory stress in yellow perch induced by subtoxic concentrations of diquat. Ohio J. Sci. 76(2):87-91. The authors measured respiratory stress in fish exposed to diquat. Two-year-old yellow perch were and obtained from an area untreated by herbicide at Chautaugua Lake, NY. Buccal catheters were implanted in the fish to monitor respiratory distress by the "cough reflex." Fish were placed in test chambers, and after a 24-hour acclimation period diquat was added at 1 and 5 ppm. Continuous recording of the cough response was made for 48-72 hours. One ppm diquat was, according to the authors, the dose commonly applied to the lake for weed control. Local concentrations of up to 10 ppm were often found in the lake immediately after spraying. At both concentrations of diquat tested, statistically significant (p less than 0.05) increases in cough frequency were observed. The authors concluded that toxic effects from diquat occur in yellow perch at concentrations routinely used in weed control. 89. Binns, W,, and Balls, L. (1971) Non teratogenic effects of 2,4,5-trichlorophenoxyacetic acid and 2,4,5-T propylene glycol butyl ester herbicides in sheep. Teratology 4:245. [Abstract, only.] 31 �90. Bionetics Research Laboratories, Inc. (1968a) Evaluation of carcinogenic, teratogenic, and ip.utagenic activities of selected pesticides and industrial chemicals. Vol I: Carcinogenic study. NTIS PB 223159. A compilation of experimental results presented in Innes et al. (1969). 91. Bionetics Research Labs, of Litton Ind. (1968b) Progress report on program of carcinogenesis studies Vol. 2 - Teratogenic study in mice and rats. National Cancer Institute. The teratology of 2,4-D and 4 esters, 2,4,5-T, monuron, diuron and a proprionate were studied in mice and 2,4,5-T was studied in rats. A/Ha, AKR, C3H, and C57BL6 strains of mice and BL6AK hybrid mice were administered test compounds in doses of 22-215 mg/kg subcutaneously in DMSO or orally by gavage in honey-water (1:1) on days 6-15 to AKR mice and days 6-14 for all other mice. Additional studies of 2,4,5-T were conducted in C57BL6 mice given 113 mg/kg 2,4,5-T in DMSO subcutaneously on days 9-17 and in rats orally administered 5-47 mg/kg 2,4,5-T in honey on days 10-15 or 22-47 mg/kg on days 6-15. On day 19 for AKR mice and day 18 for all other mice, fetuses and placentas were removed and weighed. The amount of amniotic fluid was calculated from the difference between gravid uterus weight and the sum of the placentas, fetuses, and uterine muscle weights. Fetuses were examined grossly and then fixed in Bouin's solution and examined for visceral anomalies or stained with alizarin red and examined for skeletal anomalies. Maternal liver and body weight were determined and fetal liver weights of mice treated with 2,4,5-T on days 9-17 were determined. Postnatal examinations were performed on some mice by recording body weights of neonates at 1 and 8 days of age and performing the gross examinations of 1 day-old neonates and necropsy and skeletal examinations on 8-day-old neonates. Resulto of treated animals were compared to vehicle controls. 2,4-D administration caused a decrease in fetal weight and an increased number of abnormal fetuses. No consistent, reproducible, dose-related effects were observed for the butyl, isopropyl or isooctyl esters of 2,4-D and no adverse effects followed administration of the methyl or ethyl esters. 2,4,5-T caused an increase in malformations and increases in fetal and maternal liver weight in rats and mice. Both monuron and diuron produced increases in the numbers of abnormal C57BL6 fetuses and in mortality of G3H fetuses. Alpha-(2,5-dichlorophenoxy)-propionic acid treatment yielded an increase in fetal mortality and decrease in fetal weight. The teratogenicity of a total of 48 compounds was reported in this study. The authors ranked the teratogenic potential of each compound and concluded that 2,4,5-T is probably dangerous, 2,4-D and 2,4-D isooctyl ester are potentially dangerous but need further study, 2,4-D butyl ester is fetotoxic but probably not teratogenic, 2,4-D isopropyl ester, monuron, diuron, and alpha-(2,5-dichlorophenoxy) propionic acid are unclassifiable, needing further study and the methyl and ethyl esters of 2,4-D are essentially inactive. The postnatal study was considered inconclusive because the numbers of mice of the same strain given the same treatment were too low for statistical evaluation, and in some groups no neonates survived for 8 days. The causes of death for these 32 �neonates were unknown, but cannibalism was identified as contributing to fetal loss. 92. Bionetics Research Laboratories, (1968c) Evaluation of carcinogenic, teratogenic, and mutagenic activities of selected pesticides and industrial chemicals. Vol. 3V MutagenicT Stud"y~NTIS PB-223 161. The authors evaluated the mutagenicity of 2,4-D isooctyl ester, diuron, monuron, 2,4-D and several other pesticides. Mutagenicity tests were not specified, and no results were presented for these compounds. 93. Birmingham, D. J. (1964) Occupational dermatology: Skin Feb. 1964:38-42. current problems. [Review article.] 94. Biro, P. (1979) Acute effects of the sodium salt of 2,4-D on the early developmental stages of bleak, Alburnus alburnus . J. Fish. Biol. ' The authors studied the effects of 2,4-D on fertilized eggs and freshly hatched larvae of bleak, a freshwater fish. Fish were exposed to concentrations of 0, 25, 50, 100, 200, 400, 800, 1,600 and 3,200 rag/liter 2,4-D in triplicate. Mortality in eggs and larvae was monitored every 2 hours. At 12 hours, LC,-^ for embryos was 159.4 mg/liter 111.2 mg/ liter. At 48 hours, the LD fpr embryos was 12.9 mg/liter and for larvae 51.6 mg/liter. Effects of 2,4-D exposure included slowing or stopping of development of embryos, malformations of hatched larvae, and behavioral changes. The authors concluded that to prevent injury to littoral fish the concentration of 2,4-D in shallow freshwater lakes should not exceed 0.5-1.0 mg/liter. 95. Bjorklund, N. E., and Erne, K. (1971) Phenoxy-ac id- induced renal changes in the chicken. I. Ultrastructure. Acta Vet . Scand . 12:243-256. The subacute toxicities of 2,4-D and 2,4,5-T were studied in the chicken. Cornish chickens (15 per group) were administered 1,000 ppm 2,4-D triethanolamine salt (commercial preparation) in drinking water 1,000 ppm 2,4,5-T triethanolamine salt (a combination of technical and analytical grades) in drinking water from 5 days of age to 7 months. After 20 weeks of exposure 2 chickens from each treatment group were removed from exposure for the subsequent 8 weeks. Beginning at 18 weeks of age, 2 control chickens were administered 1,000 ppm 2,4-D for 7 months. Chickens from each group were killed after 14-201 days of exposure and tissues were removed; kidney tissue was prepared for light and electron microscopic examinations and several tissues were analyzed for herbicide levels. The only clinical sign of toxicity was general weakness, observed after both treatments. Decreased food intakes and 33 �growth rates as well as increased relative kidney and liver weights of the treated groups were not compared statistically to the result for the control group. Grossly, the kidneys were enlarged in both treatment groups but not in the chickens that were returned to (control) water for 8 weeks. No other gross pathological changes occurred. Hypertrophy of the renal proximal convoluted tubules was observed. This lesion, and the renal ultrastructural changes were more severe after 2,4,5-T than 2,4-D exposure and occurred in chickens first exposed at 8 weeks of age, but not in chickens fed 2,4-D and then returned to water for 8 weeks prior to death. Ultrastructural changes included the presence of nuclear bodies and electron opaque regions of the nucleus, circular arrays and fusion of mitochondria and more microbodies than controls. Tissue levels reported for the liver, lung, kidney, gastrointestinal contents and egg yolk revealed relatively high kidney and egg levels for both phenoxy acids. The authors concluded that both compounds produce severe lesions in the proximal convoluted tubule, the region of the kidney probably involved in phenoxy acid excretion. * 96. Bjorklund, N., and Erne, K. (1966) Toxicological studies of phenoxyacetic herbicides in animals. Acta Vet. Scand. 7:364-390. The acute and subacute toxicities of 2,4-D were studied in pigs, calves, rats, and chickens. The triethanolamine salt or the potassiumsodium salt of 2,4-D, or 2,4-D-butyl ester or the triethanolamine salt of 2,4,5-T orally-as a single dose or repeatedly for up to 51 doses. Some animals were fed diets with 500-1000 ppm 2,4-D amine chronically. Peak plasma levels, general condition, and for some experiments histopathology and organ weights were determined. Two calves were given each salt and ester of 2,4-D as single doses of 100 mg/kg with a 4 week period separating each administration. The 2,4-D amine salt also was administered at doses of 50 and 200 mg/kg to each calf. The peak plasma levels of 2,4-D were 100, 150 and 250 ug/ml after 50, 100, and 200 mg/kg doses, and 24 hours after treatment, plasma levels were 10, 20, and 70 ug/kg, respectively. Dysphagia, anorexia, and hind muscle weakness were observed within the first 4 days of treatment. Two pigs were given a single 50, 100, 500 or 1000 mg/kg dose of 2,4-D amine. Peak plasma levels ranged from 120 to 525 ug/ml 2,4-D and levels after 24 hours were between 35 and 580 ug/ml in accordance with the magnitude of the dose given. Vomiting, severe muscle weakness and general depression occurred in pigs given the 2 highest doses. Autopsy 2-3 days after dosing revealed pneumonia and renal and hepatic congestion and, at all doses, gastro-intestinal irritation. Subacute doses of 2,4-D administered to pigs caused lesions of the gastrointestinal tract, lungs, and excretory organs. Plasma levels of 200-400 ug/ml after 24 hours were observed in affected pigs while pigs tolerating 50 mg/kg/day doses (the lowest given) had 24 hour plasma levels of 10 ug/ml. After chronic exposure to 500 ppm 2,4-D in feed for up to 12 months, 3 of 5 pigs developed locomotory disturbances, 2 had elongated lateral hoofs, and all showed depressed growth rates. A pregnant sow was fed 2,4-D during her seventh pregnancy which resulted in her first abnormal litter. One stillborn and 10 deaths of 15 neonates occurred 34 �within 1 day of parturition. Maternal anorexia was evident throughout the pregnancy. The surviving piglets were maintained on a diet of 500 ppm 2,4-D and developed locomotory disturbances. Small alterations occurred in blood analysis. The results of blood analyses showed.small alterations, but no statistical analysis was performed. Albuminuria occurred in treated animals and multinucleated hepatocytes were observed. No acute toxicity was observed in rats given 100 mg/kg 2,4-D or 2,4,5-T. No reproductive effects were observed in 5 rats given diets with 1,000 ppm 2,4-D. No malformations or toxicity was observed in the offspring. Chronic treatment of the 22 offspring with dietary levels of 1,000 ppm 2,4-D for 2 years resulted in decreased growth rate and food intake and an increased mortality which was not associated with any specific cause. Toxic effects of 2,4-D administered to chickens involved the same organs as those affected in other species. Plasma levels of 2,4-D were reported for rats and chickens after several dosage regimens, but no clinical signs were observed in these animals. The authors concluded that a threshold plasma level of 200-300 ug/ml of 2,4-D in the pig and calf is required to produce clinical symptoms. The butyl ester of 2,4-D was not absorbed well after oral administration. 2,4-D was concluded to produce moderate chronic toxicity. 97. Bjorn, M. K., and Northen, H. T. (1948) Effects of 2,4-dichlorophenoxyacetic acid in chicks. Science 108:479-480. A brief report of the toxicity of 2,4-D to chickens is given. White Rock chicks (5 per group) were administered from 0.3 to 280 mg/kg acid equivalent of 2,4-D alkanolamine salt by gavage 3 times per week for 4 weeks or a single dose of 380 or 765 mg/kg of the 2,4-D salt. Body weights were recorded of chicks that received subacute exposure, and mortality and gross pathology were described after acute exposure. None of the subacute doses caused a significant decrease in body weight compared to controls that did not receive 2,4-D. The lower acute dose produced no mortality and the higher dose produced 100% mortality. Hemorrhagic gastroenteritis, and fatty degeneration and mottling of the liver, spleen, kidney, and heart were observed at necropsy. The authors concluded that 2,4-D was not a cumulative poison, because the highest subacute dose, equivalent to 3,360 mg/kg total was ineffective while an acute dose of 765 mg/kg produced 100% mortality. The authors estimated that a 1 kg chicken would have to consume all of the 2,4-D applied to 72 sq. ft. at the rate of 1 Ib. per acre in 1-2 days to consume a lethal dose. 98. Blackman, G. E., Fryer, J. D., Lang, A., and Newton, M. The Effects of Herbicides in South Vietnam. Part B: Working papers: Persistence and disappearance of herbicides in tropical soils. National Academy of Sciences—National Research Council. NTIS Publication No. AD779025. The authors measured levels of herbicides including 2,4-D, 2,4,5-T and picloram in South Vietnam soils to determine persistence and disappearance of these chemicals. They sampled sites that had been sprayed 35 �during major herbicide operations during the war and sites that were sprayed at the time of the study. Analysis of surface and subsurface samples indicated levels of 0.007-0.08 Ib/acre of 2,4-P, 0.004-1.35 Ib/acre of 2,4,5-T, and 0.001-1.19 Ib/acre of picloram in soils sprayed during military operations. Water samples analyzed for picloram had levels of 0.07 - 0.03 ppb of water and 2.2 to 0.8 pp of dry weight of sediment, the picloram being detected in the sediment filtered from the water. Agricultural sites in South Vietnam and the Philippines were sprayed with herbicides and crops were planted for this study. It was found that Agent White (with picloram) had longer lasting effects than Agent Orange (with 2,4,5-T). both agents persisted longer in the Philippines. Areas of forest soil in the Philippines were sprayed with the herbicide and soil samples showed the levels of 1.6 Ib/acre of picloram applied decreasing to less than 0.02 Ib/acre in 31 days, and a more rapid disappearance rate for 2,4,5-T. The author notes that volitizaton, leaching, runoff, and uptake by plants are responsible for removing the herbicides unchanged from the system. Chemical breakdown by biological and nonbiological processes results in the decomposition of the herbicides within the system. The author concludes that herbicides applied in massive doses may still be present at levels that would induce phytotoxic symptoms in some species. Herbicide use during the Vietnam war did not result in making the soil "sterile." 99. Bleiberg, J., Wallen, M., Brodkin, R., and Applebaum, I. L. (1964) Industrially acquired porphyria. Arch. Dermatol. 89:793-797. The results of physical examinations of 29 workers with chloracne who were involved in the manufacture of 2,4,-D and 2,4,5-T were reported. Eleven of these workers had acquired porphyria cutanea tarda, evidenced by increased urinary excretion of uroporphyrins. In addition, hyperpigmentation of the sun-exposed areas of the head, neck, and hands was seen in 17 workers, and hirsutism, which always involved the temples, was seen in 14. The severity of both of these symptoms, unlike the severity of the porphyria, was proportional to the severity of chloracne. Neither the severity of chloracne or of porphyria corresponded to the degree of exposure to chemicals. Instead, previous liver damage seemed to predispose workers to porphyria, and adolescent acne predisposed workers to severe chloracne. The authors concluded that one of the chemical intermediates or final products in the manufacture of 2,4-D or 2,4,5-T caused both chloracne and porphyria. 100. Bodai, J., Tamas, L., and Vegh, A. (1974) Reproductive consequences of Dikonirt toxicosis in cattle. J. Hungarian Veterinar. 29(5):319-322. Reproductive toxicity is described for cattle that grazed on pastures or fed corn that was sprayed with 2,4-D. The authors described 8 incidents that involved inappropriate mating behavior and premature births after cattle were exposed to Dikonirt, comprised of 80% 2,4-D. Methods for examining herds, and in some instances, numbers of cows per herd or numbers adversely afflicted, amount of herbicide sprayed, and 36 �time between spray and consumption of sprayed foliage were not reported. The symptoms described in cattle resembled those caused by estrogens and not those reported from acute poisoning of 2,4-D in man. Pregnant cows exhibited behavioral and physiological characteristics of animals in heat and abortion occurred. Fetuses of various ages were aborted and no fetal or placental abnormalities were observed. In 2 of the 8 instances of poisoning described, anestrus and ovarial atrophy occurred justifying the slaughter of 14% to 42% of the herds. Disturbances in the timing of the normal estrous cycle were observed in nonpregnant animals. Samples of fodder and green feed were found to from 0.075 to 0.8 rag/kg 2,4-D but no indication was made of which herds had received this feed. The authors concluded that the reported reproductive effects resulted from ingest ion of 2,4-D and that sprayed fields were used for grazing without allotting enough time for degradation of herbicide to occur. In other instances, the authors attributed poisoning to low temperatures and high humidity which resulted in a decreased rate of degradation and increased persistence of the herbicide. Confounding exposure to other chemicals, other components in the herbicide preparation that was sprayed, and analysis of relevant pasture foliage for 2,4-D were not reported. The lack of similarity of the symptoms reported here with symptoms reported by others emphasizes the importance of residue levels and confounding factors to evaluate the present results. 101. Boeri, R., Bordo, B., Crenna, P., Filippini, G., Massetto, M., and Zecchini, A. (1978) Preliminary results of a neurological investigation of the population exposed to TCDD in the Seveso region. Riv. Pat. Nerv. Ment. 99:111-128. Clinical examinations were conducted of representative populations from the Seveso region of Italy to determine neurologic effects resulting from exposure to TCDD. This study was initiated 7 months after the accident that released TCDD into the surrounding regions. Populations from two zones were chosen for investigation: 470 subjects from zone A (high risk for acute exposure) and 152 subjects from zone R (low risk). The study populations included children and adults of both sexes. Each subject was examined twice, 6 months apart. Results reported here were mainly from the first set of examinations since the second sets were not completed. Medical histories were compiled for each subject prior to clinical examinations. Neurophysiologic examinations of motor nerve conduction volocities of the right ulnar and left perneal nerves, as well as an electronyogram of the uterosseus muscle of the right hand, were also conducted on each subject. Clinical symptoms of peripheral neurologic disorder included numbness and paresthesia of the extremities, muscle fatigue, decreased tendar reflexes and abnormal position sense of fingers and toes. Clinical symptoms were manifest in populations from both zone A and zone R, but a significantly higher prevalence of symptoms were evident in subjects from zone A. The zone A subjects also had a greater frequency of psychological symptoms and visual coordination problems than zone R subjects. In the 6 months between the first and second set of examinations of the zone A subjects, the neurologic condition of 12.7% of the adults and 11.1% of 37 �the children worsened or declined. Results of the condition velocity tests and electromyograms indicated no difference between zone A and zone R populations. The authors tentatively concluded that although both populations (zone A - high risk and zone R - low risk) exhibited a high frequency of neurologic disorders, the zone A population had a signficiantly greater incidence of polyneuropathy than the zone R population. These disorders were assumed to be caused by exposure to TCDD. 102. Boffey, P. M. (1971) Herbicides in Vietnam: spread devastation. Science 171:43-47. AAAS study finds wide- [Editorial.] 103. Bogen, G. (1979) Symptoms in Vietnam veterans exposed to Agent Orange. JAMA 242(22):2391. [Review article.] 104. Bonaccorsi, A., Fanelli, R., and Tognoni, G. Seveso. AMBIO 7(5-6):234-239. (1978) In the wake of [Review article.] 105. Bonderman, D. P., Mick, D. L., and Long, K. R. (1971) Occupational exposure to aldrin, 2,4-D and 2,4,5-T and its relationship to esterases. Ind. Med. 40(6):23-27. The levels of erythrocyte esterases in 20 herbicide formulators were determined and compared to levels in matched controls. Twenty workers and 20 unexposed controls matched for age (within 3 years), sex and race were studied. Seven formulators exposed to 2,4-D and 2,4,5-T were included. Blood drawn from each subject was separated into plasma and erythrocyte fractions. Erythrocyte esterases, including tributyrinase, acetylcholinesterase, and cholinesterase and plasma tributyrinase were assayed. No statistically significant differences between any enzymeactivities of the group of 2,4-D and 2,4,5-T exposed workers and their matched controls were observed. Data was also analyzed according to duration of employment and no differences between pesticide (including aldrin)-exposed workers and controls existed. 106. Bongso, T. A., and Basrur, P. K, (1973) In vitro response of bovine cells to 2,4-dichlorophenoxyacetic acid. In Vitro 8(5):416-417. [Abstract, only.] 38 �107. Bovey, R. W., and Baur, J. R. (1972) Persistence of 2,4,5-T in grasslands of Texas. Bull. Environ. Contamin. Toxicol. 8:229-233. [Background material.] 108. Bovey, R. W., Burnett, E., Richardson, C., Baur, J. R., Merkle, M. G., and Kissel, D. E. (1975) Occurrence of 2,4,5-T and picloram in subsurface water in the blacklands of Texas. J. Environ. Qual. 4(1):103-106. The authors describe the persistence and movement of 2,4,5-T and picloram in soil and vegetation and the occurrence of these herbicides in subsurface water after repeated herbicide application. The study areas were located near Riesel and Temple, Texas, and were composed of deep, dense, slowly permeable, montmorillonite clay soils. The first study area was 3.1 ha, consisting of a cattle feedlot, a cultivated area, and an area of native grasses. The area was sprayed five times in 3 years with a 1:1 mixture of 2,4,5-T triethylamine salts and picloram at 2.24 kg/ha (four times) and 1.12 kg/ha (one time). Determination of 2,4,5-T in soil was made by gas chromatography, while water samples were analyzed by gas-liquid chromatography. In the first study area, herbicide concentration in the soil profile immediately after treatment did not exceed 144 ppb 2,4,5-T or 162 ppb picloram. Concentrations in grass cover, however, were high soon after treatment, e.g. less than 30,000 ppb 3 days after the second spraying. Dissipation of the herbicide occurred rapidly. After 145 days, no herbicide was detected in grass. Similar results were observed after the 1st and 4th treatments. The authors believed that loss of pesticide was due mainly to photodegradation. Neither herbicide appeared to accumulate in soils or vegetation. Herbicide levels in subsurface water from the treated area were not significant 2 weeks, and 6, 7, 9 and 11 months after the last herbicide treatment. The second study area was a drainage lysimeter (213 cm diameter x 132 cm deep) which received a single application of 2,4,5-T triethylamine salt and picloram (1:1) at 1.12 kg/ha. No 2,4,5-T was detected in 122 water samples from the drainage lysimeter for 1 year after herbicide application. Picloram (1-4 ppb) was detected up to 9 months after application, but was not detected after 9 months. The authors concluded that the herbicide tested did not accumulate in plants or soil and degraded and disappeared even after repeated application. 109. Bovey, R. W., and Diaz-Colon, J. D. (1978a) Selected Bibliography of the Phenoxy Herbicides, Vol. VIII: Effects on Higher Plants. U.S. Department of Agriculture, 58 pp. [Bibliography.] 39 �110. Bovey, R. W., and Diaz-Colon, J. D. (1978b) Selected Bibliography of the Phenoxy Herbicides, Vol. VI: Methods of Extraction and Analysis. U.S. Department of Agriculture, 34 pp. [Bibliography.] 111. Bovey, R. W., and Diaz-Colon, J. D. (1978c) Selected Bibliography of the Phenoxy Herbicides, Vol. IV: Ecological Effects. U.S. Department of Agriculture, 28 pp. [Bibliography.]• 112. Bovey, R. W., and Diaz-Colon, J. D. (1977) Selected Bibliography of the Phenoxy Herbicides, Vol II: The Substituted Dibenzo-p-Dioxin. U.S. Department of Agriculture, 57 pp. [Bibliography.] 113. Bovey, R. W., Dowler, C. C., and Merkle, M. G. (1969) Pesticides in soil. The persistence and movement of picloram in Texas and Puerto Rican soils. Pestic. Monit. J. 3(3):177-181. The authors reported on the persistence and movement of picloram in sandy and clay soils. Five soil types were used, two in Texas and three in Puerto Rico: Nipe clay (Puerto Rico) in which water penetrates rapidly and is poorly retained; Fraternidad clay (PR) which drains slowly; Catano sand (PR) which is poorly drained; and Erving clay loam (TX) and Lakeland sand (TX). Picloram was applied to each plot (three replicates per soil type) at rates of 1, 3, and 9 Ib/acre. Texas plots were analyzed for picloram by gas chromatography and plant bioassay immediately after application, and at 3, 6, and 18 months after application. Puerto Rican plots were analyzed at 3, 6, and 12 months. In Texas, picloram was applied to dry soils which received little rainfall during the first 6 weeks. Loss of picloram was rapid and most likely due to photodecomposition. By gas chromatography, approximately 20% of the 1 Ib/acre application was found in the clay soil at 3 months, while no picloram was detected in the sandy soil at 3 months. At 3 and 9 Ib/acre, picloram persisted for 18 months in clay soil but only for 6 months in sandy soil. A bioassay at 18 months using beans as the indicator organism yielded similar results: in sandy soil, picloram was only detected at 9 Ib/acre, while in clay soil, picloram was detected at 3 and 9 Ibs/acre. Picloram applied at 1 Ib/acre was only detected in the top 0-6 cm of soil, while the higher picloram applications were detected at the greatest depths tested (36-48 cm) at all three application rates. In Puerto Rican soils, at 3 months picloram, occurred throughout the soil profile (0-48 cm) at all three application rates. However, in the sandy soil, picloram was only detected at 21-27 cm (1 Ib/acre), 21-39 cm (3 Ib/acre), and 6-39 cm (9 Ib/acre). Picloram in sandy soil was undetectable at all application 40 �rates 6 months after application. The authors concluded that the.rapid disappearance was probably due to the heavy rainfall occurring in this area. Picloram was most persistent in Fraternidad clay where rainfall was lowest and was detected throughout the soil profile at 3 and 9 Ib/acre applications one year after application. Nipe clay also had detectable levels of picloram after 1 year at the 9 Ib/acre application rate. The authors concluded that leaching by rainfall and soil type are important to the movement of picloram through soil. 114. Bovey, R. W., and Young, A. L. (1980) The Science of 2,4,5-T and Associated Herbicides. (New York: John Wiley and Sons.) [Review article.] 115. Boyd, E. M., and Dobos, I. (1969) Acute oral toxicity of monuron in albino rats fed from weaning on different diets. J. Agr.Food Chem. 17(6):1213-1216. The acute toxicity of monuron was compared in rats fed dietary protein levels of 0-26% casein. Male weanling Wistar rats were fed diets with no protein, 3.5%, 9.0% or 26% protein for 28 days. The control group was fed standard laboratory chow. Monuron (94% purity) in cottonseed oil was administered by gastric intubation and the subsequent effects of mortality and gross and histopathologic changes were observed. The LD^Q of monuron was 1.48 g/kg for controls, 0.25 g/kg for rats fed a protein-free diet, 0.95 g/kg for rats fed 3.5% casein, 1.58 g/kg for rats fed 9% casein, and 2.88 g/kg for rats fed 26% casein. All LD-0 values were significantly different from controls except for the value of the group fed 9% casein. The hours to death were significantly shorter (11 hours) for the rats in the protein-free diet and longer (41 hours) for rats fed 26% casein than for controls (27 hours). Monuron produced the same clinical signs of toxicity in all groups of rats, including anorexia and central nervous system disorders. Gross pathological changes included gastric ulcers, gastroenteritis, renal and splenic pallor and hepatic necrosis. Degenerative changes were observed histologically in the kidneys, liver, muscle, salivary glands and testes, congestion was observed in the brain, heart, and lungs; the spleen, adrenals and thymus showed evidence of stress. Most organ weights were decreased after monuron treatment. Survivors were free of clinical signs of toxicity within 4 days of treatment. The authors concluded that rats fed diets low or free of protein were susceptible to raonuron and in particular were susceptible to starvation while rats fed 26% casein were relatively resistant and showed early signs of recovery. The authors suggested that quantitative differences in the activities of enzymes involved in monuron detoxification were responsible for the increased susceptibility of protein-deficient rats. However, no attempt to measure monuron levels or enzyme activities were made and many other alterations in physiology occur after protein deprivation that could compromise the response of an animal to toxic substances. 41 �116. Boyd, E. M., and Krupa, V. (1970) Protein-deficient toxicity. J. Agric. Food Chem. 18(6):1104-1107. diet and diuron The acute toxicity of diuron was compared in rats fed protein deficient diets. For 28 days weanling Wistar rats were fed diets containing either 3.5% protein as casein, 26% protein as casein or 25% protein as mixed animal and plant proteins. Diuron (95% purity) was administered in cottonseed oil by gastric intubation. Animals were observed for 1 month, mortality was determined and gross and histopathologies were performed. The LD._ was 1,017 mg/kg for rats on the mixed protein diet, 2,390 mg/kg for rats fed 26% casein and 437 mg/kg for rats on the low protein diet. The mean time to death was 23-28 hours for all groups and clinical symptoms of diuron toxicity were the same for all 3 groups. Death was caused by respiratory failure. Clinical signs indicated a depression of the central nervous system and cholinergic stimulation by diuron. Gross pathology included gastritis, enteritis, dehydrated cecum, congested brain and lungs, and yellowish kidney. Histological examination revealed gastrointestinal congestion, stress reactions of the adrenals, thymus and spleen and hepatic and renal lesions. Decreases occurred in organ weights except the adrenal and salivary glands, which were hydrated. Survivors recovered from toxic effects in 72 hours. The authors concluded that the toxicity of diuron was augmented 5-fold in rats fed low protein diets relative to those fed normal, mixed protein diet and these results were compared to the relative increase in toxicity of other pesticides in rats fed low protein diets reported in another publication. 117. Bradlaw, J. A., Garthoff, L. H., Graff, D. M., and Hurley, N. E. (1975) Detection of chlorinated dioxins: Induction of aryl hydrocarbon hydroxylase activity in rat hepatoma cell culture. Toxicol. Appl. Pharmocol. 33(1):166. [Abstract, only.] 118. Bradlaw, J. A., Garthoff, L. H., Hurley, N. E., and Firestone, D. (1976) Aryl hydroearbonhydroxylase activity of twenty-three halogenated dibenzo-p-dioxins. Toxicol. Appl. Pharmacol. 37(1):119. [Abstract, only.] 119. Bradlaw, J. A., and Casterline, J. L. (1979) Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic compounds. J. Assoc. Off. Anal. Chem. 62(4):904-916. The use of TCDD as a standard for a bioassay based on enzyme induction is described. Rat H-4-11E hepatoma cells were homogenized and aryl hydrocarbon hydroxylase activity was determined in cell extracts. Benzo(a)pyrene was used as a substrate. The enzyme activity of TCDDpretreated cells was compared to the activity of non-induced cells. In 42 �this system, TCDD was a more potent inducer than 24 other halogenated dibenzo-p-dioxins, 11 dibenzoyfurans or 7 biphenyls. The concentration of TCDD that produced half-maximal induction was 0.38 nM. The potencies of various chemicals in this bioassay were compared to results reported elsewhere of toxic responses. The potencies in the enzyme induction bioassay correlated well with the relative potencies of the same chemicals in assays of other toxic responses. The authors concluded that the enzyme induction bioassay is a useful method for screening food extracts for polychlorinated planar substances. 120. Bradlaw, J. A., Garthoff, L, M., Hurley, N. E., and Firestone, D. (1980) Comparative induction of aryl hydrocarbon hydroxylase activity in vitro by analogues of dibenzo-p-dioxin. Fd. Cosmet. Toxicol. 18:627-635. The potencies of various compounds on aryl hydrocarbon hydroxylase activity in hepatoma cell extracts is described. TCDD was the most potent compound tested and was selected as the standard for the method, when used as a screening technique. The methods and results reported in this article were described in a previous publication by Bradlaw and Casterline (1980). 121. Bradley, R. L., Shoemaker, J. P., and Hoffman, R. V., Jr. (1974) Treatment of experimental mammary adenocarcinoma with herbicides. Cancer Chemother. Rep. Pt. 1. 58(5):745-748. The authors studied the chemotherapeutic potential of picloram on transplantable mammary adenocarcinomas in mice. Three separate studies were conducted. In the first experiment, male C3H mice (15 mice per group) received subcutaneous injections of picloram (10 mg/kg, 0.5 ml) or 0.5 ml saline. Picloram was dissolved in 95% ethanol diluted with sterile water, pH 7.3. Mice in both groups were injected with C3HBA tumor subcutaneously. In additon, each mouse received daily inoculations of picloram or saline for the duration of the experiment. Survival times and tumor sizes of controls and treated animals were analyzed by the student's t-test. Statistically significant decreases in tumor size were observed in picloram-treated animals on days 8 through 15 and on day 25. No signficant differences were observed on days 26 through 36 or on survival times of the animals. In the second experiment, animals received 20 mg/kg subcutaneous doses of picloram. Eight male and 10 female C3H mice were used in each group. C3HBA tumors were injected in the same manner as above. Picloram was administered daily for 12 days. Then on alternate days starting with day 14, the animals were treated with 10 mg/kg doses until death or the end of the experiment. Control mice received saline injections. A statistically significant decrease (p less than 0.001) in tumor size was observed in picloram-treated animals compared to controls on days 34 through 53. Two of the 3 picloram-treated mice that were alive on day 82 no longer had tumors. Tumor sizes did not differ according to sex. A side-effect of picloram administration, i.e., edema, was noted in 9/18 treated animals about day 14. In a third experiment, the toxicity 43 �of picloram was studied. Groups of 5 male and 5 female mice received up to 40 mg/kg picloram for up to 2 weeks. Dosing schedule was not reported. At 10 mg/kg picloram, mice became lethargic for 20-30 minutes; at 20 mg/kg lethargy lasted for 1 hour; at 30 mg/kg mice were lethargic for more than 1 hour, and 3/6 mice died on day 4. The LD,-n for picloram was calculated to be 30 mg/kg. No histologic differences were observed between control and treated mice and nontoxic effects were observed in tissues of normal mice receiving 10 or 20 mg/kg doses. In mice killed by picloram treatment, fatty metamorphosis of the liver was observed. Sex-related differences in picloram toxicity were not reported. 122. Brady, H. A. (1975) Picloram and dicamba persistence in forest environments. Proc. South. Weed Sci. Soc. 28:230-235. [Not available.] 123. Brady, H. A. (1974) Soil moisture affects absorption of 2,4,5 T sprays. Pro. Soil Sci. Soc. of America. 27:206-210. [Not available.] " 124. Braman, R. S. (1975) Arsenic in the environment. In Arsenical Pesticides, ACS Symposium Series Vol. 7 [Washington, D.C.: American Chemical Society] pp. 108-123. [Review article.] 125. Braman, R. S., and Foreback, C. (1973) Methylated forms of arsenic in the environment. Science 182(4118):1247-1249 . The quantities of cacodylic acid (dimethylarsenic acid), methylarsenic acid, arsenate and arsenite ions in environmental samples and human urine were determined. Water samples from ten lakes, bays, and rivers near Tampa, Florida, as well as seashells, bird eggshells, sandstone rock and urine samples from 3 men and 1 woman, between 27 and 42 years old, were analyzed for arsenic. The method of analysis, developed to detect arsenic in each of the 4 forms, involved an atomic absorption technique and had a lower detection level of about 1 nanogram. Water samples contained up to 3.6 ppb of arsenic, with less than 1% as methylated arsenic. The rock sample contained arsenic primarily as arsenate and arsenite ions, while the shells contained primarily methylated arsenic compounds. The urine samples contained an average of 22.5 ppb arsenic, with 66% as dimethylarsenic acid, 8% as methylarsenic acid, 8% as arsenate and 17% as arsenite. The authors concluded that environmental inorganic arsenic was reduced and methylated, eventually to dimethylarsine by bacteria, and that this biotransformation was confirmed by verifying the presence of some of the intermediates. Human biotransformat ion was proposed to be the result of 44 �methylcobalamin-methionine reactions. Since the source of the arsenic that was eventually excreted into urine is not identified in this study, there is no way to differentiate whether the methylated arsenic was derived from inorganic arsenic or from exposure to methylated arsenic pesticides. The data can be used to illustrate that methylated arsenic compounds are not necessarily degraded to inorganic arsenic prior to excretion in human urine. 126. Brandt, M. R. (1971) Herbatox poisoning: a brief review and report of a new case. Ugeskrift for Laeger 133(11):500-503. The author describes a case of intoxication by an herbicide preparation that contained 2,4-D. In a suicide attempt, a 47-year-old man ingested an estimated 300-600 mg/kg of 2,4-dichlorophenoxypropionic acid 2,4-dichlorophenoxyacetic acid (2:1, w/w) as Herbatox. Within 5 hours, most muscles were rigid. Other symptoms that developed were vomiting, shallow respiration, foaming at the mouth, and coma. In 3 days, which included therapy involving artificial respiration, forced diuresis, and drug therapy (atropy and furasemid), the patient was fully awake, but lost his memory and ability to see colors for 5 days, and had pain and paresthesia of the left arm and leg. Five months after the incident, the only symptoms that remained were hypesthesia, algesia and mononeuritis of the left leg. Laboratory tests revealed slight anemia, perturbances in the levels serum transaminases, and urine levels of 25 mg and 35 mg of dichlorophenoxypropionic acid and 2,4-D respectively per 100 ml of urine collected 1 day after the incident. The authors also reviewed literature on other instances of intoxication by Herbatox and concluded that this herbicide was more toxic than generally thought. 127. Braun, W. (1970) Chloracne. Therap. Umscham. 27(8):541-546. [Review article.] 128. Bretag, A. H., and Caputo, C. (1978) The nature of the antimyotonic action of 2,4-D on the soleus muscle of the rat. Proc. Aust. Physiol. Pharmacol. Soc. 9(2):150 pp. fAbstract, only.] I 129. Brooker, M. P. (1976) The ecological effects of the use of dalapon and 2,4-D for drainage channel management. II Fauna. Arch. Hydrobiol. 78(4):507-525. [Not available.] 45 �130. Brown E. A. B., and Haling, H. M. (1975) The effects of paraquat and related herbicides on the acetylcholinesterase of rat lung. Fed. Proc. Fed. Am. Soc. Exp. Biol. 34(3):226. [Abstract, only.] 131. Brown, J. W. (1962) Vegetational spray tests in South Vietnam. Defense Documentation Center, AD 176961. 119 pp. [Background material.] 132. Brown, M. H. (1979) Love Canal and the poisoning of America. 244(33):33-47. Atlantic [Editorial.] 133. Brown, V. K. (1968) Solubility and solvent effects as rate-determining factors in the acute percutaneous toxicities of pesticides. Soc. Chem. Ind. Monogr. 29:93-105. [Review article.] 134. Bucher, N. L. R. .(1946) Effects of 2,4-dichlorophenoxyacetic acid on experimental animals. Proc. Soc. Exper. Biol. Med. 63:204-205. A brief description of the clinical symptoms of 2,4-D toxicity in mice and dogs is reported. Strain A mice were administered the sodium salt of 2,4-D in physiological saline subcutaneously, intraperitoneally or intravenously in single doses of 150-350 mg/kg or as 1 or 2 daily doses for 3 weeks to 3 months of 1/3 to 1/5 the LD.« per day. Gross and histological necropsy examinations were performed and for chronically treated mice, growth rates, hematology analyses, transplanted sarcoma growth, and reproduction were assessed. The LD5Q was 280 mg/kg for subcutaneous treatment to mice. Myotonia which developed in mice injected (route not specified) with 2,4-D was described and included features that resembled clinical myotonia, including alleviation of muscle spasms by exercise, acerbation by rest and inducement by spasms by a local blow to the muscle. Hind limbs were more affected than forelimbs. Doses above 250 mg/kg 2,4-D produced diarrhea, inertia, coma, and sometimes death in mice. The liver, kidney and spleen of acutely treated mice were mottled and dilated blood vessels were observed in several organs. No tissue pathology, hematologic changes, tumor or body growth retardation, or reproductive problems were observed in chronically treated mice. Anorexia, vomiting and nasal and eye irritation were reported in treated dogs, but the dose, route or number of dogs treated were not reported. The descriptions of 2,4-D toxicity presented here do not include the route of administration, the doses (except related to myotonia), the numbers of animals studied or the proportions afflicted, nor methods or results for the studies of 46 �reproductive effects or tumor growth. Therefore, this study does not contribute significantly to an understanding of 2,4-D toxicity. 135. Buratowski, J., Piasecki, M. , and Warczynski, A. (1975) Effects of chemical toxic agents used in Vietnam on the visual system. Lek. Woj. 51(9):581-583. [Foreign language.] 136. Bureau of Foods, Pesticides, and Product Safety FDA (1970) Toxic ity of 2,4,5-T, 2,4-D and related eomp'ds. March 13, '80, revised April 8, "rW. [Not available.] 137. Bureau of National Standards. (1981) Second DOW Sarcoma case found; company suggests smoking as a factor. Occupational Safety and Health Reporter. 10(4):1347. [Review article.] 138. Burk, R. F., Lawrence, R. A., and Lane, J. M. (1980) Liver necrosis and lipid peroxidation in the rat as a result of paraquat and diquat administration. J. Clin. Invest. 65(5):1024-1031. The toxicity of diquat in selenium-deficient rats was assessed. Male Holtzman rats maintained on control diets and vitamin E-deficient diets were administered 78 umol/kg diquat in saline, intraperitoneally. A group of rats fed a selenium-deficient diet were administered 19.5 umol/kg diquat. Ethane in expired air was determined by gas chromatography and used as a measure of lipid peroxidation. Mortality, renal and hepatic histology and serum enyzme activities were evaluated as measures of toxicity. Increases in ethane production were 3 and 28 times the control values for the vitamin E-deficient and seleniumdeficient groups, respectively and death occurred for all animals in 24 and 2 hrs. for the 2 groups, respectively. Liver and kidney necrosis were observed at necropsy, while other organs were unaffected. Cod liver oil, which is rich in linoleic acid-related fatty acids, was used to replace corn oil in the selenium deficient diet and was found to cause a 3-fold increase in ethane production and no change in liver or kidney necrosis occurred. Neither liver necrosis nor increased ethane production occurred in control rats administered diquat. Selenium, administered as a single dose to rats on a selenium-deficient diet, had no effect on glutathione peroxidase activity in lung, plasma, liver or kidney tissues removed and assayed 10 hrs. after the injection. Both lipid peroxidation and mortality from diquat were reduced 6 and 10 hrs. after the selenium injection. Superoxide dismutase activity in several tissues was unaltered in rats fed the 1 selenium-deficient diet compared to those fed the control diet. The authors concluded that diquat 47 �toxicity was increased in selenium-deficient rats and that the biochemical role of selenium in protection against lipid peroxidation remains unknown. 139. Burk, R. F. , Lawrence, R. A., Lane, J. M., and Hatnm, D. P. (1979) Lipid peroxidation and liver necrosis in selenium-deficient rats given diquat and paraquat. Gastroenterology 76(5)Pt. 2:1109. [Abstract, only.] 140. Burton, J. A., Gardiner, T. H., and Schanker, L. S. (1974) Absorption of herbicides from the rat lung. Arch. Environ. Health 29(l):31-33. Absorption of 2,4-D, 2,4,5-T and diquat by the lung of anesthetized rats were assessed. Male Charles River rats under.sodium pentobarbital anesthesia were administered 0.1 ml of 1.0 -10 mMf C}-2,4-D, or [ C]-2,4,5-T or 0.01-0.1 mM [ C]-diquat by tracheal cannula. After 0.5-120 minutes, the trachea and lungs were removed, homogenized, digested, and counted to determine unabsorbed radioactivity. All compounds showed absorption (calculated from the unabsorbed radioactivity in the lungs) that followed first order kinetics. Half-times for absorption were 1.4, 1.7, and 51 minutes for 2,4-D, 2,4,5-T and diquat, respectively, and the corresponding rate constants were 30.1, 24.8, and 0.82 per hour. The amount of each compound absorbed was dependent on the concentration administered; the percentage of the dose absorbed in a given time was constant for various concentrations. Amitrole absorption was also studied. The authors concluded that the rates of absorption for the four compounds correlated with their lipid solubilities and not their molecular weights and the more soluble compounds probably entered the lung through membrane pores and through lipid regions of the membrane, while diquat probably only passed through pores. 141. Bus, J. S., Preache, M. M., Cagen, S. Z., Posner, H. S., Eliason, B. C., Sharp, C. W., and Gibson, J. E. (1975) Fetal toxicity and distribution of paraquat and diquat in mice and rats. Toxicol. Appl. Pharmacol. 33(3):450-460. The fetotoxicity, maternal toxicity, and fetal distribution of diquat was studied in the rat. A dose of 15 rag/kg of [ C]-diquat was given intravenously to pregnant rats on a single day of gestation from day 7 to 21. On day 22, the number of dead and resorbed fetuses was determined and compared to a vehicle control group that received saline only. Radioactivity in fetal tissues was examined 1, 3, 7, and 24 hours after a single intravenous dose of 15 mg/kg [ Cl-diquat was administered maternally on day 13, 16, or 21 of gestation. Fetal tissues were excised, dissolved in toluene and counted for radioactivity. The average percentage of dead plus resorbing fetuses was 57%, with peak fetotoxicity occurring from diquat administration on days 7-9 and on days 16-17. The incidence of maternal deaths averaged 48 �20% with 8 of the 9 deaths occurring from treatment,on or before day 13. Rat fetuses took up more radioactivity when f C]-diquat was administered late in gestation. The levels of radioactivity in the whole fetus and in the fetal liver, kidney, and lung decreased at successive times after administration. Compared to the effects of [ C]-paraguat which were studied using the same dose and route as for diquat, [ C]-diquat caused a substantially higher degree of fetor toxicity and reached the rat fetus in much larger amounts than f Cjparaquat. Neither compound was present in maternal plasma or fetal extracts in the form of metabolites. The authors concluded that the differential toxic effects of the 2 compounds were probably the result of differences in distribution of the compounds to the fetus. 142. Buselmaier, M. V,, Rohrborn, G., and Propping, P. (1972) Pesticide mutagenicity investigations by the host mediated assay and the dominant lethal test in mice. Biol. Zentralbl. 91:311-325. The authors tested 2,4,5-T in a host-mediated assay and a dominant lethal test in mice. Two bacterial strains were used as the indicator organisms on the host-mediated assay: Salmonella typhimurium G46 which reverts from histidine requiring to histidine independence upon exposure to a mutagen and Serratia marcescens a2l, a leucine requiring strain which reverts to prototrophy after treatment with a mutagen. A plate incorporation assay in S. typhimurium was also performed to compare in vivo (host-mediated assay) and in vitro (plate assay) results. In the host mediated asay, NMRI mice (10-12 weeks old, 6 animals per group) received an intraperitoneal injection of bacteria immediately followed by a subcutaneous injection of 500 mg 2,4,5-T/kg body weight or 1000 mg 2,4,5-T butylester/kg body weight. Mice were killed 3 hours later and the bacteria was recovered from the peritoneal cavity. Results of the assay were analyzed by the Wilcoxon rank test. A result was positive at p 0.01. By this criteria, both 2,4,5-T and 2,4,5-T butylester were negative in the host-mediated assay using either jS_. typhimurium or S_. marcescens as the indicator organism. Both compounds were also negative in the plate assay. In the dominant lethal assay, NMRI male mice (10 weeks old) received a single intraperitoneal injection of 100 mg/kg 2,4,5-T and were mated with untreated females of the same age. Each male was caged with three females for 1 week. The experiment continued for 6 weeks. Males were caged with new females each week to observe all stages of spermatogenesis. Females were killed 14 days after being placed with the males. Uteri were scored for numbers of dead implants. Experimental results were analyzed by the x method with p 0.0027 as the criteria for a positive result. 2,4,5-T did not induce dominant lethal mutations according to this criteria. 143. Buslovich, S. Yu., Aleksashina, Z. A., and Kolosovskaya, V. M. (1976) Embryotoxic effect of chloro-derivative phenoxyacid-herbicides. Zdravookhraneniye Belorussii (10):83-84. The teratogenicity of 2,4-D sodium, amine, and diethylamine salts and butyl ester was studied in the rats. Pregnant albino rats were exposed 49 �to half of the LD dose of the 2,4-D compound and resorptions, and fetal size, weights and malformations were determined. One of 9 rats exposed to 2,4-D-butyl ester on day 9 of gestation had 8 live fetuses and 1 of 7 rats exposed on day 10 retained 2 fetuses. All other rats exposed to the butyl ester on days 4, 5, 6, 9, 10, 11, or 12 had 100% resorptions. Administration of 2,4-D diethylamine salt on days 5, 9, 10, or 13 increased the number of post-implantation deaths. Treatment with the sodium or amine salts of 2,4-D did not alter fetal viability. The weights and size of the fetuses exposed to 2,4-D butyl ester, 2,4-D diethylamine and, for the sodium and amine salts given on day 10 or 14 only, were reduced. Enlarged brain ventricles and hemoperitoneurns were observed in some fetuses (the frequency or treatment groups with affected fetuses were not stated). The authors concluded that esters of 2,4-D with long side chains are more toxic than 2,4-D acids and should not be used near water that could result in human exposure and risk of embryotoxicity. 144. Buslovich, S. Yu., and Koldobskaya, F. D. (1972) Activity of hexokinase from the skeletal muscle of albino rats under the conditions of experimental myotonia. Voprosy meditsinskoi khimii. 18(4):403-406. The effects of administering hydrocortisone or desoxycorticosterone (DOCA) or adrenalectotny on myotonia produced by 2,4-D was studied in the rat in vivo and in vitro. Female rats were administered 400 mg/kg 2,4-D diethylamine perorally and skeletal muscle hexokinase activity was determined from 15 minutes to 72 hours after 2,4-D treatment. Adrenalectomies were performed on some rats prior to 2,4-D treatment and other rats were administered 5-10 mg/kg DOCA and 5-10 mg/kg hydrocortisone (together) subcutaneously. 2,4-D was added to skeletal muscle homogenates and the effect of hydrocortisone succinate on hexokinase levels in vitro were determined. A myotonic response was observed in rats after 2,4-D treatment, and was accompanied by a decrease in hexokinase levels to a low of 11% of control levels after 1 hour. In adrenalectotnized rats, no myotonic;effect resulted from 2,4-D administration. Hexokinase levels, which were elevated after adrenalectomy, were decreased to almost normal levels by 2,4-D treatment. A combination of 10 mg/kg.of hydrocortisone and DOCA reversed the effect of 2,4-D on hexokinase activity, but lower doses of the steroids or administration in the absence of 2,4-D produced a large decrease in hexokinase activity (to 30 to 60% of control levels). A,small decrease (about 25%) in hexokinase activity was produced by 5x10 "M 2,4-D in vitro and a substantial effect was observed in muscle homogenates from rats exposed to 2,4-D in vivo and studied in vitro. This last effect was reversed by addition of 100-200 ug of hydrocortisone succinate to the cultures. The authors concluded that hexokinase activity paralleled the development of myotonia and suggested that the change in activity was indicative of a disturbance of carbohydrate energy metabolism during myotonia. 50 �145. Buslovich, S. Yu., Voinova, I. V., and Milchina, M. G. (1973) The distribution of chloro-derivative phenoxyacid herbicides in albino rats. Gigiena truda i professionalnyye zabolevaniya. 17(3):35-37. The tissue distribution of 2,4-D and pharmacokinetics of blood clearance was studied in the rat. Groups of 10 rats were administered 555 mg/kg of 2,4-D sodium salt or 405 rag/kg of 2,4-D diethylamine salt by oral intubation and groups of 20 rats were given 50 daily oral doses of 91 or 23 mg/kg 2,4-D sodium salt or 28 or 111 mg/kg 2,4-D diethylamine salt. Tissue levels of 2,4-D were determined by degrading 2,4-D to 2,4-dichlorophenol with pyridine hydrochloride and analyzing the phenol spectrophotometrically. Peak blood levels were reached 3 hours after an acute dose and half-lives were calculated as 53 hours and 72 hours for the sodium and diethylamine salts, respectively, for the period from 3 to 72 hours after exposure and half-lives of about one-third these values were calculated for the period from 3-12 hours after exposure. The peak tissue levels were reached 3 days after 2,4-D was administered with the highest levels in the liver and lowest levels in the brain. Subsequently, the kidney retained the highest levels. After chronic exposure, the highest levels were observed for the liver and kidney and the lowest levels for the brain. No accumulation of 2,4-D was observed in any tissue. The authors concluded that the lack of tissue accumulation reflected the water-solubility and size of the dose of 2,4-D administered. 146. Buu-Hoi, N. P., Chanh, P. H., Sesque, G., Azum-Gelade, M. C., and Saint-Ruf, G. (1972a) Enzymatic functions as targets of the toxicity of "dioxin" (2,3,7,8-tetrachlorodiobenzo-p-dioxin). Naturwissenschaften 59(4):173-174. The effect of TCDD on the levels of 8 serum enzymes in days after TCDD administration was evaluated. Rats were administered 10 mg/kg of TCDD in olive oil intraperitoneally, after 10 days, blood was removed and assayed for glutamic oxaloacetic transaminase (S.G.O.T.), glutatnic pyruvic transaminase (S.G.P.T.), aldolase, lactic dehydrogenase (LDH), hydroxybutyric dehydrogenase, alkaline phosphatase, arylesterase, and cholinesterase. No changes were observed in aldolase and alkaline phosphatase between levels for treated and control groups, while statistically significant increases in levels for TCDD-treated animals occurred for S.G.O.T., S.G.P.T., LDH, and hydroxybutyric dehydrogenase, and significant decreases occurred for arylesterase and cholinesterase. Some blood serum components were present at significantly higher concentrations in treated rats than controls. These components included urea, total lipids, and bilirubin. Glucose levels were lower in blood from treated rats than control rats. The authors concluded that the liver was the main target organ of TCDD toxicity, followed by the thymus and heart. Histopathologic findings were described in a separate article. (Buu-Hoi, et al, 1972). Physical appearance, mortality, body weights, and other parameters relevant to interpreting the clinical chemistry results were not reported. Furthermore, the dose of TCDD used in this study is accessive, relative to other reports which describe lethal effects of TCDD doses in the ug range. 51 �147. Buu-Hoi, N. P. H., Chanh, P. H., Sesque, G., Azum-Gelade, M. C., and Saint-Ruf, G. (1972b) Organs as targets of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) intoxication. Naturwissenschaften 59(4):174-175. v Histopathologic evidence of toxicity of TCDD to various organs of the rat is presented. Wistar rats weighing 210 g were given 10 mg/kg of TCDD in olive oil intraperitoneally. After 10 days the treated rats were examined for hetnatologic and histopathologic effects and were compared with vehicle controls. Treated rats lost 60-80 g, while control rats gained 60-70 g. Analysis of blood samples from treated rats revealed statistically significant increases over controls in hematocrits, leukocyte counts, and polynuclear neutrophils, while the proportion of lymphocytes decreased and the bone marrow did not change (the parameters measured for evaluating bone marrow were not reported). Severe hepatic lesions were described which were evident 6 days after treatment. Involution of the thymus, severe cardiac lesions and pulmonary alveolitis were also observed in treated rats. The authors mentioned that a dose of 1 mg/kg of TCDD produced the same qualitative changes as the 10 mg/kg dose. TCDD elicits toxicity in the ug range and the dose administered in the study is well beyond those used by most investigations to study various parameters of TCDD toxicity. 148. Buu-Hoi, N. P., Saint-Ruf, G., Bigot, P., et al. (1971) Preparation, properties and identification of dioxin (2,3,7,8-tetrachlorodibenzopara-dioxin) in the pyrolysate of defoliants containing 2,4,5-T and its esters and in contaminated vegetation. C.R. Acad. Sci., Paris Ser. D. 273:708-7111. (French) [Background material.] 149. Byast, T. H., and Hance, R. J. (1975) Degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Bull. Environ. Contain. Toxicol. 14(l):71-76. The authors describe the degradation of 2,4,5-T in four South Vietnamese soil samples under laboratory conditions. Two soil samples (1 and 2) were grey podzolic soils from an agricultural area, having pH values of 4.9 and 5.0, respectively. The other two samples (3 and 4) were saline alluvial soils from a mangrove swamp, pH values of 6.6 and 7.9, respectively. In the laboratory, samples 3 and 4 were air-dried to 15% moisture content from their original moisture of 35 and 47%, respectively. Samples 1 and 2 orginally contained 15% moisture. Duplicate soil samples received acetone solutions of carboxy-labeled C-2,4,5,-T butyl ester at 1 or 15 ppm. Controls received only acetone. After solvent evaporation the soil was transferred to respiration flasks. Samples 3 and 4 were returned to their original moisture content. Evolution of C02 and residual C and 2,4,5-T were measured,in each soil sample by scintillation counting. At both 1 and 15 ppm, CO^ evolution was greatest in sample 2. The authors offered no explanation for this. At 1 ppm, soils evolved a maximum of 64-69% 52 �14 CO- ipA^9 days. However, at 15 ppm a slow degradation was observed, 74-9o% C02 in 168 days. Less than 4% of the 1 ppm application and only 1-16% of the 15 ppm application of 2,4,5-T could be extracted from the soil with or without hydrolysis. The authors speculated that the unextractable CO- had been incorporated into the soil organic matter. The authors did not state what relevance these data may have to the actual field situation. 53 �150. Calderbank, A., and Slade, P. Diquat and paraquat. In Herbicides: Chemistry, degradation, and mode of action. Vol. 2 [eds., P. C. Kearney and D. D. Kaufman] (New York: Marcel Dekker Inc., 1976) pp. 501-540. [Review article.] 151. Caldwell, R. S., Buchanan, D. V., Armstrong, D. A., Mallon, M. H., and Millemann, R. E. (1979) Toxicity of the herbicides 2,4-D, DEF, propanil and trifluralin to the Dungeness crab, Cancer magister. Arch. Environ. Contain. Toxicol. 8:383-396. The authors describe the toxicity of 2,4-D free acid to various life stages of the Dungeness crab. Technical grade 2,4-D (98 percent active ingredient) was dissolved in acetone and added to seawater with a final acetone concentration of 100 ul/1. Control vessels contained only seawater or seawater with acetone. Chromatographic analysis to determine the final concentration of 2,4-D in seawater were not performed. The effects of 2,4-D on egg hatching and early development through the prezoeal stage were measured in a 24 hour toxicity test. Duplicate beakers of 3,300, 10,000, 33,000, 100,000, or 330,000 ug/1 2,4-D each received 30 eggs. At the highest dose tested, 2,4-D inhibited egg hatching (1/58 eggs hatched). At 100,000 ug/1 less than 20 percent of the hatching prezoeae developed into the first zoeae stage. Motility of the first stage zoeae was inhibited at 100,000 ug/1. In 80 day long term toxicity assays, 2,4-D decreased survival of larvae statistically significant at the highest dose tested, 10,000 ug/1, but not at the lower doses, 1,000 ug/1. Exposure to both concentrations of 2,4-D increased the duration of larval stages. In juvenile crabs and in adults, no effects of 2,4-D were observed up to doses of 10,000 ug/1. The authors concluded that the larva was the stage most sensitive to 2,4-D. From their data, the maximum acceptable toxicant concentration (MATC) was below 1,000 ug/1 for 2,4-D free acid. 152. California - Department of Food and Agriculture. aerial use of phenoxy herbicides. 75 pp. (1978) Report on the [Background material.] 153. Cant, J. S. and Lewis, D. R. H. (1968) Ocular damage due to paraquat and diquat. Brit. Med. J. 2:224. A case report of ocular damage after accidental contact of a mixture of diquat and paraquat to the eye is presented. A 36-year-old man accidentally splashed Preeglone Extra fluid (comprised of paraquat and diquat in the equal amounts and an unspecified surface-active agent) in one eye and on his eyelids as he was diluting the concentrate with water. He washed his eye with water. Irritation of the exposed eye occurred over the next 3 days, and after 1 week, 50% of the tar sal conjunctiva of the lower eyelid and a small amount of that of the upper eyelid were lost. The corneal epithelium was lost in some regions and 54 �edematous in other regions. Mild uveitis was also present. Treatment with chloramphenicol and atropine was initiated (1 week after exposure) and adhesions between the denuded surfaces were repeatedly separated. In 11 days, the corneal epithelium was healed and the uveitis had subsided. The authors concluded that both dipyridilium compounds contributed equally to the effect and that the delayed nature of the response and difficulty of removal of the splashed chemicals when a surface-active agent is present create a significant hazard which should be guarded against by proper warning and proper handling. 154. Carere, A., Cardamone, G., Ortali, V., Bruzzone, M. L., and Di Giuseppe, G. (1976) Mutational studies with some pesticides in Streptomyces Coelicolor and Salmonella typhimurium. Mutat. Res. 38:136. [Abstract, only.] 155. Carere, A., Ortali, V. A., Cardamone, G., Torracca, A. M., and Raschetti, R. (1978) Microbiological mutagenicity studies of pesticides in vitro. Mutat. Res. 57:277-286. The authors tested dalapon, picloram, a,nd 12 other pesticides for mutagenicity in Salmonella typhimurium and Streptomyces coelicolor. Four strains of S. typhimurium, TA1535, TA1536, TA1537, and TA1538, were used in the assay with and without rat liver S-9 metabolic activation. Upon exposure to a mutagen these strains revert from histidine requiring prototrophy. A streptomycin sensitive strain of S. coelicolor A3(2), hisAl, was used in a forward mutation assay. Mutagenic effects of a chemical are measured by induction of streptomycin resistance. Both assays were performed as a "spot test" i.e., absorbent paper saturated with solutions of the chemical to be tested were placed on agar which was seeded with bacteria. In the S_. typhimurium assay, dalapon-Na (85% pure) was tested at 2000 ug/plate while picloram (99% pure) was tested as 200 ug/plate. Neither compound induced increased numbers of revertants in any of the 4 tester strains with or without metabolic activation. The experiment was repeated 3 times using triplicate plates for each chemical. Results of both positive and negative controls were reported. In the S_, coelicolor forward mutation assay, both dalapon and picloram were tested at 200 ug/plate without metabolic activation. Picloram treated plates had appoximately 40 times the number of resistant colonies compared to controls while dalapon treatment did not induce an increase in numbers of resistant colonies. Testing of these compounds was repeated three times and results reported were an average of triplicate platings. Both positive and negative controls were reported. However, no statistical analyses or criteria for a positive result were presented by the authors. 55 �156. Carlson, M., Nolting, A. R., and Fullerton, R. W. (Attorneys for the Secretary of Agriculture of the U.S.) (1974) In re: 2,4,5-trichlorophenoxyacetic acid. Statement of position of the Secretary of Agriculture of the U.S. FIFRA Docket No. 295. U.S. Environmental Protection Agency; Before the Administrator. 26 pp. [Not available.] 157. Carlstedt-Duke, J. M. B. (1979) Tissue distribution of the receptor for 2,3,7,8 tetrachlorodibenzo-p-dioxin in the rat. Cancer Res. 39(8):3172-3176. The concentrations of TCDD cytosol receptor for various rat tissues are presented. Male Sprague-Dawley rats were killed and tissues were removed and homogenized. The cytosol fractions were prepared by centrifugation and incubated with [ H]-TCDD. After mild proteolysis (to increase resolution at a subsequent step in the purification), free TCDD was removed onto dextran-coated charcoal and the supernatant was analyzed for the receptor complex by isollectric focusing in polyacrylamide gel. For each tissue, the radioactivity on the purified receptor was displaced by addition of 100 fold higher concentration of 2,3,7,8tetrachlorodibenzofuran. TCDD receptor concentrations, reported in fentomol per mg protein were thymus, 25.2; lung, 20.8; liver, 13.9; kidney, 12.5; testis, 3.5; brain, 2.8; muscle, 0.3; adrenal and pancreas, none. The TCDD-binding species of ventral prostate cytosol was not affected by protease treatment and focused at a lower pH. The author concluded that TCDD-receptor distribution correlated with the sites of TCDD effects, especially of aryl hydrocarbon hydroxylase induction; prostate tissue was one exception, and the receptor characteristics of this tissue were different from those for all other tissues studied. 158. Carlstedt-Duke, J. M. B., Elfstrom, G., Hogberg, B., and Gustafson, T. (1979) Ontogeny of the rat hepatic receptor for 2,3,7,8 tetrachlorodibenzo-p-dioxin and its endocrine independence. Cancer Res. 39:4653-4656. The level of hepatic cytosol receptor for TCDD was determined in rats at various ages. Livers were removed from male and female SpragueDawley newborn rats and aged 7, 21, 42 and 56 days old. [3H1-TCDD was added to liver cytosol and the receptor was isolated by isoelectric focusing in polyacrylamide gel. Cytosol receptor levels were also determined in adult rats after an orchiectomy, ovariectomy, adrenalectomy or hypophysectomy was performed. Male and female rats of the same age had the same number of receptors. The lowest receptor concentrations were observed in 7-day-old and 56-day-old rats, with 13 fmol/mg protein. Twenty-one day old rats had the highest concentration, with 36 fmol/mg protein. Rats that underwent surgical removal of an endocrine gland or other organ had the same number of hepatic cytosol receptors as intact rats. The authors concluded that the peak TCDD-receptor concentration occurred at puberty, at the same time that maximum inductive responses were observed (by others). 56 �159. Carrier, J. M. (1974) The effects of herbicides in South Vietnam. Part B. Working papers: The location of herbicide missions and Rickey's informants in South Vietnam: An appraisal. National Academy of Sciences - National Research Council. AD-779 028. 15 pp. [Background material.] 160. Carter, C. D., Kimbough, R. D., Liddle, J. A., Cline, R. E., Zack, M. M., Barthel, W. F., Koehler, R. E., and Phillips, P. E. (1975) Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188(4189):738-740. The consequences are described of an incident in which TCDDcontaminated industrial wastes were sprayed on three horse arenas and one road to control dust. Of the exposed humans (total number was not, reported), one 6 year-old female developed cystitis and pyelonephritis, and three other children and one adult developed skin lesions, which resembled chloracne in two persons. Two adults developed arthralgia. At one farm, 48 of 85 horses exposed to the sprayed arena died 1 month to 2.5 years after the arena was sprayed, despite several attempts to remove the contaminated soil. Horses exposed to the other two arenas also died. Symptoms in horses included weight loss, skin lesions, intestinal colic, dark urine, hematuria, conjunctivitis, and joint stiffness. Birds, dogs, cats, and rodents in the area died within a month of the spraying, and 70 chickens exposed to the sprayed farm road died 2 weeks after it was sprayed. The presence of TCDD in the soil was confirmed by gas-liquid chromatography and by gas chromatographymass spectrometry, and the toxicity of the soil was demonstrated in a dermal bioassay in rabbits. The concentration of TCDD in the arena soil was 32 (u)g/g and in a distillate residue suspected of being the source of TCDD in the salvage oil, 330 (u)g/g. The authors concluded that the incident described here resulted from improper disposal of the toxic chemical waste TCDD. 161. Case, A. A. (1976) Tetrachlorodibenzodioxin (TCDD) - clinical aspects of poisoning. Clin. TqxicQl. 9(6) :963-967. [Review article.] 162. Case, A. A., and Coffmann, J. R. (1973) Waste oil: Vet. Clin. North Am. 3(2) :273-277. toxic for horses. The clinical symptoms of horses that became ill after exposure to waste oil was described. General descriptions of symptoms were reported, without indicating the frequency of the observed symptom or quantitating the severity of the lesions (for example, average weight loss or body weights or serum protein levels were not given). Horses were exposed to waste oil sprayed on three arenas in Missouri. No reports of toxicity were issued for exposed animals or people from 13 other arenas that were sprayed during the same period of time by the same 57 �company. The toxic substance in the waste oil had not been identified at the time of the report. The affected horses were characterized by severe weight loss, listlessness, nasal discharge suggestive of respirating infection, ulcerative dermatitis, loss of hair including the mane and tail, inflammation of oral and nasal mucous membranes, decreased serum gamma globulin levels, prolonged bromsulphalein clearance time and decreased cholinesterase activities of red blood cells. Changes observed at necropsy included mucous membrane inflammation, atrophy of adipose tissue, generalizing edema, and severe biliary lesions including dilation of bile ducts filled with bile-stained mucous. The spleen and lymph nodes were reduced in size. Loss of body fat and of hair was observed in affected dogs, cats, and mice. 163. Centen, A. H. J., Strik, J. J. T. W. A., and Colombi, A. (in press 1979-80) "Coproporphyrinuria and chronic hepatic porphyria type A in people from Seveso (Italy) exposed to 2,3,7,8 - TCDD." In: The Diagnosis and Occurrence of Chronic Hepatic Porphyria in Man Caused by Chonls, ed. J.J.T.W.A. Strik and J. H. Koeman. (North-Holland: Amsterdam.) [Not available.] 164. Chang, H., Rip, J. W., and Cherry, J. H. (1974) Effects of phenoxyacetic acids in rat liver tissues. J. Agr. Fd. Chetn. 22(l):62-65. The hepatic response of rats to subchronic administration of 2,4-D or 2,4,5-T was studied. Male Long-Evans rats were administered 2,4-D or 2,4,5-T (less than 0.05 ppm TCDD contaminant) in paired feeding experiments 28 to 49 days. The average doses of each herbicide were 130 mg/week for rats initially 4 weeks of age and 220 mg/week for rats initially 7 weeks of age. Liver weights and hepatic levels of carbohydrate, glycogen, protein, DNA and RNA polymerase activity were measured in liver preparations from treated and control rats. A few 2,4,5-T treated rats showed evidence of toxicity and weight loss. Two deaths occurred among the 2,4,5-T treated groups of 52 rats and no deaths occurred among the group of 19 rats treated with 2,4-D. Younger rats were more susceptible to the toxic effects of 2,4,5-T than the older rats. None of the results were analyzed for statistical significance. Increased liver weights and concomitant increases in hepatic glycogen, reducing sugar, RNA and protein content occurred in 2,4,5-T treated rats. Liver glycogen was increased in 2,4-D treated rats. Hepatic nuclear DNA isolated from 2,4,5-T treated rats decreased compared to controls, while cytosol DNA was not altered. A technical problem of recovery of DNA in the relevant fraction of sucrose gradients was offered to explain these differences. Analogous to effects reported elsewhere for plants, hepatic RNA polymerase activity was stimulated 20-30% by either herbicide. The magnitude of this response was much less than the stimulation observed in plants. The inability to establish the statistical significance of changes observed in this study preclude any conclusions from being drawn from the study. 58 �165. Charles, J. M., Abou-Donia, M. B., and Menzel, D. B. (1978) Absorption of paraquat and diquat from the airways of the perfused rat lung. Toxicology 9(1-2) -.59-67. The pharmacokinetics of transfer of diquat from the airways to the vasculature was determined in perfused rat lung. Lungs from female Sprague-Dawley rats were isolated, suspended in an artificial glass thorax, respired artificially, cannulated through the pulmonary artery and perfused. A dose of 1.86 nmol/lung of [ C]-diquat in isotonic sucrose was administered into the lung, and radioactivity in the effluent was monitored for the subsequent 30 min. Diquat introduced into the airways was cleared from the lung in a biphasic pattern with half-lives of 4 and 75 minutes. The initial rate of absorption of diquat introduced into the vasculature and taken up into lung tissue was calculated at 18.4 pmol/sec and efflux rate was 22.1 pmol/sec. The kinetics of paraquat uptake and removal were also reported. The authors concluded that low pulmonary absorption of diquat reflected its lipid insolubility and rapid efflux of diquat indicated that diquat was unlikely to be stored in the lung. 166. Chiou, C. T., Freed, V. H., Schmedding, D. W., and Kohnert, R. L. (1977) Partition coefficient and bioaccumulation of selected organic chemicals. Environ. Sci. and Techno!. 11(5):475-478. [Background material.] 167. Chou, C., Montgomery, M. L., and Yu, T. L. (1971) Methodology and analysis for residues of MCP and 2,4,5-T in wheat. Bull. Environ. Contamin. Toxicol. 6:576-580. [Background material.] 168. Clark, D. G., and Hurst, E. W. J. Ind. Med. 27(l):51-55 (1970) The toxicity of diquat. Brit. The acute and chronic toxicities of diquat are described for several species. Diquat dichloride or dibromide (99% purity) was administered in water or physiological saline by stomach tube or subcutaneous injection for acute studies. Diquat was administered to Friesian cows, Alderley Park strain dogs, guinea pigs, rats and mice, albino rabbits, and Rhode Island hens. Male mice and females of all other species were used. The oral LDc« values were: cows, 30 mg (of cation)/kg; dogs, 100-200 mg/kg; rabbits, 101 mg/kg; guinea pigs, 100 mg/kg; rat, 231 mg/kg, mouse, 125 mg/kg; and hen, 200-400 mg/kg. The subcutaneous LDcn for both salts was 10-11 mg/kg in the rat. Death occurred 2-14 days after dosing and was preceded by lethargy, respiratory difficulty, weight loss, and after injection, pupillary dilatation. Histological changes were minimal. Dermal toxicity and irritation were studied in the rabbit. A dermal dose of 400 mg/kg of diquat produced no ill effects or irritations. Daily doses of 20 mg/kg only produced mild 59 �erythema and thickening of the skin, and 20 daily applications of 40 rag/kg was lethal to 4 of 6 rabbits end produced weight loss and muscle weakness. Slight eye irritation occurred for 2 days after one drop of a 20% solution of diquat was injected into the conjunctival sac of one eye of a rabbit. Rats (50 per group) were fed diets of 0.100-0.1% diquat dichloride for 2 years and dogs (6 per group) were fed 1.75 mg/kg daily for 4 yrs., 0.4 and 0.8 mg/kg for 3 yrs., and 5 mg/kg for 2 yrs. Weight gain was decreased only in the highest dosage rat group and no deaths resulted. Cataracts occurred in all groups of rats except the lowest dosage group and in dogs in the two highest dosage groups. No other changes were detected by gross or histological examinations, or clinical blood and urine analyses. Rats fed diets of 0.05% diquat for a few months did not develop cataracts in the next 2 yrs. Protecting diquat-treated rats from light or giving supplemental ascorbic acid (200 mg/ml in drinking water) did not alter the development of cataracts. Nasal inflammation and bleeding from human exposure to diquat powder and nail damage and delayed healing of cuts on the hand from exposure to diquat herbicide concentrate were mentioned. The authors concluded that diquat produced moderate toxicity, and did not readily penetrate or irritate the skin. 169. Clark, D. E., and Palmer, J. S. (1971) Residual aspects of 2,4,5-T and an ester in sheep and cattle with observations on concomitant toxicological effects. J. Agric. Fd. Chem. 19(4):761-764. The distribution of 2,4,5-T in sheep tissues after subacute exposure is described. Four daily doses of 250 mg/kg 2,4,5-T (100% purity) were administered in gelatin capsules to one Delaine ewe. After the exposure period, tissue was removed and analyzed for 2,4,5-T content by gas chromatography. Recoveries by this method were 95% for 2,4,5-T in urine samples, 93% in blood, 71% in lean tissue and 78% in fat. (Data were not corrected for losses from low recoveries.) Tissue levels were 34 ppm 2,4,5-T in omental fat, 40 ppm in muscle, 20 ppm in liver, 176 ppm in kidney and 60 ppm in renal fat. Toxic effects observed in this sheep included anorexia, dehydration, and muscle spasms and, at necropsy, hepatitis, rumen stasis and enteritis. The distribution, metabolism and excretion of 2,4,5-T propylene glycol ether esters in cows and sheep were also reported. The authors concluded that tissue levels of 2,4,5-T after 4 daily doses of 250 mg/kg each were very high, especially in the kidney, the site of elimination. 170. Clark, D. E., Palmer, J. S., Radeleff, R. D., Cruckshank, A. R., and Farr, F. M. (1975) Residues of chlorophenoxy acid herbicides and their phenolic metabolites in tissues of sheep and cattle. J. Agr. Fd. Chem. 23(3):573-8. Distribution of 2,4-D and 2,4,5-T in tissues of cattle and sheep are described. 2,4-D was administered at 300, 1,000 or 2,000 ppm in feed (equivalent to 9, 30 and 60 mg/kg body weight daily) to cattle and at 2000 ppm to sheep for 28 days. 2,4,5-T (less than 0.5 ppm TCDD contamination) was administered as 2000 ppm in feed to sheep for 28 60 �days. Muscle, fat, kidney and liver samples were removed 1 day after the last exposure from each treatment group and 1 week later from animals that received the 2,000 ppm doses of each compound. Tissue samples were analyzed for 2,4-dichlorophenol and 2,4,5-trichlorophenol in the 2,4-D and 2,4,5-T-treated groups, respectively, by 2 methods. Recovery was above 80% for both methods and 0.05 ppm was the lowest detectable level reported. Fat and muscle samples from all treatment groups had less than 0.05 ppm of the corresponding phenol. 2,4-dichlorophenol levels detected by one method were 0.08-0.16 ppm in liver and kidney from both species, and were 2-5 times higher by the second method, which included an alkaline and acid hydrolysis steps (which would cleave any conjugated phenols, releasing free phenols). Liver 2,4,5-trichlorophenol levels were .4 and 6.1 ppm by the 2 methods and for kidney were .8 and .9 ppm by both methods, in cattle. 2,4-D levels were 1 ppm in sheep liver and 9 ppm in sheep kidney 1 day after the 2,000 ppm dosage ended. All 2,4-D, 2,4-dichlorophenol, and 2,4,5trichlorophenol levels were substantially lower in both species and all tissues 1 week after exposure ended than after 1 day, except for 2,4,5-trichlorophenol levels in the liver and kidney of sheep. All treatments produced temporary reductions in weight gain. Chlorophenol metabolites were not produced by tissue homogenates mixed with 2,4-D or 2,4,5-T or by the hydrolysis procedures used to analyze phenols. The authors suggested that hydrolysis was carried out by rumen microorganisms or by enzymatic processes within the animal. Silvex metabolism was also studied in these investigations. The authors concluded that high residues of the phenoxy herbicides were not likely to be encountered in the tissues of grazing animals from herbicides used for agricultural purposes. -171. Clark, D. E., Young, J. E., and Younger, R. L., Hunt, L. M., and McLaran, J. K. (1964) The fate of 2,4-dichlorophenoxyacetic acid in sheep. J. Agric. Food Chem. 12(l):43-45. The tissue distribution and rate of elimination of by 2,4rD sheep was assessed. One yearling ewe was administered 4 mg/kg of [ Cl-2,4-D in 95% ethanol in a gelatin capsule. Blood, urine and fecal samples were collected for the subsequent 4 days and tissues were removed at necropsy (on day 4). All samples were extracted with ethanol and analyzed for radioactivity. The level of detection of 2,4-D in tissue was 0.05 ppm by the radiometric method used. Radioactivity in extracts of blood reached the peak value 75 min. after dosing and was not detectable 24 hours after exposure. At 8.5 hours, 50% of the dose was recovered in the urine and in 28 hrs. 90% was recovered in the urine. By 70 hrs., 95.8% of the dose was excreted in the urine and 1.4% was recovered in the feces. Only 1 radioactive compound in the urine corresponding to 2,4-D standards was separated by paper chromatography and paper electrophoresis. The only tissues were detectable levels of 2,4-D were the thyroid with 0.56 ppm and the urinary bladder with 0.50 ppm; no detectable amounts were recovered from any edible tissues. The authors concluded that 2,4-D was excreted unchanged in urine by sheep with no detectable residual compound in edible tissue. 61 �172. Cleland, M. (1980). Statement of Administrator of Veterans' Affairs before the Subcommittee on Medical Facilities and Benefits of the Veterans1 Affairs Committee, House of Representatives. Feb. 25, 1980, 44 pp. [Testimony.] 173. Cobb, L. M. and Grimshaw, P. (1979) Acute toxicity of oral diquat (1,1'-ethylene-2,2'-bipyridinium) in cynomolgus monkeys. Toxicol. Appl. Pharmacol. 51(2):277-282. The acute oral toxicity of diquat was described in monkeys. Diquat (dichloride monohydrate) in water was administered by stomach tube to Macaca fascicularis monkeys. Single doses of 100, 300 or 400 mg/kg of diquat were given to two monkeys per dose and 200 mg/kg was administered to four monkeys. After 14 days, all survivors were killed and necropsies were performed by gross and histologic examination. Blood and urine samples collected before treatment and during the observation period were analyzed for biochemical and hematologic parameters. Tissue slices were stained for glycogen and fat. Within 4 days of treatment one monkey each that received 100 and 300 mg/kg doses and both monkeys given the highest dose died. No other death occurred during the observation period. Diarrhea and vomiting occurred in all monkeys. All monkeys that died and no survivors showed increase in polymorphonuclear leukocyte counts which persisted to day 4 after treatment. Other•changes that were transcient in survivors and persisted in fatal cases were increases in serum urea, plasma glucose, SCOT and SGPT levels and in urinary protein, glucose, and blood cells. Abnormal findings at necropsy included necrotic gastrointestinal epithelia, renal lesions, necrosis and exfoliation of the epithelium of the kidney tubules, and minimal hepatic necrosis of single hepatocytes and fat droplets. Vacuolation of the lens observed in some monkeys was not attributed to diquat treatment. Survivors were found to be normal at necropsy, except for one monkey that displayed mild hepatic necrosis. The authors attributed death to the gastrointestinal and renal lesions and estimated the LDcn to be between 100-300 mg/kg diquat ion. The authors concluded that diquat toxicity in the monkey resembles toxicity observed after human ingestion. 174. Cocucci, S., DiGerolamo, F. , Verderio, A., Covallaro, A., Colli, G., et al. (1979) Absorption and translocation of tetrachlorodibenza-p-dioxin by plants from polluted soil. Experientia 35(4):482-484. [Background material.] 62 �175. Cohen, G. M., Bracken, W. M., Lyer, R. P., Berry, D. L., Selkirk, J. K., and Slaga, T. J. (1979) Anticarcinogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dixoin on benzo(a)pyrene and 7,12-dimethylbenzfa)anthracene tumor initiation and its relationship to DNA binding. Cane. Res. 39:4027-4033. The authors investigated the role of TCDD in the suppression of tumors induced by benzo(a)pyrene (BP) and 7,12-dimethyl benz(a)anthracene (DMBA) by studying its metabolic fate: specifically, covalent binding to epidermal DNA, RNA, and protein, and the hydrocarbon-deoxyribonucleoside adducts after treatment of mouse skin. Female Sencar mice aged 7 to 9 weeks were tested for tumorigenesis. All chemicals were applied in 0.2 ml of acetone. Groups of 30 were given a single application of either 100 nmol BP or 10 nmol DMBA, applied topically to their shaved backs. One week later they were given the first of twiceweekly doses of 2 ug 12-0-tetradecanoylphorbol-13-acetate. Additional groups were also pretreated with 1 ug TCDD 72 hours before BP or DMBA initiation. The mice were observed for carcinomas and papillomas, and development was recorded weekly. Carcinomas and papillomas were removed at random for histologic verification. For mice pretreated with TCDD, there was a significant decrease in the incidence of DMBAor BP-induced papillomas, and in the number of papillomas per mouse. For DMBA, TCDD pretreatment decreased the percent of mice with papillomas at 15 weeks from 100 to 10 percent, and the actual number of papillomas per mouse from 9.1 to 0.1. For BP, TCDD pretreatment reduced the percentage of mice with papillomas at 15 weeks from 85 to 25 percent, and the number of papillomas from 3.8 to 0.3. To study in vivo macromolecular binding, female CD-I mice were used to test one compound as well as the Sencar. Treatment was similar to the-tumor experiments, except that the initiating compounds used were f H]BP (approximately 250 uCi/animal; 100 and 200 nmol of the compound for Sencar and CD-I, respectively) and [ HlDMBA (10 uCi/animal, and 10 nmol of the compound for Sencar). Certain animals were pretreated topically 72 hours before initiation with 1 ug TCDD. In some of the pretreated mice, an epoxide hydratase inhibitor 1,2-epoxy-3,3,3-trichloropropane (TCPO) was administered topically 10-15 minutes before initiation with [ H]BP. Three or 24 hours after the initiator was applied, the mice were killed. Using heat treatment, the epidermal material was removed from the animal, and DNA, RNA, and protein were extracted, and the amount of covalently bound radioactivity was determined. The DNA samples had an A260:A28Q ratio of 1.89 +_ 0.01, and an A26Q:A230 ratio of 2.34 +_ 0.05 (mean +_ S.E. of 16 determinations). In animals pretreated with TCDD, the" binding of [HlDMBA to both DNA and RNA was markedly decreased at both 3 and 24 hours after treatment. The effects of TCDD on binding of [ H]DMBA correlate with the decrease in the incidence of tumors, as previously reported. On the other hand, with TCDD pretreatment there was an increase in the binding of f H BP in the Sencar mice. In CD-I mice, there was a marked increase in binding to DNA and protein, and a smaller increase in binding to RNA. Thus, it appeared to the authors that the decrease in tumorigenesis in TCDDpretreated mice was associated with an increase in \ H]BP binding to DNA. The application of TCPO 10-15 minutes before initiation of pretreated mice caused an increase in f H]BP binding to DNA, RNA, and 63 �protein which was notably higher than,when only [ H]BP was given, but which was similar to the effects of [ H]BP initiation with only TCDD pretreatment. Using DNase I, phosphodiesterase, and alkaline phosphatase, the authors also enzymatically hydrolyzed the extracted DNA to deoxyribonucleosides and separated the enzymatic hydrolysates. Fraction volumes were measured for radioactivity. A Sephadex LH-20 color chromatography elution profile 24 hours after [ H]BP treatment showed that the four deoxyribonucleosides eluted between 100 and 200 ml; some radioactive material eluted in this region, and between 30 and 90 ml. A radioactive hydrocarbon-deoxyribonucleoside adduct, apparently BP-7,8-dihydrodiol-9,10-epoxide, bound predominantly to the exocyclic NH- group of quanine (and to a lesser extent to the exocyclic NH_ group of adenine) eluted at between 516 and 574 ml. In mice pretreated with TCDD alone or in combination with TCPO, there was no hydrocarbon-deoxyribonucleoside adduct corresponding to this; most radioactivity eluted before 84 ml. There was a correlation noted in the pretreated mice between significant tumor inhibition and the absence of the diol-epoxide-guanosine adduct from DNA. To trace the metabolism by epidermal homogenates of [ H]BP, Sencar mice were pretreated with either 1 ug TGDD or 100 nmol BP 24 or 72 hours, respectively, before sacrifice. They were killed by cervical dislocation, and their shaved backs were depilated with a cream that was washed off with water after 7 minutes. The scraped-off epidermis (4 mice/ml) was removed and suspended in a solution of 0.05 ra Tris-0.25 M sucrose buffer (pH 7.5) and then homogenized. This solution was then incubated with [ H]BP at 37° for 30 minutes, after which the reaction was stopped with 1 volume acetone. Ethyl acetate extracts were dried with sodium sulfate, concentrated by vacuum rotary evaporation, separated by highpressure liquid chromatography, and eluted with a 30-70 percent methanol-water gradient. Metabolites which eluted included 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene (9,10-diol), 8-dihydrodiol, quinones, 9-hydroxybenzo(a)pyrene, and 3-hydroxybenzo(a)pyrene. Just before 9,10-diol a major band of radioactivity eluted. Metabolites formed to a greater extent with TCDD pretreatment than with BP pretreatment. Untreated CD-7 mice tested in the same way showed only minimal metabolism in epidermal homogenates. The major dihydrodiol formed in all cases was 7,8-dihydrodiol, a precursor of the highly reactive mutagenic and carcinogenic . (jO-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. In homogenates from TCDD pretreated mice, the ratio of monohydroxybenzo(a)pyrenes to 7,8-dihydrodiol was much greater than in BP pretreated mice. 176. Cohn, W. E. 55(35):5. (1977) Validity of animal testing. Chem. Eng. News [Editorial.] 64 �177. Colburn, W. A. (1978) A model for the dose-dependent pharmacokineti.es of chlorophenoxy acid herbicides in the rat: The effect of enterohepatic recycling. J. Pharmaco. Biopharmac. 6(5):417-426. A two-compartment kinetic model of the distribution and elimination of 2,4,5-T is presented and used to analyze previously published pharmacokinetic data for these 2 herbicides. The concentrations of herbicide in the 2 compartments, representing the gut (compartment 2) and the remainder of the organism (compartment 1) are dependent upon 4 firstorder rate processes. These processes include biliary excretion (k._), fecal excretion (k2g), intestinal reabsorption (kop and renal excretion (Vmax/km). At high plasma concentrations, renal excretion mechanisms become saturable and the kinetics of this process then become zero-order with a rate constant equal to Vmax. Another model which was the same as the 2-compartment model except that a third compartment was inserted to represent the kidney. This compartment was described by 2 first-order rate constants, Vmax/km to represent transfer between the plasma and kidney, and k-Q, urinary excretion. Values for the rate constants from data on 2,4,5-T in the rat from another investigator was used to simulate the time course of plasma and urinary clearance of 2,4,5-T. For a low dose of 5 mg/kg 2,4,5-T, the simulated data matched the actual data for urinary and fecal excretion and kidney to plasma ratios. At a higher dose of 50 mg/kg, the model did not simulate the initial, non-linear portion of the plasma or urine curves. The model was modified to simulate kinetics after bile duct cannulation by eliminating the k~, component and the non-linear portion of the clearance curves were tnereby reduced. The pharmacokinetics of 2,4,5-trichlorophenoxy propionic acid was also studied. The author concluded that the non-linear portion of the pharmacokinetic profiles for urine and plasma were dependent upon enterohepatic recycling of 2,4,5-T as well as saturation of renal mechanisms at high doses. The author also concluded that both the biological half-life and the toxicity of 2,4,5-T would be reduced by inhibiting biliary recycling. 178. Colby, C. (1978) Miscarriages suffered: dioxin. Congressional Record - Senate. Monkeys show effects of 124(118):S12271. [Testimony.] 179. Collins, C. V. (1967) Herbicide operations in Southeast Asia, July 1961-June 1967. DTEC 67-0020. Pacific Air Force, APO San Francisco DTIC No. AD 779796. 76 pp. [Background material.] 65 �180. Collins, T. F. X., Williams, G. H., and Gray, G. C. (1971) Teratogenic studies with 2,4,5-T and 2,4-D in the hamster. Bull. Environ. Contam. Toxicol. 6(6):559-567. The teratogenic effects of 7 commercial preparations of 2,4,5-T with various levels of TCDD contamination and 3 commercial preparations of 2,4-D were studied in the hamster. The test compounds were administered orally on days 6 to 10 of gestation. On day 14 of gestation, corpora lutea and fetuses were counted. Fetuses were stained with alizarin red and fixed in Bouin's fluid and observed for skeletal and visceral malformations, respectively. 2,4-D, which was tested at doses of 20-100 mg/kg, produced no dose-related alterations in fetal viability, fetal weights or frequency of malformations. Administration of 2,4,5-T preparations without detectable TCDD contamination at daily doses of 20-100 mg/kg, produced dose-related increase in fetal mortality and incidences of malformations and decreases in fetal weights. These effects were augmented in animals that received 2,4,5-T with 0.1, 0.5, 2.9 or 45 ppm TCDD. Exposure to the highest 2 levels of TCDD contamination, however, resulted in edematous fetuses which masked the decrease in fetal weights that resulted from the exposure. The malformations in 2,4,5-T treated fetuses were related to cranial development5 but included only 2 cases of cleft palate. Exposed fetuses were observed with hemorrhagic gastrointestinal tracts, which were attributed to direct toxicity on the fetal organ rather than an effect on development. 181. Committee on Veterans' Affairs. (1979) Herbicide "Agent Orange" Hearing before the Subcommittee on Medical Facilities and Benefits of the Committee on Veterans' Affairs, House of Representatives, 95th Congress - Second Session, Oct. 11, 1978. Washington, DC.: U.S. Government Printing Office, 62 pp. [Testimony.] 182. Commoner, B. 59. (1978) Toxicologic time bomb. Hosp. Prac. 13(6):56 and [Editorial.] 183. Commoner, B. (1977) Seveso: ll(4):479-482. The tragedy lingers on. [Editorial.] 66 Clin. Toxicol. �184. Commoner, B., and Scott, R. E. (1976) US Air Force studies on the stability and ecological effects of TCDD (dioxin): An evaluation relative to the accidental dissemination of TCDD at Seveso, Italy. Louis, Mo,: Center for the Biology of Natural Systems, Washington University, 51 pp. St. [Review article.] 185. Commoner, B., and Scott, R. E. Accidental contamination of soil with dioxin in Missouri: effects and countermeasures. (no ref.) 23 pp. [Not available,] 186. Comptroller General of the United States. (1979) U.S. ground troops in South Vietnam were in areas sprayed with Herbicide Orange. U.S., Washington, DC., No. FPCD-80-23. 12 pp. [Background material.] 187. Comptroller General of the United States. (1979) Health effects of exposure to Herbicide Orange in South Vietnam should be resolved. U.S., Washington, DC,, No. CED-79-22. 38 pp. [Background material.] 188. Conaway, C. C., and Matsumura, F. (1977) Alteration of cellular utilization of thymidine by TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). Bull. Environ. Contam. Toxicol. 13(l):52-56. Alterations in thymidine incorporation by hepatic slices from TCDDtreated rats are described. Male Sprague-Dawley rats were administered 5 ug/kg TCDD in acetone-corn oil (1:9) by stomach tube. Controls received vehicle only. Ten days later, the_liver was excised, and liver slices were incubated in medium with H-thymidine or H-uridine for 16 min. and then incubated in nonradioactive medium for 1 hr. Tissue was then homogenized and subfractionated by centrifugation. Radioactivity of each subcellular fraction was determined. The nuclei of treated cells contained 24% of total cellular radioactivity, compared to 12% for controls. No significant changes occurred in any other fraction or for H-uridine incorporation. Incorporation of -H-thymidine by isolated nuclei from TCDD treated rats was higher than for controls and 78% of the nuclear radioactivity was associated with the chromatin material after the TCDD treatment, compared to 51% for controls. The authors concluded that TCDD disrupted nuclear utilization of thymidine and suggested that TCDD might disrupt DNA synthesis as its general mechanism of action in living cells. 67 �189. Conning, D. M., Fletcher, K., and Swan, A. A. B. (1969) Paraquat and related bipyridyls. Br. Med. Bull. 25(3)-.245-249. [Review article.] 190. Cook, R. R., Townsend, J. C., Ott, M. G. (1980) Mortality experience of employees exposed to tetrachlorodibenzo-p-dioxin (TCDD). J. Occup. Med. 22:47-50. 191. Cooper, P. (1977) Dioxin: 15(5):481-483. A new biological probe? Fd. Cosm. Toxicol. [Review article.] 192. Corthay, J., Medilanski, P., and Benakis, A. (1977) Induction of hepatic microsomal enzymes by diuron, phenobenzuron, and metabolites in rats. Ecotoxicol. Environ. Safety 1(2):197-202. The effect of subacute administration of diuron to rats on microsomal liver enzyme activity is presented. Male Wistar rats (4 per group) were administered feed with 691 pptn diuron for 3, 7 and 14 days. Rats were weighed weekly. One day after the exposure period ended, rats were killed and hepatic microsoraes were isolated and assayed for protein, cytochrome P-450, aminopyrine N-demethylation and aniline para-hydroxylation activities. Weight gain for treated and nontreated rats were comparable. The only parameters that were altered in treated rats were the microsomal protein content and cytochrome P-450 content which increased transiently at 7 days to 25-50% above control values. Other compounds were also studied in these experiments. The authors concluded that the high dose of diuron tested, which would probably be encountered only from occupational accidents showed weak effects on parameters related to microsomal enzyme induction. 193, Coulston, F. (1970) Working paper: 2,4,5-T. Institute of Experimental Pathology and Toxicology. Albany Medical College, Albany, N.Y. 26 pp. [Review article.] 194. Courtney, K. D. (1979) Postpartum CPK and LDH cardia and serum isozymes after 2,4,5-T, carbaryl or aniline treatment. Toxicol. Appl, Pharmacol. 48(1):A139. [Abstract, only.] 68 �195. Courtney, K. D. (1977) Prenatal effects of herbicides: Evaluation by the prenatal development index. Arch. Environ. Contain. Toxicol. 6:33-46. The teratogenicity fetotoxicity and embryolethality were evaluated in rats administered various esters of 2,4,5-T and 2,4-D by various routes in various vehicles. About 45 protocols were used for these experiments, in which various esters of 2,4-D, 2,4,5-T (with less than .05 ppm TCDD contaminant), and their structural analogs were administered during the second trimester to pregnant CD-I mice. The compounds were administered by oral gavage or subcutaneous injection in a corn oilacetone mixture (90% oil), dimethyl sulfoxide (DMSO), or 15% sucrose. On day 18 of gestation, fetuses were weighed, stained with Bouin's solution, and examined for malformations. Maternal weight gains and liver weight-to-body weight ratios were also recorded. The results for experimental groups were compared with corresponding vehicle control groups. Results were expressed in terms of a prenatal development index. 2,4,5-T, administered in doses from 0.45 to 1 mM/kg in different vehicles by different routes did not produce a statistically significant change in maternal weight but caused a consistent increase in maternal liver weight to body weight ratio. Three fetal effects were elicited by different dosages of 2,4,5-T. Cleft palate was observed at the lowest dosages and at higher doses was observed concomitant with increased fetal mortality. In addition to these effects, the highest dosages produced fetotoxicity which was detected as a decrease in fetal weight. The magnitude of the response elicited by the same dose of 2,4,5-T administered in various vehicles reflected the degree of solubility of the administered dose and corresponding bioavailability. The esters of 2,4,5-T produced the same types of maternal and fetal effects as 2,4,5-T, although cleft palate was produced at the same doses that produced fetal toxicity and mortality. 2,4-D and its esters elicited vehicle-dependent changes in maternal weight gain, and increased maternal liver weight to body weight ratios. Fetal weights were decreased at low dosages of 2,4-D which did not cause fetal mortality or cleft palate. In general, the structural analogs did not alter maternal liver to body weight ratios or fetal parameters,except Silvex which adversely altered all 3 fetal parameters although at doses higher than the active doses of 2,4,5-T. Agent Orange, administered orally at 1 mM/kg on days 12-15 resulted in an increased maternal liver to,body weight ratio, decreased fetal weight and cleft palate (which was absent in the control group). The authors suggested that 2,4-D and 2,4,5-T act by different mechanisms because their combination in Agent Orange did not produce an additive effect, but rather had a potency intermediate between 2,4-D and the more toxic compound, 2,4,5-T. 196. Courtney, K. D. (1976) Mouse teratology studies with chlorodibenzop-dioxins. Bull. Environ. Contarn. Toxicol. 16(6):674-681. The teratologic effects of TCDD were studied in mice. On days 7 through 16 of gestation, pregnant CD-I mice were administered doses of 69 �25-400 ug/kg/day of TCDD in 5% anisole in corn oil by gastric intubation or 25-200 ug/kg/day of TCDD in DMSO subcutaneously. On day 18, fetuses were removed, weighed, and fixed in Bouin's solution for pathologic examination. Adult female CD-I mice administered 10 ug/kg TCDD in anisole-corn oil orally for 14 days, showed no lethal effects or weight loss. Doses of 200 and 400 ug/kg/day to pregnant mice produced marked edema, vaginal bleeding and spontaneous abortions. At 100 ug/ kg/day, TCDD adversely affected survival and weights of fetuses and maternal weight gain. At 25-50 ug/kg/day, TCDD produced an increase in the maternal ratio of liver to body weight. Subcutaneous doses of 25 ug/kg TCDD incidences of 82% for cleft palate, 53% for renal malformation and 11% for clubfoot. Control incidences were zero for cleft palate, 1% for kidney malformations and 4% for clubfoot. Oral doses of 50 ug/kg TCDD produced 19% cleft palate, 72% kidney malformations and 7% clubfoot. Other chlorodibenzo-p-dioxins were studied in these experiments. The authors concluded that TCDD was the most fetotoxic and teratogenic of the compounds they studied and that the bioavailability of TCDD given orally is less than that of subcutaneously administered TCDD. 197. Courtney, K. D. (1970) In Pesticides Symposia, Hales & Assoc., Florida. pp. 277-283. [Not available.] 198. Courtney, K. D., Ebron, M. T., and Tucker, A. W. (1977) Distribution of 2,4,5-trichlorophenoxyacetic acid in the mouse fetus. Toxicol. Letters 1(2):103-108. The distribution of radioactivity in pregnant mice administered f Cl2,4,5^ was reported. Pregnant CD-I mice were administered 100 mg/kg of [ C]-2,4,5-T orally on day 13 or daily from day 10 through 13 of gestation. From 0.5 to 72 hrs. after the last dose, mice were killed and maternal tissues and blood, placentas, and fetuses were analyzed for radioactivity. The location of radioactivity in fetal tissues was visualized by autoradiography. All tissues showed peak levels of radioactivity 8 hours after a single dose was given. At 48 hours only traces of radioactivity remained in maternal tissues. After 4 doses, maternal blood levels were 10 times higher than after the single dose, skeletal muscle had the same concentration and other organs in general as well as the fetuses had 2-3 fold higher levels. The autoradiographs revealed that the liver, eye, and ventricles contained most of the fetal radioactivity 30 minutes after a single dose. By 2 hours, radioactivity was distributed among most of the internal organs and by 8 hours, radioactivity was increased in the liver and brain. At later times, radioactivity was reduced. 2,4,5-T was deposited on the skin and could have come from the amniotic fluid. No radioactivity was associated with the palates at any time. Urine samples analyzed spectrophotometrically contained only 2,4,5-T. Half of the dose was excreted in 24 hr. Although the method used to identify urinary metabolites was not described sufficiently to convincingly demonstrate that 70 �likely metabolites of 2,4,5-T could be separated, the results clearly demonstrate that 2,4,5-T administered to pregnant mice reaches the fetus and seems to be eliminated from both organisms at similar rates. 199. Courtney, K. D., Gaylor, D. W., Hogan, M. D., Falk, H. L., Bates, R. R., and Mitchell, I. (1970) Teratogenic evaluation of 2,4,5-T, Science 168:864-866. The teratogenic effects of 2,4,5-T contaminated with 30 ppm TCDD were evaluated on 2 strains of mice and on the rat. The herbicide was administered subcutaneously in dimethylsulfoxide or orally in an aqueous honey solution. The test preparation was administered daily to C57BL/6 mice from days 6 to 14 or days 9 to 17 of gestation, to AKR mice from days 6 to 15 and to rats from days 10 to 15. C57BL/6 mice were killed on day 18 of gestation, AKR mice on day 19 and rats on day 20. Both mothers and fetuses were examined for gross histopathological effects and one-third of each mouse litter was stained with alizarin red and examined for skeletal anomalies. Daily doses of 113 mg/kg or 46 mg/kg administered by either route to both strains of mice or to rats produced substantial increases in the proportion of litters with at least 1 malformed fetus (up to 100%) and in the proportion of abnormal fetuses per litter (30-80%) compared to untreated controls or controls administered vehicle only. Cleft palate and cystic kidney accounted for almost all fetal abnormalities. Oral administration qf 113 mg/kg 2,4,5-T caused a significant increase in fetal mortality per litter of rats or-either strain of mice. Lower doses of 2,4,5-T did not produce teratotoxic or fetotoxic effects in mice, although rats were susceptible to these effects from doses of 10 mg/kg 2,4,5-T. The ratio of liver weight to body weight was elevated significantly in fetuses and in mothers of C57BL/6 mice treated on days 9 to 17 with 113 mg/kg 2,4,5-T. Although 2,4,5-T treatment of AKR strain mice did not induce cystic kidney malformations in fetuses, hybrid litters of C57BL/6 females mated with AKR males were susceptible to this defect from 2,4,5-T exposure. A dose-related increase in the incidence of hemorrhagic gastrointestinal tract in rat fetuses from 2,4,5-T exposure was attributed to direct toxicity on the fetal organ rather than a developmental defect. 200. Courtney, K. D., and Moore, J. A. (1971) Teratology studies with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-pdioxin. Toxicol. Appl. Pharmacol. 20:396-403. The teratogenic effects of 2,4,5-T preparations of known purities and of TCDD was evaluated in 3 mouse strains and in the rat, TWO inbred strains of mice, DBA/2J and C57B1/6J and CD-I, a random-bred mouse strain were tested, as well as CD strain rats. Pregnant rats were administered TCDD and 2,4,5-T in dimethylsulfoxide (DMSO) subcutaneously on days 6 through 15 of gestation and fetuses were removed and examined on day 17 (CD-I) or day 18. Pregnant rats were administered 2,4,5-T in a 15% sucrose solution by gastric intubation and TCDD in DMSO from days 6 through 15. Rat fetuses were examined on day 20 of 71 �gestation. Vehicle controls but no untreated controls were included. Neither an analytical grade nor a technical grade of 2,4,5-T nor TCDD were fetocidal in mice or rats at doses below thoae which produced maternal toxicity. A dose-related decrease in fetal weight was observed in CD-I mice administered 2,4,5-T which was considered a toxic effect on the fetus. This effect was not seen in the rat or with TCDD treated animals. An increased maternal liver to body weight ratio and decreased maternal weight gain occurred in some groups of rats and some mouse strains from 2,4,5-T or TCDD administration. TCDD and 2,4,5-T each produced cleft palate in all mouse strains but not in the rat. Administration of both agents together did not result in a potentiation of the effect in the mouse. Renal anomalies, including unilocular cystinephrotic kidney and hydronephrosis, were observed in CD-I mice treated with 2,4,5-T and in all strains of mice and in rats treated with TCDD with no potentiation observed when both agents were administered. C57B1/6J showed the highest sensitivity to TCDD-induced kidney anomalies. In a postnatal study, rats delivered from 2,4,5-Ttreated mothers showed the same weight gains and mortality to 21 days of age and timing of eye-opening as those delivered from vehicle controls. The authors suggested that differences in teratologic responses of the various strains to each agent may reflect differences in distribution or metabolism of the compounds. 201. Courtney, K. D., Putnam, J. P., and Andrews, J. E. (1978) Metabolic studies with TCDD (dioxin) treated rats. Arch. Environ. Contain. Toxicol. 7 ( ) 3135-396. 4: Food and water consumption and changes in body weight were monitored in rats given a single dose of TCDD and fed modified diets. Female Wistar rats were administered 100 ug/kg TCDD (99%+ pure) in 5% anisole in corn oil by oral intubation. Control rats received the vehicle only. Supplemental diets were administered by oral intubation given in 5 ml portions 3-7 times per day. Diets were supplemented daily with either 15 ml of water, 15 or 35 ml of an oral electrolyte solution, or 15 ml of a nutrient supplement dissolved in electrolyte solution. The typical pattern of weight loss in treated rats were biphasic, with an abrupt loss during the first 7-10 days followed by an abrupt gain during the next 4-5 days and then a sustained gradual weight loss. Concomitant changes in food and water consumption occurred. The TCDDtreated rats administered supplemental diets showed the same mortality and the same weight changes which occurred at the same times as TCDDtreated rats fed ad libitum. The rats on supplemental diets voluntarily consumed almost as much water as non-supplemented rats. In general, the TCDD-treated rats that survived had smaller weight losses, gained more weight during the 4-day period between the two phases of weight loss, and had normal organ weights, all of which contrasted with those of rats that did not survive. Brain, adrenal, and kidney weights did not change in any TCDD treatment group. Urinalysis revealed high protein content during the first week after TCDD treatment which returned to normal in rats that survived. A transient orange color of the urine observed 2 weeks after TCDD treatment was not accompanied by fluorescence under UV light. Other control rats 72 �deprived of water or food for 4 days lost as much weight as TCDDtreated rats and became irritable and restless. They regained the weight after 4 days on a normal diet, in contrast to TCDD-treated rats which were listless and achieved their initial body weight in 20-30 days. No changes in oxygen uptake by hepatic mitochondria were observed 7 or 14 days after TCDD treatment. The authors concluded that some of the observed changes resulted secondary to the primary effect of cessation of food and water consumption or to non-utilization of nutrients, but the present studies could not distinguish between primary and secondary effects. 202. Coutselinis, A., Kentarchou, R., and Boukis, D. (1977) Concentration levels of 2,4-D and 2,4,5-T in forensic material. Forensic Science 10:203-204. Human tissue levels of 2,4-D and 2,4,5-T are presented for a fatal case of poisoning. Blood, liver, spleen and kidney samples were removed at autopsy from a woman who consumed an unknown amount of Tributon 60 (containing a 3:2 mixture of 2,4-D; 2,4,5-T) 16 hours before death occurred. These samples were analyzed for 2,4-D and 2,4,5-T by thinlayer chromatography. The levels of 2,4-D were 82.6 mg/100 ml blood, and 2.1, 1.2 and 8.2 mg/lOOg of liver, spleen and kidney, respectively. 2,4,5-T levels were 18.2 mg/100 ml blood and 0.5, 0.5, and 2.2 mg/lOOg of liver, spleen and kidney, respectively. The authors concluded that a large amount of herbicide was ingested but did not have enough information to estimate the exact amount. 203. Crabtree, H. C., Lock, E. A., and Rose, M. S. (1977) Effects of diquat on the gastrointestinal tract of rats. Toxicol. Appl. Pharmacol. 41(3):585-595. The effects of oral diquat administration on redistribution of water from the blood to the gastrointestinal tract of rats is described. Male Alderley Park rats were administered 900 umol/kg of diquat in saline by stomach tube or 90 umol/kg in saline, subcutaneously. Controls received saline vehicle only. Body weights were determined at the time of dosing and 24 hr. later. Mortality was recorded for 8 weeks. Water content was determined for the gastrointestinal (GI) tract by clamping both ends and weighing the tissue and its contents before appreciable evaporation occurred. Hematocrits were determined and tissue water was estimated as the amount of weight loss that occurred during drying to a constant weight. Within 24 hrs. of oral diquat dosing, water intake was reduced to 19% of controls. After 24 hr., signs of toxicity, including subdued activity, piloerection, and characteristic green feces were evident. Within 3 days, half of the total deaths occurred and these animals, unlike the 3-dayfailed to gain weight. After subcutaneous dosing, water intake and signs of toxicity resembled those after oral dosing but the symptoms appeared within 4-6 hr. The earliest deaths occurred after 5 days and some rats died as late as 8 weeks after dosing. All rats that died after 14 days (but no survivors) had severely extended terminal ileums and cecums. Water 73 �content of the GI tract was maximum 1 day after oral dosing and the magnitude was dose-related, with twice the water content of control after 900 umol/kg was administered. The same effect occurred after subcutaneous dosing but did not appear until 3 days after dosing and was maximum at 8 days (the last day water content was measured). This elevation did not occur in the survivors who also did not have distended abdomens. Significant blood, muscle and liver dehydration occurred 24 hr. after oral dosing. Hemoconcentration after subcutaneous dosing was transient and peaked 17 hr. after iniection. The authors concluded that deaths after oral diquat administration could be attributed to rapid fluid loss to the GI tract and later deaths and deaths after subcutaneous dosing appeared to have unrelated and unidentified causes. Information on volumes of urine excreted by rats in this experiment would help in understanding the mechanisms of water balance that are crucial to this study and concurrent histology of tissues from the GI tract would also help to elucidate the source of fluid shifts. i 204. Crabtree, H. C., and Rose, M. S. (1978) Effect of diquat dichloride on gastric emptying and fluid accumulation in the rat stomach. Toxicol. Appl. Pharmacol. 45(1):259-260. [Abstract, only.] 205. Crabtree, H. C., and Rose, M. S. (1976) Early effects of diquat on plasma corticosteroid concentrations in rats. Biochem. Pharmacol. 25(22):2465-2468. The effect of diquat administration on corticosteriod production in vivo and in vitro was studied in the rat, Male Sprague-Dawley rats were administered 250 ug/kg dexamethasone (subcutaneously) and 2 hrs. later were anesthetized with phenobarbital (150-200 mg/kg, subcutaneously). Two hrs. after the anesthetic was given, diquat dichloride monohydrate in saline was administered, an oral dose of 900 u moles/kg or an intraperitoneal or subcutaneous dose of 10-260 u moles/kg. From 1 to 4 hrs. later, the animals were killed by decapitation and blood was removed and analyzed fluoremetrically for corticosteroids and by radioimmunoassay for adrenocorticotrophic hormone (ACTH). In vitro incubation of rat adrenals was performed in the presence of diquat for 2 hrs., with ACTH added during the last 30 min. Adrenal tissue was then homogenized and analyzed for corticosteroid content. Plasma corticosteroid levels (in non-dexamethasone-pretreated rats) were increased maximally within 30 min. of a subcutaneous or intraperitoneal dose of 90 u moles/kg diquat to four times above levels of vehicle controls. This effect was sustained for 4 hrs. and occurred after oral administration, with a delay in the onset of the effect. The effect was maximum after 4 hrs. with doses of 50 u moles/kg of diquat and greater. The effect was blocked by pretreatment with dexamethasone and the ACTH levels were significantly elevated in diquat treated rats after 1 hr., compared to ACTH levels in rats administered dexamethasone 74 �and diquat. ACTH administration in vivo did not alter the plasma corticosteroid levels in diquat treated or control rats. In vitro, diquat at 25-50uM inhibited corticosteroid synthesis significantly in the presence of ACTH but not in its absence. The authors concluded that diquat caused increased adrenal steroid synthesis by stimulating release of ACTH from the pituitary. 206. Craig, D. A. (1975) Use of herbicides in Southeast Asia. Historical report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 pp. {Background material.] 207. Creso, E., De Marino, V., Donatelli, L., and Pagnini, G. (1978) Effetti neuropsicofarmacologic: della TCDD. Boll. Soc. It. Biol. Sper. LIV:1592-1596. [Foreign language.] 208. Croft, W., Yang, K. H., and Peterson, R. E. (1977) Pathological effects of 2,3,7,8-tetrachlorodibenzo-p-dixoin on the common bile duct of rats. Fed. Proc. Fed. Am. Soc. Exp. Biol. 36(3):1060. [Abstract, only.] 209. Crosby, D. G. (1976) Nonbiological degradation of herbicides in the soil. In Herbicides: physiology, biochemistry, ecology. Vol. 2. ed., L. J. Audus, (New York: Academic Press) pp 65-97. [Review article.] 210. Crosby, D. G., Moilanen, K. W., and Wong, A. S. (1973) Environmental generation and degradation of dibenzodioxins and dibenzofurans. Environ. Health Perspec. 5:259-265. The authors studied the degradation and generation of TCDD and other dioxins in a laboratory simulating a field situation. Photochemical generation of TCDD was measured by ultraviolet irradiation of 2,4,5-T, 2,4,5-trichlorophenol, or sodium 2,4,5-trichlorophenate, in a "sunlight simulator" equipped with F40BL fluorescent lamps. The authors did not report the details of the experiment. No TCDD was detected as a photogeneration product of any of the three chemicals. The authors explained their failure to detect TCDD by its extreme instability in sunlight. TCDD (5 mg/ml) in purified methanol, was nearly 60^ degraded by 6 hours irradiation in the "sunlight simulator." The applicability of this system to a field situation was not known by the authors. 75 �211. Crosby, D. G., and Tutass, H. 0. (1966) Photodecoraposition of 2,4-dichlorophenoxyacetic acid. J. Agric. Food Chetn. 14:5%-601. [Background material.] 212. Crosby, D. G., and Wong, A. S. (1973) Photodecomposition of 2,4,5trichlorophenoxyacetic acid (2,4,5-T) in water. J. Agric. Food Chem. 21(6):1052-1054. [Background material.] 213. Crosby, D. G., and Wong, A. S. (1977) Environmental degradation of TCDD. Science 195(4284):1337-1338. The authors studied the photodecomposition of TCDD in a model environmental system. Residual TCDD was measured by gas-liquid chromatography. Thin layers of Agent Orange containing 15 ppm TCDD on glass petri dishes were exposed to summer sunlight. Identical plates were shielded from light and served as dark controls. After 6 hours of exposure to sunlight, less than half of the applied TCDD was present on plates. Agent Orange was also applied in droplets (size unspecified) over excised leaves of a rubber plant and on the surface of loam soil and exposed to sunlight. Less than 50% of the TCDD was present on leaves 2 hours after irradiation, while 80% of the TCDD was present on the soil 6 hours after irradiation. Dark controls remained unaffected. From these and previous experiments, the authors concluded that TCDD photodecomposition requires 1) dissolution in a light transmitting film, 2) the presence of an organic hydrogen donor, such as solvent or pesticide, and 3) ultraviolet light. All three conditions would have been met during Agent Orange spraying in southeast Asia. 214. Crosby, D. G., Wong, A. S., Plimmer, J. R., and Woolson, E. A. Photodecomposition of chlorinated dibenzo-p-dioxins. Science 173(3998):748-749. (1971) The authors studied the photodecomposition of TCDD. When TCDD was diluted in raethanol (5 mg/1) and irradiated by summer sunlight or a "sunlight simulator", photodecompsition occurred in 24-36 hours. Photolysis products included 2,3,7-trichlorodibenzo-p-dioxin and a dichloro homolog identified by mass spectrometry. However, when TCDD was added as a spot (2.4 ppm in ethanol) on glass plates coated with sandy soil or silty clay loam soil, irradiation with an ultraviolet lamp for 96 hours produced negligible decomposition. The same result was achieved when the soil was moistened with water. Similarily, when TCDD was applied as a dry film to glass dishes or in a suspension of water, quantitative recovery of TCDD was observed after 14 days irradiation. This study suggests that TCDD is photodecomposed in the presence of organic hydrogen donors such as organic solvents, but not on surfaces of soil and water. 76 �215. Crossen, P. E., Morgan, W. F., Koran, J. J., and Stewart, J. (1978) Cytogenetic studies of pesticide and herbicide sprayers. New Zeal. Med. J. 88(619):192-195. The authors studied chromosomal damage in 57 pesticide and herbicide sprayers by the sister chromatid exchange (SCE) test. The test group was composed of healthy males from a wide variety of occupations that involved spraying pesticides and herbicides. Exposure to specific pesticides was not reported. A control group of 15 males with no known history of exposure was included in the study. Peripheral blood samples were obtained from each test subject and lymphocytes cultured from the samples for 72 hours followed by 4 hour incubation with colchicine. Twenty well spread metaphases were examined for each subject. Scoring of sister chromatid exchanges was done using coded samples. No positive controls were reported by the authors. Only a one time sampling was done by the investigators. When the means of sister chromatid exchanges of control and exposed subjects were compared no significant differences in the two groups were observed. However, 5 sprayers had an SCE rate 3 standard deviations outside the mean of the controls. The data were then analyzed according to length of exposure and use of protective clothing. Only the group of sprayers with greater than 1 year exposure and no use of protective clothing showed an elevated SCE ratte. The authors concluded that these data suggest a possible genetic hazard from pesticide and herbicide spraying. However, because of the small number of subiects, the unknown nature of exposures, and the one time sampling, no real conclusion can be made about this study. 216. Crow, K. D. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA Docket No. 415 et al., Nov. 14. [Testimony.] 217. Crow, K. D. 1:982. (1979) Lipid profiles in dioxin-exposed workers. Lancet [Editorial.] 218. Crow, K. D. (1978a) Chloracne - an up to date assessment. Ann, Occup. Hyg.. 21:297-298. [Review article.] 77 �219. Crow, K. D. pp. 78-80. (1978b) Chloracne: the clinical disease. New Scientist [Review article.] 220. Crow, K. D. (1977) Effects of dioxin exposure, Lancet 2(8028):82-83. [letter to the editor] [Editorial.] 221. Crow, K. D. (1970) Chloracne. Transactions of the St. Johns Hospital Dermatological Society 56:79-99. [Review article.] 222. Cunningham, H. M., and Williams, D. T. (1972a) Effect of tetrachlorodibenzo-p-dioxin on growth rate and the synthesis of lipids and proteins in rats. Bull. Env. Contain. Toxicol. (U.S.) 7:45-51. The effects of dioxin treatment on hepatic protein and lipid synthesis was studied in the rat. Single doses of 0.1-10.0 ug/kg of TCDD in corn oil were given to.weanling male Wistar rats by oral ^ntubation. From 1-7 days later, C-leucine and H-sodium acetate were injected intraperitoneally and 1 hour later liver, adipose, and muscle tissues were removed and homogenized. Lipids were extracted by a method described elsewhere and protein in the aqueous phase was precipitated with trichloroacetic acid, solubilized and counted for radioactivity by liquid scintillation methods along with radioactivity incorporation into lipid. Thin-layer chromatography was used to fractionate various lipid fractions. At 10 ug/kg, TCDD caused a statistically significant decrease in weight gain after 3 days and in radioactivity (per mg liver) associated with total lipids, triglycerides, phospholipids, and epididymal lipids while liver protein synthesis increased. The effects on synthesis and body weights were not consistently reproduced in subsequent experiments presented in this report. The effect on hepatic total lipid incorporation was greater 7 days after TCDD treatment than from 1-3 days after treatment. Both the liver weight and the total lipid content of the liver increased with TCDD treatment, so that the total incorporation of radioactivity into lipids (DPM per liver) did not change, but the specific activity of the liver lipids (DPM per mg lipid) decreased substantially. The authors concluded that TCDD restricted the transport of lipids out of the liver. The adequacy of biochemical techniques in separating incorporated from unincorporated counts, in separating and recovering quantitatively the protein and lipid fractions, and difficulties in reproducing effects in each experiment limit the interpretation of these data. Changes in specific activity of the isotope, effects of protein and lipid degradation during the 1 hour pulse period, and large variability in data preventing a. two-three fold changes in incorporation from being statistically significant difference further weaken this study. 78 �223. Cupello, J. M. , Young, A. L., and Smith, J. C. H. (1977) A method for simulating subsurface disposal of herbicides. Weed Sci. 25(4):36S-372. The authors describe a laboratory model to simulate field disposal practices of phenoxy herbicides using subsurface injection and to investigate the effects of massive quantities phenoxy herbicides to undercut and uncut sorghum. Special growth boxes were designed for use in the experiment so that root systems of the plants could be reached. Four treatment variables were used: 1) under cut control, 2) undercut treated, 3) uncut control, and 4) uncut treated. Three treated replicates and two control replicates were used in the study. Herbicide treatment was 20.1 ml of a 50:50 mixture of the butyl esters of 2,4-D and 2,4,5-T (equivalent to 2,240 kg/ha) and occurred on day 3 for plants with uncut root systems and on day 22 for plants with cut root systems. Due to variations within the model system undercut boxes could not be compared to uncut ones. Treatment of both cut and intact root systems with herbicides caused a statistically significant reduction in plant growth. The authors concluded that this was a reliable model for assessment of herbicidal effects in the laboratory. 224. Cutting, R. K., Phuoc, T. H., Ballo, J. M., Benenson, M. W., and Evans, C. H. (1970) Congenital malformations, hydatidiform moles and stillbirths in the Republic of Vietnam 1960-1969.(Washington, DC.: Department of Defense. U.S. Government Printing Office No. 903.233.) 29 pp. To determine the incidence of stillbirths, hydatidiform moles, and congenital malformations, data were collected from 22 hospitals throughout the Republic of Vietnam. The primary data source for all but 4 hospitals was the Daily Summary Ledger, prepared by the chief midwives. The following criteria were used to record stillbirths, malformations and moles: 1) stillbirths were determined by fetal weights of 750 grams or more with a gestation period of at least 24 weeks; 2) malformations were recorded for both live births and stillbirths and, 3) hydatidiform moles were counted only if delivered. The following is a breakdown of the 488,852 birth events from 1960-1969: 470,200 live births; 15,812 stillbirths; 2,840 hydatidiform moles; and 2,355 congenital malformations, of which 80% were anencephaly, cleft lip/palate, clubfoot, and hydrocephaly. Data were sorted into two five-year time periods, 1960-1965 a period of eight spraying and, 19661969 a heavy spray period, to identify incidence trends associated with herbicide usage. Data show the following: for the period 1960-1965: mole rate - 6.6 per thousand, stillbirth - 36.1 per thousand, malformation - 5.5 per thousand; for the period 1966-1969: mole rate - 5.6 per thousand, stillbirth - 32.0 per thousand, malformation - 4.5 per thousand. The following biases limit the use of the findings: 1) the majority of the data were obtained from population centers and large hospitals; 2) private clinics and hospitals were not surveyed; 3) data were collected only on ethnic Vietnamese where a large segment of the population were Chinese; 4) data on home delivery and morbidity characteristics were not collected; 5) gaps in data appeared due to destroyed records. Authors concluded that "meaningful correlation of any province's annual abnormal birth events to quantitative herbicide data were precluded by inconsistent sampling of birth data." 79 �225. Dalderup, L. M. (1974) Safety measures for taking down buildings contaminated with toxic material. J. Soc. Geneesk. 52:582-623. Results are described of clinical laboratory tests of workers who were involved in dismantling a factory in Amsterdam, the Netherlands, that was contaminated with TCDD. Twelve healthy workers participated in the project and each worker wore a suit with its own oxygen supply while working in the contaminated building. Physical measurements that were performed included lung function tests, X-ray photographs of the thorax, and photographs of the exposed skin areas. Nine different enzyme levels and hematologic parameters were monitored, and urinalysis was performed. Each worker was given a physical examination and his medical history was studied for conditions or medications that would complicate interpretation of the results. Apart from 1 case of slight glucosuria which was noted prior to exposure, no serious abnormalities were detected by any of the tests. Two additional men entered the contaminated area, without protective head gear. Both men had abnormal liver function tests at least once in the 11 week period after exposure (the type of test or magnitude of the deviation from normal was not stated). These workers were not part of the demolition team, so no pre-exposure values for liver function tests were available for these men. The authors concluded that the air-supplied suits used in this decontamination operation provided adequate protection of the workers from the adverse effects of TCDD. 226. Dalgaard-Mikkelsen, S. V., and Poulsen, E. herbicides. Pharmacol. Rev. 14:225-250. (1962) Toxicology of [Review article.] 227. Daniel, J. W., and Gage, J. C. (1966) Absorption and excretion of diquat and paraquat in rats. Brit. J. Industr. Med. 23:133-136. The excretion of diquat in urine, feces and bile was determined in the rat. Male Wistar rats were administered 14-85 mg/kg [ C]-diquat dibromide or dichloride in aqueous solution by gastric intubation or subcutaneous injection. Urine, feces, and bile (from cannulated bile ducts collected for 24 hr. from restrained rats) were collected and analyzed for radioactivity and for diquat and metabolites by a colorimetric procedure after separation by ion exchange chromatography. Recovery of the administered radioactivity was reported to be 90-101%. From 84 to 97% of an oral dose of either diquat salt was excreted in the feces during the 3 days following exposure and 4 to 11% appeared in the urine. From 0.5 to 5.4% of the oral dose of diquat dibromide was excreted as urinary metabolites, 68-81% as fecal metabolites, and 1.1-4.8% was excreted in the bile. After diquat was injected, 88-98% of the dose of dibromide salt was excreted in the urine and up to 2% in feces, and 81-90% of the dose of dichloride salt was recovered in the urine in 2 days. After fecal homogenates were incubated for 24 hr. at 37°C, 52% of the diquat content was degraded, compared to 8% when the microbial content was heat-denatured prior to incubation. Paraquat 80 �excretion was also studied. The authors concluded that diquat was poorly absorbed from the gut and that biotransformat ion resulted only from intestinal microbial degradation and reabsorption of metabolites, as no metabolites were detected in the urine after subcutaneous dosage. 228. Danon, J. M., Karpati, G., Carpenter, S. (1978) Subacute skeletal myopathy induced by 2,4-dichlorophenoxyacetate in rats and guinea pigs. Muscle and Nerve (1) Mar/Apr:89-102. The histochemical and histopathological changes associated with myopathy after 2,4-D administration are described. Sprague-Dawley rats and Hartley guinea pigs were administered 200-250 mg/kg/day of 2,4-D in three separate doses per day, by intraperitoneal injection. After 2-5 days of treatment, biceps muscles were removed and prepared for histochemical analyses, phase microscopy, or electron microscopy. During the day the treated animals remained myotonic and approximately 20% of the animals died and were not studied further. A total of 14 rat biceps and 12 guinea pig biceps were studied. Histochemical changes in muscles from treated rats and guinea pigs included extensive Z-disc streaming in type 2 fibers, masses of proliferating sarcotubular profiles, neutral lipid droplets in muscle fibers, and depletion of type 28 fiber glycogen and amylphosphorylase. Increases in the numbers of necrotic and of regenerating fibers were seen in muscles after 3 days of treatment. Regenerating fibers were also seen 2 and 7 days after treatment was terminated. Fat droplets, sacromere smudging, necrotic fibers, and vacuoles were also identified in both species by phase microscopy. Electron microscopy revealed, in addition, proliferation and dilatation of the sarcoplasmic reticulum, proliferation of myofilaments in cytoplasm and nuclei, loss of myofilament organization, and rounded mitochondria in both species. The authors concluded that in both species 2,4-D produced proliferative effects, possibly related to auxin activity, and degenerative effects that may have occurred secondarily, after a significant amount of cellular energy was diverted to the proliferative activities. 229. Danow, R. A., Truchelut, G. B., and Bartlett, C. M. (1966) OCONUS defoliation test program. US Army Biological Center, Fort Detrick, Frederick, MD. Technical Report No. 79. [Background material.] 230. Davidson, J. H. (1980) Update of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Down to Earth 36(2);19-22. [Background material.] 231. Davis, D. E. pp. 58-59. (1979a) Dioxin and the Vietnam veteran. The Bulletin. [Editorial.] 81 �232. Davis, D. E. 91-94. (1979b) Herbicides in peace and war. BioSci. 29(2):84, [Editorial.] 233. Davis, G. G. (1974) Fluometuron and 2,4,5-T residues in soil, sediment, runoff water and percolation water. Dissertation Abstracts Internat ional: Sect ion B 34(11):5285B-5286B. [Abstract, only.] 234. Davring, L. (1975) Effects of a 2,4,5-T ester on early oogenesis, fertility, and development in Drosophila melanogaster. Hereditas 80:255-262. The authors studied the effects of a commercial preparation of 2,4,5-T butoxyethyl ester (500 g/1 spraying fluid) on oogenesis, fertility, and development in female Drosophila melanogaster. The 2,4,5-T used in this study contained less than 0.1 ppm TCDD as a contaminant. Twentyfour-hours-old Canton-S strain adult flies were fed on wheat agar medium containing 0, 50, 100, 250, or 500 ppm 2,4,5-T for 4 days. Eight replicate cultures containing 10 males and 10 females were set up for each dose level. Development of eggs from exposed females was studied. Decreased fertility (adults/eggs laid) was observed as low as 50 ppm 2,4,5-T and was dose dependent. However, fertility of controls in this experiment was unusually low. The author's explanation for this phenomenon was that flies were grown on wheat agar medium instead of the usual corn agar medium. The number of eggs laid by treated flies also decreased with increasing doses of 2,4,5-T. The authors speculated that the decrease in oviposition may reflect disturbances in oogenesis or an aversion to laying eggs in the treated medium or both. Chromosome disturbances during oogenesis were also studied by examining defective egg follicles. Technical routine handling of ovaries from control flies resulted in a loss of approximately 30% of the egg follicles. The authors did not report an estimate on the loss of egg follicles from treated flies. Egg follicles were collected from freshly eclosed (hatched) females and females that were more than 24 hours old at the beginning of treatment. Flies were grown on medium containing either 1 or 250 ppm 2,4,5-T for 5 days. On the 6th day, the flies were killed and ovaries fixed on slides for microscopic evaluation. The percentage of defective egg follicles increased with 2,4,5-T treatment and was dose dependent. Freshly eclosed females treated with 2,4,5-T had more defective egg follicles than the older females. In addition, about 95% of the first metaphase chromosomes in stage 14 oocytes became a single compact chromosome plate, a normal event. However, in stage 14 oocytes from 2,4,5-T treated females first metaphase chromosomes were distributed in two or three groups in about 26% of the oocytes examined. No effects of 2,4,5-T on the male were presented in this study. 82 �235. Davring, L., and Hultgren, K. (1977) Cytogenetic effects on in vivo bone-marrow cells of Mus musculus induced by a commercial 2,4,-T ester product. Hereditas 85:123-134. The authors studied the cytogenetic effects induced by 2,4,5-T on mouse bone marrow cells. The effects of 2,4,5-T butoxyethyl ester and its formulation components was also studied. For this experiment the high concentration of 2,4,5-T that could be obtained in water was tested. Male mice 15 weeks old 5 per group were given a single intraperitoneal injection of 2,4,5-T or 5 daily injections. Mice receiving only 1 injection were killed 6, 24, and 48 hours after treatment, while those receiving 5 injections were killed 6 hours after the last injection. Two strains of mice were used: (Swiss x CBA) x Swiss, called strain 9, and (DBA x CBA) x DBA, called strain 13. In other experiments, strain 9 mice were more resistant to the effects of chemical exposure than strain 13 mice. Both negative and positive controls were included in the study. Fifty metaphases were scored for chromosome aberrations. Results were analyzed by analysis of variance, students' t- and ranktest. In strain 9 mice receiving a single injection of 2.5 mg 2,4,5-T/kg body weight of metaphases with aberrations was 1.2% at 6 hours, 0% at 24 hours, and 1.5% at 48 hours. Control mice had 0.7% metaphases with aberrations. Similarly strain 13 mice injected once had 0.4% metaphases with aberrations at 6 hours, 0.8% at 24 hours, and 1.0% at 48 hours. Negative controls had no aberrations. In mice receiving multiple injections, strain 9 had 2% metaphases with aberrations, strain 13 had 2.4% metaphases with aberrations and negative controls-had 1.2%, Mice receiving 2.4 mg 2,4,5-T ester in complete herbicide had 2.8% cells with chromosome aberrations at 6 hours, 5.2% at 24 hours and 4.2% at 48 hours, which is an increase in chromosome aberrations compared to controls. Furthermore, mice which received injections of the solvent and emulsifier also had increased chromosome damage. Similar results were obtained using strain 13. In mice receiving multiple injections, 2,4,5-T ester commercial preparation as well as the solvent and the emulsion produced increased chromosome aberrations. 236. Debate continues over Dow's dioxin theory. Sept 24, 1979:27-28. (1979) China and Eng. News [Editorial.] 237. DeBruin, A. (1976) "Chapter 9: Biological Exposure Tests," In Biochemical Toxicology of Environmental Agents. (New York: Elsevier/N. Holland Inc.) [Review article.] 238. Dedrick, R. L. (1971) Extrapolation of teratogenic effect. News 49(20):5-6. [Editorial.] 83 Chem. Eng. �239. Defoliant, cancer: Studies show link. (1980) Science News 117:230. [Editorial.] 240. de Larrard, J., and Barbaste, M. (1969) Intoxication suicidaire mortelle agro-chimique a 1'hormone desherbante 2,4-D. Arch. Mai. Prof. Med. Trav. 30:434. [Abstract, only.] 241. Dencker, L. (1976) Chapter IV: The herbicide 2,4,5-T. Tissue localization of some teratogens at early and late gestation related to fetal effects. Acta Pharmacol. Toxicol. (Suppl) 39(l):59-72. The uptake of radioactivity by C57BL mouse fetuses and hamster fetuses was determined after pregnant animals were administered [ C]-2,4,5-T. Mice were administered 24 ug (5uCi) of F C]-2,4,5-T in 50% alcohol intravenously and hamsters received twice this dose. Autoradiography was performed on tissues removed 4-24 hours after the dose was given. The concentrations of radioactivity per gram of fetal tissue and per ml of maternal serum were determined. Mice and hamsters dosed between day 8 and 10 and autopsied within 12 hours of treatment, contained radioactivity which was localized in the gut. Autoradiographs of mice treated after day 10 showed radioactivity distributed in all tissues except the gut, with increased amounts of activity from treatment later in pregnancy. No.radioactivity was observed in fetal hamster tissue from treatment on day 10 or 11. Radioactivity in mice treated on day 16 was highest in the kidney and visceral yolk sac, followed in descending order by the blood, liver, and muscular tissues. The brain was free of radioactivity. Comparing results of treatments from days 11-16, the concentration of radioactivity was always higher than in the chorio-allantoic placenta and the concentration in the amniotic cavity was always low. The ratio of the concentration of radioactivity between fetal tissue and maternal serum increased 20-fold from day 12 to day 18 which was an estimated 500 fold increase in 2,4,5-T transported to the fetus when correlated with the fetal growth weight at this time. The author postulated that the dramatic increase in uptake of 2,4,5-T by fetuses after day 11 of gestation may reflect an increased permeability of the choriovallantoic placenta which functionally replaces the yolk sac placenta at this time in development. The authors did not comment on the inability of 2,4,5-T to be transported to the fetal hamster. The latest treatment was given on day 12 and if this trend in sustained through day 16, no teratologic effects would be predicted in this species. 84 �242. De Reuck, J., De Coster, W., Willens, J., and vander Eecken, H. (1979) Influence of 2,4-dichlorophenoxyacetate and of dantrolene sodium on the target phenomenon in tenotomized rat gastronemius muscle. Acta Neuropathol. 46:167-168. The effect of 2,4-D on the formation of target fibers in tenotomized rat muscle with and without neurotomy was assessed. Female Wistar rats were subjected to unilateral tenototay (10 rats) or bilateral tenotomy (10 rats) of the Achilles tendon. The sciatic nerve was sectioned (on the side opposite the unilateral tenotomy) in all rats. Starting the day after surgery, half of the rats in each group were administered 90 rag/kg 2,4-D subcutaneously daily for 12 days. The remaining rats were administered only vehicle (triethanolamine buffer). One rat from each group was killed after 1, 2, 5, 7, and 12 days. The gastrocnemius muscles were excised and examined histopathologically and histochemically for succinate and lactate dehydrogenase, cholinesterase, and nonspecific esterase activities. In control tenotomized muscles, type I target fibers developed by day 5 and increased in numbers at later times; type II target fibers first appeared on day 12. Target fibers were preceded by contraction bands. Neither the fibers nor the bands were present in intact muscles or muscles that underwent tenotomy and neurotomy. Treatment with 2,4-D (or dantrolene, which was also described) did not affect the number or time course of target fiber formation in any group. The authors concluded that 2,4-D increased resting membranes resistance in association with a reduced chloride conductance to produce muscle toxicity. 243. Desi, I., and Sos, J. (1962a) Central nervous injury by a chemical herbicide. Acta. Medica. Acta. Sci. Hung. 18:429-433. The authors investigated the effects of acute and sub-acute administration of 2,4-D on the electroencephalographic activity and conditioned reflex responses of 24 rats, 4 cats, and 2 dogs. 2,4-D, at a dose of 200 mg/kg, was administered intraperitoneally only once in the acute experiments and once daily until death in the sub-acute experiments. The animals usually died on the 6th day of 2,4-D treatment. Electrodes were inserted into the cortex and reticular formation of the brains of the experimental animals and EEC readings were recorded before and after 2,4-D administration. Response to a previously learned and reinforced conditioned reflex (sound and electric shock) was measured in animals treated with 2,4-D for 5 consecutive days. In acute experiments, EEC recordings revealed an inhibition of desynchronization within 15 minutes of administration of 2,4-D. The inhibition lasted about 60 minutes then returned to normal. In the sub-acute experiments, EEC measurements revealed a gradual decrease in the frequency of spontaneous electrical activity. In addition, the duration of desynchronization decreased as the 2,4-D treatment continued. By the third day of treatment, desynchronization was reduced 81% while on the fourth day it had further decreased to 91% of the normal duration. By the fifth day no desynchronization was evident. Response to a previously learned conditioned reflex also decreased steadily with continued 2,4-D treatment. All response to the conditioned reflex was lost by the fifth day of treatment. Histologic 85 �examination of the spinal cord showed detnyelinization of the pyramidal tract. The authors concluded that humans working with 2,4-D herbicides should use caution and be specifically examined for symptoms of neurologic disorders. The data presented in this report is identical to data presented in another paper by Desi et al (VA 1429/0). It appears that both papers discuss the same set of experiments. Therefore, the short-comings of the experimental procedures in the study have already been discussed in the annotation of VA 1429/Q. 244. Desi, I., and Sos, J. (1962b) The effect of 2,4-dichlorophenoxyacetic acid and triorthocresyl phosphate on the function of the upper part of the central nervous system (Bioelectric activity and conditioned reflexes). Gig. Sanit. (12):38-46. The effect of 2,4-D on the EEC activity in the brain of adult rats, cats and dogs was investigated. Acute and subacute experiments were conducted. Eight rats were given a single subcutaneous infection of 2,4-D at a dose of 200 mg/kg. These animals were wired for EEC examination. Another set of 8 rats, along with 4 cats and 2 dogs, received a daily dose of 200 mg/kg of 2,4-D for several consecutive days. EEC examinations were also conducted on these animals. A third set of 8 rats received the same consecutive daily dose of 2,4-D but were tested for retention of a previously learned conditioned-reflex response. When the experiments were concluded, usually after 6 days when the animals died, brain and spinal cord tissues were examined histologically. The EEC pattern in the 8 rats that received a single dose of 2,4-D became abnormal within 24 hours of the administration. A reduction or a complete absence of the desynchronization pattern was noted in these animals. In animals that received consecutive daily doses of 2,4-D, electrical activity in the brain decreased steadily as the experiment progressed. A decrease in wave frequency and a drastic reduction in the duration of desynchronization were the main abnormalities. By the fifth day of 2,4-D administration, the desynchronization period had disappeared completely. In the rats that had learned a conditioned response, consecutive doses of 2,4-D caused a deterioration of the conditioned-reflex function. The number of correct responses diminished by 21% after 24 hours, by 58% after 2 days, by 63% after 3 days and 79% by the end of the fourth day. The conditioned response disappeared completely by the fifth day of the experiment. Histologic examination of brain tissue revealed no pathological change, but the pyramidal tract of the spinal cord exhibited signs of demyelinization. The effect of triorthocresylphosphate on the EEC activity of rats, cats, and dogs was also investigated and compared with the effects produced by 2,4-D. The authors concluded that 2,4-D inhibited the electrical activity of the brain and the site of action was in the reticular formation and cerebral cortex. They stated that EEC examination of humans working with 2,4-D could be used to detect early symptoms of intoxication. Control animals were not used in these experiments, so the effect of anesthesia, surgery and brain tissue manipulation on the EEC results is not known. EEC abnormalities in the heavily intoxicated animals were stated to be the result of 2,4-D administration, but the authors did not consider that the extreme debilitation of the animals may have effected the EEG patterns as well. 86 �245. Desi, I., Sos, J., and Nikolits, I. (1962a) New evidence concerning the nervous site of action of a chemical herbicide causing professional intoxication. Acta. Physiol. 22:73-80. The effect of 2,4-D on the electroencephalographic activity in thyroidectonized and thyroxine-substituted cats was investigated. Fifteen adult cats, 11 experimental and 4 controls, were used. Following attachment to the EEC electrodes, the cats were subjected to thyroidectonies. The experimental cats were given 100 ug/kg of thyxorine daily while the controls received no treatment. After 10 days of observation, the experimental cats were given a daily dose of 100 mg/kg of purified 2,4-D. The electrical activity of the cerebral cortex and reticular formation was recorded daily. The duration of desynchronization following stimulation of the reticular formation and the cardiac rate were also measured daily. EEC activity in the control cats decreased 24 hours after thyroidectomy and remained at a low level. EEC activity in the thyroxine-substituted experimental cats remained normal after thyroidectomy, but became abnormal 24 hours following administration of 2,4-D. EEC activity in the experimental cats included decreased frequency combined with increased amplitude of the EEC wave patterns. After 5 days of treatment with 2,4-D, the EEC tracing displayed exclusively big, slow waves indicative of brain dysfunction, Desynchronization time in the brain of experimental cats was drastically reduced following administration of 2,4-D. By 24 hours after administration, the time of desynchronization decreased by 50% and by 4 days later, little or no desynchronization response was evident. Treatment with 2,4-D also decreased the cardiac rate by 25% after the first day and 36% after the fifth day. The authors concluded that 2,4-D reduced central nervous excitability directly and was not due to alterations in thyroid function. In particular, they stated that 2,4-D probably damaged the reticular formation and that functional disorders in different organs were secondary to the central nervous effect. 246. Desi, I., Sos, J., Olasz, J., Sule, F., and Markus, V. (1962b) Nervous system effects of a chemical herbicide. Arch. Environ. Health 4:101-108. Acute neurotoxicity of 2,4-D in rats, cats, and dogs was studied. In one set of experiments, a single dose of 200 mg/kg of 2,4-D was administered intraperitoneally. For the other set of experiments, this dose was repeated daily and continued until the death of the animal. Electroencephalograms of treated and control animals were recorded, A single dose of 200 mg/kg of 2,4-D in rats caused a decrease in electrical activity and time of desynchronization in the brain. Desynchronization in these animals was completely abolished 25 minutes after administration of 2,4-D. This effect was reversible, however, since normal EEC patterns were regained by 50 minutes after treatment. Rats, cats, and dogs given consecutive daily doses of 2,4-D also displayed a decrease in brain electrical activity and desynchronization that steadily worsened with each day of treatment. By the fifth day of treatment, EEC examination revealed no desynchronization activity in 87 �these animals. Rats that had been conditioned to respond to a loud noise lost all continued learning by the fifth day of continuous doseage of 2,4-D. Histologic examination of brain tissue revealed no observable alterations, but demyelinization of nerve fibers of the pyramidal tract in the spinal cord was noted. The authors concluded that the reversible desynchronization observed in treated animals was caused by 2,4-D effecting cerebral cells via the bloodstream. They concluded that intraperitoneal administration of 2,4-D primarily damaged cells in the reticular formation, which in turn induced electrical dysfunctions in the cerebral cortex. This study is of limited value since the authors did not describe the preparation of the 2,4-D compound or their use of control animals. In addition, it is nuclear whether identical EEC irregularities were observed in all species of animals that were examined (rats, dogs, cats). Biological effects noted in the dogs could particularly have been correlated to effects observed in humans exposed to 2,4-D chemicals. 247. DeSilva, D. P., and Michel, H. B. (1974) Effects of mangrove defoliation on the estuarine ecology and fisheries of South Vietnam. National Academy of Sciences - National Research Council. NTIS Publication No. AD779014. [Background material.] 248. Diaz-Colon, J. D.j and Bovey, R. W. (1976) Selected Bibliography of the Phenoxy Herbicides, Vo1. I: Fate in the Environment. U.S. Department of Agriculture, 61 pp. [Bibliography.] 249. Diaz-Colon, J. D., and Bovey, R. W. (1977) Selected Bibliography of the Phenoxy Herbicides, Vol. Ill: Toxicological Studies in Animals. U.S.Department of Agriculture, 105 pp. [Bibliography.] 250. Diaz-Colon, J. D., and Bovey, R. W. (1978a) Selected Bibliography of Phenoxy Herbicides, Vol. V: Interrelations with Microorganisms. U.S. Department of Agriculture, 88 pp. [Bibliography.] 251. Diaz-Colon, J. D., and Bovey, R. W. (1978b) Selected Bibliography of the Phenoxy Herbicides, Vol. VII: Military Uses. U.S. Department of Agriculture, 26 pp. [Bibliography.] 88 �252. Diaz-Colon, J. D., and Bovey, R. W. (1980) Selected Bibliography of the Phenoxy Herbicides, Vol. IX: Toxicological and Physiological Effects of 2,4-D. U.S. Department of Agriculture, 92 pp. [Bibliography.] 253. Diaz-Colon, J. D., Bovey, R. W., and Young, A. L. (1979) Selected bibliography of recent literature on the substituted dibenzo-p-dioxins. Texas Agricultural Experiment Station, Texas A & M University System. 50 p. [Bibliography.] 254. Dietz, Jr., E. A., and Moore, L. 0. (1978) Monomethylarsonic acid, cacodylic acid, and their sodium salts. In Analytical Methods for Pesticides and Plant Growth Regulators, Zweig, G., ed. Volume X.TNew York:Academic Press.] pp. 385-401. [Review article.] 255. Dilling, W. L. (1977) Interphase transfer processes II. Evaporation rates of chloromethanes, ethanes, ethylenes, prepares, and propylaxes from dilute aqueous solutions. Comparisons with theoretical predictions. Environ. Sci. and Technol. 116(4):405-409. [Background material.] 256. Di Giovanni, J., Juchau, M. R., Berry, D. L., and Slaga, T. J. (1979) 2,3,7,8-tetrachlorodibenzo-p-dioxin: potent anticarcinogenic activity in CD-I mice. Biochem. Biophys. Res. Commun. 86:577-584. The effects of TCDD-pretreatment on tumor induction by benzo(a)pyrene (BP) and 7,12-dimethylbenz(a)anthracene (DMBA) were studied. Groups of 30 female CD-I mice 7-9 weeks old received topical applications to the shaved skin of the back of .01, .1, or 2 ug TCDD/kg in .2 ml acetone. After varying intervals of time (5 minutes or 1, 3, 5, or 10 days) they were given tumor-initiating doses of DMBA. Tumor promotion with 10 ug 12.0-tetradecanoylphorbol-13-acetate (TPA) applied topically began one week after initiation and continued twice weekly for 20 or 24 weeks. Mice were examined for the presence of papillomas. TCDD was found to inhibit significantly the initiation of tumors by DMBA; mice treated with 1 ug TCDD 5 days before DMBA initiation showed only 6 percent as many tumors as mice receiving no TCDD. Tumor incidence increased with decreasing pretreatment time. The effect of TCDD pretreatment on BP tumor initiation was also examined in female CD-I mice. TCDD (1 ug) was applied to shaved skin 1, 3, or 5 days prior to BP initiation. TCDD inhibited papilloma formation in mice although the effect was not as strong as that observed in DMBA induced mice. The group of mice receiving TCDD 5 days before BP initiation had 35% as many tumors as 89 �mice receiving no TCDD. As with DMBA, the inhibition effect of TCDD decreased with decreasing pretreatment time. The authors were aware that TCDD pretreatment is known to raise aryl hydrocarbon hydroxylase, (AHH) activity in mice sensitive to tumor induction. They then investigated the effect of TCDD pretreatment on metabolism of DMBA. In a test similar to the tumor induction study, mice were given a topical application of 1 ug TCDD or .2 ml acetone only, 3 days prior to sacrifice. Metabolite profiles on DMBA from epidermal homogenates showed a greatly increased rate of formation of hydroxylated DMBA products in TCDD-pretreated mice, and proportionately greater increases in polyhydroxylated metabolite. The authors noted both qualitative and quantitative changes in the metabolism of DMBA under conditions similar to the tumor experiments. To study further the inhibitory effects of TCDD pretreatment, the authors traced the covalent binding of [ H] DMBA in mouse epidermal DNA, RNA, and protein. Topical application of 1 ug TCDD was followed 3 days later by administration of [ H] DMBA. Mice were killed 3 and 24 hours later; it was found that TCDD pretreatment reduced covalent binding of DMBA to epidermal DNA by 60-70 percent, and the RNA by 45-55 percent, but that there was no effect on binding to proteins. The authors suggest that TCDD-pretreatment increases the rate of inactivation of the DMBA molecule relative to the rate of AHH activation in mouse skin. 257. DiGiovanni, J., Viaje, A., Berry, D. L., Slaga, T. J., and Juchau, M. R. (1977) Tumor initiating ability of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and Aroclor 1254 in a two-stage system of mouse skin carcinogenesis. Bull. Environ. Contam. Toxicol. 18(5):552-556. The authors investigated the ability of TCDD to initiate tumors in the two-stage system of mouse skin carcinogenesis. Female Charles River CD-I mice, 7-9 weeks old, were shaved 2 days prior to treatment; only mice in the resting phase of the hair cycle were chosen for the experiment. Groups of 30 preshaved mice received either 2 ug/TCDD (98.6 % purity) in 0.2 ml acetone mouse, or TCDD at the same dose 5 minutes prior to initation with dimethylbenz(a)anthracene (DMBA) (2.56 ug/mouse), a known tumor initiator. No untreated group was included. The dose of TCDD used killed nearly one-third of the animals at 32 weeks, the time of termination of the experiment. One week after initiation with TCDD or TCDD and DMBA, mice received twice-weekly application of 5 ug TPA in 0.2 ml acetone for 32 weeks. Incidences of papillomas and carcinomas were observed and recorded weekly. No statistical analyses of results were presented. When TCDD was administered alone, it showed weak tumor initiating ability (0.1 papillomas/mouse, 14 % survivors with papillomas) after 32 weeks. Concurrent application of TCDD with DMBA resulted in a small increase in tumor initiating ability compared to DMBA alone (2.2 papillomas/ mouse TCDD and DMBA, 1.9 papillomas/mouse DMBA alone). However, since no statistical evaluation of results and no control data were presented, it is not possible to assess the significance of this study, 90 �258. The dioxin curse. (1977) Atlas World Press Review, pp. 13-15. [Editorial.] 259. Dioxin: The 10-year battle that began with Agent Orange. Nature 278:108-109. (1979) [Editorial.] 260. A discussion on herbicides. Statement on 2,4,5-T and TCDD. [Journal interview] J. For. 73(7):410-412. (1976) [Background material.] 261. Dispute resolution conference on 2,4,5-T. 22(l):40-42. (1980) Vet. Hum. Toxicol. [Review article.] 262. Dmitriyev, V. I. (1974) Harmful effects of chemical substances used by the U.S. Army in Indochina. Voenno-Med Zh. 1:88-90. [Review article.]• 263. Dougherty, R. C., and Piotrowska, K. (1976) Screening by negative chemical ionization mass spectrometry for environmental contamination with toxic residues: Application to human urines. Proc. Natl. Acad. Sci. USA 73(6):1777-1781. The frequency of detecting 2,4,5-T in urine samples collected from healthy college students and in food samples is reported. Urine samples were collected from 11 men on a college swimming team, 21 male football players and 25 dorm residents. Urine samples were analyzed by negative chemical ionization mass spectrometry and in some cases the chemical identification of 2,4,5-T was confirmed by gas chromatographymass spectrometry. Seminal fluid samples from 7 sexually active men and fingernail scrapings for 2 people were collected and analyzed for polychlorophenoxy acid by mass spectrometry. Other products, including 4 paper products, 8 beverages, 10 food products and liquid detergent were also analyzed by mass spectrometry. 2,4,5-T was detected in 92, 24% and 36% of the samples from swimmers, football players, and dorm residents, respectively, and in beef fat. No other samples had detectable levels of 2,4,5-T. The authors concluded that low levels (in the range of parts per billion) of 2,4,5-T in biological and food samples were within the range of detection of the method used. 91 �264, Dougherty, W. J., Coulston, F., and Golberg, L. (1976) The evaluation of the teratogenic effects of 2,4,5-trichlorophenoxyacetic acid in the Rhesus monkey. Environ. Qual. Saf. 5:89-96. The teratogenicity of 2,4,5-T was evaluated in the monkey. Pregnant Rhesus monkeys (10 per group) were administered 0.05, 1, or 10 mg/kg 2,4,5-T (with 0.05 ppm TCDD contaminant) in gelatin capsule by oral intubation on days 22 through 38 of gestation. The control group received the vehicle only. At birth, the offspring were weighed, examined, and returned to the mother. Two infants were scheduled to be killed and examined after birth, at 6 months and at 1 year. The stillborn rate was 13.3% for all groups combined and no statistically significant changes were observed for any treatment group compared to the control group for numbers of stillborns, abortions, live births, gestation length, or initial body weight or growth rate. No teratogenicity was observed in any offspring examined, although all infants had not been killed and examined at the time of the report. Although clinical chemistry, histology, and skeletal X-ray examinations were scheduled for all infants, none of the results were included in the report. The authors concluded that 2,4,5-T was non-teratogenic in the monkey. A premature conclusion from an incomplete study seems inappropriate, since apparently half of the infants had not been examined when the report was prepared. 265. Dougherty, W. J., Coulston, F., and Golberg, L. (1973) Non-teratogenicity 2,4,5-trichlorophenoxyacetic acid in monkeys (Macaca mullatta). Toxicol. Appl. Pharmacol. 25:442. [Abstract, only.] 266. Dougherty, W. J., Herbst, M., and Coulston, F. (1975) The nonteratogenicity of 2,4,5-trichlorophenoxyacetic acid in the Rhesus monkey (Macaca mulatta). Bull. Environ. Contain. Toxicol. 13(4)":477-482. The reproductive effects of 2,4,5-T were examined in the Rhesus monkey. Forty pregnant monkeys were divided into 4 equal groups which were administered 0.05, 1.0 or 10.0 mg/kg of 2,4,5-T (with 0.05 ppm TCDD contaminant) in gelatin capsules or, for the control group, empty gelatin capsules by stomach tube on day 22 to 38 of pregnancy. No signs of maternal toxicity were observed in any treatment group. Nine of the 10 control monkeys delivered live offspring and the tenth fetus aborted on day 68 of gestation. One premature infant born on day 143 of gestation died 21 days later. The low treatment group produced 10 live infants with one death at 37 days of age. The other 2 groups each had 8 live births. One abortion and one stillborn on day 119 occurred in the middle dose group and 2 abortions (prior to day 50) occurred in the high dose group. The lengths, weights, clinical chemistry values, and hematology for all infants were within normal values for the duration of the study (1 year of age). No malformations were observed in the aborted fetuses, stillborns, or infants (which were sacrificed 92 �at birth, 6 months, or 1 year). The highest dose used in this study was 80% of the dose that produced toxicity in adult Rhesus monkeys. Despite the small number of infants per group, the data give no trends toward adverse fetal effects and suggests that the monkey is not susceptible to the effects seen in other species when 2,4,5-T was administered from day 22-38 of gestation. 267. Dow Chemical Co. 39 pp. (1978) Chlorinated dibenzo-p-dioxins - Bibliography. [Bibliography.] 268. The DOW Chemical Company. (1974) 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T). FIFRA Docket No. 295. EPA before the Administrator. Dow prehearing memo No. 2. 208 p. [Background material.] 269. Dragsnes, L., and Helgoland, K. ^1974) EffectSgOf the herbicide 2,4,5-T on the incorporation of C-thymidine, H-uridine and H-L-leucine into L929 cells. Acta Pharmacol. Toxicol. 35(2):103-112. The uptake and incorporation of radioactive precursors of RNA, DNA, and protein by L929 cells was studied in vitro in the presence of,2,4,5-T. Mouse L929 fibroblast cells were cultured in the presence of Cthymidine, H-uridine, or H-1-leucine for 20 minutes. The cell layer was then treated with cold 10% trichloroacetic acid and the acid soluble supernatant and insoluble fractions were counted for radioactivity. Radioactive components in acid-soluble extracts were separated by thin-layer chromatography. 2,4,5-T was added at 2.25 mM to the medium (a 100% pure preparation and a stock 2,4,5-T preparation with an estimated 2-3 ppm dioxin were mentioned but-not specified for individual experiments). The effect of 2,4,5-T on H-leucine uptake (acid-soluble counts) was variable over a 10 hour exposure period, while protein synthesis (acid-insoluble counts) was strongly inhibited to a maximum of 23|,of control incorporation. Uptake and incorporation of H-uridine and C-thymidine were inhibited by 2,4,5-T. Above 0.75 mM, 2,4,5-T produced a dose-dependent increase in H-leucine uptake and a concomitant decrease in incorporation uptake and incorporation of H-uridine and C-thymidine were inhibited by doses above 0.5 mM of 2,4,5-T. The rate of H-uridine uptake as a function of uridine concentration was biphasic. The more rapid rate at low concentrations of uridine appeared more sensitive to inhibition by 2,4,5-T than the slower phase at high uridine concentrations. The increased uridine uptake was not associated with increased uptake of any specific phosphosorylated uridine nucleotide pool. The authors concluded that 2,4,5-T produced the observed effects by producing alterations in plasma membrane permeability. 93 �270. Dragsnes, L., Helgeland, K., and Jonsen, J. (1975) Effects of the herbicide 2,4,5 trichlorophenoxyacetic acid on growth and morphology of L929 cells. Acta Pharmacol. Toxicol. 36(2):97-102. The effects of 2,4-D and 2,4,5-T added to culture medium of mouse fibroblasts on cell growth rate and cell morpohology are described. 2,4,5-T (with an estimated 2-3 ppm dioxin contamination) in culture medium at a final concentration of 0.25 to 2.25 mM was added to L929 strain mouse fibroblast cells in monolayer culture. Medium with 2.25 mM 2,4,5-T was replaced with control medium for some cultures after 3 or 6 days. After 1-14 days of culture, cells were trypsinized and counted. Cell morphology was evaluated by phase microscopy. 2,4,5-T produced a dose-dependent inhibition of cell growth over 6 days of culture. The effect was reversible, with an increase in cell number observed 1 day after treatment medium had been replaced by control medium, and with complete recovery approached 9 days after control medium was introduced. Accumulation of cytoplasmic particles occurred with treatment. The number and time of appearance of particles were dependent on 2,4,5-T dose, and they disappeared after 2,4,5-T was removed. At high 2,4,5-T concentrations, cell rounding and detachment were evident. The authors concluded that the effect of 2,4,5-T on L929 cells resembled the 2,4-D effect reported by other investigators with 2,4,5-T showing more potency than 2,4-D in producing this effect. 271. Drill, V. A., and Hiratzka, T. (1953) Toxicity of 2,4-dichlorophenoxyacetic acid and 2j4,5-trichlorophenoxyacetic acid. Arch. Ind. Hyg. Occ. Med. 7:61-67. The acute and subchronic oral toxicities of 2,4-D and 2,4,5-T were examined in dogs. Both compounds (98.5-98.9% purity) were administered in capsules once for the acute studies (to a total of 22 days) and daily for 5 days per week over 13 weeks to 26 dogs for the subchronic studies. Body weights and hematology were monitored and autopsies were performed at the end of the 90 day subchronic treatment period or 14 days after a single acute dose. The lung, heart, liver, kidney, adrenals, spleen, thyroid, and ovary or testes were examined histologically, as well. The LD was about 100 mg/kg for both compounds and death was delayed until 2-9 days after the dose was administered. Both compounds caused anorexia. 2,4,5-T treatment did not cause marked effects in treated animals even after a fatal dose was given. Myotonia, in some dogs limited to mild ataxia and stiffness of the hind limbs, was observed in 2,4-D treated dogs. Hind limbs were affected first and sometimes solely. Sneezing, rubbing of the eyes, and diarrhea were occasionally observed after 2,4,-D treatment. Dogs treated with either compound were observed with pneumonia or with redness in the small intestine which histologically was associated with inflamed and necrotic areas. Other pathologic findings were hepatic necrosis and renal tubular degeneration in one to two dogs that received high doses of 2,4,5-T. Hepatic congestion was commonly observed in dogs that received fatal doses of either compound. All dogs survived that were given 2-10 mg/kg of either dose subchronically and none survived that were given 20 mg/kg of either compound once 94 �daily. The dogs that survived were free of symptoms. Body weights were decreased only from fatal doses and the effect was first se'en 7-12 days prior to death. Other symptoms observed prior to death in dogs given either compound were weakness, stiffness in the hind legs, difficulty in swallowing, and bleeding from the gums. The only alteration in blood counts was a terminal fall in the percentage of lymphocytes in three dolgs that died. Redness of areas of the gastrointestinal tract were noted after treatment with 2,4-D and with 2,4,5-T, and slight increases in heart and kidney weights were observed after fatal doses of either compound. No treatment-related microscopic changes were observed. The authors suggested that the delayed death resulting from subchronic dosing of 2,4-D indicated a cumulative effect of 2,4-D. 272. Dudley, A. W,, and Thapar, N. T. (1972) Fatal human ingestion of 2,4-D a common herbicide. Arch. Path. 94:270-275. The pathology of acute 2,4-D poisoning in man is described. A 76-yearold man with senile dementia ingested an unknown quantity of pure 2,4-D in kerosene. He was found in a comatose condition and never recovered consciousness before his death, 6 days later. High concentrations of 2,4-D were found in the tissues removed at autopsy. Cardiac dilatations, pulmonary edema, and passive congestion of the kidneys were observed at autopsy and led the authors to conclude that death resulted from cardiac arrest caused by auricular fibrillation. Other pathological findings included widespread plaques of acute demyelination in the brain, peripheral neuropathy, gastroenteritis, and hepatic necrosis, with normal endocrine and musculoskeletal systems. 273. Dugois, M. M. P., Amblard, P., Aimard, M., and Deshors, G. (1968) Acne chlorique collective et accidentelle d'un type nouveau. Bull. Soc. Fr. Derm. Syph. 75:260-261. : The skin lesions that occurred in workers following an industrial explosion are described. The explosion occurred in a factory that manufactured 2,4,5-trichlorophenol, from excessive pressure in the reactor pipelines. Twenty-one of the workers that were in the vicinity of the explosion site within 3 days of accident and that developed chloracne were examined. Details of the accident and total' number of exposed workers were not reported. The authors indicated that the majority were exposed for only a few hours, but did not explain what decontamination steps were taken. The characteristics of the lesions were described, without indicating how common these characteristics were among the affected individuals. Edema and burning occurred during the 2 days after exposure, and regressed after several days. Folliculitis developed on the face and extremities during the next 2 weeks, followed by classic chloracne lesions and keratosis which persisted for the next year. The only other symptom described was painful paroxysmic pressure over the hypochondrium, which the authors assumed originated in the liver. The authors concluded that the skin lesions resulted after the absorbed toxic substance was released from body stores and eliminated through the skin. They did not know the complete identity of the toxic substance. 95 �274. Dugois, P., Marechal, J., and Colomb, L. (1958) Chloracne due to 2,4,5-trichlorophenol. Arach. Mai. Prof. 19:626-627. Generalizations are presented related to 17 cases of chloracne in workers at a 2,4,5-trichlorophenol plant. Histologically, lesions were characterized as follicular keratin cysts and atrophied sebaceous glands. Inflammatory reactions and secondary formation of blackheads occurred during the course of the disease. Clinically, mild forms in which only the face and arms were involved were distinguished from moderate cases that involved half of the body and severe cases that persisted for many months. Moderate and severe cases were not completely curable because elimination of the keratinized material from the cyst left atrophic scars. A strong chlorine odor was reported to be associated with the cysts. General disorders observed in the patients included asthenia, anorexia, weight loss, headache, and digestive and hepatic disorders. Incidences of these symptoms or further descriptions of the patients or their health was not provided. The chemical substance in the manufacturing process which was responsible for chloracne could not be identified; (the methods used were not reported). The authors concluded that no therapy was effective for the chloracne and modifications in the manufacturing process successfully prevented new outbreaks of chloracne. 275. Dunachie, J.F., and Fletcher, W.W. (1967) Effect of some herbicides on the hatching rate of hen's eggs. Nature 215:1406-1407. In a brief report, the effects of diquat, dalapon and 2,4-D on hatching rates are presented. Hen's eggs (25 per group) were injected with each herbicide by a previously published method that was not described in this report. Diquat was dissolved in water and dalapon and 2,4-D were administered in acetone. Three doses of dalapon and 2,4-D tested: 200, 100, and 10 ppm, corresponding to 10, 5, and 0.5 rag/egg, respectively, diquat was tested at 100, 10, and 5 ppm. The percentages of eggs that hatched were: 100% for all 3 doses of dalapon; 50%, 70%, and 80-90% for 200, 100, and 10 ppm of 2,4-D, respectively; and 10%, 10%, and 60% for 100, 10, and 5 ppm diquat, respectively. None of the compounds produced any deformities, although methods used for detecting deformities were not described. The authors used for detecting deformities were not described. The authors concluded that their results for herbicides contrasted with results for organophorphorus compounds, which produced malformations. 276. Durham, W. P., and Williams, C. H. (1972) Mutagenic, teratogenic and carcinogenic properties of pesticides. Annu. Rev. Entomol. 17:123-148. [Review article.] 277. Dussart, L. (1946) Acne chlorique, dermatose professionelle. beiges Derm. 3:218. [Not available.] 96 Arch. �278. Dux, E., Toth, I., Dux, L., and Joo, F. (1978) The localization of calcium by X-ray microanalysis in myopathic muscle fibers. Histochemistry 56(3-4):239-244. The location of calcium in muscles from rats treated subacutely with 2,4-D was described on the electron microscopic level. Adult Wistar rats were administered 50 mg/kg 2,4-D intraperitoneally, daily for 3 weeks. The soleus muscles were then removed, immersed in the presence of ammonium oxalate to precipitate calcium, and prepared for electron microscopy. Calcium identification was verified by energy-dispersive X-ray microanalysis. Calcium precipitates were localized in the vesicles of the sarcoplasmic reticulum of control rat muscle. In muscle from 2,4-D treated rats, calcium was not observed in the vesicles, but rather, just beyond the vesicles and in the myofibrils at the Z line of the A-I junction. The authors concluded that 2,4-D blocked reuptake of calcium by the sarcoplasmic reticulum and the higher levels of calcium outside the vesicles could cause long-lasting activation of the contractile system. 279. Dwyer, J. H., and Smith, R. (1980) Statement before the President's Task Force—the Interagency Work Group to Study the Possible Long Term Effects of Phenoxy Herbicides and Contaminants (the IAG), Sept. 22., 1980. 15 pp. [Testimony.] 97 �280. Eberstein, A., and Goodgold, J. in denervated muscles by 2,4-D. (1979) Experimental tnyotonia induced Muscle and Nerve. 2:364-368. The authors investigated the effect of 2,4-D administration on contraction responses in muscles of rats that were denervated for 10 days or longer. The right hind limb of each of thirty male Wister rats was surgically denervated by excision of a 0.5 centimeter segment of the sciatic nerve. The dnervated rats were divided into three groups: 5 rats remained untreated; 19 rats were injected with 2,4-D one hour prior to anesthesia and subsequent contraction experiments; and 6 rats received 2,4-D 30 to 40 minutes after anesthesia. Animals received a single intraperitoneal injection of 2,4-D at a dose of 225 mg/kg. The purity of the 2,4-D compound was not stated. Electromyographic measurements of the anterior tibialis muscle were made before and after administration of 2,4-D. The contractual properties of the denervated extensor digitorium longus muscle were tested by attaching the proximal tendon of the muscle to the strain gage of a myograph. Denervated muscles of the untreated rats were tested in the same manner. Administration of 2,4-D produced a statistically significant increase in the relaxation time of muscle denervated 15 days prior to the administration. This prolongation did not occur until 50 minutes after administration; prolongation was not evident at the 20-minute interval. Tetanic prolongation reached its maximum 100 minutes after treatment with 2,4-D. Electromyographic examination of the denervated muscles showed fibrillation activity prior to 2,4-D treatment. After 2,4-D administration, no appreciable change was noted. No unusual electrical discharges and no-repetitive fering were evident. The authors concluded that 2,4-D produced a prolonged relaxation time in muscles by slowing the rate of calcium uptake and producing a higher calcium concentration in the sarcoplasmic reticulum of the muscle. The absence of repetitive firing in denervated muscle treated with 2,4-D was tentatively thought to be ue to increase of the threshold for action potentially caused by prolonged denervation. No comparisons were made regarding how these findings related to 2,4-D induced myotonia in humans. In addition, the authors did not fully discuss the role of peripheral nerve and neuromuscular involvement in 2,4-D induced myotonia. 281. Ebron, M., and Courtney, K. D. (1976) Difference in 2,4,5-T distribution in fetal mice and guinea pigs. Toxicol. Appl. Pharmacol. 37:144-145. [Abstract, only.] 282. Eckardt, R. E., and Scala, R. A. (1978) Toxicology: assessing the hazard. Occ. Medicine 20(7):490-493. [Not available.] 98 �283. Edson, E. F. 185:361-367. (1960) Applied toxicology of pesticides. Pharm. J. [Review article.] 284. Effects of herbicides in Vietnam and their relation to herbicide use in the United States. (1975) Council for Agricultural Science and Technology, Report No. 46, 14 pp. [Review article.] 285. El-Dib, M. A., Aly, 0. A. (1976) Persistence of some phenylamide pesticides in the aquatic environment-Ill. Biological degradation. Water Res. 10(12):1055-1059. The authors report on the biological degradation of Monuron in Nile River water, which contained a mixed microbial population derived from natural water and sewage. Monuron was added to Nile River water at 10 mg/liter. Water samples were removed over a period of 4 months and analyzed for Monuron and its metabolites by thin layer chromatography. Monuron remained undegraded by the microbial population of the water. 286. Ellgehausen, H., Guth, J. A., and Esser, H. 0. (1980) Factors determining the bioaccumulation potential of pesticides in the individual compartments of aquatic food chains. Ecotoxicol. Environ. Saf. 4:134-157. [Review article.] 287. Elo, H. A., and Ylitalo, P. (1979) Distribution of 2-raethyl-4chlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid in male rats; evidence for the involvement of the central nervous system in their toxicity. Toxicol. Appl. Pharmacol. 51(3):439-446. The tissue distribution of [ C]-2,4-D was studied in rats after pretreatment with 2,4-D. Male Sprague-Dawley rats (4) were administered 250 mg/kg of 2,4-D sodium salt subcutaneously and after 3 hr., [ C]-2,4-D was administered intravenously. Cerebrospinal fluid (CSF) was collected for 1 hr. (starting 30 min. after the isotope injection) and plasma and tissue samples were removed at the end of the CSF collection period and counted for radioactivity. Control rats (5) were administered saline instead of the 2,4-D pretreatment dose. The largest increases in tissue radioactivity for 2,4-D pretreated rats over controls was seen for the brain, and CSF followed by the liver and then the muscle, heart, testes and lung. The plasma and kidney levels were decreased. (All*tissue levels were expressed as a percentage of the tissue to plasma C ratio). The distribution of 2-methyl-4chloro-phenoxyacetic acid was also studied. The authors concluded that 99 �cerebral [ C]-2,4-D concentrations were increased substantially in 2,4-D pretreated rats than in saline pretreated rats, but was unable to attribute this effect to increased 2,4-D influx, decreased efflux, or both. 288. Elo, H., and Ylitolo, P. (1977) Substantial increase in the levels of chlorophenoxyacetic acids in the CNS of rats as a result of severe intoxication. Acta Pharmacol. Toxicol. 41:289-294. Levels of 2,4-D in various rat tissues were determined. Male SpragueDawley rats were administered 250 mg/kg 2,4-D sodium salt subcutaneously, followed in 3 hours by an intravenous dose of [ C]-2,4-D. 1.5 hours later, radioactivity was determined in the brain, cerebro-spinal fluid, liver, kidney, muscle and plasma. 2-methyl-4 chlorophenoxyacetic acid was also studied and details of the protocol and results for 2,4-D were reported to be similar to those for MCPA, but were not specified. Compared with rats that received only the radioactive dose of 2,4-D, in pretreated rats the brain and cerebrospinal fluid contained higher 2,4-D levels (relative to plasma levels). Muscle, liver and kidney levels showed slight or no increase after pretreatment, compared to non-pretreated rats. The authors concluded that 2,4-D treatment may have impaired function of the blood-brain barrier. 289. Elovaara, E., Savolainen, H., Parkki, M. G., Aitio, A., and Vainio, H. (1977) Neurochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Wistar and Gunn rats. Res. Commun. Chem. Pathol. Pharmacol. 18(3):487-494. [Background material.] 290. Emerson, J. L., Thompson, D. J., Gerbig, C. G., and Robinson, V. B. (1970) Teratogenic study of 2,4,5-trichlorophenoxyacetic acid in the rat. Toxicol. Appl. Pharmacol. 17:317. [Abstract, only.] 291. Emerson, J. L., Thompson, D. J., Strebing, R. J., Gerbig, C. G., and Robinson, V. B. (1971) Teratogenic studies on 2,4,5-trichlorophenoxyacetic acid in the rat and rabbit. Fd. Cosmet. Toxicol. 9:395-404. The teratogenic effects of 2,4,5-T were examined in the rat and in the rabbit. Pregnant Sprague-Dawley rats were administered from 1 to 24 mg/kg of 2,4,5-T (with 0.5 ppm dioxin contaminant) in a suspension of hydroxy propylmethyl cellulose by gavage on days 6 through 15 of gestation. Control rats were administered the vehicle, only. Pregnant New Zealand white rabbits received from 10 to 40 mg/kg 2,4,5-T in gelatin capsules on days 6 through 18 of pregnancy. Control rabbits received empty capsules. On day 20 of gestation, rats were killed and corpora lutea and resorptions were counted. Fetuses were weighed, examined for 100 �gross malformations, and then stained with Bouin's fluid and examined for visceral malformations or stained with Alizarin red and examined for skeletal abnormalities or examined histologically. Rabbits were killed on day 29 of gestation and the same types of fetal examinations were performed as those for the rat study except that the rabbit fetuses were incubated for 24 hours to evaluate survival rate prior to sacrifice for evaluation of malformations. Although the group of rats treated with 1 rag/kg 2,4,5-T had significantly fewer implantations and a smaller mean litter size, the decreases were small and the higher doses did not produce this effect. No increases in the incidences of corpora lutea or resorptions or in the mean weight of offspring occurred in any group. No major abnormalities were observed by gross or histologic examination and no increases in skeletal deviations in the experimental groups compared to controls were observed in the rat fetuses. Hydronephrosis was observed in both the control and high dosage groups. No significant changes in incidences of implantations, corpora lutea, resorptions, litter size, or fetal weights were observed except for an increase in litter size at the highest dose and a decrease in corpora lutea per dam in the middle dosage group. Neonatal mortality resulted primarily from respiratory insufficiency. The rates for the control, 10, 20, and 40 mg/kg treatment groups were 7, 26, 11, and 16%, respectively. The authors concluded that the observed neonatal mortality, which was caused by a failure of the mares to dialate, was caused by inappropriate incubation conditions rather than by 2,4,5-T. They concluded that 2,4,5-T is not teratogenic in the strains of rat or rabbit studies. 292. Environmental cancers: Humans as the experimental model. Environ. Sci. and Tech. 10(13):1190-1195. (1976) [Editorial.] 293. Environmental Sciences Laboratory. Dioxins. 48 pp. (1980) Bibliographic references: [Bibliography.] 294. EPA suspends the tnaior uses of two herbicides. (1979) Environ. Sci. and Tech. 13(6):640-641~. [Editorial.] 295. Epstein, S. S. (1973) Teratological hazards due to phenoxy herbicides and dioxin contaminants. Environmental Science Research Series. 2:708-729. [Review article.1 101 �296. Eriksson, M., Hardell, L., Berg, N. 0., Holler, T., and Axelson, 0. (1979) Case-control study on malignant mesenchymal tumors of the soft tissue and exposure to chemical substances. Lakartidningen 76:3872-3875. A case-control study of a male study population of 110 cases of which 38 were deceased and 219 controls was conducted to determine the relationship between the incidence of mesenchymal soft-tissue tumors and exposure to chemical substances. Controls were matched to cases by age and municipality. Exposure was determined through blind telephone and mail questionnaire survey techniques. Data collected included occupational history, exposure conditions in work environment, smoking, etc. To avoid bias special attention to phenoxy acids or chlorophenols were avoided. Specific data on occupational exposures were obtained from persons reporting agricultural, forestry or horticultural employment. Chi-square values and relative risk for matched case-control pairs were calculated using the methods developed by Miettinen (1969) and (1970). Confidence intervals were constructed utilizing the method proposed by Mittlinen (1976). In one analysis, persons exposed to phenoxy acids alone had relative risk of 6.8 and a dose-effect relationship was indicated although insignificant. The authors concluded that exposure to phenoxy acids probably constitutes a risk factor with respect to occurance of malignant mesenchymol soft tissue tumors and that the risk is associated not only with those phenoxy acids which, like some chlorophenols, may contain polychlorinated dibenzodioxins and dibenzofurans, but with other phenoxy acids as well. 297. Erne, K, (1975) Phenoxy herbicide residues in Swedish fish and wildlife. Environ. Qual. Saf. 3(suppl.) Pesticides Issue:192-195. The author assayed fish and wildlife samples for phenoxy herbicide residues by gas chromatography (detection limit 0.05 ppm). Three hundred samples of apparently healthy freshwater fish were removed from 120 localities in Swedish watersheds. Extent of herbicide application in these areas was not reported. Analysis of the samples showed that 30% of the samples contained 2,4-D or MCPA at levels up to 0.8 ppm. Of 50 samples of other aquatic ecosystem organisms (crustaceans, birds, mammals), none contained detectable phenoxy acid residues. Of 250 samples of wildlife (deer, hare, birds) found dead, 25% contained 2,4-D and/or 2,4,5-T. Residue levels in liver and kidneys, those organs that would contain the highest concentration of herbicide, did not exceed 6 ppm and most fell below 1 ppm. Tn healthy wildlife shot in herbicidetreated forests, phenoxy acid residues were found in 32% of the samples. Liver and kidney levels did not exceed 4.5 ppm and most were below 1 ppm. In addition, the author reported on feeding studies in hares and reindeer. Mountain hares (sex, age, and number unspecified) were fed leaf and bark vegetation from sprayed forest areas containing 2-4 ppm 2,4-D and 2,4,5-T (ratio of 1) for 8 weeks. No signs of toxicity were observed in any of the animals. Pregnant reindeer fed about 40 ppm 2,4-D and 2,4,5-T (ratio unspecified) during the last 4-6 weeks of gestation exhibited no signs of toxicity. In addition, no 102 �abortions or fetal abnormalities occurred. Tissue levels of 2,4-D and 2,4,5-T were 1.1 and 1.6 ppm in the liver and up to 8.9 and 13.3 ppm in the kidneys, respectively. The ratio of 2,4,5-T to 2,4-D increased from 1.0 in the feed to 2.0 in reindeer plasma, suggesting a slight accumulation. 298. Erne, K. (1966a) Studies on the animal metabolism of phenoxyacetic herbicides. Acta Vet. Scand. 7:264-271. The formation of 2,4-D conjugates was studied in the rat and pig and protein binding was studied in horse plasma. Pigs (numbers not given) were orally administered 3 or 23 doses of 50 mg/kg 2,4-D or were fed a diet supplemented with 500 ppm 2,4-D for 5 months. Urine samples collected at the end of the exposure period were hydrolyzed with acid and with alkali to determine to proportion of urinary 2,4-D present as acid-labile and alkali-labile conjugates. The identification of conjugates was confirmed by thin-layer chromatography. Plasma from the pig fed the 500 ppm 2,4-D diet was analyzed for acid- and alkali-labile conjugates. Blood from one rat administered a single oral dose of 100 mg/kg 2,4-D butyl ester and 1 pig administered 3 oral doses of 50 mg/kg 2,4-D butyl ester and blood, urine, and liver tissue from 1 pig given 23 oral doses of 50 mg/kg 2,4-D butyl ester were extracted and analyzed for free and esterified 2,4-D content by thin-layer chromatography. Binding of 500 ug of 2,4-D equilibrated with 5 ml of 50% horse plasma was determined by the elution pattern of the mixture on Sephadex G-25. Less than 20% of the 2,4-D in any urine sample was conjugated and none of the plasma 2,4-D was conjugated. Urine, plasma and tissue from animals given 2,4-D butyl ester contained 2,4-D acid and only traces of the ester. 2,4-D in the presence of horse plasma eluted with partition ratio (K.) of 0.95 when the column was equilibrated and eluted with 50% of horse serum. Two peaks with K. values of 0 and 2.2 were obtained for 2,4-D when buffer without horse serum was used in the equilibration and elution buffers. The authors concluded that urinary 2,4-D was conjugated to only a minimal extent and 2,4-D butyl ester was metabolized to the acid rapidly. A weak protein interaction was evidenced by the presence of 2,4-D in the protein peak (K, = 0). The methods used to determine protein-binding and conjugation have been superseded by more sensitive methods. 299. Erne, K. (1966b) Distribution and elimination of chlorinated phenoxyacetic acids in animals. Acta Vet. Scand. 7:240-256. The tissue distribution and elimination of 2,4-D, 2,4-D butyl ester, and 2,4,5-T were studied in the cow, pig, rat and chicken. An aqueous emulsion of 2,4-D butyl ester in petroleum solvent and aqueous solutions of the other test herbicides were administered to albino rats, pigs, calves (6-8 weeks old), male chicks (1 week old) and White Leghorn chickens orally by stomach tube or, for long term exposure in water or feed. At selected intervals, animals were killed, and tissues were examined for histopathological changes and were analyzed for phenoxyacid content. Plasma levels of 2,4-D were determined in calves 103 �(2), pigs (5-10 per group), rats (35 per group) and chickens (5-10 per group) given 50-200 mg/kg 2,4-D and in calves (2) and pigs (4) given 100 mg/kg 2,4-D butyl ester (amine or sodium-potassium salt). Plasma levels of 2,4,5-T were determined in rats (10) and pigs (2) given 100 mg/kg 2,4,5-T. Half-lives for all the compounds were similar within a species, with values of 8-10 hr. for calves, 10-12 hrs. for pigs, about 3 hr. for rats and 8 hr. for chickens. Oral doses of 50 mg/kg/day of 2,4-D and 2,4-D butyl ester to pigs (4-5 per group) or 300 mg/kg/day to 2 hens resulted in 2,4-D plasma levels that gradually decreased over 23 doses and urine levels that increased (exception for 1 pig per group, in which plasma levels rose and intoxication developed). After 2,4-D acid or butyl ester was combined with blood in vitro, almost all of the 2,4-D was recovered in the plasma and not in the cells. Tissue levels after a single dose in all species were highest in excretory organs and low in the brain. Tissue half-lives were 5-10 hrs. in the rat and 10-30 hrs. in the other species. No sex differences were seen for the patterns of distribution and elimination. Clinical symptoms for all species fed diets or water with 500-1000 ppm for 2 mo. to 2 yrs. were anorexia and reduced weight gain. No evidence of tissue accumulation of 2,4-D was observed. A pregnant pig fed a diet with 500 ppm 2,4-D throughout gestation delivered 15 piglets, of which 10 died within 24 hrs. of birth. The authors concluded that both phenoxy acids were absorbed well, distributed rapidly to tissues and excreted rapidly in the urine by all species. In the pig indirect evidence suggested placental transfer of 2,4-D. 300. Erne, K., and Sperber, I. (1974) Renal tubular transfer of phenoxyacetic acids in the chicken. Acta Pharmacol. Toxicol. 35(3):233-241. The rates of renal tubular excretion of 2,4-D and 2,4,5-T were studied in the chicken. 2,4-D or 2,4,5-T was infused over 3 min. into a leg vein of White Leghorn or broiler chickens. Urine was collected from each urethra separately over the subsequent hour and analyzed, for phenoxy acid by thin-layer chromatography and gas chromatography. Phenol red excretion was determined as well for each kidney and urine was analyzed spectrophotometrically for phenol red. The difference in renal excretion by the separate kidneys referred to as the apparent excretion fraction (EF) was expressed as a percentage of the dose,. Only chickens with EF values below 10% for phenol red were used. A dose of 46 umol of 2,4-D per chicken (about 1.5 kg body weight) yielded EF values of 8.9 to 15.7% and recoveries of 18-41% of the dose in the collected urine. A dose of 90 umol of 2,4-D gave EF values of 4.6-5.4% with 23-36% recovery. A dose of 38-39 umol of 2,4,5-T produced EF values of 1.6-7.2% and doses of 55-58 umol given with 200 ug phenol red produced EF values of 0.5-1.2%. When phenol red (200 ug) infusion was initiated 30 min. after the test compound was introduced, the EF values for 2,4-D and 2,4,5-T were reduced by 90-100%. Other chlorinated phenoxy acid compounds were studied in this experimental system, as well. The authors concluded that all compounds they studied except 2,4,5-T underwent tubular excretion because they were associated with EF values above 10%. The transport mechanism was saturable at high doses and was shared by phenol red. The authors also suggested that 104 �affinity of the phenoxy acids to the transport mechanism was related to its degree of chlorination. 301. Espir, M. L. E., Hall, J. W., Rhirreffs, J. G., and Stevens, D. L. (1970) Impotence in farm workers using toxic chemicals. Br. Med. J. 1:423-425. Four out of a team of five males engaged in spreading agricultural chemicals reported difficulty in achieving and maintaining penile erections. Three men were married and had children. This type of information was not provided for the fourth man. The men, aged 35 to 46, were all treated with methyltestosterone and advised to discontinue working with chemicals. For three subjects, the symptom was alleviated within 3 months of the termination of exposure and for the fourth subject within 1 year. No other health problems were identified in the workers. The authors concluded that the chemicals used on the farm were responsible for producing impotence, but the active chemical(s) could not be identified. Among the chemicals the men used were fertilizers and pesticides, including substituted phenoxy compounds (dichloroprop, 2,4-D, and M.C.P.A). 302. Esposito, M. P., Tiernan, T. 0., and Dryden, F. E. (1980) Dioxins. U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory, Office of Research and Development. US-EPA, Cincinnati, Ohio. 351 pp. [Review article.] 303. Evans, J. 0., and Duseja, D. R. (1973) Herbicide contamination of surface runoff waters. U.S. Environmental Protection Agency, Office of Research and Development, No. EPA-R2-73-266. 99 pp. [Not available.] 304. Ewing, A. (1980) Few long term effects from dioxin - Monsanto. News 6(1);3. Chem. [Not available.] 305. Eyzaguirre, C., Folk, B. P., Zierler, K. L., Lilienthal, J. L. (1948) Experimental myotonia and repetitive phenomena: the veratrinic effects of 2,4-dichlorophenoxyacetate (2,4-D) in the rat. Am. J. Physiol. 155:69-77. The authors studied the effects of 2,4-D administration on muscle tension and electrical activity of muscle and nerve in the rat. The triceps surae muscle of Whelan and Sherman rats was exposed and attached to the strain gage of a myograph. Muscle tension was recorded 105 �under isometric conditions after 100-250 rag/kg 2,4-D was administered to the rat intraperitoneally. Action potentials and electrical activity of the muscle and sciatic nerve were recorded as well. Various other compounds were administered and their effects on the response of muscle and nerve to 2,4-D were recorded. 2,4,-D produced tnyotonia muscles of conscious, pentobarbital-anesthetized, and curare-treated rats and in denervated muscle. Repetitive spikes of changes in electrical potential were generated in 2,4-D treated muscle by a single shock applied to the nerve or muscle. The myotonic response decreased with repeated stimulation but recovered completely after 10 minutes of rest. Muscle with 2,4-D induced myotonia showed elevated sensitivity to mechanical stimuli. The myotonic effect of 2,4-D was potentiated in rats administered potassium intraperitoneally and was diminished after quinine, or alpha-tocopheryl phosphate, magnesium, or calcium was injected. 2,4-D administration potentiated the electrical response of muscle to intra-arterially injected acetylcholine. The authors concluded that the myotonic effect of 2,4-D resembled the effect produced by many other compounds as well as the myotonic effect of 2,4-D observed in man and in the goat. 106 �306. The facts about Agent Orange: A time for reason. Congressional Record, March 3.1, H 2394-H 2397. (1980) The [Editorial.] 307. Fahrig, R. (1974) Comparative mutagenicity studies with pesticides. IARC Sci. Pub. 10:161-179. [Review article.] 308. Faith, R. E., and Luster, M. I. (1979) Investigations on the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on parameters of various immune functions. Ann. N.Y. Acad. of Sci. 320:564-571. Humoral and cell-mediated immunity were evaluated in rats after perinatal exposure to TCDD. Female fischer rats were bred to male Wistar rats and were administered 5 ug/kg TCDD (99+% purity) on day 18 of gestation and on postnatal days 0, 7 and 14 or only on postnatal days 0, 7, and 14. Offspring tymus and spleen lymphocytes were cultured with phytohemagglutinin (PHA) or conconavalin A (Con A) and prqliferative responses were measured as an increase in the level of [ H]thytnidine incorporation into DNA. The procedure used to measure delayed hypersensitivity response to tuberculin challenge was not described in this report. Antibody titers were determined by passive hemagglutination method in serum of rats that were immunized twice with bovine gamma globulin. Lymphocytes were labeled with CR and injected into recipients. The concentration of radioactivity in the recipient thymus, liver, lymph nodes and spleen were used as a measure of homing patterns. Thymus and body weights of rats treated postnatally with TCDD had recovered to normal by 128 days of age, while rats treated pre- and postnatally took longer to recover. Lymphocytes responses to mitogens and delayed hypersensitivity responses were diminished in both treatment groups and these effects, observed in cell from 25-128 dayd old rats were absent by 270 days of age. TCDD treatment had no effect on antibody titres. Alterations in homing patterns were observed in treated recipients injected with untreated donor cells and in untreated recipients injected with treated donor cells, the direction of the effect varied with the tissue and treatment regimen. The authors concluded that TCDD was equally immunosuppressive in Fisher/Wistar rats which represent a strain that elicits good immunologic responses, as in Fisher rats, which are poor immunologic responders (and were studied in experiments reported elsewhere). 309. Faith, R. E., Luster, M. I., and Moore, J. A. (1978) Chemical separation of helper all functions and delayed hypersensitivity responses. Cellular Immunol. 40:275-284. The effects of TCDD on cell-mediated and on humoral activity in the rat are described. The data were presented in a previous publication, see Faith and Moore, 1977. 107 �310. Faith, R. E., and Moore, J. A. (1977) Impairment of thymus-dependent immune functions by exposure of the developing immune system to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). J. Toxicol. Environ. Health. 3:451-465. The effects of perinatal TCDD treatment of rats on parameters of humoral and cell-mediated immune functions are described. Fischer-344 rats were administered 5 ug/kg TCDD (99+% purity) in acetone-corn oil by gavage, on day 18 of gestation and on postnatal days 0, 7, and 14 or only on postnatal days 0, 7, and 14. Spleen and thymic lymphocytes were isolated from the offspring and cultured with phytohemagglutinin (DHA) or conconavalin A (Can A) mitogen and proliferature response was measured as the level of [ H]-thymidine incorporation into DNA. Delayed hypersensitivity was measured in oxazolone-sensitized rats by injecting [ H]-thymidine, followed in 24 hours with a dermal application of oxazolone to 1 ear. Radioactivity in plugs from each ear 24 hours later was determined and their rates was used as an index of reactivity. Antibody liters were measured in rats administered 2 bovine gamma globulin injections; Passive hemogglutination was used to measure antibody liters, by a microtiter technique. Selected tissues are examined by light microscopy. Thymic, and body weights were depressed for 39 days after postnatal exposure and for 145 days after pre- and postnatal exposure. TCDD-treated groups showed reduced responsiveness of spleen and thymus lymphocytes to PHA and to Con A; and reduced delayed hypersensitivity responses; the reduction was usually larger after pre- and postnatal treatment than postnatal treatment only, although the effect was observed in both groups at 59 days of age (the last age tested). TCDD treatment did not reduce human antibody response to bovine gamma globulin. The authors concluded that TCDD-treated rats resembled rats treated with corticosteroids, allogenic lymphocytes or thymectomy and that TCDD-induced immune suppression was long lasting, with only certain T-cell subpopulations affected. The rats that were treated pre- and postnatally received 1 more dose than the rats treated only postnatally; this difference in total dosage, could account for the differences wich the authors attribute to an increased sensitivity of fetal rats. 311. Fanelli, R. (1977) TCDD determination in human samples (cerebrospinal fluid and mesenteric fat). 28th Technical Report of the Mario Negu Institute of Pharmacological Research to the Seveso Authority. Milan, Italy. 28 pp. [Not available.] 312. Fanelli, R., Castelli, M. G., Martelli, G. P., Noseda, A., and Garattini, S. (1980) Presence of 2,3,7,8- tetrachlorodibenzo-p-dioxin in wildlife living near Seveso, Italy: A preliminary study. Bull. Environm. Contam. Toxicol. 24:460-462. The authors presented preliminary results of a study of TCDD residue levels in wildlife in the contaminated areas around Seveso,Italy. 108 �Where a chemical plant exploded all animals except hares were collected from areas known to have considerable environmental contamination. Hares were collected in various places inside and around the contaminated area. Animal tissues were analyzed by gas chromotography and low resolution mass fragmentography. Detection limits were not reported. All 14 field mice sampledhad TCDD residues, ranging from 0.07-49 ppb (mean 4.5 ppb). Most of these animals came from an area where TCDD in the top 7 cm of soil ranged from 0.01-12 ppb, suggesting that rodents living on polluted soils accumulate whole body TCDD concentrations up to the same order of mangitude of the soil. Three of 5 hares sampled were positive for TCDD contamination and had an average liver concentration of TCDD of 7.7 ppb (2.7-13 ppb range). One toad sampled had a whole body TCDD concentration of 0.2 ppb, while one snake had 2.7 ppb liver TCDD accumulation and 16 ppb TCDD in adipose tissue. Of 2 samplings (5 g each) of earthworms, 1 sample contained 12 ppb TCDD. No levels of TCDD in animals outside the contaminated areas were reported. The results from these few animals suggest an accumulation of TCDD in wildlife, but no conclusions can be drawn without more information. 313. Fang, S. C., Fallin, E., Montgomery, M. L., and Freed, V. H. (1973) The metabolism and distribution of 2,4,5-trichlorophenoxyacetic acid in female rats. Toxicol. Appl. Pharmacol. 24:555-563. The pharmacokinetics of 2,4,5-T and tissue distribution were studied in pregnant and non-pregnant rats. Female Wistar rats (4 pregnant and 13 non-pregnant) were administered 0.17, 4.3, or 41 mg/kg [ C]-2,4,5-T in corn oil by stomach tube. Urine and feces collected for 3 days following treatment.were analyzed for radioactivity by liquid scintillation counting and C0« in expired air was monitored continuously over the 3-day period with an electrometer. Tissue accumulation of 2.4,5-T was determined in a group of 9 non-pregnant rats given 1 mg of f C]2,4,5-T per rat 1-72 hours prior to tissue removal and analysis for radioactivity and in a group of 6 pregnant rats given 0.043 mg f Cl2,4,5-T per rat 4-24 hours prior to tissue removal and analysis. Radioactive tissue and urinary metabolites were identified by thin layer and paper chromatographics. No C0« was detected in expired air. The rate of elimination of 2,4,5-T was the same for pregnant and non-pregnant rats and for non-pregnant rats in all three dosage groups. After 1 and 7 days, 75% and 85%, respectively, of the dose was excreted in the urine and 8% and 11%, respectively in the feces. Only 10% of the urinary radioactivity was in the form of metabolites, including two benzene-extractable compounds and one water-soluble compound. Maximum tissue levels were reached 6-12 hours after exposure, with the highest levels in the kidney. About 90% of the tissue radioactivity was in the form of 2,4,5-T acid. Maternal distribution of radioactivity in pregnant rats resembled the distribution in non-pregnant rats. The placental level was higher than all other tissues, except the kidney. Fetal transfer of radioactivity was higher at more advanced stages of pregnancy than earlier stages. The average half-life for 2,4,5-T in adult rat tissues was 3.4 hours and for newborns exposed to 2,4,5-T in milk was 97 hours. Tissue levels of 2,4,5-T for neonates exposed to milk containing 2,4,5-T were substantially higher than levels for 109 �neonates that resulted from placental transfer. The authors concluded that 2,4,5-T was eliminated rapidly from the adult rat and at a. much slower rate from the newborn rat. 314. Fara, G. M. (1977a) Seveso: Studies on teratogenic and other chronic effects of chemical pollutants following an accident in a chemical plant. Teratology 16:365. [Abstract, only.] 315. Fara, G. M. (1977b) Quaderno di Documentazioni 28:5. [Not available.] 316. Fedorova, L. M., and Belova, R. S. (1974) Incorporation of 2,4-dichlorophenoxyacetic acid into the organs of animals: paths and dynamics of its excretion. Gig. Sanit. 2:105-107. The distribution and excretion of 2,4-D was studied in the rat. A single dose of 0.05 rag/kg 2,4-D in water was administered to rats by gastric intubation. After 1-35 days, rats (3 per group) were killed and tissues, as well as urine and feces collected during the postexposure period, were analyzed for radioactivity. Female rats were administered 0.04-0.06 mg/kg 2,4-D immediately after they gave birth and several offspring were killed daily for 7 days and their stomach and intestines were analyzed for radioactivity, to estimate 2,4-D transfer in milk. Transpacental transfer was measured administering 0.04 mg/kg TCDD on day 19 of gestation and on the next day analyzing radioactivity inthe placenta, uterus, fetuses and amniotic fluid. Some rats were administered 100 mg/kg 2,4-D, urine andf eces of males were collected for 25 days, and the stomach and intestines of offspring of treated nothers were removed from 1-7 days after maternal treatment. These urine, fecal and (indirect) milk samples were analyzed for 2,4-dichlorophenol by thin-layer chromatography. After 1 hr., 28-37% of the administered dose of 2,4-D was in the digestive tract; 7.7% and 6.3% of the dose remained in rats 26 and 35 days, respectively, after treatment. Over half of the administered dose is excreted in urine on the second day and 58-70% is excreted in urine on the first and second days. From 2.5-6.1% is excreted in the feces in 2 days. 2,4-D was excreted in milk, with peak levels excreted 2-3 days after treatment. 2,4-Dd distirbution in the placenta, uterus, fetuses and ammiotic fluid was uniform and each accounted for 2.7-4.9% of the dose, (all 4 tissues contained about 17% of the dose). (§,4-Dichlorophenol was not detected in any of the analyzed samples. The authors concluded that about 17% of 2,4-D administered crossed the placenta and 1% was transferred in milk to offspring; these levels were considered by the authors to be important factors in establishing threshold doses for embryo-or gonadotoxic effects of 2,4-D. The indirect methods used to measure levels of 2,4-D in milk is unlikely to produce accurate results, since fetal absorption rates from the intestine are not considered. Placental 110 �transfer was calculated inaccurately, as uterine and placental contents do not necessarily reach the fetus. 317. Fee, D. C., Hughes, B. M. , Taylor, M. L., Tierman, T. 0., and Hill, C. E. (1975) Analytical methodology for herbicides orange. Volume II: Determination of origin of USAF stocks. Aerospace Research Laboratories. Technical report ARL TR 75-0110. 30 p. [Background material.] 318. Feldmann, R. J., and Maibach, H. I. (1974) Percutaneous penetration of some pesticides and herbicides in man. Toxicol. Appl. Pharmacol. 28:126-132. The extent of absorption of 2,4-D and diquat applied to the forearm of volunteers was examined. The extent of urinary excretion of Cradioactivity from C-labeled pesticides was determined after each compound was administered intravenously to 6 subjects. All of the dose of 2,4-D was excreted into the urine within 5 days, with a half-life £gr excretion of 13 hours. Only 61.2% of intravenously administered C-diquat was.excreted in the urine in 5 days, and the half-life was 4 hours. Each C-labeled compound was applied to the forearm and che volunteers were asked not to wash the forearm for 24 hours. Absorption was calculated from the amount of radioactivity excreted in 5 days and, for diquat, this value was corrected for the proportion of the dose found to be excreted in urine after intravenous injection. Only 5.8% of the dermally administered dose of 2,4-D and 0.3% of the dose of diquat was absorbed. Absorption of 10 other herbicides was also measured with these methods. Diquat was the least absorbed compound. The authors suggested that these rates of absorption will be influenced by the degree of sweating and condition of the skin. 319. Fetisov, M. I. (1966) Occupational hygiene in the application of herbicides of the 2,4-D group. Gig. Sanit. 31:383-386. Acute toxicity in rats and mice, and the health conditions of occupationally exposed workers for 2,4-D and 2,4-D butyl ester were briefly summarized. For 2,4-D the LD for the mouse and rat were 300 and 1000 mg/kg, respectively, and for 2,4-D butyl ester were 380 and 920 mg/kg, respectively. (The route of administration or other details of the protocols used were not provided). After one-fifth the LD5Q was administered repeatedly, the cumulation coefficients were determined to be 2.6 for 2,4-D and 2.1 for 2,4-D butyl ester. Subacute dermal application of 2,4-D produced marked local irritation without systemic toxicity. For mice tail immersion into 50% 2,4-D butyl ester for 4 hours daily for 3-5 days produced 70% mortality. The threshold concentration for inhalation exposure (4 hr, daily for 4 mo.) was 0.01 mg/1 for ^,4-D. The concentration of 2,4-D and its butyl ester exceeded 10 mg/m in 55% of the locations surveyed in two chemical factories. A 111 �study of the disease incidence and physiological functions was undertaken in a group of 105 herbicide factory workers and in 45 aviation workers involved in herbicide spraying. Comment complaints of aviators were fatigue, headaches, poor appetite, abdominal pain and taste sense impairment. Olfactory and taste impairments were confirmed by a test protocol. No details of protocols or results were provided and the health of the herbicide factory workers was not described. The authors concluded by recommending 2 mg/m as the maximum permissible concentration of 2,4-D in the work zone of agricultural workers. 320. Field, B., and Kerr, C. (1979) Herbicide use and incidence of neuraltube defects. Lancet. 1(8130):1341-1342. [Abstract, only.] 321. Firestone, D. The 2,3,7,8-tetrachlorodibenzo-para-dioxin problem: a review. In Chlorinated Phenoxy Acids and their Dioxins; ed. C. Ramel, (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978) p. 39-52. [Review article.] 322. Firestone, D. (1973) Etiology of chick edema disease. Perspec. 5:59-66* Environ. Health [Review article.] 323. Flamm, B. R., and Cravens, J. H. (1971) Effects of War Damage on the Forest Resources of South Vietnam. J. For. 69(11):784-789. [Editorial.] 324. Flick, D. F., Firestone, D., and Higginbotham, G. R. (1972) Studies of the chick edema disease 9. Response of chicks fed or singly administered synthetic edema-producing compounds. Poult. Sci. 51:2026-2034. The toxic effects of various dioxin preparations were evaluated in the chicken. A mixture of 58% TCDD, 42% 2,3,7-trichlorodibenzo-p-dioxin and traces of di- and pentachlorodibenzo-p-dioxins was fed to White Leghorn chickens for 21 days. Feeding was initiated at 1 day of age at dietary levels of 0.01, 0.1 and 10 ppra dioxins in feed. These doses corresponded to total dioxin intakes of 1.9 17.1 and 51.9 ug. All eight chickens fed the highest dose died, seven deaths occurred within the first 7 days and the remaining chickens that died on the 17th day showed a severe edema. Four of five and three of five chickens fed 0.01 ppm and 0.10 ppm diets, respectively, died. These chickens showed severe decreases in body weight gain. Tissue changes among the three treatment groups included enlarged adrenals, and pale liver, pancreas 112 �and kidney. Other dioxins mixtures were evaluated for toxic effects in the chicken. The authors concluded that the tri- and tetra-chloro derivatives of dibenzo-p-dioxin were the most lethal compounds tested and produced much tissue damage and moderate edema while the hexa- and heptachloro derivatives were more potent in producing edema and t'issue damage. 325. Flint, G. W., Alexander, J. J t > Funderhurk, 0. P. (1968) Vapor pressures of low volatile esters of 2,4-D. Weed Sci. 16:541-544. [Background material.] 326. Florsheim, W. H., and Velcoff, S. M. (1962) Some effects of 2,4dichlorophenoxyacetic acid on thyroid function in the rat: effects on iodine accumulation. Endocrinology 71(l):l-6. Alterations in thyroid function after 2,4-D administration to rats is described. Male Sprague-Dawley rats were administered daily subcutaneous doses of 2,4-D in aqueous solution. Body weights, organ weights, and thyroid function tests were carried out (length of treatment or time from the end of treatment to sacrifice were not reported). Statistically significant effects of.I.daily doses of 80 mg/kg 2,4-D on thyroid function included increased I uptake over 24 hours and decreased serum protein-bound iodine levels, while weights of the thyroid, pituitary,.adrenals, testes, and whole body, thyroid cell height and thyroid I half-life, renal histology, protein excrer, tion, and serum electrolyte patterns were unchanged. Thyroidal I uptake was not altered by 2,4-D in hypophysectomized rats or in rats fed an iodine depleted diet. Goiter development or the ratio of thyroid to serum radioiodine concentrations in rats fed a goitrogenic diet for 2 weeks were not affected by 2,4-D administration. Other parameters that were not altered by 2,4-D exposure were pituitary and serum thyroid-stimulating hormone levels, and the proportion of thyroxine among total iodinated thyroid amino,acids. The authors concluded that 2,4-D produced an increase in I uptake by a direct effect on the peripheral iodide pool and not an alteration in pituitary thyrotrophic hormone production. 327. Foissac-Gegoux, P., LeLievre, A., Basin, B., and Warot, P. (1962) Polyneuritis after use of a herbicide: 2,4-D acid. Lille. Med. 7:1049-1051. A case report is presented of polyneuritis which developed in a man after he sprayed 2,4-D. Two solutions, containing 235 g/1 and 410 g/1 of 2,4-D had been applied over several days from a sprayer, towed by a tractor. The tractor was open and heavy winds resulted in herbicide being blown into the cab, exposing the operator. The day following the last day of the spraying operation, the 52-year-old male worker experienced pain on the right side of the face that developed into paresthia and anesthesia and on the next day developed hypoesthesia of the right 113 �leg. Two weeks later, symptoms of the right leg improved while anesthesia and weakness of the left leg developed and the facial symptoms remained. The patient presented at a clinic after an additional 2 weeks, at which time Achilles reflex and all but deep sensations were found to be absent. No disorders of the arms or of other organ systems were detected. Polyneuritis and disorders of the right trigeminal nerve were treated with vitamin B and in 6 weeks the patient was discharged. The condition of his face and left leg had improved and responses to electrodiagnostic stimulation were normal. Neurological impairment of the right muscle still remained and the Achilles reflex was still absent, but atrophy did not develop. The authors concluded that the cause of polyneuritis was exposure to 2,4-D. 328. Folmar, L. C. (1976) Overt avoidance reaction of rainbow trout fry to nine herbicides. Bull. Environ. Contam. Toxicol. 15(5):509-514. The author describes the avoidance reaction of rainbow trout to water containing dalapon, 2,4-D dimethylamine salt, and diquat. Fish (10 per trial) were placed in the holding area of a Y-shaped avoidance maze. Tests at each concentration were repeated five times. Avoidance of herbicides was evaluated statistically by the chi-square test on the assumption that if a fish could not discriminate between treated and untreated water they would enter each section of the maze with equal frequency. Herbicides were tested at 0.1, 1.0, or 10.0 mg/1. These concentrations were within the range of expected water concentrations of each herbicide-under recommended application rates. Fish avoided concentrations of 1 and 10 mg/1 dalapon and 2,4-D. According to the author, avoidance reactions occurred well below the 96 hr LC,- values for these herbicides. No avoidance was observed in fish exposed to diquat. The author concluded that the fish would probably not be killed by and would not avoid low levels of dalapon and 2,4-D. 329. Forth, W. (1977) 2,3,7,8-tetrachlorodibenzo-l,4-dioxin (TCDD): Seveso accident. Dentsches Arzteblatt. 44(3):2617-2628. the [Review article.] 330. Fowler, B. A., Hook, G. E. R., Lucier, G. W. (1977) Tetrachlorodibenzo-p-dioxin induction of renal microsomal enzyme systems: Ultrastructural effects on pars recta (S-)proximal tubule cells of the rat kidney. J. Pharmacol. Exp. Therap.' 203(3):712-721. Induction of 2 microsomal enzymes by TCDD was localized to specific regions and cells of the rat kidney and was correlated with ultrastructural changes in these tissues. Male Charles River rats were administered a single oral dose of 25 ug/kg TCDD (98% purity) in acetone-corn oil (1:6) 18 hours to 16 days before they were killed. Kidney tissue was prepared for electron microscopy and renal microsomes were isolated and assayed for benzpyrene hydroxylase activity by a 114 �fluorometric method and for glucuronyl transferase by a spectrophotometric method. Other kidneys were separated into the outer strip of the medulla (rich in S3 proximal tubular cells), the inner medulla and the cortex and each region was assayed for both enzymes. The only oxert sign of TCDD toxicity was a mild decrease in growth rate of treated rats. Both enzyme activities were elevated 18 hours after TCDD treatment and reached plateau levels within 3 days. Benzpyrene activity was induced 40 fold at day 7 and glucuronyl transferase was induced 4-fold. Ultrastructural changes in kidneys of treated rats were limited to a massive increase in the smooth endoplasmic reticulum of the terminal straight ( , segments of the proximal tubule (parsrecta). S) No changes in microsomal protein levels or tissue weight occurred in any region of the kidney after TCDD treatment while the activities of both enzymes were induced substantially in all 3 regions. The highest specific activities for the enzymes were associated with the 105,000 xg pellet which contained both smooth and rough microsomes. The authors concluded that renal microsomal enzymes are highly inducible by TCDD, with induction localized to the smooth endoplasmic reticulum of S, proximal tubule cells. 331. Fowler, B. A., Lucier, G. W., Brown, H. W., and McDaniel, 0. S. (1973) Ultrastructural changes in rat liver cells following a single oral dose of TCDD. Environ. Health Perspec. 5:141-148. The histological changes in rat liver tissue following an acute dose of TCDD are described. Charles River rats (30 per group) were administered 5 or 25 ug/kg TCDD in acetone-corn oil by gavage. Five rats per group were killed at each of six time points between 1 and 28 days after treatment. Liver tissue was removed and prepared for light microscopy. The only changes observed in hepatic tissue samples from treated rats compared to controls was a proliferation of the smooth and rough endoplasmic reticulums of parenchymal cells. The authors concluded that these changes were consistent with a cellular attempt at detoxification and were probably related to induced changes in RNA and protein synthesis. 332. Fowler, D. L., and Mahan, J. N. (1978) The Pesticide Review 1977. S. Department of Agriculture, Agricultural Stabilization and Conservation Service. 44 pp. U. [Review article.] 333. Foy, C. L. Picloram and related compounds. In Herbicides: Chemistry, degradation, and mode of action. Vol. 2. eds., P. C. Kearney and D. D. Kaufman.(New York:Marcel Dekker Inc., 1976) pp. 777-813. [Review article.] 115 �334. Fox, J. enzymes explain June 4, L. (1979) Research solving body's detoxifying system. P-450 rival the immune system of the body in complexity and may how toxins such as dioxin exert their effects. Chem. Eng. News 1979, pp. 24-26. [Review article.] 335. Fox, R. P. 1961-1973. (1979) Air Base Defense In The Republic of Vietnam Washington, B.C., U.S. Air Force, 278 pp. [Review article.] 336. Frank, R., Sirons, G. J., and Ripley, B. D. (1979) Herbicide contamination and decontamination of well waters in Ontario, Canada, 1969-1978. Pest. Monit. J. 13(3):120-127. The authors report on suspected herbicide contamination of 239 wells in Ontario, Canada. Well contamination was divided into 5 categories: 1) a spill of concentrated herbicide in or near the well, 2) a spill of diluted herbicide in or near the well, 3) herbicide drift during spraying, 4) contamination as a result of storm runoff, and 5) subterranean movement of herbicide into a well. One liter composite samples from several pumpings from each well were collected and analyzed by gas-liquid chromatography. A total of 61 wells were contaminated by 2,4-D primarily as drift from spraying and storm runoff. Concentrations of 2,4-D found in wells ranged from 0.1 ug/1 to 14.6 mg/1. Twenty-five wells were contaminated with 2,4,5-T primarily from herbicide spraying. Six wells received picloram contamination from spraying, storm runoff and subterranean movement which ranged from 0.1 - 1.5 ug/1. Only one well was found to be contaminated by dalapon as a result of a spill of diluted herbicide. The authors noted that contamination was usually due to improper handling of herbicides. In cases where wells were decontaminated, 2,4-D was removed rapidly while picloram was difficult to remove. Ease of removal of dalapon and 2,4,5-T were not reported. 337. Fries, G. F., and Marrow, G. S. (1975) Retention and excretion of 2,3,7,8 tetrachlorodibenzo-p-dioxin by rats. J. Agr. Fd. Chem. 23:265-269. The pharmacokinetics of TCDD elimination and tissue distribution were studied in the rat. Spragug-Dawley rats were fed a diet which contained 7 or 20 ppb of [ C]-TCDD for 42 days and then were fed a control diet for the subsequent 28 days. Rats were killed at 2 week intervals during the experiment and the liver and carcass were each homogenized and analyzed for radioactivity. Neither dose of TCDD was lethal to any rats. Food intake and weight gain were decreased during TCDD exposure and recovery occurred when the control diet was reinstituted. Liver weights were increased to a greater extent in the low dosage group than the high dosage group. TCDD concentrations were 116 �highest in the liver and the liver contents accounted for 85% of the dose administered to male rats and 7Q% of the dose administered to females. Half-lives for TCDD were calculated for liver clearance and whole body clearance as 11-15 days and steady state retention by the whole body was estimated at 10.5 times the daily intake. The authors concluded that fluctuations in their data resulted from low specific activity of the F Cj-TCDD preparation and inherent assumptions applied to the kinetic models probably introduced deviations in their calculated parameters from actual values. 338. Frohberg, H., Gleich, J., and Hofmann, A. (1975) Investigation on the embryotoxic effect of 2,4,5-T in NMRI mice. Teratology 10(3):309. [Abstract, only.] 339. Fujita, K., Fujita, H. M., and Funazaki, Z. (1975) Chromosomal abnormality caused by 2,4,5-T. J. Jpn. Assoc. Rural Med. 24(2).-77-79. The authors studied the clastogenic effects of 2,4^5-T in hyman cell culture. Aduet human leuhocytes cultureed with 10 to 10 2,4,5-T having 0.09 ppm TCDD as a contaminant. Apprxomately 100-200 cells were examined at each dose level tested and chromated breaks, isochromated breaks, deletions + free fragment dicentrics, + rings were measured. Chromated breaks appeared to increase with increasing doses of 2,4,5-T. the authors concluded that 2,4,5-T induced chromosome aberrations in human cells in culture. However, the authors could not determine whether this effect was due to 2,4,5-T or to the TCDD contaminant. 340. Fullerton, P. M. (1969) Toxic chemicals and peripheral neuropathy: Clinical and epidemiological features. Proc. Roy. Poc. Med. 62:201-204. [Review article.] 341. Fullerton, R. W., Carlson, M. B., and Nolting, A. R. (1974) Final report on the 2,4,5-T scientific workshop. Washington, DC, Mar. 8-9, 45 pp. [Background material.] 342. Funderburk, H. H., Jr., and Bozarth, G. A. (1967) Review of the metabolism and decomposition of diquat and paraquat. J. Agr. Food Chem. 15(4):563-567. [Review article.] 117 �343. Furst, J. R. (1980) Statement Before the White House Interagency Work Group on Long Term Health Effects of Phenoxy Herbicides and Contaminants. Sept. 22, 1980. 8 pp. [Testimony.] 118 �344. Gage, J. C. (1968a) The action of paraquat and diquat on the respiration of liver cell fractions. Biochem. J. 109(5) :757-761. The effect of diquat on rat liver cell oxidation-reduction systems was studied in vitro. Subcellular fractions were prepared from male Alderley Park rat liver cells and incubated in the presence of diquat and various metabolic substranes and cofactors. The products formed were quantitated spectrophotometrically. In the presence of ADP, 1.0 mM diquat produced a slight (25%) increase in oxygen consumption which was not blocked by adding atnytal or antimycin A. Diquat (1.0 mM) also stimulated oxygen uptake by mitochondrial fragments in the presence of antimycin A or amytal, with certain substrates, including NADH and beta-hydroxybutyrate, but not succinate. Diquat stimulated NADH oxidase and NADPH oxidase activities in microsomal preparations and in the soluble cell fraction. The raicrosomal effect was not inhibited by carbon monoxide or para-chloromercuribenzoate, and the hydrogen peroxide generated during diquat oxidation by molecular oxygen was degraded by raicrosomal catalase. Paraquat was also studied in these experiments. The authors concluded that diquat caused stimulation of oxygen utilization by interacting with microsomal NADPH dehydrogenase and transient dipyridilium-free radicals generated during the reaction were likely to be responsible for diquat toxicity in animal cells. The authors also concluded that diquat probably did not penetrate intact mitochondrial membranes. 345. Gage, J. C. (1968b) Toxicity of paraquat and diquat aerosols generated by a size-selective cyclone: effect of particle size distribution. Br. J. Ind. Med. 25(4):304-314. The toxicity of diquat from inhalation exposure was evaluated in 5 laboratory species. Diquat dichloride aerosol, with mean particles sizes of 2.5 micron or greater, was delivered to an exposure chamber. Two male Alderley Park rats were exposed to 25 ug/1 of diquat in air (1 rat for 15 minutes and 1 for 30 minutes). Other groups of 8 rats (4 per sex) were exposed to 0.5 ug/1, 1.06 ug/1, or 2 ug/1 diquat concentrations. Exposure to each atmosphere was for 15.daily 6 hr. periods. Groups of 10 mice (5 per sex), 8 guinea pigs (4 per sex), 2 female rabbits and 1 male beagle were exposed to 1.06 ug/1 diquat for 15 daily 6 hour periods. The general condition of the animals and after subacute exposure of rats, hematological analyses, and necropsies were performed and organs were weighed. No signs of toxic effects were observed after acute exposure of rats- or subacute exposure of any species except rats. Female rats that were exposed to the highest concentration initially showed signs of noisy breathing and histopathological signs of pulmonary irritation, edema, hyperplasia and macrophage infiltration. No other organs were affected. The toxic effects of paraquat aerosol exposure were also described. The authors concluded that a concentration of 0.5 mg/m of respirable diquat aerosol would not constitute an excessive exposure. 119 �346. Gaines, T. B., Holson, J. F., Nelson, C. J., and Schumacher, H. J. (1974) Analysis of strain differences in sensitivity and reproducibility of results in assessing 2,4,5-T teratogenicity in mice, Tox. Appl. Pharmacol. 33:174-175. [Abstract, only.] 347. Gale, T. F., and Ferm, V. H. (1973) Effects of the herbicide 2,4,5-T and Pyrazon embryogenesis in the hamster. Anat. Rec. 175(2):503. [Abstract, only.] 348. Galston, A. W. 29(2):85-90. (1979) Herbicides: A mixed blessing. Biosci. [Review article.] 349. Galston, A. W. (1974) The ungreening of South Vietnam. 83(6):10,12,14. Natur. Hist, [Editorial.] 350. Galston, A. W. (1971) Some implications of the widespread use of herbicides. Bioscience 21:891-892. [Editorial.] 351. Gandenberger, G. F. (1980) Letter on the Proposed Statement at Hearing, 22 Sept., to Office of the General Counsel, U. S. Dept. of Health and Human Services, Washington, DC. 2 pp. [Background material.] 352. Garattini, S. (1977) TCDD poisoning at Seveso. Biomedicine 26:28-29. [Editorial.] 353. Garcia, J. D., and Rhodes, M. J. (1979) Residues of 2,4,5-T in the American coot (Fulcia americana). Bull. Environm. Contam. Toxicol. 23:231-235. The authors report on levels of 2,4,5-T and the American coot, a waterfowl. Twenty-nine August through December 1976 at White River which receives runoff from a watershed that June 1976. The application rate of 2,4,5-T 120 TCDD found in tissues of coots were collected from Lake, Texas, a reservoir was treated with 2,4,5-T in was 0.56 kg 2,4,5-T acid �equivalent/ha. Coot breast muscle, fat, gizzard, liver, brain, and heart were analyzed for 2,4,5-T and TCDD residues by gas chromatography (detection limit unspecified). No TCDD was detected in any of the samples. In addition, no 2,4,5-T was detected in brain and heart samples. Residues of 2,4,5-T were detected in the breast muscle of 14 birds, in the fat of 12 birds, and in the liver and gizzards of 5 birds. The authors did not present a breakdown of contamination by individual bird. The range of 2,4,5-T in breast tissue varied from 8-1,338 ppb. Levels higher than 1,000 ppb were found in only 2 of the 14 birds that had 2,4,5-T residues. All other birds had levels below 500 ppb. The authors speculated that the 2 birds with the highest residue levels probably received 2,4,5-T exposure elsewhere. Vat residue levels in birds ranged from 5-30 ppb; liver residues ranged from 2-30 ppb in 3 birds and were 68 and 118 ppb in 2 birds. Gizzard residue levels ranged from 6-21 ppb in 3 birds and were 36 and 41 ppb in 2 birds. When residue levels among tissues were compared, residues in liver in August were significantly higher (p 0.05) than in other tissues. Residue levels in breast tissue were significantly higher (p 0.05) than other tissues in October and November. No differences among tissues were observed in December. The authors concluded that use of 2,4,5-T at recommended rates would not present any ,serious hazard to coots. 354. Gardiner, J. A. Substituted uracil herbicides. In Herbicides: Chemistry, degradation, and mode of action. Vol. 1., P. C. Kearney and D. D. Kaufman, eds., (New York: Marcel Dekker, Inc., 1975) pp 293-321. [Review article.] 355. Gasiewicz, T. A., and Neal, R. A. (1978) Tissue distribution and excretion of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and effects upon clinical chemical parameters in the guinea pig. Fed. Proc. Fed. Am. Soc. Exp. Biol. 37(3):501. [Abstract, only.] 356. Gasiewicz, T. A., and Neal, R. A. (1979) 2,3,7,8-Tetrachlorodibenzo-pdioxin tissue distribution, excretion, and effects on clinical chemical parameters in guinea pigs. Toxicol. Appl. Pharmacol. 51:329-339. The tissue distribution and kinetics of excretion of TCDD in the guinea pig are described. Adult male guinea pigs were administered 2.0 ug/kg of [ Hj-TCDD, intraperitoneally, as a single dose. Animals were killed within 1-15 days after dosing and tissues were analyzed for radioactivity. Other groups of guinea pigs were administered 0.5 ug/kg of TCDD, intraperitoneally. Excretion of radioactivity was monitored in urine and feces, collected for 23 days after the injection and these data were analyzed pharmacokinetically. Groups of guinea pigs were administered 1.0 ug/kg of TCDD and blood chemistry analyses were 121 �performed on blood samples collected during the 14-day period after dosing. The results of these analyses were compared to those of pair-fed controls. Tissue levels of TCDD were greater than 0.3% of the dose per gram tissue for adipose, adrenals, liver, spleen, intestine and skin, 1 day after administration. Liver contained 3 fold higher levels on day 15, and levels for the adrenals, kidneys, and lungs also increased. TCDD excretion was linear over 23 days, with a calculated half life of 30.2 days. TCDD treatment produced elevated albumin, total protein, iron, urea nitrogen, cholesterol and triglyceride levels, compared to levels for pair-fed controls. The authors concluded that the increasing tissue levels they observed resulted from mobilization of TCDD from fat deposits to other tissues. 357. Gehring, P. J., and Betso, J. E. Phenoxy acids: effects and fate in mammals. In Chlorinated Phenoxy Acids and Their Dioxins, ed. C. Ramel (Ecol Bull. No. 27, Stolkholm: Swedish Natural Research Council, 1978) pp. 122-133. [Review article.] 358. Gehring, P. J., Kramer, C. G., Schwetz, B. A., Rose, J. Q., and Rowe, V. K. (1973) The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral administration to man. Toxicol. Appl. Pharmacol. 26:352-361. The pharmacokinetics of 2,4,5-T clearance was described for five male volunteers. Five mg/kg of 2,4,5-T (with less than 0.05 ppm TCDD) was ingested by each subject and blood, urine, and fecal samples collected during the subsequent 96 hours were analyzed for 2,4,5-T content. The data for the log of plasma levels plotted as a function of time suggested first order kinetics for absorption and for clearance. The half-lives were calculated as 0.75 hours for absorption of 2,4,5-T and 23.1 hours for plasma clearance, and the volume of distribution was calculated to be 0.079 liter/kg. To determine plasma binding of 2,4,5-T, plasma samples were dialyzed against solutions of 2,4,5-T and the concentration of 2,4,5-T in the plasma fraction after 8 hours at 37°C was measured. Over 98% of 2,4,5-T was recovered bound to plasma protein, yielding an apparent volume of distribution (adjusting for fact that only a small proportion of 2,4,5-T is in plasma water) of 6.1 I/kg. Neither recovery of 2,4,5-T and ether extract of acid hydrolyzed urine nor chromatograms of the extracts revealed any metabolites. The urinary excretion rate was the same as the rate of plasma clearance. Over 88% of the dose was recovered in the urine while less than 1% was excreted in the feces. The pharmacokinetic data were used to extrapolate theoretical plasma levels of 2,4,5-T after various exposure regimens after repeated administrations of 2,4,5-T, plasma levels were estimated to plateau in 3 days. 122 �359. Geissbuhler, H., Martin, H., and Vos, G. The subtituted ureas. In Herbicides: Chemistry, degradation and mode of action. Vol. 1 P.C. Kearney and D.D. Kaufman, eds,(New York: Marcel Dekker Inc., 1975) pp. 210-291. [Review article.] 360. Geldmacher-V., Mallinckrodt, M., and Lautenbach, L. (1966) Two cases of fatal poisoning (suicide) with chlorinated phenoxyacetic acids (2,4-D and MCPA). Archiv. Toxikol. 21:261-278. [Foreign language.] 361. Geldmacher-V., Mallinckrodt, M., and Schussler, F. (1971) Toxicity of diuron l-(3,4-dichlorophenyl)-3,3-dimethylurea (diuron) and its metabolism in man. Arch. Toxikol. 27(3-4):187-192. The acute toxicity and metabolism of diuron is described following a case of human ingest ion. A 39-year-old woman ingested 38 mg/kg diuron and 20 mg/kg aminotriazole as the herbicide preparation, Ustinex PA, in a suicide attempt. No signs of poisoning were observed in the patient upon admission to a hospital and no sugar or albumin were detected in the urine collected a few hours (exact number not given) after ingestion of herbicide. Urinary metabolites detected in the urine sample by thin layer chromatography were l-(3,4-dichlorophenylurea) and l-(3,4-dichlorophenyl)-3-methylurea, and a small amount of 3,4dichloroaniline. Non-altered diuron and non-conjugated phenolic metabolites were not detected and no analysis of conjugated metabolites was performed. Unmetabolized aminotriazole was excreted. The authors concluded that diuron metabolism in man involved sequential demethylation of the N-methyl groups and then hydrolysis. 362. Gianotti, F. (1977) Chloracne in children from 2,3,7,8tetrachlorodibenzo-p-dioxin. Ann. Dermatol. Venereol. (Paris) 104:825-829. Clinical and histologic descriptions are given of dermatologic lesions observed in children exposed to TCDD from the Seveso accident. In general, the children had erythematous hyperkeratotic lesions on the face and limbs several days after the accident, which developed into chloracne in 2 months. Histologically, the nodular lesions showed epidermal hyperplasia. Hypercanthosis, metaplasia of the sweat glands, and horny cysts on the extremities were observed. The skin also contained foreign body granulomas. The total number of children affected, or even the ages of all the children whose clinical descriptions were presented, were not provided. The authors concluded that these symptoms resembled epidemic follicular keratosis, which was observed in about 2,000 children in Switzerland in 1946, and suggested that the 30-year follow-up of the Swiss children would provide useful predictive information for the Seveso children. 123 �363. Gile, J. D., Collins, J. C., and Gillett, J. W. (1980) Fate of selected herbicides in a terrestrial laboratory microcosm. Environ. Sci. Technol. 14(9):1124-1128. The authors describe the fate of 2,4,5-T and bromacil in a model terrestrial laboratory ecosystem. Model ecosystems were prepared using sand, clay, and Jiffy Mix, and water. After allowing 1 week for equilibration, Douglas fir seedlings, red alder seedlings, and rye grass were planted. When growth of rye grass was established, earthworms, pillbugs, and mealworm larvae were added, followed by snails and crickets. A.vole was added after treatment. Twenty-six days after planting, 2- C-labeled bromacil or ring-labeled C-2,4,5-T isooctyl ester were applied as a foliar spray (0.28 kg/ha) to the ecosystem model. Both animals and plants were analyzed for uptake of herbidides or their metabolites. The experiment was terminated after 22 days for bromacil and 20 days for 2,4,5-T. In the experiment, 91% of the applied 2,4,5-T was recovered, while only 69% of the applied bromacil was recovered. The measurement of recovered bromacil was complicated by the location of the C label: the C-2 carbon position is readily converted to COj. Concentrations of C materials were greatest in the upper 5 cm of soil: 0.12 ppm bromacil and 0.19 ppm 2,4,5-T. In ryegrass plants, at experiment termination, 2.5 ppm 2,4,5-T and 15 ppm bromacil were detected. 2,4,5-T was mainly present as extractable metabolites, while bromacil recovered was mostly extractable parent. In Douglas fir seedlings, most of the bromacil and 2,4,5-T detected was present in the roots with only a small amount in the stems. However, bromacil was present as extractable parent, while 2,4,5-T was present as extractable metabolites. Residues of both herbicides were greatest in snail feces and snails. In voles, the heart, lung, liver, GI tract, kidney, brain, and carcass were examined, and the majority of C-herbicide was found in the gastrointestinal tract (0.18 ppm bromacil, 0.20 ppm, 2,4,5-T). Bromacil was present mainly as bound residue, while the majority of 2,4,5-T recovered was metabolites. The authors did not make any conclusions on the environmental effects of these pesticides. However, it appears that neither herbicide accumulates in any of the plants or animals studied under the conditions of the experiments. 364. Glass, B. L., and Edwards, W. N. (1974) Picloram in lysimeter runoff and percolation water. Bull. Environ. Contam. Toxicol. 11(2):109-112. The authors investigated the transport of picloram in surface runoff and percolation water from a lysimeter treated with 2.24 kg/ha in March 1970. Surface runoff and percolation water were analyzed for picloram by gas liquid chromatography. Cumulative rainfall in the area was 62.5 cm for the experimental period. Picloram was detectable in runoff water up to 7 months after treatment. No picloram was detected in runoff 1 year after treatment. The total picloram removed by this means was 0.13 mg, 0.007% of the total applied treatment. The largest amount (14.5 ppb) of picloram occurred in runoff water collected during the first month after application and decreased during each successive month. In percolation water, picloram was initially detected 2.4m 124 �beneath the soil at 1.0 ppb one year after application. The highest amount of picloram detected in percolation water was 1.2 ppb observed during months 13 and 17. The authors concluded that movement through the soil column does not appear to be a major route of transport for picloram. 365. Goldmann, P. J. (1975) Initial study of persons exposed to dioxin after an accident on November 13, 1953 in Ludwigshaven, Germany. Working Paper, joint NIEHS/IARC Working Group. [Not available.] 366. Goldmann, P. J. (1973) Severe, acute chloracne, a mass intoxication due to 2,3,7,8-tetrachlorodibenzo-dioxin. Per Hausarzt 24(4):149-152. A summary is provided of the health problems observed in workers that contacted TCDD following an accident in the Baden Aniline and Sofa Factory (BASF) in Germany in 1953. A total of 55 patients developed chloracne, including a worker who was only in contact with exposed animals and the son of another worker. Only 42 of the patients were included in this study (the reasons for eliminating the remainder were not provided). Dermal lesions were the only symptom in half of the 4?. persons. The symptoms observed along with the dermal lesions in the other 21 persons were: central nervous system disorders (7 cases), tracheobronchitis (5), and. hepatitis (4). Symptoms were described for the 14 most severe cases of the 42 cases studied; these 14 workers were unable to work for 5-24 months. Common symptoms for this group were bronchitis (6 cases), pain in arms and legs (6), liver damage (6), and headaches (5). These 14 workers were unable to work for 5-24 months. One person who visited the facility briefly in 1958 developed fatal pancreatic necrosis. This person also developed chloracne, but the authors did not suggest an etiology for the fatal lesion. The authors concluded that TCDD was the acnegenic agent released during the factory accident. 367. Goldmann, P. J. (1972) Extremely severe acute chloracne due to trichlorophenol decomposition products. A contribution to the perna problem. Arbeitstnedizin Socialmedizin Arbeitshygiene 7f.l): 12-18. A summary is presented of the health of workers exposed to vapors released during an accident in a 2,4,5-trichlorophenol factory (Badischen Aniline and Soda Factory) in Germany in 1953; selected aspects of specific cases were also presented. A group of 21 patients had chloracne only. This group included the son of one worker (the son never entered the plant); one case in which comedones and sebaceous cysts remained 18 years later; two cases of chronic conjunctivitis and blepharitis; one case with involvement of the Meibomian glands, which lubricate the margin of the eyelid and are ontogenetically related to the sebaceous glands and their involvement was considered a subsymptom of chloracne. Some patients with only chloracne were unable to work 125 �for up to 5 years. Skin and internal organ disorders were observed in an additional 14 persons. Disorders of internal organs involved the liver in four cases and inflammation of respiratory passages in five cases. One fatal case of pancreatitis occurred in the group. In general, an increased susceptibility to infection was noted in this group. The third group of seven patients had dertnatologic and central nervous system disorders. Overall tiredness and neuromuscular weakness were characteristic of all members of this group. Included in this group were three cases of polyneuritis. Cases of neurasthenia occurred in all groups. Residues of TCDD were detected in the factory, and in 1969 the facility was demolished, because all previous decontamination procedures had failed. The demolition team showed no adverse symptoms. The authors concluded that TCDD exposure was responsible for chloracne and some of the internal and neurological disorders. 368. Goldstein, H. E., and Long, J. F. (1958) Observations on domestic animals exposed to herbicide spray applications of 2,4,5-T and dalapon. Proc. North Cent. Weed Control Conf. 15:28-29. [Not available.] 369. Goldstein, J. A., Hickman, P., Bergman, H., and Vos, J. G. (1973) Hepatic porphyria induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Res. Com. Chem. Path. Pharmacol. 6(3):919-928. Porphyrogenic properties of TCDD in the mouse are described. Male C57BL/6 mice (12-18 per group) were administered weekly doses of 1, 5 or 25 ug/kg TCDD in acetone-corn oil (1:6) orally for 4 weeks. Seven days after the last dose, mice were killed and the livers were analyzed for delta-aminolevulinic acid (ALA) synthetase by column chromatography, for porphyrins by thin-layer chromatography or fluorometry, and for iron by a spectrophotometric method. Liver tissue was also examined for histopathologic changes. A 2,000 fold increase in uroporphyrin levels was observed in livers of the highest dosage group, as 8- and 7- carboxyporphyrins. Half of the mice in this group died 2-3 days before they were scheduled to be killed, and no difference in uroporphyrin levels were observed in the 2 subgroups. Twelve mice were given a single oral dose of 150 ug/kg TCDD and 21-25 days later their liver uroporphyrin levels were elevated 4,000 fold over control levels. ALA synthetase was elevated 225% and iron by 160% in the 25 ug/kg dosage group and 260% in the single dosage group. The only changes observed in the 2 low dosage groups were increased liver weights. Hepatic necrosis and lipid accumulation were observed in the groups in which porphyria occurred, but no increase in liver weight occurred (liver weights were not given relative to body weight) nor any evidence of hemosiderosis. The authors concluded that TCDD was the most potent porphyrogen known. 126 �370. Goldstein, N. P., Jones, P. H., and Brown, J. R. (1959) Peripheral neuropathy after exposure to an ester of dichlorophenoxyacetic acid. JAMA 171(10):1306-1309. Three case reports are presented of peripheral neuropathy which developed in 2 men and 1 woman after exposure to 2,4-D. The patients ranged in age from 50 to 65 years. Exposure occurred while spraying weeds with 2,4-D herbicide; their legs or arras had become wet with the liquid herbicide. Health problems in all three patients developed within 2 days of exposure. Symptoms included nausea, numbness, and weakness of fingers and toes, muscle aches and fatigue, and fasiculation of limb muscles. The fatigue and muscle pain continued to worsen until all 3 patients were no longer able to walk normally. Neurologic examination revealed total or partial lack of tendon reflexes, absence of joint sensation, and, in one case, fasiculation of arm and leg muscles. Electromyographic tests on all 3 patients showed denervation in several extremity muscles. Conduction velocities in the ulnar nerve were measured and found to be slower than normal. Other physiologic function tests were performed, and all results were normal. These clinical findings were compared to results of animal experiments that found similar peripheral nerve dysfunction following exposure to 2,4-D. The authors concluded that the peripheral neuropathy observed in these patients was a direct result of cutaneous exposure to 2,4-D. 371. Grant, W. F. 65:83-119. (1979) The genotoxic effects of 2,4,5-T. Muta. Res. [Review article.] 372. Grant, W. F. (1973) Cytological effects of environmental mutagenspesticides. Mutat. Res. 21:221-222. [Abstract, only.] 373. Grant, W. F. (no date) Cytological effects of environmental mutagens pesticides. Abstract. Proc. Am. Environ. Mut. Soc., 1st International Conference: pp. 221-222. [Abstract, only.] 374. Green, S., and Moreland, F. S. (1975) Cytogenetic evaluation of several dioxins in the rat. Toxicol. Appl. Pharmacol. 33:161. [Abstract, only.] 127 �375. Green, S., Moreland, F., and Sheu, C. (1977) Cytogenetic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on rat bone marrow cells. FDA By-Lines 6:242-294. The authors studied the in vivo cytogenetic effects of TCDD on rat bone marrow cells. Male and female Osborne-Mendel rats (8 per group) received doses of 0.25, 0.5, 1, 2, or 4 ug TCDD/kg body weight by gavage two times per week for 13 weeks. No control group was included in the experiment. Bone marrow cells were obtained from the test animals and 50 metaphases per animal were examined for chromosome aberrations. Mitotic inhibition was scored in 1000 cells per animal. Numbers of chromosome aberrations at 1, 2, or 4 ug/kg were compared with numbers of aberrations at 0.25 ug/kg in a t-test. Significant increases in chromosome aberrations were observed at 2 and 4 ug/kg in males and at 4 ug/kg in females. However, the values of 2.36% chromosome aberrations (2 ug/kg, males) and 2.75% (4 ug/kg, females) are within the usual control range of 2-3%. Only the value of 4.65% for the group of males treated with 4 ug/kg can be regarded as biologically significant. The authors concluded that TCDD does produce chromosome aberrations in rats, that this is a weak effect. 376. Greenlee, W. F., and Poland, A. (1979) Nuclear uptake of 2,3,7,8 tetrachlorodibenzo-p-dioxin in C57BL/6J and DBA/2J mice. J. Bio. Chem. 254(19):9814-9821. Uptake of TCDD by-hepatic nuclei of methylcholanthrene (MC)-responsive and non-responsive mice in vivo and in vitro is described. C57BL/6J and DBA/25 mice were administered 10 mmol/kg of [ H]-TCDD and were killed 0.5h to 2 weeks later. Radioactivity associated with the nuclear and cytosol fractions of hepatic cells and microsomal 7-ethoxycoumarin 0_-deethylase activity were determined. The specific binding capacity of the cytosol fraction for f H]-TCDD was determined in vitro as the amount of radioactivity that could be displaced from the receptor by adding excess cold TCDD (or active congener). In vitro nuclear binding was studied by incubating hepatic nuclei with cytosal that had been pretreated with f H]-TCDD. Specific nuclear binding capacity was estimated as the difference between total nuclear uptake of radioactivity and nonspecifically bound nuclear radioactivity. Non-specific radioactivity was measured as the amount of radioactivity that was taken up by nuclei exposed to cytosol whose f Hl-TCDD content was displaced from the-receptor by an access of cold TCDD congener. From 1-8 hours after [ H]-TCDD was administered to C57BL mice, the specific cytosol binding capacity was half of its initial value; by 16 hours recovery to control values occurred. Nuclear uptake peaked at 8 hours and at 2 weeks remained elevated by 25%. Microsomal enzyme activity was elevated between 1 and 14 days after TCDD was administered. In vivo uptake of [ H]-TCDD in C57BL hepatic nuclei was 5 times the uptake by DBA nuclei and was inhibited almost completely by administering an excess nonradioactive active TCDD congeners, but not by inactive analogs in vitro. Nuclear uptake from I Hj-TCDD bound cytosol did not occur at 0°C and at 25 °C was proportional to the amount of [ Hj-TCDD bound to cytosol. In vitro uptake by C57BL nuclei was 14 128 �3 times greater when [ H]-TCDD-bound C57BL cytosol rather than DBA cytosol was the [ H]-TCDD source and was reduced by 80-90% by inactivating charged C57BL cytosol with heat or with hypsin. The ability of structural analogs of TCDD to compete for nuclear uptake in vitro correlated highly with the ability of the same compounds to compete with specific cytoplasmic binding. The authors concluded that nuclear binding of TCDD is an essential event in the process of monooxygenase enzyme induction and nuclear TCDD uptake and binding are mediated by the cytosol receptor. 377. Greer, G. S. (1977) The effects of 2,4,5- trichlorophenoxyacetic acid on Swiss-Webster mice. Proc. Ark. Acad. Sci. 31:46. [Not available.] 378. Greig, J. B. (1979) The toxicology of 2,3,7,8-tetrachlorodibenzo-pdioxin and its structural analogues. Ann. Occup. Hyg. 22:411-420. [Review article.] 379. Greig, J. B. (1972) Effect of 2,3,7,8-tetrachlorodibenzo-l,4-dioxin on drug metabolism in the rat. Biochem. Pharmacol. 21:3196-3198. The effect of TCDD administration to the rat on zoxazolamine paralysis time and hexabarbital sleeping time are described. A dose of 200 ug/kg TCDD in arachis oil was administered orally to male Porton rats. The duration of paralysis induced by 100 rag/kg zoxazolamine hydrochloride intraperitoneally 1-3 days after TCDD was determined. Male and female rats were administered 200 ug/kg TCDD in dimethyl sulfoxide and after 1-3 days sleeping times induced by 150 mg/kg hexobarbital in males and 75 mg/kg in females were measured. Zoxazolamine paralysis time was reduced about 54% and 75%, 1 and 3 days after TCDD treatment, respectively. Hexobarbital sleeping times were significantly extended 1 day after TCDD treatment and by 3 days were doubled in treated rats of both sexes. The increase in hexobarbital sleeping times occurred in starved rats treated with TCDD and was not evident in rats 89 days after TCDD treatment. The authors concluded that TCDD caused an increase in zoxazolamine metabolism and decrease in hexobarbital metabolism and were unable to determine whether these effects were related to dioxin toxicity. 380. Greig, J. B., and De Matteis, F. (1973) Effects of 2,3,7,8tetrachlorodibenzo-p-dioxin on drug metabolism and hepatic microsomes of rats and mice. Environ. Health Persp. 5:211-219. The effects of TCDD on hexabarbital and zoxazolamine metabolism and on cytochrome P-450 levels and difference spectra are described for the rat and mouse. Portland rats and C57BL/6 and DBA/Z mice were 129 �administered 200 ug/kg TCDD in arachis oil, orally and hexabarbitalinduced sleeping times were measured at intervals between 12 hours and 2 weeks after TCDD was administered. Plasma barbital levels at waking were measured in female rats. Microsomes from livers of rats taken 3 days after TCDD treatment were assayed for zoxazolamine and hexabarbital metabolism and the cytochrome P-450 protein levels and difference spectra were established. Within 1 day of TCDD treatment, barbital sleep time in rats was increased 46% over times for vehicle controls and by 2 weeks this increase was 870% over controls. In C57BL mice 20 days after TCDD treatment, barbital sleep times were 300% of controls and zoxazolamine paralysis time was 15% of controls. Concomitant changes in barbital and zoxazolamine metabolism were demonstrated in vitro. The effect on sleeping time, but not the effect on paralysis time was blocked by administering ethionine to rats prior to barbital or zoxabolamine. Waking barbital levels were the same in rats 3 days after TCDD treatment and in controls. Liver weights and microsomal cytochrome P-450 levels increased in TCDD treated rats, while microsomal protein levels (per gram of liver) were unchanged. The pH dependence of the pyridine difference spectra was the same for microsomes from methylcholanthrene- and TCDD-treated rats but not for controls. TCDD-treated microsomes showed a type II difference spectra in the presence of aniline and of hexabarbital. The authors concluded that TCDD produced changes in cytochrome P-450 that resembled the effects of methylcholanthrene and the TCDD effects on hexabarbital and zoxazolamine metabolism resulted from separate modes of action. 381. Greig, J. B., Jones, G., Butler, W. H., and Barnes, J. M. (1973) Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fd. Cosmet. Toxicol. ll(4):585-595. The acute toxicity of TCDD was studied in rats, guinea pigs, and chickens. TCDD was synthesized and administered by oral intubation in DMSO or arachis oil to White Leghorn chickens, Porton rats, and Porton guinea pigs, while control animals received only the vehicle. Mortality, growth rate, food consumption, hematology, liver weight and histology of major organs were evaluated. A single dose of 25-50 ug/kg of dioxin was lethal to chickens causing death 12-27 days later. (The number of chickens tested was not reported.) The treated animals showed diminished weight gain, labored breathing, and serous fluid in the pericardial sac, characteristic of chicken edema syndrome. Three groups of six guinea pigs received 2, 4, or 10 ug/kg TCDD orally. One animal in each of the two highest dosage groups died within 24 days and others (number not stated) in poor condition were killed. The treated guinea pigs showed a 15-30% decrease in body weight and although the gastrointestinal tract was distended with gas and contained little or no food, no other visceral abnormalities were observed. Rats given 200 ug dioxin/kg in DMSO also showed a loss in body weight and decreased food intake and weanling female rats given 25-200 ug/kg TCDD had lower or zero growth rate compared to vehicle control. Single doses from 30 to 500 ug/kg of dioxin were administered to groups of six rats. The LDcQ was not estimated. The mean time to death was 40.4 days in this experiment. No consistent changes were noted in treated rats that 130 �died. Common findings included gross hemorrhage, pulmonary changes involving hemorrhages, edema or infection, iaundice, and bile duct enlargement. Toxic effects, weight loss, and death did not occur earlier in rats administered a 10 fold higher dose of TCDD orally (in arachis oil). Elevations in red cell count, hematocrit, hemoglobin content and leucocyte counts were observed in rats given 200 ug/kg dioxin. No plasma assays related to liver function were altered 1-3 days after dioxin treatment, while ratios of liver weight to body weights and water content of the liver were increased and bilirubin levels rose after 3 weeks. Hepatic lesions were observed 3 weeks after treatment and included the formation of multinucleate parenchymal cells. The degree of severity of hepatic lesions was the same after all doses of TCDD. Acute inflammatory lesions in the lung and congestion of the gastrointestinal mucosa were observed. The authors concluded that TCDD elicited its toxicity by interfering with the capacity of the liver cells to maintain their correct organization. 382. Greig, J. B., Taylor, D. M., and Jones, J. D. (1974) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the stimulated DNA synthesis in the liver and kidney of the rat. Chem. Biol. Interactions 8:31-39. The effect of TCDD on DNA synthesis in the kidney after treatment with folic acid or lead in vivo or in the liver after partial hepatectomy were evaluated in the rat. Porton strain rats were administered 10 or 200 ug/kg TCDD in dimethylsulphoxide (DMSO) in arachis oil by oral intubation and August-strain rats were administered 10 ug/kg TCDD in DMSO intraperitoneally. Lead acetate trihydrate (40 mg/kg) was administered in water, intraperitoneally. Folic acid (250 mg/kg) was administered intraperitoneally and for some rats was preceded by 24 and 48 hr. with doses of 20 mg/kg of 3-methylcholanthrene i.p. or by 24 hr. with 1 ml/kg carbon disulphide orally. [ H]-thymidine was administered 1 hour before the animals were killed. DNA was extracted from the kidney and liver and counted for incorporated radioactivity. Pretreatment with 10 or 200 ug/kg TCDD 1 or 3 days prior to performing a 70% hepatectomy did not alter DNA synthesis in the liver 24 hours after the operation, compared to controls not treated with TCDD. DNA synthesis was decreased in folate-stimulated kidneys by pretreatment 0-9 days earlier with either dose of TCDD. The effect disappeared in an experiment in which folic acid-treated rats were pair-fed with TCDDtreated rats. Pretreatment of 200 ug/kg TCDD 1 day prior to lead treatment caused a depression in DNA synthesis to the same extent (50%) as occurred in folate-treated kidneys. Peak H incorporation into DNA occurred 34 hr, after lead was administered for controls and TCDDtreated rats. Unlike TCDD, methylcholanthrene or carbon disulfide pretreatment did not produce a depression in DNA synthesis. The authors concluded that dioxin did not interact directly with DNA or block protein synthesis to exert its inhibitory effect but suggested that mediation by a hepatic humoral factor was possible. 131 �383. Greim, H., and Loprieno, N. Urnsehau 2:53. (1978) No permanent injuries at Seveso? The health effects observed in people exposed to TCDD released from an industrial accident in Seveso, Italy in July, 1976 and distributed as a cloud to the surrounding countryside is summarized. Half of the 734 people who lived in the zone (A) of highest contamination (surface soil concentration, 10 mg/kg TCDD) had ingested contaminated animal and vegetable food and 50% worked in agricultural occupations in zone A during the 15-20 day period after the accident and prior to evacuation. The 5,000 residents of zone B (0.01 mg/kg of TCDD in soil) were not evacuated. In the summer, chloracne was diagnosed in 607 of 30,000 children. In the fall, chloracne was unchanged in 278 of 467 children re-examined, had regressed in 66 cases, and was not classified for the remainder. The authors expected chloracne to heal within several months and that conditions other than chloracne or repeated exposure to toxic substances occurred. One hundred eighteen people were treated for ocular irritation and ocular disorders (of unspecified nature) occurred in four cases. No adverse effects to the liver, kidney, gastrointestinal tract, peripheral nervous system or to carbohydrate or porphyrin metabolism were detected in clinical blood analyses performed on all inhabitants of zones A and B and all workers at the factory where the explosion occurred. No chromosomal aberrations were detected in peripheral blood tests on 90 of the factory workers and 180 zone A residents. No malformations were observed in fetuses from 30 therapeutic abortions and 4 spontaneous abortions. Animal teratology was reported, in which births of 1 normal calf and 1 malformed calf, 8 stillbirths and 5 premature births occurred among 12 pregnant cows that fed on contaminated grass through the end of August. The authors concluded that severe embryotoxic effects should be taken into consideration in light of the animal effects, despite lack of observed effects in exposed people. 384. Grolleau, G., deLavaur, E., and Siou, G. (1974) Effects of 2,4-D on the reproduction of quail and partridge after application of the product by spraying on eggs. Ann. Zool.-Ecol. Anim. 6(2):313-331. [Not available.] 385. Gross, M. L. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA Docket Nos. 415 et al. [Testimony.] 386. Grover, R. (1976) Relative volatilities of ester and amine forms of 2,4-D. Weed Sci. 24:26-28. [Background material.] 132 �387. Grover, R., Maybank, J., and Yoshida, K. (1972) Droplet and vapor drift from butyl ester and dimethylamine salt of 2,4-D. 20C4) -.320-324. [Background material.] 388. Grunow, W. , and Boehme, C. (1974) Metabolism of 2,4,5-T and 2,4-D in rats and mice. Arch. Toxicol. 32(3):217-225. [Foreign language.] 389. Grunow,'W., Boehme, C., and Budczies, B. (1971a) Renal excretion of 2,4,5-T in the rat. Health Aspects Pestic. 5:130. [Abstract, only.] 390. Grunow, W., Bohme, C., and Budczies, B. (1971b) Renal Ausscheidung von 2,4,5-T bei Ratten. Fd. Cosmet. Toxicol. 9:667-670. The rate of urinary excretion and metabolites excreted after a single dose of 2,4,5-T was administered to rats is described. Male Wistar rats were administered 50 mg/kg 2,4,5-T in peanut oil by gastric intubation. Urine was collected over the subsequent 7 days and was analyzed for unchanged 2,4,5-T by gas chromatography, for bound 2,4,5-T by acid-o hydrolyzing urine samples prior to gas chromatographic analysis and for N-(2,4,5-trichlorophenoxyacetyl)-glycine by thin-layer chromatography, following partial purification by ion exchange chromatography. Maximum rates of 2,4,5-T excretion occurred on day 2 in 5 of 7 rats. From 30-58% of the dose was excreted as free 2,4,5-T, 12-31% as bound 2,4,5-T and a total of 45-70% of the dose was recovered in the urine in 7 days. The glycin bound derivative was positively identified in the urine samples. The authors concluded that urinary excretion of 2,4,5-T was slower than the values published elsewhere for rats of 2,4-D excretion. .-* 391. Guarino, A. M., James, M. 0., and Bend, J. R. (1977) Fate and distribution of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5- trichlorophenoxyacetic acid (2,4,5-T) in the dogfish shark. Xenobiotica 7(10):623-631. The tissue distribution, biotransformat ion, and kinetics of excretion of 2,4-D and 2,4,5-T were studied in the dogfish shark. Squalus acanthias. Female dogfish sharks were administered f C]-2,4-D or iu]-2,4,5-T intravenously. Urine, bile, and blood were collected and analyzed for radioactivity, along with tissues. Urinary metabolites were separated and identified by thin-layer chromatography and confirmed by paper chromatography and amino acid analysis. Urine contained 53% of the administered dose of 2,4-D in 4 hr., 68% in 1 day, and 90% in 6 days. Two percent of the dose was excreted in bile in 6 days. From 94 to 98% of the radioactivity in urine and bile was 133 �conjugated to taurine, 2-4% was present as 2,4-D acid and traces, 4% as two unidentified metabolites. The half-life of plasma clearance of 2,4-D was 44 min. Tissue levels for the kidney and liver were 2-40 times the plasma, and were lower than plasma levels for muscle, brain, and cerebrospinal fluid. Urine contained 24% of the administered dose in 4 hr. , 57% in 1 day, and 70% by 4 days. Biliary excretion accounted for 7% of the dose in 4 days. From 91-98% of the biliary and urinary radioactivity was in taurine conjugates, 2-6% as 2,4,5-T, and traces to 5% in two unidentified metabolites. The half-life of plasma clearance was 41 min. and tissue distribution showed the same pattern of distribution as described for 2,4-D. Plasma binding of both compounds was about 57% for concentrations up to 50 ug/ml herbicide. The authors concluded that the pharmacokinetics of 2,4-D and 2,4,5-T in the dogfish shark resembled those reported for some mammals, although only the dogfish shark excreted the compounds as conjugates. 392. Guenther, T. M., Fysh, J. M. , and Nebert, D. W. (1979) 2,3,7,8tetrachlorodibenzo-p-dioxin covalent binding of reactive metabolic intermediates, principally to protein in vitro. Pharmacol. The binding of TCDD to protein and to DNA in vitro is described. 2.0 uM [3H]-TCDD (58 Ci/mmol) in p-dioxane was added to 20 mg of deproteinized salmon sperm DNA and 5 mg of microsomal protein from 3-methylcholanthrene-pretreated C57BL/6N or DBA/2N mice. After incubation at 37°C for 45 minutes, DNA was isolated from the mixture, treated with degradative enzymes and separated on a Sephadex LH20 column as TCDDmetabolite-nucleoside complexes. Some mixtures were treated with 500 uM alpha-naphthof lavone or the isolated DNA was additionally digested with proteinase K. Protein was isolated from some mixtures by methanol precipitation and analyzed for radioactivity. More radioactivity bound to protein or DNA in the presence of microsomes from C57BL than DBA mice. Binding to the DNA fraction was decreased by 80% in proteinase K digests and was abolished when NADPH was not added to the incubation medium. NADPH was also required for protein binding and binding to both DNA and protein were decreased by more than 60% in the presence of naphthof lavone. The ratio of TCDD metabolite protein binding: DNA binding ratio was 120-440 and the rate of TCDD metabolism was calculated from its covalent binding to proteins at 100-250 f mol/min/mg microsomal protein. Two metabolites were identified by chromatography . The authors concluded that TCDD was metabolized to radioactive intermediates that bound covalently to cellular macromolecules . The authors proposed that this metabolite was an arene oxide produced by cytochrome P-450 and bound to the cytochrome protein so rapidly that a significant amount of further metabolism was precluded. The relative reactivity of TCDD in vitro to protein from the two mouse species corresponded well to both its relative teratogenicity and the levels of P-450 in the two species. The authors further suggested that TCDD toxicity in different species is related to the P-450 level for each species. 134 �393. Guenther, T. M., and Nebert, D. W. (1978) Evidence in rat and mouse liver for temporal control of two forms of cytochrome P-450 inducible 2,3,7,8-tetrachlorodibenzo-p-dioxin. Europ. J. Biochem. 91:449-456. TCDD induction of 2 hepatic enzymes in maternal and perinatal liver were correlated with the simultaneous appearance of 2 specific proteins. Sprague-Dawley rats and C57BL/6 mice were administered 20 ug/kg TCDD in para-dioxane, intraperitoneally 40 hours before they were killed, (in some cases mothers were treated 1 day before birth and livers of their 1-day-old offspring were examined). Livers were removed and microsomes were isolated and essayed for cytochrome P-450 sprectrophotometrically by 2 methods, and for aryl hydrocarbon hydroxylase (AHH) and acetanilide 4-hydroxylase (A4-H) activities. Microsomal proteins were isolated by polyacrylamide gel electrophoresis. Between days 18 and 22 of gestation, AHH activity (per mg microsomal protein) doubled and A4-H activity increased 4 fold in TCDD-treated rats. Based on mg cytochrome P-450 protein, AHH activity decreased between day 20 and 22 and A4-H activity increased at this time. Concomitant with an increase in AHH between days 18 and 20 were increases in cytochrome P^-450 and in a protein-staining band of 54,000 molecular weight, identified by electrophoresis. The increase in A4-H activity between day 20 and 22 was accompanied by increases in a cytochrome P-450 and a 56,000 molecular weight protein. Compared to activities of non-TCDD-treated rats of the same age, AHH was induced 200 fold and 35 fold in 18 day fetuses and mothers, respectively and A4-H was induced 15 fold and 4 fold in 22 day fetuses and mothers, respectively. Analogous changes occurred in mice studied with the same protocol. The authors concluded that TCDD induced 2 structurally distinct forms of cytochrome P-450 which correspond to 2 separate enzyme activities which are expressed at different points in fetal development (separated by 2 days). 394. Guenzi, W. D., and Beard, W. E. (1976) Picloram degradation in soils as influenced by soil water content and temperature. J. Environ. Qual. 5(2):189-192. The authors studied the degradation of picloram in five soils: Glendale (sandy clay loam), Panoche (clay loam), Palouse (silty clay loam), Molakai (clay), and Ephrata (sandy loam). Roil samples (20g each) were air-dried and passed through a,2-mm sieve. Two replicate flasks received 0.2 mg.carboxyl-labeled C-picloram for each soil sample. Evolution of CO. was measured as an indication of microbial activity. Picloram degradation in Glendale, Panoche, and Palouse soils was very low at 5°C, increased gradually at 10°C, increased rapidly at 30°C, and decreased at 508C. In Molokai and Ephrata soils, degradation of picloram increased to 50°C. Little degradation of picloram occurred in sterile soils indicating that microbial activity was probably responsible for picloram degradation. When soil samples were exposed to alternating wet and dry cycles designed to simulate soil conditions, degradation decreased with successive "dry" cycles. No relationship was found between soil factors (pH, surface area, organic matter, (texture) and picloram degradation. Picloram degradation also 135 �decreased as soil water content decreased. The authors concluded that picloram degradation seemed to be primarily microbial. 395. Guiney, P. D., Yang, K. H., Seymour, J. L., and Peterson, R. E. (1978) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the distribution and biliary excretion of polychlorinated biphenyls in rats. Toxicol. Appl. Pharmacol. 45:403-414. The effects of TCDD on polychlorinated biphenyl distribution and biliary excretion are described. Male Holtzman rats were administered a single oral dose of 10 or 25 ug/kg TCDD in acetone-corn oil (1:19). Ten days later, 6 mg/kg of 2,5,2',5'-[ H]-tetrachlorobiphenyl (4-CB) or 2,4,5,2',4',5'-f H]-hexachlorobiphenyl (6-CB) was administered intravenously to TCDD-treated rats and blood, and bile samples were collected and liver biopsies were removed periodically during the next 4 hours. Fat, muscle and skin tissue were removed at the end of 4 hours and all samples were analyzed for radioactivity. Some experiments were terminated 1 hour after the PCB injection and the liver was analyzed for radioactive metabolites by hexane extraction procedure. In other experiments bile collected 2 hours after the PCB injection was extracted for polar radioactive PCB metabolites; these metabolites were administered intravenously and biliary excretion over 4 hours was followed by the methods used for 4-CB and 6-CB. TCDD treatment resulted in dose-related increases in liver weight and increased liver retention of PCB radioactivities, decreases in PCB biliary excretion and bile flow, and no effect on PCB plasma clearance and body weight. The ratio of hexane-extracted PCB and metabolites to the unextracted polar metabolites, for 4-CB was unaltered and for 6-CB was decreased by TCDD. Radioactivity in the skin was lower and in the liver was higher in TCDD-treated rats than in controls. The rate of biliary excretion after 4-CB metabolites were administered was the same as the rate when 4-CB was administered. Hepatic retention of hexane-extractable PCBs and of polar metabolites were increased by TCDD treatment. The authors concluded that TCDD inhibited biliary PCB excretion, the major route of PCB elimination; this effect could increase the half-life of PCB in the body and could increase apparent toxicity by delaying elimination of toxic metabolites. 396. Gunby, P. (1979a) Dispute over some herbicides rages in wake of Agent Orange. JAMA 241(14):1443-1444. [Editorial.] 397. Gunby, P. (1979b) Plenty of fuel for Agent Orange dispute. 242(7):593-597. [Editorial.] 136 JAMA �398. Gupta, B. N., Vos, J. G., Moore, J. A., Zinkl, J. G. , and Bullock, B. C. (1973) Pathological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Persp. 5:125-140. Pathological changes resulting from acute, daily and weekly exposures of rats, guinea pigs, and mice were compared. CD rats were administered a single dose of 5-100 ug/kg TCDD or 3-31 daily doses of 0.110.0 ug/kg or 6 weekly doses of 0.2-5.0 ug/kg TCDD. Hartley guinea pigs were administered 1 dose of 3 ug/kg or 8 weekly doses of 0.008-1 ug/kg TCDD and CD-I mice were administered 1 dose of 1-50 ug/kg TCDD. All doses were administered in acetone-corn oil by gastric intubation. Necropsies, performed on all animals, included histological examination of most tissues and liver, kidney, bone, and whole body examinations under ultraviolet light to detect porphyrin accumulation. A single dose of 100 ug/kg TCDD was lethal to 43% of rats, with death occurring within 18-21 days—50 ug/kg caused 7% mortality. Over 90% of rats given 16-31 daily doses of 10 ug/kg and guinea pigs given 1 dose or 1.0 ug/kg weekly for 4-5 weeks died, all within 15-32 days. No mortality resulted in groups given lower doses under these regimens or in mice. The thytnus of all 3 species were atrophied. Liver lesions were severe only in the rat. These lesions included degenerative changes and the appearance of multinucleated giant hepatocytes. Hemorrhages were seen in various organs of rats and guinea pigs that received lethal doses of TCDD. Other lesions in the rat included renal and thyroid degeneration, spleen and ovarian follicle megakaryocytes, and lymphoid depletion. In guinea pigs, hyperplasia of the urinary bladder mucosa and adrenal atrophy occurred. Porphyria did not occur in rats or guinea pigs. The authors concluded that the hepatotoxic and porphyrogenic actions of TCDD were varied in different species. 399. Guseva, Y. N. (1956) On the pharmacology of 2,4-dichlorophenoxyacetic acid. Farmakologiya i toksikologiya. 19(4):41-44. The acute and subacute toxicities of 2,4-D were examined in cats, rabbits, rats, mice, and frogs. 2,4-D sodium salt in aqueous solution was administered subcutaneously as a single dose to groups of 10 mice or as 22 daily doses of 5 mg/kg to groups of 6 mice. The LD was 220 mg/kg and no clinical signs of toxicity were observed for 2 months after subacute treatment. Rabbits were administered doses of up to 5 ml of a 5% solution of 2,4-D per kg body weight intraveneously. The only changes observed were temporary muscle stimulation, slower respiration, and dilation of the pupils. Urethane-anesthetized cats and rabbits were administered 20-30 mg/kg or 250-300 mg/kg 2,4-D and blood pressure and the rate of respiration were recorded. Both parameters were decreased transiently, and atropinization occurred. The functioning of the heat in situ of immobilized frogs was inhibited by a minimum of 2.5-5.0 mg of 2,4-D per frog and the effect was not blocked by 0.5 mg of atropine. Vasoconstriction of peripheral vessels of rabbit ears at concentrations of at least 0.4% 2,4-D applied dermally. At concentrations above 0.5 mg/ml, 2,4-D applied directly to isolated rabbit intestine produced a myotropic effect. No other details of methols were provided for these frog and rabbit experiments. The remaining 137 �studies were mentioned without details of methods provided. 2,4-D at doses of 0.1-10 rag/kg did not alter efferent function in mice, determined by actography and at doses of 1-30 mg/kg did not alter frog sciatic nerve fatigue curve. At doses of 10-50 mg/kg, swimming time in mice was not changed. The latent time between administration of 10 mg/kg of strychnine to mice (10 per group) and the onset of convulsions was prolonged from 9 minutes in control group to 12 and 14 minutes in groups that received 50 and 100 mg/kg 2,4-D respectively. Strychnine was lethal to all mice. Sensitivity of rats to pain from electric current was unaltered by subcutaneous doses of 10-100 mg/kg 2,4-D. Blood catalase activity was increased and serum cholinesterase was decreased by 25-250 mg% 2,4-D in vitro. The authors concluded that the toxicity of 2,4-D was comparatively low. 400. Gutenmann, W. H., and Lisk, D. J. (1970) Metabolism and excretion of Bromacil in milk of dairy cows. J. Agric. Food Chem. 18(1):128-129. Biotransformat ion and excretion of bromacil by the cow was studied. Diets that contained 5 and 30 ppm bromacil were administered to Holstein cows (one cow per dosage) for 4 days. Milk, urine, and feces was collected from the cow that was fed the 5 ppm diet and milk was collected from the second cow for 10 days after the experimental diets were introduced. Fresh rumen fluid and the 10,000 G supernatant fraction of homogenized beef liver were each incubated with bromacil. Bromacil and metabolites were analyzed in the biological fluids and incubation mixtures by gas chromatography. A total of 113.5 mg and 681 mg of bromacil were recovered in the milk of the cows given the 5 ppm and 30 ppm diets, respectively. No bromacil was detected in milk collected more than 1 day after the experimental diet was discontinued or in any urine or fecal samples. No bromacil metabolites were detected in incubates with rumen fluid for 7 hours or liver homogenate for 1 hour. The authors concluded that bromacil was excreted in the milk within 1 day of intake and was not metabolized. The proportion of the dose that was recovered in milk was not calculated. 401. Guthrie, F. E., Shah, P. V., and Moreland, D. E. (1974) Effects of pesticides on active transport of glucose through the isolated intestine of the mouse. J. Agr. Fd. Chem. 22(4):713-715. The effects of 2,4,5-T, diuron, and about 30 other pesticides on intestinal glucose transport were studied in vitro. Midgut sections from 6 week old male mice were filled with the pesticide in a buffer containing [ C]-glucose as well as 0.3% acetone and 0.03% tween 80. After incubation at 30°C for 80 min., the radioactivity in the serosal fluid, luminal fluid, and gut tissue was analyzed. In the presence of 0.1 mM of 2,4,5-T or diuron, which were both reported elswhere to uncouple oxidative phosphorylation, the percentage of radioactivity associated with the gut was significantly higher than for the glucose control, while the amounts in the luminal fluid were not significantly altered from controls and only diuron caused a significant decrease in radioactivity in the serosal fluid. The authors concluded that in general, 138 �pesticides that inhibited oxidative phosphorylation also usually inhibited glucose transport. The significance of these preliminary findings in vitro with a single dose of each compound in vivo toxicity has yet to be established. 402. Gyrd-Hansen, N., and Dalgaard-Mikkelsen, S. (1974), The effect of phenoxy herbicides on the hatchability of eggs and the viability of the chicks. Acta Pharmacol. Toxicol. 35(4):300-308. The effects of 2,4-D (dimethylamine salt), 2,4,5-T (dimethylamine salt), mechlorprop, and dichlorprop on chicken egg hatchability, teratogenicity, and chick survival were studied. The effects of pure compounds of over 99% purity were compared with those of commercial products of 93% purity (except for 2,4,5-T, with 58% purity). Two methods of administration were used. Aqueous solutions of compounds were injected into the egg yolks of unincubated eggs (35 per dose). Other eggs were immersed in 1% or 5% aqueous solutions of the test compound for 10 seconds at 10 or 10°C. For each treatment group, 6 unhatched, fully developed chicks were stained with alizarin red and examined for skeletal malformations. The hatched chicks were weighed weekly and on the fourth week were killed and observed macroscopically. For the injection study in general, all herbicides showed the same pattern of effects; at least 1 mg per egg of pure compound produced a minimal effect of hatchability and at least 2 ing/egg was required to decrease survival of hatched chicks through 4 weeks of age. The weights of surviving chicks in treated and control groups were similar at 4 weeks. The lowest concentration used in the immersion studies failed to produce any adverse effects in hatchability or chick survival and the higher dose of each compound had minimal effects. The most common anomalies of the embryos treated by injection of the 4 compounds were gastroschisis and growth retardation. The embryos of immersed eggs showed few anomalies. No skeletal malformations were observed. The only malformation in hatched chicks were bended toes, which occurred more frequently in treated than control groups. No maior differences in survival or hatchability were seen in groups treated with commercial products compared with those treated with pure compounds of 2,4-D or 2,4,5-T. The commercial product of mechlorprop and the pure chemical dichlorprop each were more toxic than the other preparation of the same product. Two other compounds were tested, 4-chloro-2-methylphenoxy-acetic acid and 2,7-dichloro-dibenzo-p-dioxin. The former showed similar toxicity to the other phenoxy compounds, whereas the dioxin was about 100 times more potent. The. authors estimated that a 1% solution of herbicide spray would expose a 60 gm egg to 2 mg of herbicide if the entire surface were exposed, but from the current results, no deleterious effects would be expected from this exposure. Furthermore the embryotoxic response of chicken embryos by phenoxy herbicides corresponds to the effects observed by others in rodents. 139 �403. Haag, D., Goerttler, K. , and Preiss, D. (1975) The influence of non-cytotoxic concentrations of the herbicide 2,4-dichlorophenoxyacetic acid on the DNA synthesis in cultured vertebrate cells. Arch. Toxicol. 33:91-102. The effects of 2,4-D on DNA synthesis by embryonic muscle cells are described. Primary cultures of embryonic chicken skeletal muscle cells were maintained for 8 days, and then cultured in medium with 2,4-D sodium salt for 22 or 44 hr. Controls were cultured with sodium chloride for up to 44 hr. The cells were then fixed, dried, and stained for fluorescence microscopy. DNA fluorescence was measured with a photometer and then used to derive curves of DNA synthesis. After 44 hr., but not 22 hr., cultures treated with 2.5-50 mM 2,4-D showed a lack of polar orientation and increased nuclear to cytoplasmic area ratio, compared to controls. After treatment medium was replaced with control medium, these cells resumed a normal morphology. Treated cells contained nucleoli that did not fluoresce and heteropycnotic nuclear chromatin. At 0.5 mM, and above, 2,4-D produced significantly more nuclear necrosis by 44 hr. than observed in controls. The number of DNA synthesizing cells increased after 22 and 44 hrs. of treatment with 2.5mM, 2,4-D, but not in controls. In treated cultures, the number of cells in G_ phase decreased. The authors concluded that 2,4-D exerted its effects on the chromosomal level. 404. Hall, S. M. (1972) Effects on pregnant rats and their progency of adequate low protein diets containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T or p,p'-DDT). Fed. Proc., Fed. Am. Soc. Exp. Biol. 31:726. [Abstract, only.] 405. Halprin, K. M. (1980) Chloracne recognition and its significance. Presented at the 2d Continuing Education Conference on Herbicide Orange, Washington, DC, May 28-30. A description of the pathogenesis and clinical symptoms of acneiform lesions is provided. The author also commented on chloracne related to the use of "Agent Orange" in Vietnam. The only feature that distinguishes chloracne from acne is the development of the characteristic acneiform eruption within 1-3 months after exposure to certain halogenated compounds. Chloracne that developed after industrial exposure to these compounds cleared within 1-5 years. The authors indicated that the type of acne condition that would have been likely to develop from herbicide exposure in Vietnam should have been obvious to a physician, but was not observed by a team of physicians who examined troops in the field in the last 3 years of the war to assess skin problems. This condition was likewise not observed in Air Force RANCHHAND personnel who were heavily exposed to "Agent Orange" and were examined annually. The authors concluded that acneiform eruptions from "Agent Orange" use in Vietnam would have appeared within 1-3 months of exposure, and in 1980 (the time of the report), would remain in 10 percent of the patients. In 90 percent of the patients only scars, 140 �which are indistinguishable from those of other types of acne, would remain. 406. Hamada, N, and Peterson, R. E. (1978) Effect of microsotnal enzyme inducers on 2,3,7,8-tetrachlorodibenzo-p-dioxin induced depression in the biliary excretion of ouabain in rats. Drug Metal. Dispos. 6(4):456-464. The effects of various drugs on TCDD-inhibit ion of biliary excretion of ouabain are described. Male Holtzman rats were administered 10 or 25 ug/kg TCDD or 25 ug/kg [ H]-TCDD in acetone-corn oil (1-19) as a single oral dose. Other drugs were administered in propylene glycol, intraperitoneally on days 6 through 9 or only day 10 following TCDD administration, or 4 days prior to TCDD. These drugs included phenobarbital, PB(50 mg/kg/day); 3-methycholanthrene, 3-MC (20 mg/kg/day); pregnenolone-16-alpha-carbonitrile, PCN (75 mg/kg/day); and spironolactone, S(75 mg/kg/day). On day 10 (or day 20, following drug administration on day 10), following TCDD administration, [ H]-ouabain was injected intravenously and blood and biliary samples and liver samples were collected and counted for radioactivity. Mortality was determinedfor rats administered 100 ug/kg TCDD and then 75 mg/kg/day of PCN or S daily for 19 days, starting 1 day after TCDD was given. TCDD treatment caused a decrease in biliary excretion and of blood clearance of ouabain. Neither PB nor 3-MC altered these parameters. PCN treatment on days 6-9 caused both parameters to be elevated, in control and in TCDD-treated rats, while S treatment on days 6-9 reversed the TCDD effect but did not change the control rates. When PCN or S were administered on day 10, no effects on TCDD-inhibited biliary ouabain excretion were observed 6 hours later. PCN, but not S, reversed the TCDD effect on day 20; likewise, PCN, but not S, when administered 4 days prior to TCDD, reversed the TCDD effect. Survival times for rats were 14.5 days for 50% of rats treated with 100 ug/kg TCDD, 14.3 for the TCDD and S-treated group and 10.0 for the TCDD and PCN-treated group. Body weight reductions occurred in all 3 groups. PCN and S treatments before or after TCDD did not influence the concentration of [ H]-TCDD in the liver. The authors concluded that TCDD inhibition of biliary excretion, by PCN and by S, was the only known instance of drug reversal of an effect of TCDD. 407. Handler, P. (1980) Testimony before the Subcommittee on Medical Facilities and Benefits of the Committee on Veterans' Affairs, U. S. House of Representatives, Sept. 16., 1980. 11 pp. [Testimony.] 141 �408. Hanify, J. A., Metcalf, P., Nobbs, C. L., And Worsley, K. J. (1981) Aerial spraying of 2,4,5-T and human birth malformations: an epidemiological investigation. Science_: 2.12. Hanify et al (1981) investigated the incidence of birth malformations in relation to regions of New Zealand where spraying of 2,4,5-T had occurred during the same time period. Hospital records were used to identify births in specific areas of New Zealand where spraying had taken place during 1972 to 1976. Areas of low, intermediate, and high averaged annual spray density were determined. Time periods with spraying (1972-1976) versus those without spraying (1959-1965) were identified. Two tests of the data were performed. In the first, a control group of all babies born between 1 January 1960 and 31 August 1966 who were not exposed to 2,4,5-T and a study group of all babies born between 1 September 1972 and 31 August 1977 were identified. Incidence ratios and 90 percent confidence interval were constructed for commoner malformations. Defects of the heart, hypospadias and epispadias, talipe, and "all birth malformations" exhibited incidence ratios significantly different from 1. In the second test, incidence rates of "all birth malformations" were plotted against average annual exposure to putative teratogen with a decay factor of f = 1.0. Regions of high, low, and intermediate spray density were distinguished as were the years of spraying and non-spraying. The authors noted that incidence rate appears to be positively associated with exposure both across areas and across years. The authors also conducted an analysis utilizing binary regression to further investigate the correlation between individual malformations and exposure, where a malformation was considered to be a dichotomous variable (1 if malformation present, 0 if malformation absent). Exposure was considered in terms of three variables: (1) year, (2) month of the year, and (3) hospital catchment area. The difference between the average exposure value for malformed babies and the exposure value for all babies, suitably normalized, was used as the test statistic. Correlation between malformations exposure were derived in terms of three variables: year, month of the year, and hospital catchment area. The authors decided that a necessary condition for evidence of a correlation between spray and exposure would be tests with year and hospital catchment area significant at the p is less than .05 level. Given this criteria, the results of the tests conducted by the authors showed (1) no identifiable association for talipes when the decay factor, f, equals 0.25, but not when the decay factor, f, equals 1.00 and (3) no identifiable association for cleft lip with or without cleft palate, for isolated cleft palate, for malformations of the heart as a group, or for malformations of the male genitalia. 409. Hansen, W. H., Quaife, M. L., Habermann, R. T., and Fitzhugh, 0. G. (1971) Chronic toxicity of 2,4-dichlorophenoxyacetic acid in rats and dogs. Toxic. Appl. Pharmacol. 20:122-129. The authors attempted to determine the effects of long term feeding of 2,4-D to dogs and rats. Technical grade 2,4-D was obtained from Dow Chemical Company and contained no detectable TCDD (1 ppm detection 142 �limits) and had a purity of 99.12 %. In the rat feeding study, groups of 25 male and 25 female 3-week old Osbourne-Mendel rats received 0, 5, 125, 625, or 1,250 ppm 2,4-D in their diet for 2 years. No analytical check on the 2,4-D content of the diet was reported by the authors. Storage conditions for the diet formulation also were not reported. Statistical analyses applied to the data were not presented. During the 2-year study, no significant differences in survival rates were observed between control and treated animals. Mean body weights and organ-to-body weight ratios of major organs were not significantly different at the time of termination of the experiment. Hemoglobin, hematocrit, and white blood cell count of both control and treated animals were within the normal range. However, at the end of the study, rats treated with 5, 625, or 1,250 ppm 2,4-D showed a tendency toward macrocytosis and slight polychromasia which was not present in the control animals. Tumor incidence in the rats did not reveal any "target organ" types according to pathological interpretation. Statistical analysis of tumor incidence showed a statistically higher incidence of tumors in male rats fed 1,250 pp 2,4-D and a trend toward increased tumor formation with log dose in females. Pathological interpretation of the data, however, did not show a carcinogenic effect. While trends towards increased tumor incidence in this study show a possible carcinogenic effect, no clear conclusions can be drawn. Only a small number of animals were used in the study and the dose range was not broad enough to show any kind of toxicological effect. In the dog feeding study, 3 male and 3 female 6-8 month old Beagles were fed 0, 10, 50, 100 or 500 ppm 2,4-D in their diet for 2 years. None of the lesions occurring in the dogs was believed to be due to 2,4-D ingest ion. However, only small numbers of animals were used and the length of time of the study was not sufficient to prove a negative effect. In the rat reproductive study, male and female rats were fed 0, 100, 500, or 1500 ppm 2,4-D in their diet for 3 generations and a total of 6 litters. Twenty female and 10 male Osbourne-Mendel rats composed each test group. Fertility, numbers of pups surviving to weaning and weanling weights were measured as well as liver aliesterase and acy.lamidase in animals (10 males and 10 female) from the F-, litter. Both enzymes were assigned in the 0, 100, and 500 ppm groups only. No effects on fertility or average litter size were observed in 2,4-D treated rats. However, the percentage of pup surviving to weaning decreased in the 1500 ppm group. Lower dose levels did not appear to effect fertility, litter size, or survival to weaning. The measured liver enzymes were not altered by 2,4-D treatment. 410. Haque, R., Deagen, J., and Schmedding, D. (1975) Binding of 2,4-dichloro- and 2,4,5-trichlorophenoxyacetic acids to bovine serum albumin. A proton magnetic resonance study. J. Agric. Food Chem. 23(4):763-766. The nature of the binding of 2,4-D and 2,4,5-T to protein was studied using nuclear magnetic resonance (NMR). Bovine serum albumin (BSA) was dissolved in deuterated water ^ ) and 2,4-D and 2,4,5-T (as the O potassium salts were also dissolved in DjO and the "H NMR spectra of 143 �all three compounds were obtained. BSA at 4 mg/ml, was added to solutions of 3-12 mg/ml 2,4-D and to solutions of 3-12 mg/ml 2,4,5-T in deuterated water and the changes in the line widths in the resonance peaks were determined. All line widths for the phenoxy-acids broadened in the presence of BSA, with the smallest increase in the peak corresponding to HOD and the largest increase in the methyl peak. No changes in the chemical shifts of any of the resonance peaks occurred when BSA was added to either herbicide. The authors concluded that a weak binding occurred between the non-ring methyl group of each herbicide and BSA. 411. Hardell, L. (1979) Malignant lymphoma of histiocytic type and exposure to phenoxyacetic acids or chlorophenols. Lancet 1(8106):55-56. The author describes a pilot study of male patients with exposure to phenoxyacetic acids or chlorophenols who were admitted to the University Hospital in Umea, Sweden, from January to September of 1978. Fourteen out of 17 men who were admitted for treatment of malignant lymphoma of the histiocytic type were employed in occupations that were likely sources of exposure to the chemicals being studied. Eleven of them reported that they had been repeatedly or chronically exposed to phenoxyacetic acids or chlorophenols. All but one patient were exposed to chemicals that often are contaminated by dibenzodioxins and dibenzofurans. A 64-year-old man who had been exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) developed a retroperitoneal tumor. Six men aged 83, 68, 66, 56, and 56, who had been exposed to both 2,4-D and 2,4,5trichlorophenoxyacetic acid (2,4,5-T) developed tumors of the sinus frontalis; left parotic region; ileum; left femur; and right groin, pelvis, and paravertebrae; respectively. A 38-year-old man who had been e-xposed to 2,4-D, 2,4,5-T, and chlorophenol developed a tumor in the left groin. Two other men, aged 75 and 63, who had been exposed to chlorophenol only, developed tumors of the left side of the neck and the left submandibular region, respectively. A 72-year-old man who had been exposed to pentachlorophenol had developed a tumor of the bladder, and a 55-year-old man who received exposure to 4-chloro-2-methylphenoxyacetic acid (MCPA) had a retroperitoneal tumor. The author postulated that the immunosuppressive effect of dioxins, particularly TCDD, may be related to the occurrence of malignant lymphomas of histiocytic type. 412. Hardell, L. (1977) Malignant mesenclrymal tumours and exposure to phenoxy acids - a clinical observation. Lakartidningen 74:2753. [Foreign language.] 144 �413. Hardell, L., Eriksson, M., and Lenner, P. (1980) Malignant lymphoraa and exposure to chemical substances, especially organic solvents, chlorophenols and phenoxy acids. Lakartidningen 77(4):208-210. A case-control study examined exposure to phenoxy acids or chlorophenols contaminated by polychlorinated dibenzodioxins or dibenzofurans, and to a lesser extent organic solvents, in men aged 25-85 with malignant lymphoma who were admitted to the Department of Oucology in Omea, Sweden, from 1974-1978. Two controls for each case were chosen based on age, sex, occupation, place of residence, and in the case of deceased cases, year of death. Controls for deceased cases watched within 5 years of age; for cases who died in 1978, controls had 1977 deaths lymphoma were histopathologically verified; classification as Hodgkin's disease was done according to Lukes and Butler (1966); as non-Hodgkin's lymphoma, by a modification of Lukes and Collins (1975). Total number of cases was 169; (62 deceased) 60 of these had Hodgkin's disease and 109 non-Hodgkin1s lymphoma (4 of them unclassifiable). Total number of controls was 335. Questionnaires sent to patients, next of kin and employers inquired as to leisure time activities, exposure to chemicals, medicine intake, and smoking habits. Employers verified both the ingredients of compounds and actual exposure. Phenoxy acid exposure (primarily 2,4-D, 2,4,5-T, and picloram) occurred in forestry and agriculture. Chlorophenol exposure occurred in work with cutting oils, leather products, and wood protection agents; the last group was classified as either low-grade (less than 1 week or repeated brief exposure for less than 1 month) or high-grade exposure. For organic solvents, special consideration was given to so-called "high-grade" solvents (e.g., benzene, trichlorocrhylene, styrene) with demonstrated mutagenic effect. Exposures to any combination of the three groups were considered separately. Possible latency periods between exposure and development of tumors called for two separate analyses 5 for one, cases with less than 5 years' exposure were excluded. The following numbers of exposures were determined. For exposure to phenoxy acids only, 108 cases were unexposed, 31 were exposed for less than 90 days, and 10 were exposed for more than 90 days. Corresponding controls were: unexposed, 303; less than 90 days, 20; and more than 90 days, 4. For exposure to chlorophenols only, 83 cases were unexposed, 14 had low-grade exposure, and 25 high-grade. Controls had 277 unexposed, 19 low-grade, and 9 high-grade. For exposure to organic solvents only, 60 cases were unexposed, 10 had low-grade exposure, and 40 had high-grade. Controls had 222 unexposed, 33 low-grade, and 48 high-grade. In addition, 23 controls had exposure to both organic solvents and either phenoxy acids or chlorophenols; 7 controls had the same exposure. Exposure to either phenoxy acids or chlorophenols comprised 36.1 percent of the cases and 9.6 percent of the controls. The authors indicate that there is no decisive proof that malignant lymphoma can be chemically induced. They could show no significant dose-response relationship for phenoxy acids; there seemed to be a relative risk of 9.3 for high-grade exposure to chlorophenols, and of 2.5 for low-grade exposure; exposure to high-grade organic solvents alone or in combination with either phenoxy acids or chlorophenols, did show increased risk compared to exposure to other solvents. No noticeable difference was seen between the incidence of 145 �Hodgkin's and non-Hodgkin's lymphoma. After accounting for congenital factors, induced immunity deficiencies, and known links between benzene and malignant lymphoma, the authors suggest that there may be a relationship between the incidence of malignant lymphoma and exposure to chlorophenols, or phenoxy acids that are contaminated with PCDDs or PCDFs. 414. Hardell, L., and Sandstrom, A. (1979) Case-control study: Soft-tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols. Br. J. Cancer 39:711-717. The authors performed a retrospective study to determine a causal relationship between phenoxyacetic acid and the development of soft tissue carcinoma. The case population consisted of 21 living and 31 deceased male patients. Four matched controls were selected for each case. The living patients were matched to persons from the National Population Registry of Sweden for sex, age, and place of residence. The deceased patients were matched to controls obtained from the National Registry for Causes of Death by sex, age, and year of death. Information on exposure, work environment, and health habits i.e. smoking were obtained through mail and telephone questionnaires, 36.5% of the patients were exposed to chlorophenols and/or phenoxyacetic acids. Calculation of relative risk in the matched material was based upon principles by Miethinen 1970. The effect of matching for control of confounding factors was estimated as the quotient of the relative risk in the matched to unmatched material. The relative risk for exposure to chlordphenol and/or phenoxyacetic acids was found to be 6.2 in the matched group and 5.7 in the unmatched. The relative risk for phenoxyacetic acid only was found to be 5.3. In addition the author states no apparent differences were observed in patient or control populations between smokers and non-smokers. The author concluded an increased risk for soft tissue sarcoma related to the use of phenoxyacetic acids or chlorophenols and that "it was unlikely that" confounding factors influenced the results. It is not possible however to correlate the development of soft tissue sarcoma to either phenoxyacetic acid or chlorophenol exposure due to the mixed exposure of these and other herbicides. 415. Hardell, L., and Sandstrom, A. (1978) Malignant meseuchymal softtissue tumors and exposure to phenoxy acids or chlorophenols. Lakartidningen. 75:3535-3536. This is the first report of a formal investigation prompted by a pilot study (Hardell, 1977) suggesting a relationship between exposure to phenoxyacids or chlorophenols and development of mesenchyraal soft tissue tumors. The population under study consisted of all males hospitalized between 1970 and 1977 at the Oncological clinic at Umeaa, Sweden, and diagnosed as having mesenchymal soft tissue tumors. Eight controls were chosen for each living case based on age, sex, and residence. For deceased cases, ten controls were matched according to age, sex, and year of death based on the cause of death register. 146 �Investigation of occupation, chemical exposure, smoking habits, etc., was made by using responses to questionnaires by patients, next of kin, and employer. Of 260 people studied (52 cases, 208 controls; 31 deceased cases) 36.5 percent of cases had documented exposure to phenoxy acids or chlorophenols as compared to 9.2 percent of controls. The authors reported that phenoxy acids in wide use during the study period included 2,4-D, 2,4,5-T, and 4-chloro-2-methylphenoxyacetic acid. Exposure to phenoxy acids was difficult to evaluate; replies from employers were obtained for less than half of the patients. After subtracting the number of patients and controls with documented exposure to chlorophenols, it was found that of 45 patients, 13 had been exposed to phenoxy acids, compared to 14 of 201 controls. Exposure times varied from 3 to 27 years. The relative risk to exposure was calculated to be 5.3. Confounding factors, such as smoking or exposure to DDT or chain saw fumes did not, according to the authors, contribute to the increased relative risk, but no data was provided that supported this conclusion. The influence of exposure to diesel oil or pesticides could not be evaluated. The authors concluded that the use of chlorophenols or phenoxy acids contributed to an increased risk from soft tissue sarcoma, although no evaluation of the effect of specific substances could be made. 416. Harraan, L. E. (1971) "Chapter 21: Skin Diseases in United States Military Personnel Serving in Vietnam," In The Skin. (Amsterdam: International Academy of Pathology) pp. 423-434. Dermatology problems seen in a field hospital in Vietnam in 1967 are described. Of the total new patients with skin problems, 3.8 percent were diagnosed as having acne. Soldiers often indicated that preexisting acne worsened about 6 weeks after arriving in Vietnam. The author did not mention chloracne, nor did he associate acne with any particular military assignment or location. [Other common skin lesions in Vietnam were caused by fungus and bacterial infections.] 417. Harrigan, E. T. (1970) Calibration Test of the UC-123K/A/A45Y-1 Spray System. Technical Report ADIG-TR-70-36. Armament Development and Test Center, Eglin AFB, Florida. 160 p. [Background material.] 418. Harris, M. W., Moore, J. A., Vos, J. G., and Gupta, B. N. (1973) General biological effects of TCDD in laboratory animals. Environ. Health Perspect. 5:101-109. The effects of acute and subchronic administration of TCDD were studied in the guinea pig, rat, and mouse. TCDD (more than 99% pure) in acetone-corn oil was administered by gastric intubation to Hartley guinea pigs, CD rats and CD-I mice. Mortality, body weight gains, food consumption, and organ weights were recorded. Single doses of 1 or 5 ug/kg TCDD to rats had no effect on body weight during the next 8 147 �weeks, while a 25 ug/kg dose caused a transient decrease in body weight gain compared to controls during the first 1-2 weeks after treatment. Doses of 50-100 ug/kg and in some experiments 25 ug/kg had a doserelated deleterious effect on body weight gain and food consumption and caused death in some rats. Daily doses of 1 or 10 ug/kg of TCDD for 31 days or weekly doses of 5 ug/kg for 6 weeks administered to rats caused significant decreases in weight gain, while 0.1 ug/kg daily doses or 0,02 or 1.0 ug/kg weekly doses were ineffective. Mortality and significant weight loss were observed in guinea pigs that received a single dose of 3 ug/kg or 1 ug/kg weekly doses for 8 weeks. At 25 ug/kg weekly doses, C57B1/6 mice showed significant decreases in weight gain and one of seven mice in the group died, whereas single doses of up to 50 ug/kg TCDD were ineffective in CD-I mice. Decreased thymus weights were observed in all three species at doses below those which elicited decreases in body weight. Repeated doses of TCDD were cumulative and did not increase the threshold amount required to elicit an effect, Death was delayed in all species and female rats appeared more sensitive to TCDD than males. Although food consumption was usually reduced in animals exposed to TCDD, the authors indicated that this reduction was not adequate to account for the severe losses in body weight. 419. Harrison, D.D., Miller, C.I., and Crews, R.C. (1979) Residual levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) near herbicide storage and loading areas at Elgin AFB, Florida. Air Force Armament Laboratory. Technical report AFATL-TR-79-20. [Background material.] 420. Hart, E. R., and Valerio, M. G. (1972) Teratogenic effects of 2,4,5-T in mice. Toxicol. Appl. Pharmacol. 22:317. [Abstract, only.] 421. Hawkins, S. F. (1980) The in vivo effect of paraquat and diquat on intermediary metabolism in mouse lung. Dissertation Abstracts International: Section B 40(10):4763-4764. [Abstract, only.] 422. Hawkins, S. F., Medina, M. A., and Stavinoha, W. B. (1979) The acute in vivo effect of paraquat and diquat on intermediary metabolism in mouse lung. Fed. Proc. 38(3) Pt. 1:582. [Abstract, only.] 148 �423. Hay, A. (1979) Accidents in trichlorophenol plants: A need for realistic surveys to ascertain risks to health. Ann. N.Y. Acad. Sci. 320:321-324. [Review article.] 424. Hay, A. (1978a) Dioxin meeting recommends cancer study. 271:202. Nature [Editorial.] 425. Hay, A. (1978b) Dioxin source is "safe." No reference. [Editorial.] 426. Hay, A. (1978c) Vietnam's dioxin problem. Nature 271:597-598. [Editorial.] 427. Hay, A. (1977a) Seveso solicitude. Nature 267:384-385. [Editorial.] 428. Hay, A. (1977b) Tetrachlorodibenozo-p-dioxin release at Seveso. Disasters 1:289-308. [Review article.] 429. Hay, A. (1976a) Seveso: the aftermath. Nature 263:836-838. [Review article.] 430. Hay, A. (1976b) Toxic cloud over Seveso. Nature 262:636-638. [Review article.] 431. Hayes, W. J. (1975) Toxicology of Pesticides (Baltimore: & Wilkins Co.) pp. 138-148. The Williams [Review article.] 432. Hayes, W. J. (1964) Toxicological problems associated with use of pesticides. Industry and Tropical Health 5:118-132. [Review article.] 149 �433. Heene, R. (1968) Histochemical and morphological findings in experimental 2,4-Dichlorophenoxy Acetate (2,4-D) myopathy in warm blooded animals. Acta Neuropath. 10:166-169. [Foreign language.] 434. Helling, C. S., Isensee, A. R., Woolson, E. A., Ensor, P. D. J., Jone8, J. R., Plimmer, J, R. , and Kearney, P. C., (1973) Chlorodioxins in pesticides, soils, and plants. J. Environ. Qual. 2(2):171-178. [Not available.] 435. Henck, J. W., et al. 2,3,7,8-tetrachlorodibenzo-pdioxin - acute oral toxicity in hamsters. Toxicol. Appl. Pharmacol. (pre pub. copy) [Not available.] 436. Henig, R. M. (1979) Congress calls for 2,4,5-T ban after dramatic herbicide hearings. BioSci. 29:453-454. [Editorial.] 437. Herbicide Assessment Commission Amer. Assoc. for the Advancement of Science. (1970) Summary of Presentations. [Not available.] 438. Herbicides: More research on 2,4,5-T. Chem. Eng. News. 49(20):11. 1971. [Editorial.] 439. Hewitt, W. R., Pegg, D. G., and Hook, J. B. (1976) Effect of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on renal function in rats in vitro. Toxicol. Appl. Pharmacol. 37(l):177. [Abstract, only.] 440. Hickey, G. C. (1974) The effects of herbicides in South Vietnam. Part B. Working papers: Perceived effects of herbicides used in the highlands of South Vietnam. National Academy of Sciences - National Research Council. AD-779 027. 23 pp. [Background material.] 150 �441. Higginbotham, G. R. , Huang, A., Firestone, D., Verrett, J., Ress, J. and Campbell, A. (1968) Chemical and toxicological evaluations of isolated and synthetic chloro derivatives of dibenzo-p-dioxins. Nature 220:702-703. The effects of TCDD in the chick embryo bioassay were briefly reported. The report also described results of the isolation and identification of compounds produced from the pyrolysis of chlorophenols and their biological activity in relation to chick edema factor, hexachlorodibenzo-p-dioxin. TCDD was prepared by chlorinating dibenzo-p-dioxin and 0.05 ug/egg was injected into the air space of fertilized eggs. After 21 days of incubation, 100% of the embryos were dead compared to 10-15% of the non-treated and vehicle controls. Other compounds tested showed only a fraction of the potency of TCDD. The authors suggested that chick edema factor could arise in fat samples during heating operations used to produce commercial fats from chlorophenols present as contaminants from their use as pesticides. 442. Highman, B., Gaines, T. B., and Schumacher, H. J. (1977) Retarded development of fetal renal alkaline phosphates in mice given 2,4,5trichlorophenoxyacetic acid. J. Toxicol. Environ. Health 2(5):1007-1018. The level of alkaline phosphatase detected histochemically in fetal mouse kidneys was determined to evaluate renal functional development in fetal mice which develop cystic kidney in response to maternal 2,4,5-T treatment.' Pregnant CD-I and hybrid mice were administered 60-120 mg/kg 2,4,5-T (less than 0.05 ppm dioxin contaminant) by gavage in acetone-corn oil (1:9) on days 6-14 of gestation. On day 17 or 18 a region of each fetus which included the kidneys was excised, prepared histologically and slices were stained by a cobalt sulfide method to visualize alkaline phosphatase. The numbers of resorptions were recorded and fetuses were examined for cleft palate. Maternal blood was analyzed for hematologic and blood chemistry parameters by automated methods. Maternal toxicity was observed at the highest dose. Alkaline phosphatase was localized principally in clusters of tubules in the inner cortex and along the brush border of the convoluted tubules in day 17 (untreated) fetal kidneys. Groups treated with 2,4,5-T had a statistically significant increase in the frequency of fetuses with diminished or no alkaline phosphatase present. The renal pelvis was described as dilated or cystic in some fetuses of 2,4,5-Ttreated mice but not of control mice, but the incidence was not reported. No cleft palates occurred in fetuses from control groups. For each dose group, about 20% of the mice with normal alkaline phosphatase levels had cleft palate, while 40 to 70% of the fetuses with low alkaline phosphatase levels had cleft palate. Fetuses with cleft palate tended to weigh less than fetuses from the same treatment group with normal palates. Hematologic and clinical chemistry tests of maternal blood revealed no differences among treatment groups. The incidence of viable fetuses decreased from 93% in control groups to 74% and 59% for the 90 and 120 mg/kg dose groups. The frequently concomitant occurrence of cleft palate and decreased fetal weight indicated to the authors that 2,4,5-T produces toxic effects at several indepen- 151 �dent loci. The incidence of lowered alkaline phosphatase levels in treated fetuses on day 18 was more lowered than on day 17 and the authors concluded that renal abnormalities caused by 2,4,5-T resulted from a general retardation in development. This conclusion would be greatly strengthened by a demonstration of a stage when renal development is complete in 2,4,5-T-treated fetuses or neonates. 443. Highman, B., Gaines, T. B., and Schumacher, H. J. (I976a) Sequential histopathologic, hematologic and blood chemistry changes induced in mice by a technical and purified preparation of 2,4,5-trichlorophenoxyacetic acid. J. Toxicol. Environ. Health 1(3):469-484. The effects of 2 preparations of 2,4,5-T administered to pregnant mice on maternal health was evaluated. Dihybrid female mice derived from a cross of (C57BL X A/JAX) hybrids and (C3H X BALE) hybrids were administered 60 or 120 mg/kg 2,4,5-T in acetone-corn oil (1:10) by gavage on day 6-15 of gestation. Two preparations of 2,4,5-T were administered: a technical preparation (97.0% purity, with less than 0.05 ppm TCDD) was administered to 314 mice and a purified preparation (99% purity, with less than 0.005 ppm TCDD) to 64 mice. Of the control mice, 23 received no treatment and 73 received vehicle only. Nonpregnant mice were included in the study in treatment and control groups. Mice were killed from 6 hours to 11 days after the first dose was administered; moribund mice were also killed. Tissues were prepared for light microscopy and blood was analyzed for hematological parameters and blood chemistries. The frequencies of lesions were tabulated for mice in various groups by-clinical condition (normal, sick, or moribund). In general, lesions were most frequent and severe in moribund mice and mild or absent in mice that appeared normal. Differences between treated and control mice were not analyzed for statistical significance. Histopathologic changes included rarefaction of myocardial fibers, and necrosis of the outer myocardium, thymus atrophy (involution was considered to be associated with pregnancy), splenic atrophy, bone marrow and lymph node lesions, thyroid hyperplasia (by day 17 of gestation), hepatic glycogen depletion during treatment, and increased leukocyte counts, urea nitrogen levels and lactate dehydrogenase and glutamic oxaloacetic transaminase activities. Plasma calcium levels fell in moribund mice with severe myocardial rarefaction. The incidences of lesions of the spleen, bone marrow, and lymphatic organs was higher in mice treated with purified 2,4,5-T than with the same dose of the technical preparation. The incidence of fetal deaths or malformations were not given. The authors concluded that the observed hemoconcentration in treated mice indicated that 2,4,5-T caused a hemolytic effect (in addition to its effect on bone marrow; 2,4,5-T and not contaminants of the technical preparation were responsible for the observed toxicity; and the maternal toxicity was not a factor in causing fetal abnormalities because they only occurred immediately prior to maternal death. 152 �444. Highman, B., Gaines, X. B., and Schumacher, H. J. (1976b) Renal alkaline phosphatase activity in fetal offspring of maternal mice given 2,4,5-trichlorophenoxy acetic acid. Toxicol. Appl. Pharmacol. 37:145. [Abstract only.] •f 445. Highman, B., Gaines, T. B., Schumacher, H. J., and Haley, T. J. (1976) Strain differences in histopathologic, hematologic and blood chemistry changes induced in mice by a technical and purified preparation of 2,4,5-trichlorophenoxyacetic acid. J. Toxicol. Environ. Health 1(6):1041-1054. The toxicity of 2,4,5-T was described in pregnant and nonpregnant mice. Pregnant and nonpregnant CD-I mice from several laboratories and dihybrid mice from a mating of (C57BL X A/JAX) hybrids with (C3H X BALE) hybrids were administered 60-140 mg/kg 2,4,5-T by gavage daily "on days corresponding to days 6 through 14 of pregnancy" (which was not explained further for nonpregnant mice). Two preparations of 2,4,5-T were used, including a technical preparation of 97.9% purity and less than 0.05 ppm dioxin, and a purified preparation of 99% purity with less than 0.005 ppm dioxin. Controls received only the acetonecorn oil (1:9) vehicle. Animals were killed 1-11 days after the first dose and were necropsied. Blood samples were removed from some CD-I mice for hematology and blood chemistry analyses. Histopathological lesions included myocardial fiber rarefaction; myocardial necrosis; thymic and splenic atrophy; bone marrow hypocellularity, congestion and hemorrhages, and lymph node lesions and thyroid hyperplasia. In males, urothelial hyperplasia and testicular degenerative changes were also observed. CD-I mice from 1 laboratory (NCTR), showed toxicity from 60 mg/kg doses while CD-I mice from another laboratory (CRBL) were insensitive to doses of 90-120 mg/kg 2,4,5-T, which the authors attributed to a latent epizootic infection in NCTR mice. Another group of CD-I mice (with comparable response to 2,4,5-T as CRBL mice) were more resistant to 2,4,5-T than nonpregnant female dihybrids or male dihybrids and severe lesions in CD-I mice were restricted to moribund mice. Although no fetuses were examined in this study, the incidences of cleft palate in these strains after 2,4,5-T exposure obtained in an unpublished study were reported to be divergent from the relative maternal sensitivity to 2,4,5-T toxicity. In general, pregnant mice showed no increased sensitivity to 2,4,5-T compared to non-pregnant mice. In clinically normal, treated mice hemoconcentration and elevated lactate dehydrogenase levels were observed. The authors concluded that maternal toxicity by 2,4,5-T was unlikely to have caused fetal abnormalities reported in other studies; 2,4,5-T and not other components in the preparation probably cause the observed toxic effects; large differences in sensitivity to 2,4,5-T were demonstrated for different strains of mice; and development of bladder epithelial hyperplasia in dihybrid treated males indicated a potential for carcinogenicity of 2,4,5-T. 153 �446. Hiles, R. A., and Bruce, R. D. (1976) 2,3,7,8-tetrachlorodibenzo-pdioxin elimination in the rat: first order or zero order? Fd. Cosmet. Toxicol. 14(6):599-600. The authors challenge the validity of the assumption made in previous reports that TCDD elimination followed first-order kinetics. Data presented previously (Allen et al., 1975; Piper, et al., 1973) was analyzed using 2 models: 1 representing zero-order kinetics, in which the rate of TCDD clearance was assumed to be independent of TCDD concentration and the concept of half-life is not applicable, and a model for first-order kinetics. The data fit the model based on zeroorder kinetics better than the model for first-order kinetics. The point at which the 2 models predicted very divergent values was for TCDD concentrations measured after 24 days after administration. Neither report presented data for this time period. The authors concluded that the order of the elimination process for TCDD could not be determined by the previously published data and the presentation of biological half-lives was invalid until first-order kinetics are verified. 447. Hill, E. V., and Carlisle, H. (1947) Toxicity of 2,4-dichlorophenoxyacetic acid for experimental animals. J. Indust. Hyg. and Toxicol. 29(2):85-95. The acute and subchronic toxicities of 2,4-D and several salts were studied in several species after oral, parenteral, and inhalation exposures. The oral LD-Q of the sodium salts of a purified 2,4-D preparation was 375 mg/kg in the mouse, 666 mg/kg in the rat, 1000 mg/kg in the guinea pig, and 800 mg/kg in the rabbit. Intraperitoneal LDjg values were the same as the oral values for the mouse and rat and lower for the other species. The maximum tolerated doses in monkey were 214 mg/kg orally and 428 mg/kg intraperitoneally. In the rabbit, an intravenous dose of 400 mg/kg caused acute ventricular fibrillation in half the animals' myotonia. Smaller animals showed symptoms of muscular incoordination, stiffness and paralysis, followed by stupor, coma, and death. Monkeys developed vomiting after large oral doses were administered. The sodium and ammonium salts of a crude (commercial) preparation of 2,4-D and the purified 2,4-D (acid) showed the same toxicity as the sodium salt of purified 2,4-D. Toxicity was the same for 2,4-D when dissolved in either n-butyl alcohol or tributylphosphate. Various subchronic doses of 2,4-D were administered to dogs. The doses were cumulative and symptoms included stiff gait, large and sudden drops in leukocyte, lymphocyte and platelet counts, and bleeding gums. Dogs that died received a cumulative dose of 300 mg/kg or more. Large subchronic oral doses of 0.1 percent 2,4-D in feed to rats, 1.0 gm or 2,4-D to guinea pigs over 12 days or 6000-8000 rag-rain per liter of vapors in air to guinea pig were survived. Gross and histologic examination of rats, guinea pigs that received fatal doses of 2,4-D, and rabbits exposed subchronically revealed renal lesions and pulmonary edema and hemorrhages. Exposed dogs were susceptible to liver damage and also had renal lesions and lymphoid necrosis. The authors concluded that 2,4-D has a low order of toxicity and estimated that a 75 kg man would tolerate a dose of 15 grams and calculated a human LDc0 of 28 gm. 154 �448. Hinsdill, R. D., Thomas, P. T. , and Couch, D. Immunotoxicity assessment of dioxins. In: Inadvertent modification of the immune response. The effects of foods, drugs and environmental contaminants. Office of Health Affairs, FDA, Washington, B.C. pp. 76-79. [Background material.] V 449. Hobson, L. B. (1980) Remarks presented at the 2d Continuing Education Conference on Herbicide Orange, Washington, DC, May 28-30. [Background material.] 450. Hodge, H. C., Downs, W. L., Fanner, B. S., Smith, D. W., Maynard, E. A., Clayton, J. W., and Rhodes, R. C. (1961) Oral toxicity and metabolism of diuron (N-(3,4-dichlorophenyl)-N',N'dimethylurea) in rats and dogs. Fd. Cosmet. Toxicol. 5:513-531. Animal studies of the acute, subchronic, chronic and reproductive toxicities, oncogenicity, potential for skin irritation and sensitivity, and metabolism of diuron are presented. Male rats were administered single doses of diuron in suspension in peanut oil orally and the LDcn was calculated. Wistar-derived rats were used for a series of feeding experiments, except for the 2-week and 3-month studies in which Charles River rats were used. For the 2-week study, male rats were administered 10 daily oral doses of 1 g/kg diuron. For the other oral rat studies, diets that contained 25-8000 ppm diuron were fed for 1, 3, 15, 23 or 24 months. Food consumption, hematological parameters and mortality were assessed during the study. Rats were necropsied and organs were weighed at the end of the feeding period. Dogs (6 per group) were fed 25-2400 ppm diuron for 24 months and for a preliminary study 1 dog was administered 8 mg/kg diuron orally in a gelatin capsule daily for 1 month and another dog was administered diuron for 2 months in doses that increased from 8 mg/kg/day to 120 mg/kg/day over a 2-month period. For a 3 generation reproductive study, rats were fed 125 ppm diuron in the diet and numbers of births, and weanlings and weight gains during lactation were recorded for all generations and histology was evaluated in the second litter of the third generation. Tissue levels of diuron were determined in rats and dogs fed diuron for 2 yrs. and urinary metabolites were identified in urine from dogs by thin layer chromatography and ultraviolet spectrophotometry. The LD-~ was 3.4 g/kg and 10 doses of Ig/kg to rats resulted in reduced weight gains. The spleens of rats, after 1-10 doses, were large and dark, with numerous foci of blood formation in the spleen and bone marrow. In the short-term rat feeding studies, mortality (60%) occurred only at the highest dose and reductions in hematocrits and growth retardation occurred in groups given at least 2000 ppm diuron. Histology resembled findings after acute administration. The only effect in dogs fed diuron for 1-2 months was weight loss and anemia from the 120 mg/kg feeding regimen. In the long-term studies, growth retardation occurred at the highest dose only. The incidences of mortality in treated groups were not compared to controls statistically. The authors did not attribute any observed increase in mortality to diuron treatment. 155 �Sporadic decreases in hemoglobin levels were observed in rats over the 2-year study and the only changes observed in tissues of treated groups were increased splenic weights. No deleterious cellular effects were observed in spleen and bone marrow cells. No adverse effects on reproduction occurred. One litter (F . ) showed growth depression which the authors did not attribute to diuron treatment. In chronically treated dogs, only the highest dosage group showed persistent weight losses, anemia, increased liver weights and erythroid hyperplasia of the spleen and bone marrow. Sulphemoglobin was detected in blood from dogs and rats fed high dietary doses of diuron. No diuron accumulation was observed in tissue, as only a minute amount of the dose was recovered in tissues. Tissue levels were related to the dosage administered and only about 10% of the estimated daily doses were recovered in urine and feces . (The fate of the ingested dose was not indicated.) The principal urinary metabolite was identified as N-(3,4-dichlorophenyl)-urea, and others present in smaller amounts were N~(3,4-dichlorophenyl)-Nfmethylurea, 3,4-dichloroaniline, 3,4-dichlorophenol and unmetabolized diuron. The distribution of metabolites in urine were the same from rats after 1 year of feeding as after 2 yr. The authors mentioned that skin irritation and sensitization tests were negative but did not present the methods or results from these experiments. The authors concluded that diuron did not produce adverse effects at dietary doses below 500 ppm fed for 1-3 months or toxicity or oncogenicity or reproductive effects over 3 generations at doses of 125 ppm or less fed chronically. The metabolic pathways for diuron were concluded to be the same as those found in soil and plants, with sequential removal of methyl groups, followed by hydrolysis of the substituted urea to its aniline derivative. 451. Hodge, H. C., Maynard, E. A., Downs, W. L., and Coye, R. D. (1958) Chronic toxicity of 3-(p-chlorophenyl)-l,l-dimethylurea (Monuron). Am. Arch. Ind. Health 17:45-47. The authors studied the effects of monuron on rats and dogs in a 2-year feeding study. Weanling albino rats (Rochester strain, 30 males and 30 females per group) were fed 0, 0.0025%, 0.025%, and 0.25% monuron in their diet for 2 years. Purity of the compound, storage of formulated diet, and an analytical check on the diet formulation were not reported. Male rats fed 0.25% showed a slight depression in growth after 1 month. Approximately a 20 g difference was observed between treated and control males; females also had a slight growth depression but this was less than seen in the males. No numerical data were presented to evaluate this conclusion. According to the authors, approximately 70-90% of both treated and untreated animals died by the end of the second year. The high mortality was due to viral epidemics within the animal colony. Therefore, results reported are data from a severely limited number of animals. Because of this, the study cannot be used as a good indicator of the effects of monuron. Hematological examinations showed slight anemia in both males and females exposed to 0.25 percent monuron. Urine sugar and protein levels were within the normal range. Incidence of tumors were also within the historicalmormal colony occurrence. No numerical data of results were presented. In the young adult Beagle dog feeding study, groups of 1 male and 1 156 �female dog were given 0, 2,5, 12.5, and 25 rag/kg monuron in their feed for one year. Neither the number of animals nor the length of the study was sufficient to make a reliable interpretation of this study. According to the authors, urine sugar and protein, blood profiles and weights of major organs were within the normal range during the study. No increases in tumor incidence were observed. 452. Hofmann, H. T. (1957) Neure Erfahrungen mit hochtoxischin chlorkoleminasextoffin. Naumiun. Schnidebergys Arch. Exp. Path. Pharmak. 232:228-230. [Foreign language.] 453. Holden, C. (1979) Agent Orange furor continues to build. 205:770-772. Science [Editorial.] 454. Holmberg, B. (1975) Biological aspects of chemical and biological weapons. Ambio 4(5-6):211-216. [Review article.] 455. Homberger, E., Reggiani, G., Sambeth, J., and Wipf, H. K. (no date) The Seveso accident: its nature, extent and consequences. Ann. Occup. Hyg. 22:327-366. The authors describe the environmental effects of TCDD contamination that resulted from the factory accident in Seveso, Italy, in 1976 and present human birth rate and morbidity data for the affected region. A constant decline in the birth rate from 17.1 per thousand in 1973 to 12.6 per thousand in 1977 was found in the Seveso region, which the authors attributed to psychological factors that led to an increase in the use of voluntary birth control methods. No increase in the death rate occurred in the Seveso region after the accident. Three days after the accident, animal deaths occurred near the factory, and animals in a.progressively larger area died in the subsequent months. In general, herbivores were affected first, probably from ingesting contaminated vegetation. Birds appeared to die rapidly, while rabbits and small animals suffered from anorexia, apathy, gastrointestinal hemorrhages, pulmonary edema, and depletion of fat stores before death. Small animals succumbed before sheep and goats, and cattle and horses were affected last. As part of the decontamination program, animals were slaughtered, and traces of TCDD were detected in the livers and fat of some, although no systemic effects were observed. Wildlife in the area closest to the factory were exterminated to prevent contamination of other areas. In the autumn when the grass died, TCDD was transported to the soil. Damage to the flora from the TCDD cloud released during the accident was limited to phytotoxic damage to some broad-leaved plants. An estimated 80 percent of the released TCDD 157 �adhered to foliage, grass, and crops. In a defined study area in the most contaminated sector (zone A), from 28 percent to 49 percent of the deposited TCDD was cleared from the soil in an 18-month period, while minimal transfer of TCDD from the soil into crops was observed. Decontamination procedures were described; natural decontamination is estimated to take from 6 to 8 years to complete. The authors concluded that the only significant health effect identified to date as attributable to TCDD exposure was mild to moderate chloracne in a sensitive segment (youth) of the population. The area remains contaminated with TCDD, although the levels are lower than immediately after the accident. 456. Honchar, P. A., and Halperin, W. E. (1981) 2,4,5-T, trichlorophenol, and soft tissue sarcoma. Lancet Jan. 31:268-269. The authors reviewed studies conducted by Zack and Suskind (1980); Cook, Towsend, and Ott (1980); Ott Holder, and Olson (1980); and the US Department of Health, Education, and Welfare's National Center for Health Statistics (1975). The four studies dealt with mortality rates among workers at Dow Chemical USA or the Monsanto Company who were exposed to 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) or 2,4,5-trichlorophenol (TCP), both of which are contaminated with TCDD. Comparing the four cohorts, the authors found that three of 105 deaths, or 2.9 percent, were due to soft tissue sarcoma. This compares to a rate of 0.07 percent of all US males aged 20-84 in 1975. One man (Zack and Suskind) died in 1978 with malignant fibrous histiocytoma of soft tissue origin; he'was 58. He had been exposed to TCP at Monsanto in 1949 and developed chloracne, a standard symptom of TCDD intoxication. Another man (Cook et al.), who died at 53 of fibrosarcoma, had worked on TCP production at Dow Chemical and had facial dermatitis; no diagnosis of chloracne was made, however. The third case (Zack, unpub.) involved a man who had worked on 2,4,5-T synthesis at Monsanto and had no history of chloracne. In 1972, at the age of 49, he died of generalized liposarcoma. According to Ott et al., one cohort of 2,4,5-T workers at Dow contained no case of soft tissue sarcoma. When considered separately, the rates of soft tissue sarcoma occurrence in the four cohorts do not show an excess risk. However, the authors feel that these three cases may represent a common pattern and suggest that their method of analysis may prove valuable in detecting trends among small cohorts of workers with common occupational exposure. 457. Hood, R. D., Patterson, B. L., Thacker, G. T., Sloan, G. L., and Szczech, G, M. (1979) Prenatal effects of 2,4,5-T, 2,4,5-trichlorophenol and phenoxyacetic acid in mice. J. Environ. Sci. Health (3013:189-204. The teratogenic effects of 2,4,5-T were studied in the mouse. Pregnant CD-I mice were administered 800-900 rag/kg of 2,4,5-T (with 0.01 ppm TCDD contaminant) in honey-water (1:1) by gastric intubation on a single day from day 8 to 15 of gestation. Other pregnant mice received 250-300 mg/kg on 3 consecutive days (days 7-9, 10-12, or 13-15) by the same route. On day 18 of gestation fetuses were removed, weighed, and 158 �examined grossly. Some fetuses were also examined for visceral malformations or for skeletal malformations of alizarin red stained and cleared fetuses. Fetuses of mice treated on day 14 or 15 or days 13-15 with 2,4,5-T weighed 35% (significantly) less than vehicle-treated controls or non-treated controls. Treatment on day 14 also resulted in a 4 fold increase in incidences of deaths and of resorptions over both control groups. Treatment on day 11 or days 13-15 consecutively produced gross malformations in 22-24% of the fetuses, including decreased jaw length and exencephaly, while less than 1% of the controls were affected. Fused ribs and malformed vertebrae were observed in 13% of the fetuses treated on days 13-15 but this value was not (statistically) significantly different from both control groups which had no skeletal malformations or cleft palates. Treatment on days 9 or 11 or 14 or days 10-12 or 13-15 all caused incidence of cleft palate of over 35%. Phenoxyacid and 2,4,5-trichlorophenol were tested in these experiments as well, but caused none of the effects reported for 2,4,5-T. The authors concluded that both the carboxyl group and the chlorinated aromatic ring are necessary for teratogenic and fetotoxic activity. The statistical tests applied to the data include arcsin transformation of means and ANOVA and Gabriel's multiple range tests and rank sum methods. These tests are relatively insensitive as they did not distinguish between frequencies of zero percent incidence of cleft palate (in both controls) and 35.3% from day 14 treatment or 10% frequency of prenatal mortality (controls) and 48.2% from day 12 treatment. Assuming the standard deviations were not excessive, these differences have biological significance and the statistical tests do not aid in interpreting the data for risk assessment. 458. Hook, G. E. R., Baseman, J. K., and Lucier, G. W. (1975) Induction and suppression of hepatic and extrahepatic microsomal foreign-compoundmetabolizing enzyme systems by 2H3,7,8-tetrachlorodibenzo-p-dioxin. Chem. Biol. Interactions 10:199-214. The effects of TCDD on several enzyme systems that metabolize foreign compounds were studied in the rat, rabbit, and guinea pig. TCDD was administered by stomach tube. Subsequently, microsomes were isolated from various tissues and assayed for different enzyme activities. At doses of TCDD that did not elicit any aberrant health effects (0.2-5 ug/kg), TCDD induced benzpyrene hydroxylase activity and cytochromes P-450 and b5 in the rat liver, which persisted for 73 days after a single dose was given. The response in enzyme induction was independent of the age of the treated rat. Actinomycin D partially blocked the inductive effect of TCDD. TCDD treatment inhibited N demethylases in adult male rats, but not in females or in young males. The effect was not observed 35 days after the single dose of TCDD was given. TCDD treatment did not alter cholesterol or phospholipid concentrations in hepatic microsomes, but increased the proportions associated with rough endoplasmic reticulum relative to smooth endoplasmic reticulum. Extrahepatic enzymes of the rat, rabbit and guinea pig were induced by TCDD, but the specific enzymes and tissue sources of the enyzmes that responded to TCDD were different for the 3 species. The authors suggested that TCDD either operates through a receptor or interacts �directly with an operator gene to explain the ability of minute amounts of TCDD to induce enzymes. 459. Hook, G. E.'R., Orton, T. C., Moore, J. A., and Lucier, G. W. (1975) 2,3,7,8 tetrachlorodibenzo-p-dioxin-induced changes in the hydroxylation of biphenyl by rat liver microsomes. Biochem. Pharmacol. 24(3):335-340. TCDD-induced biphenyl 2- and 4-hydroxylase activities of rat liver microsomes are described. Charles River rats were administered a single dose of TCDD in acetone-corn oil by stomach tube. Microsomes were prepared from the liver and assayed for cytochrome P-450 content, biphenyl hydroxylation in the 2 and 4 positions, and for testosterone hydroxylation in the 2 and 6 beta positions and 7 and 16 alpha positions TCDD at a dose of 0.2 ug/kg in females and 1.0 ug/kg in males produced increases in 2- and 4-hydroxylation and in cytochrome P-450 content. The inductive effect of 25 ug/kg on biphenyl hydroxylases persisted 73 days after TCDD treatment, the last time point examined. Biphenyl-2- and 4-hydroxylase levels were induced to the same absolute levels in rats between 10 and 335 days of age, even though 10 day old rats had lower initial biphenyl-2-hydroxylase levels than older rats. Actinomycin D administration partially blocked the TCDD effect 18 hours after the 2 agents were administered. TCDD treatment resulted in 4-17 fold elevations in the Vmax values of both biphenyl hydroxylases and an increase in the apparent Kn for the 2-hydroxylase to the same value as was shown for the 4-hydroxylase. TCDD treatment also resulted in decreased testosterone hydroxylation at the 2 beta- and 16 alpha positions. The authors concluded that TCDD is one of the most potent and longlasting inducers of biphenyl hydroxylases in the rat and suggested that separate enzymes may be responsible for biphenyl hydroxylation in the 2- and 4-positions. Hook, J. B., Bailie, M. D., Johnson, J. T., and Gehring, P. J. (1974) In vitro analysis of transport of 2,4,5- trichlorophenoxyacetic acid by rat and dog kidney. Fd. Cosmet. Toxicol. 12(2):209-218. 2,4,5-T uptake by renal cortical slices of dogs and rats was characterized. Renal cortical slices from dogs and Sprague-Dawley rats were incubated in the presence of C-labelled compounds and uptake of radioactivity into tissue was determined at the end of the exposure period. The presence of 2,4,5-T did not alter N-methylnicotinamide (NMN) uptake, but caused a dose-dependent competitive decrease in paraaminohippurate (PAH) uptake. The slice to medium (S/M) ratios for 2,4,5-T were 14 in rat tissue and 9 in dog tissue. Both ratios were decreased by addition of probenecid and PAH, but only the ratio for rat tissue was dependent upon potassium concentration. Maximum uptake of 2,4,5-T by kidney slices from 10 day-old rats (S/M ratio » 10) was below the level for adult to rat tissue. The authors concluded that prolonged half-lives for 2,4,5-T in young rats compared to adult rats and in dogs can be explained by their differences in rates of uptake by the kidney, the major route of 2,4,5-T excretion. 160 �461. Hook, J. B., Cardona, R., Osborn, J. L., Bailie, M. D., and Gehring, P. J. (1976) The renal handling of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in the dog. Fd. Cosmet. Toxicol. 14:19-23. Renal excretion of 2,4,5-T by the dog and the rat was studied in vivo and in vitro in the presence of metabolic substrates and inhibitors. Sodium pentabarbitone-anesthetized mongrel dogs were administered 2,4,5-T by intravenous infusion and urine was collected from urethral catheters. Blood samples were collected at the midpoint of each clearance period and inulin, para-aminohippurate (PAH) and 2,4,5-T clearances were determined. PAH secretion was significantly reduced by administration of 5-10 mg/kg 2,4,5-T with no change in glomerular filtration rate. Infusion of sodium acetate increased 2,4,5-T clearance in a dose-related manner, but 2,4,5-T clearance never reached the rate of inulin clearance. Administration of acetazolamide, mannitol and saline also caused increases in 2,4,5-T clearance (below the rate of inulin clearance). Pretreatment of dogs with ammonium chloride for 1-3 days before the experiment and infusion of sodium bicarbonate to acidify the urine resulted in a substantial enhancement in 2,4,5-T clearance at urine pH values above 6.0. Renal slices were prepared and incubated with 2,4,5-T and PAH. Plasma added to the incubation medium increased PAH uptake but reduced 2,4,5-T uptake by half. The authors concluded that factors that influenced 2,4,5-T clearance included, along with active renal transport, a urinary pH-dependent process as passive diffusion, and plasma-protein binding of 2,4,5-T with a higher affinity for 2,4,5-T than the affinity for the kidney transport system. 462. Hook, J. B., McCormack, K. M., and Kluwe, W. M. (1978) Renal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In Pentachlorophenol; Chemistry, pharmacology and environmental toxicology.K. R.Rao, ed., (New York:Plenum Press).pp. 381-388. The effect of TCDD on chloroform-induced renal toxicity was studied in the mouse. Male ICR mice were administered 1.6 or 16 ug/kg TCDD intraperitoneally. After 72 hours, from 0.5-25 ug/kg of chloroform was administered and 24 hours after chloroform treatment, blood was collected and analyzed for blood urea nitrogen (BUN) and serum glutamate oxaloacetate transaminase (SCOT) activities. Other mice were killed 3 days after TCDD treatment and liver and kidney microsomal preparations were assayed for epoxide hydratase, aryl hydrocarbon hydroxylase, and biphenyl-2- and -4-hydroxylases. TCDD treatment resulted in significant elevations in all enzyme activities in both tissues; all control and induced enzyme activities were higher in liver microsomes than in kidney microsomes. BUN and SCOT levels were unaltered by TCDD. TCDD-treated mice (number of mice not reported) had increased liver-to-body weights, but not kidney-to-body weights. Chloroform administered to control mice or TCDD-treated mice did not alter kidney-to-body weight ratios. Other effects of TCDD on renal function presented in previous publications were reviewed. The authors concluded that TCDD produced metabolic changes in the kidney but not physiological effects or potentiation of chloroform toxicity; any decrease in renal function was considered nonspecific and was attributed to a decline in the general health of the animal. 161 �463. House, W. P., Goodson, L. H., Gadberry, H. M., and Dockter, K. W. (1967) Assessment of ecological effects of extensive or repeated use of herbicides. Advanced Research Projects Agency, Department of Defense, ARPA No. 1086, DDC No. AD 824314. 372 pp. [Background material.] 464. Huddle, F. P. (1969) A technology assessment of the Vietnam defoliant matter: a case history. Report to the Subcommittee on Science Research and Development of the Committee on Science and Astronautics. US House of Representatives, 91st Congress, Prepared by the Science Policy Research Division, Legislative Reference Service, Library of Congress, Washington, DC. 73 pp. [Background material.] 465. Hudson, A. J. (1977) Amyotrophic lateral sclerosis and toxic hydrocarbons, [letter to the editor] Arch. Neurol. 34:721. [Editorial.] 466. Huff, J. E., Moore, J. A., Saracci, R., and Tomatis, L. (1980) Longterm hazards of polychlorinated dibenzodioxins and polychlorinated dibenzofurans. Environ. Health Perspec.' 36:221-240. [Review article.] 467. Huff, J. E., and Wassom, J. S. (1974) Health hazards from chemical impurities: Chlorinated dibenzodioxins and chlorinated dibenzofurans. Inter. J. Environmental Studies. 6:13-17. [Review article.] 468. Huff, J. E., and Wassom, J. S. (1973) Chlorinated dibenzodioxins and dibenzofurans. Environ. Health Perspec. 5:283-313. [Bibliography.] 469. Hughe-s, J, S., and Davis, J. T. . (1963) Variations in toxicity to bluegill sunfish of phenoxy herbicides. Weeds 11:50-53. [Background material.] 162 �470. Hughes, R. D., Millburn, P., and Williams, R. T. (1973) Biliary excretion of some diquaternary, ammonium cations in the rat, guinea pig and rabbit. Biochem. J. 136(4):979-984. Biotransformation and urinary and biliary excretion of diquat by the rabbit, guinea pig, and rat are described. Anesthetized female animals with cannulated bile ducts were administered an intraperitoneal dose of 14 umol (cation)/kg of diquat dichloride to Dutch rabbits, 13 umol/kg to English guinea pigs and 40 umol/kg to Wistar rats. Rats were maintained under anesthesia for 24 hr. and the remaining animals for 3 hr. Bile and urine samples were collected and analyzed for radioactivity. Metabolites were separated from the parent compound by paper chromatography and by ion exchange chromatography, followed by colorimetric analysis. In 3 hrs. the percentages of administered diquat excreted were 3% and 5% in bile of rabbits and guinea pigs, respectively and 64% and 45% in urine, respectively. Excretion by the rat was 1.4% in bile at 3 hr., 2.2% in bile at 24 hr., and 82% in urine in 24 hr. Half of the radioactivity in rat bile and all in the urine was unmetabolized diquat. Two other biliary metabolites were separated but not identified. In the rabbit and guinea pig, 89 and 72% respectively of urinary radioactivity was recovered as unmetabolized diquat and the remainder chromatographed with the metabolite in rat bile. After renal ligation, 6.1% of diquat administered to rats was excreted in bile in 3 hr., compared to 1.4% in non-ligated controls. Excretion of 4 other compounds were studied. The authors concluded that biliary excretion of dications with molecular weights below 500-600, accounted for excretion of less than 10% of the administered dose. 471. Hull, A. C., Jr. (1971) Effect of spraying with 2,4-D upon abundance of pocket gophers in Franklin Basin, Idaho. J. Range Manage. 24:230-232. [Background material.] 472. Hunter, J. H., and Young, A. L. (1972) Vegetative succession studies on a defoliant-equipment test area. Technical Report AFATL-TR-72-31. Air Force Amament Laboratory, Eglin Air Force Base, Fla. [Background material.] 473. Hurtt, W., and Danoro, R. A. (1968) Biological effectiveness of stull bifluid and orange. Air Force Armament Laboratory, Air Force Systems Command, Eglin AFB, Fla. Technical Report No. AFATL-TR-68-122. [Background material.] 163 �474. Hurtt, W. , and Darroro, R. A. Comparison Test of defoliants. Vol II. Appendix III. Biological effectiveness of stull bifluid and Orange. Air Force Armament Laboratory, Air Force Systems Command, Eglin AFB, Fla. Publication No. ADTC-TR-69-30. [Background material.] 475. Hussain, S., Ehrenberg, L., Lofroth, G., and Gejvall, T. (1972) Mutagenic effects of TCDD on bacterial systems. Ambio 1:32-33. The authors evaluated the mutagenicity of TCDD in three bacterial systems: 1) reversion to streptomycin independence in Escherichia coli Sd-4, 2) reversion to histidine independence in Salmonella typhimurium strains TA 1530 and TA 1532 which respond to mutation by base pair substitution and frame shift mutation, respectively and 3) prophage induction in E_. coli K-39. TCDD (99% purity) in DMSO was added to suspension cultures of lag phase E_. coli Sd-4 at 0.5, 1, 2, and 4 ug/ml for 1 hour. Mutation frequencies appeared to increase at 2 ug/ml. However, the two replicate cultures reported had a wide variation in frequencies of mutation (34 x 10~ and 256 x 10~ ), but not in survival, 18% and 11% respectively. Since the variation was so wide it is difficult to make conclusions about the result of the study. No further testing was done. In addition, lag phase cells were exposed in this experiment. Other investigators have found that this is not the most sensitive stage for mutagenic effects to occur. In S. typhimurium TA1530, no increased numbers of revertants were observed at 1 and 10 ug/ml TCDD for 1 hour. Survival values were 90 and less than 1% respectively. In strain TA1532 increased mutation frequency was observed when TCDD caused bacterial survival to decrease to about 1%. No numerical data were presented to analyze the author's results. However, in this test results which have survival rates of less than 10% are not generally recognized as being a reliable indicator of a positive result. In the prophage induction experiment TCDD in DMSO was incubated at 0, 0.5, 1, 1.5, and 2.5 ug/ml for 30 minutes with E_. coli K-39 ( ) cells. E^. coli K-49 cells were used as indicator cells and incubated with the treated, washed K-39 ( ) cells for 2 hours. Numbers of replicate cultures were not reported. The solvent DMSO appeared to have an effect on prophage induction. In the controls without DMSO, 7.3 x 10 plaques/ml were observed while only 1.9 x 10~ plaques/ml were observed with DMSO. The significance of this effect could not be calculated because of lack of data. At 0.5 ug/ml, TCDD induced 5.4 x 10 plaques/ml, about a two-fold increase compared to DMSO controls. No other concentration of TCDD increased the numbers of plaques observed. As a result of lack of data from replicate cultures, no statistical analysis of results, and poor presentation of experimental design, the conclusion of the authors i.e., that TCDD is mutagenic in S^. typhimurium TA1532 and is a weak inducer of prophage, cannot be relied on. 164 �476. Hwang, S. W. (1973) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the biliary excretion of indocyanine green in rat. Environ. Health Perspec. 5:227-244. Hepatobiliary function was evaluated in rats administered TCDD. Male CD rats were administered a single dose of 5 or 25 ug/kg TCDD perorally in acetone-corn oil. Controls received vehicle only. Biliary excretion and bile flow were studied in 4 rats per treatment group 1, 7 and 16 days after treatment by measuring indocyanine green (ICG) excretion. Rats were anesthetized and after indocyanine green was administered intravenously bile was collected from cannulated bile ducts for 20 minutes and plasma was collected periodically over the 20 minute collection period. The levels of ICG in the blood and bile samples and in the liver at the end of the experiment were determined spectrophotometrically. Significant increases in bile flow and in liver weights of treated rats over controls occurred 7 and 16 days after TCDD treatment. The rate of ICG excretion was decreased in treated rats 7 and 16 days after treatment. All of these effects were greater after the 25 ug/kg dose of TCDD than after the 5 ug/kg dose. ICG concentrations in the blood and liver were higher in treated rats than controls and bile concentrations were lower in treated rats. Liver-to-plasma concentration ratios of ICG were decreased by the high dose, indicating a dosedependent inhibition of hepatic uptake of ICG. Bile-to-liver ratios, an indication of ICG excretion into bile, was inhibited to the same extent by both doses. Plasma ICG clearance decreased in a dosedependent manner after TCDD treatment. The decrease in clearance was more marked after 16 days than after 7 days. The authors concluded that TCDD produced a long-lasting decrease in hepatobiliary excretion which could alter the elimination of actively secreted anions. 477. Hwang, S. W., and Schanker, L. S. (1973) Absorption of organic arsenical compounds from the rat small intestine. Xenobiotica. 3(6):351-355. The absorption of cacodylic acid by the small intestine of the rat was determined. The small intestines of entobarbital-anesthetized male Charles River derived rats were exposed, ligated and injected luminally with sodium cacodylate. The incision was then closed and absorption was allowed to take place for up to 5 hours. After the absorption period was terminated, the intestinal content was assayed for the proportion of the dose that was not absorbed and the tissue samples were analyzed for arsenous oxide. The half time for the apparent firstorder absorption of cacodylic acid (administered at a concentration of 5mM) was 1 calculated at 201 minutes. Other arsenicals that were studied included carbarsone and tryparsamide. The rates of absorption for the 3 compounds correlated with the chloroform-water partition coefficients for these compounds, but not with the molecular weights. Absorption was not saturated in the concentration range of 1-100 mM. The authors concluded that cacodylic acid was absorbed at a moderate rate compared to rates for other drugs, and that absorption was probably by simple diffusion through a lipoid phase. 103 �478. Innes, J. R. M., Ulland, B. M., Valerio, M. G., Petrucelli, L., Fishbein, L., et al. (1969) Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary note. J. Natl. Cancer Inst. 42(6):1101-1114. The authors evaluated 120 pesticides for carcinogenicity in a bioassay in mice. Test compounds included the following: 2,4-D isoopropyl ester, 2,4-D isooctyl ester, 2,4-D, diuron, 2,4,5-T, and monuron. All compounds tested were commercial formulations. No purity information on these preparations was presented by the authors. Males and females of two strains of mice, (C57BL/6 x C3H/Anf)F, and C57BL/6 x AKR)F., were used in the study and received the maximally tolerated dose for each compound: 46.4 mg/kg, 2,4-D isopropyl ester, butylester, isooctyl ester or acid, 21.5 mg/kg 2,4,5-T, 464 mg/kg diuron, 1000 mg/kg 2,4-D acid, or 215 mg/kcg monuron. Starting at 7 days and until 4 weeks of age, each mouse received daily doses of the compound in 0.5% gelatin by stomach tube. After weaning, 18 mice of each sex per strain received approximately the maximally tolerated dose in their diet for 18 months. Chemical analysis of diet formulations for correct amounts of test compound was not reported by the authors nor was storage length of treated feed reported. Both positive and negative controls were included in the study. None of the herbicides mentioned above caused a significant increase in tumors in either sex or strain of mice. 479. Interagency Work Group to Study the Possible Long-term Health Effects 'of Phenoxy Herbicides and Contaminants. (1980a) [February, 1980 report] 18 pp. [Background material.] 480. Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants. (1980b) [March, 1980 report] 109 pp. [Background material.] 481. Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants. (1980c) [August, September, and October, 1980 report] 139 pp. [Background material.] 482. Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants. (1980d) Summary Report of the Public Meeting of the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants. Sept. 22, 1980. 72 pp. [Background material.] 166 �483. International Agency for Research on Cancer. (1977) IARC monographs on the evaluation of the carcinogenic risk of chemicals to man: Some fumigants, the herbicides 2,4-D and 2,4,5-T, chlorinated dibenzodioxins and miscellaneous industrial chemicals. 15:41-299. [Review article.] 484. International Agency for Research on Cancer, (1976) IARC monographs on the evaluation of carcinogenic risk of chemicals to man: Some carbamates, thiocarbamates and carbazides. 12:167-176. [Review article.I 485. Irish, K. R., Darrow, R. A., and Minarck, C. E. (1969) Information manual for vegetation control in Southeast Asia. Mis. Pub. 33. Department of the Army, Fort Detrick, Frederick, Md. 71 pp. [Not available.] 486. Isensee, A. R. Bioaccumulation of 2,3,7,8-tetrachlorodibenzo-paradioxin. In Chlorinated Phenoxy Acids and Their dioxins. C. Ramel, ed., (Ecol. Bull. No 27, Stolkholm: Swedish Natural Science Research Council, 1978) pp. 255-262. [Review, article.] 487. Isenseej A. R., and Jones, G. E. (1971) Absorption and translocation of root and foliage applied 2,4-dichlorophenol, 2,7-dichlorodibenzo-pdioxin, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Agric. Food Chem. 19(6):1210-1214. [Background material.] 488. Isensee, A. R., and Jones, G. E. (1975) Distribution of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in aquatic model ecosystems. Environ. Sci. Techno1. 9(7):668-672. The authors studied the bioaccumulation of TCDD in an aquatic ecosystem. Concentrations of 0.0001-7.45 ppm ring-labeled C-TCDD were added to soil samples in aquaria which were filled with 4 liter water. Duplicate tanks were used for each concentration. One day later about 100 daphnids, 8 snails, a few strands of algae, and 10 ml of water containing aquatic microoganisms were added to the tanks. Water samples were removed at 2-day intervals for scintillation counting. At 30 days, samples of daphnids were removed and 2 mosquito fish added to each tank. On day 33, all organisms were removed and analyzed for C-TCDD. Two fingerling catfish were then added to each tanks and 167 �were harvested 6 days later. At 3.17 ppm, TCDD in water reached equilibrium in 4 days while and 0.1 and 0.001 pptn TCDD did not reach equilibrium until day 15. No toxicity to aquatic organisms was observed. However, fish were only treated for 3-6 days which may not be a long enough; since TCDD toxicity is delayed in most organisms. TCDD concentrations in the organisms approached concentrations that were in the soil samples. Water concentrations averaged 0.015% that in the soil. Bioaccumulation averaged from 2-2.6 x 10 for snails, mosquito fish, and daphnids, and 4-9 x 10 for duckweed, algae, and catfish, indicating that TCDD has the potential to accumulate in the environment. Highly significant correlation coefficients (r 0.94) were obtained between TCDD in organisms and in water, suggesting that bioaccumulation of TCDD by aquaticorganisms is dependent on TCDD available in the water. 489. Isensee, A. R., Kearney, P. C., Woolson, E. A., Jones, G. E., and Williams, V. P. (1973) Distribution of alkyl arsenicals in model ecosystem. Environ. Sci. Technol. 7:841-845. The authors describe the behavior of cacodylic acid in a model aquatic ecosystem. C-Cacodylic acid (10.6 ppb) was added to aquarium tanks containing 4 liters of "standard reference water", and 10 of sandy loam soil was added to each treated and control tank. Organisms added to each tank included 3 fish, 10 snails, a few strands of algae, 30 Daphnia, and a few ml of old aquarium water that contained diatoms, protozoa, and rotifers. Fish, Daphnia, snails, and algae were exposed to cacodylic acid for 3, 29, 32, and 32 days, respectively. Lower food chain organisms (algae and Daphnia) bioaccumulated more cacodylic acid than did higher food chain organisms, indicating that cacodylic acid does not biomagnify in the food chain. Bioaccumulation ratios were 1,635, 1,658, 419, and 21 for algae, Daphnia, snails, and fish, respectively. According to the authors, bioaccumulation of cacodylic acid was probably due to adsorption by the organisms. Algae and Daphnia have a higher surface area to mass ratio than fish or snails and therefore could adsorb more cacodylic acid. The authors concluded that these results show that cacodylic acid does not have a high potential to biomagnify in the environment. 168 �490. Jackson, S. (1980) Agent Orange - Bibliography, (National Library of Medicine Literature Search, No. 80-30) 9 pp. [Bibliography.] 491. Jensen, N. E. 65:21-22. (1972) Chloracne: 3 cases. Proc. Roy. Soc. Med. Chloracne was described in two men exposed to 2,4,5-trichlorophenol and in the son of one of the men. Three years before this incident, an explosion had occurred in a factory that produced 2,4,5-trichlorophenol, and the factory had been dismantled and buried when 70 maintenance workers at the site developed chloracne. (In these workers, Chloracne remained severe for 1-1.5 years and still persisted in some after 3 years). Several large tanks, however, were repeatedly cleaned and put into use again. Two men who set up one of these tanks for a new use developed chloracne on the face, trunk, and arms 1 to 2 months after working with the tank. No other clinical symptoms were reported, and both still had chloracne 11 months after it was initially diagnosed. Three months after his father's symptoms developed, the 4-year old son of one worker developed chloracne with the same clinical pattern. The authors suggested that the child developed chloracne after touching his father's work clothes. The serum lipid levels and liver function tests of the patients were normal. Rabbits housed in the decontaminated tanks before their re-use failed to develop skin lesions. In an addendum, the authors mentioned that the wife of the other worker developed facial chloracne 11 months after her husband was exposed to TCDD. 492. Jensen, N. E., Sneddon, I. B., and Walker, A. E. (1972) Tetrachlorodibenzodioxin and chloracne. Trans. St. Johns Hosp. Derm. Soc. 58(2):172-177. A brief description of four cases of chloracne is given, and a brief review of circumstances that caused chloracne in the past is presented. The four cases were described in another publication (Jensen, N,E. Proc. Roy. Soc. Med. 65:21-22) and involved two maintenance workers and two members of their families. The workers were exposed to equipment from a trichlorophenol plant in England where an explosion had occurred. An analysis of sebum from the forehead of one patient 1 year after exposure did not reveal TCDD. The analysis was by gas liquid chromatography, which can detect 1 part TCDD per 10 million of sample. Based on the simultaneous 1-day exposure of both workers to the contaminated equipment and the simultaneous development of symptoms, the authors concluded that chloracne resulted from exposure to a pocket of residual TCDD. 169 �493. Jenssen, D., and Renberg, L. (1976) Distribution and cytogenetic test of 2,4-D and 2,4,5-T phenoxyacetic acids in mouse blood tissues. Chem. Biol. Interact^. 14:291-299. The authors tested 2,4-D and 2,4,5-T in a micronucleus test in mice, a test indicating chromosome breakage. Male CBA mice (8-12 weeks old) received a single 100 mg/kg intraperitoneal infection of 2,4-D or 2,4,5-T (TCDD less than 1 ppm). Three animals were used per treatment in the micronucleus test and killed 24 hours or 7 days after injection. Positive and negative control animals were included in the study. Approximately 2,000 bone marrow cells from each animal were analyzed for numbers of polychromatic erythrocytes with micronuclei. Plasma and cells of bone marrow and peripheral blood at 4 and 24 hours post injection were analyzed for 2,4-D and 2,4,5-T by gas chromatography in another group of two mice per treatment. No increase in polychromatic erythrocytes with micronuclei was observed in animals treated with 2,4-D or 2,4,5-T 24 hours or 7 days. However, a weak toxic effect on mitotic activity was observed in treated animals. Both compounds appeared in peripheral blood and bone marrow within 4 hours after injection. 2,4-D and 2,4,5-T levels in blood plasma declined by 24 hours. No more than 50% of the plasma levels of the herbicides were found in cell fractions. The authors concluded that results of the test were not a reliable indicator of a lack of mutagenicity because the 2,4-D and 2,4,5-T do not enter the target cells to an appreciable extent. However, since the data were in agreement with the known rapid excretion of these compounds, the authors also concluded that no cytogenetic hazard is connected with 2,4-D and 2,4,5-T. 494. Jirasek, L., Kalensky, J., and Kubec, K. (1973) Acne chlorina and porphyria cutanea tarda during the manufacture of herbicides. Part I. Cesk. Dermatol. 48(5):306-315. The pattern of dermal toxicity that developed in 76 workers involved in the manufacture of 2,4,5-T and pentachlorophenol was described. All workers but two were male, and 66 percent were under 30 years old. Only six cases of chloracne began with acute dermatitis and sensitivity to the sun; the remainder of the cases involved development of chloracne in an "occult" manner. Inflammation occurred only in the most severe cases of acne. Porphyria cutanea tarda was detected in 11 persons and in two additional patients who were free of acne. In most patients, acne began with the appearance of comedones on the face above the cheek bones, but in 17 cases the first signs were papulopustules on the legs. In general, the eyebrows, eyelids and scalp were not affected. In five persons, the skin of the entire body was affected. In 19 cases, hypertrichosis, hyperpigmentation of the face, or both occurred without any evidence of a disorder of porphyrin metabolism. The extent of exposure and latency period between exposure and the appearance of symptoms varied considerably among workers. Fatigue, muscle pains, loss of appetite, headaches, and other subjective disorders were more frequent among patients with extensive skin symptoms. These and other symptoms were described in another report and are only mentioned in this report. Details of the manufacturing process were 1V70 �described. The authors concluded that TCDD was responsible for the observed toxicity; the plant remained closed at the time of the report and construction of a new factory was being considered. 495. Jirasek, L., Kalensky, J., Kubec, K., Pazderova, J., and Lukas, E. (1974) Acne clorina, porphyria »cutanea tarda and other manifestations of general intoxication during the manufacture of herbicides, Part II. Cesk. Dermatol. 49(3):145-157. A description of symptoms observed in 50 patients involved in the manufacture of 2,4,5-T was presented. The patients (except two) were among the group of 78 patients whose dermatological conditions were described in a previous report by the authors (Jirasek, et al., 1973). About 80 percent of the cases became ill during their period of employment in the manufacture of 2,4,5-T, while the remainder became ill 1-18 months after exposure had ended. Illnesses were identified between 1965 and 1968 and physical examinations were performed between 1967 and 1973. The average age of the study group was 35.9 years and over half of the patients with porphyria cutanea tarda were over 40 years old. In 11 patients uroporphyrin levels were elevated consistently and in 12 others were elevated intermittently. The levels gradually decreased from a mean of 1,043 (u)g/24 hours in 1969 to 235 (u)g/24 hours in 1973. Other porphyrin levels were not elevated. In 10 patients with elevated uroporphyrin levels, other symptoms of porphyria cutanea tarda also occurred primarily hyperpigmentation and hypertrichosis. Fluorescence of several tissues removed at autopsy was observed under UV light. Elimination of delta-amino levulinic acid was twice as high for exposed patients as for a control group in 29 patients. Lipid metabolism was altered in half of the patients and liver function tests were abnormal in 11 cases. The mean total blood protein levels were higher in all years for the exposed group than for a control group that was not described further. Mild to moderately severe neurological disorders were observed in 17 cases. All cases involved central neurological symptoms and 13 involved peripheral neuronal damage, usually in the legs. In 27 percent of the patients electroencephalogram results were interpreted as abnormal and in 1 patient as markedly abnormal. Five people had died at the time of the report, including two (ages 47 and 59) from bronchogenic carcinoma, which were too few cases to establish a causative link with exposure to TCDD. Arterioschlerosis occurred in one patient and the authors suggested that the disturbance in lipid metabolism in exposed patients may have created a predisposition for this disease. The authors concluded that TCDD exposure produced toxicity in many organ systems. 496. Jirasek, L., et al. (1976) Chloracne, porphyria cutanea tarda and other intoxications by herbicide. Per Hautarzt 27:328-333. The author presents a summary of the illnesses in workers from a herbicide factory in Czechoslovakia that was contaminated with TCDD. All of the findings were presented in more detail in a previous report (see Jirasek et al., 1974). 171 �497. Johnson, E. F., and Muller-Eberhard, U. (1977) Multiple forms of cytoxchrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxins. Fed. Proc. Fed. Am. Soc. Exp. Biol. 36(3):833. [Abstract, only.] 498. Johnson, J. E. (1971) The public health implications of widespread use of the phenoxy herbicides and picloram. BioSci. 21(17):899-905. [Review article.] 499. Joint NIEHS/IARC Working Group Report. (1978) Long-Term Hazards of Polychlorinated Dibenzodioxins and Polychloririated DibenzofuranlT World Health Organization - International Agency for Research on Cancer, Lyon. No. 78/001. 57 pp. [Review article.] 500. Jones, E. L., and Kizek, H. (1962) A technique for testing acnegenic potency in rabbits, applied to the potent acnegen, 2,3,7,8 tetrachlorodibenzo-p-dioxin. J. Invest. Dermatol. 9:511-517. The histology and keratin content of rabbit ears treated with TCDD were studied. Doses of 0.3-10 (u)g of TCDD (synthesized from 2,4,5-trichlorophenol in the laboratory) were applied on 3 successive days to the inner surface of one ear of rabbits, while acetone only was applied to the other ear. Three biopsy samples were removed with a punch from the anterior, posterior, and middle regions of the ear 14 days after the first application was made. The biopsies were cleared of cartilage and digested with pepsin, and the dried weight after digestion was reported as the keratin content. Other biopsy specimens were stained for histopathological examination. A dose-related increase in keratin occurred after TCDD treatment, with three times more keratin in biopsy plugs from rabbit ears treated with 10 (u)g TCDD compared to the keratin levels in the other ear. The lowest dose, 0.3 (u)g, was ineffective in altering keratin levels but 'produced follicular dilatations. Large comedones were produced by 3 (u)g of TCDD in the rabbits that showed the largest increases in keratin content from this dose. Histologically, TCDD-treated ears showed a characteristic hyperkeratosis and hyperplasia of the surface epidermis. Few subaceous cells were present in tissues treated with high doses of TCDD, and the follicles from these tissues were filled with a keratinous mass. Keratin masses took the form of tree structures at lower doses and were oval, of typical comedone shape, at high doses. The authors concluded that the experimental system presented was appropriate for evaluating the dermatologic effects of acnegens. The method used to quantitate keratin is non-specific and probably does not remove 100 percent of non-keratin components, while leaving 100 percent of the original 172 �amount of keratin. However, the results clearly confirm an acnei-form response of this animal model to TCDD and support the contention that keratin production is increased in afflicted tissue. 501. Jones, G. (1975) A histochemical study of the liver lesion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) in rats. J. Pathol. 116:101-105. Hepatic plasma-membrane associated enzymes were evaluated histochemically and hepatic morphology was examined histopathologically in rats administered TCDD acutely. Male Portland rats were administered 200 ug/kg TCDD in arachis oil orally. Between 1 day and 9 months later, groups of three treated and one control rat were killed and liver tissue was prepared for histology. Acid phosphatase, adenosine triphosphatase (AT Pase), 5-nucleotidase, and alkaline phosphatase were identified in liver slices and adjacent slices were stained with hematoxylin and eosin for histopathological examination. Typical TCDDinduced hepatic lesions were observed at 34 and 42 days, but not earlier. These changes included alterations in trabecular and sinusoidal patterns and cell size, the appearance of multi-nucleate cells, and fibrosis around the central vein. At 9 months, partial correction of these lesions was observed. AT Pase was diminished around the central vein along the canalicular borders of the parenchymal cells 3 days after TCDD treatment and by 5-8 days AT Pase was reduced in the midzone with normal localization of this enzyme only around the portal.tracts. The sinusoids in the centrilobular zone showed dense staining. These changes in the AT Pase reaction persisted at 34 days and 42 days only in animals that also showed clinical signs of intoxication at this time. At 9 months, when surviving rats showed improvement of their clinical state, the AT Pase reaction was restored to normal. No significant and consistent changes in the quantity or distribution of the other enzymes were observed. The authors suggested that an increase in the sinusoidal AT Pase levels was indicative of an inflammatory reaction and that the loss of parenchymal AT Pase activity indicated loss in membrane function that precedes morphological changes and correlates with the clinical state of the animal. The authors concluded that the site of TCDD toxicity is the parenchymal cell plasma-membrane. 502. Jones, G., and Butler, W. H. (1974) A morphological study of the liver lesion induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin in rats. J. Pathol. 112(2):93-97. The development of multinucleate cells in the centrilobular zone of the liver of rats after TCDD administration is described. A dose of 200 ug/kg TCDD in dimethylsulfoxide or arachis oil was administered orally to groups of four Porton rats and one group was killed each week during the subsequent 10 weeks. Two vehicle-control rats were killed with each treatment group. Prior to death, the liver was perfused in situ with glutaraldehyde and then sliced and prepared for light and electron microscopies and at 1 week degeneration of parenchymal cells was 173 �evident by light microscopy. Throughout the experiment nuclear morphology was normal and damage was restricted to the cytoplasm of the hepatic parenchymal cells of the centrilobular zone. Proliferation of smooth endoplasmic reticulum was evident, as well as fusion of plasma membranes, which were interpreted in relation to enzyme induction and the formation of multinucleate cells, respectively. Mural fibrosis of the central veins was also observed. The endothelial cells that line the sinusoids appeared normal. The authors concluded that TCDD induces membrane abnormalities at the canalicular borders of hepatocytes. 503. Jones, G., and Greig, J. B. (1975) Pathological changes in the liver of mice given 2,3,7,8-tetrachlorodibenzo-p-dioxin. Experientia 31:1315-1317. Histopathological changes of the liver were described for mice. Male C57BL/6 mice were administered 250 ug/kg of TCDD by oral intubation in arachis oil and were killed 1 to 35 days later. The liver was weighed, homogenized, and analyzed for protein DNA, fatty acid, and cholesterol. The body weights of treated mice fell while the liver weight increased relative to body weight, which was attributed to the concomitant increase in hepatic lipid content. The protein and water contents of the liver decreased and the DNA content remained unaltered and was attributed to inflammatory infiltration. The hepatic lesions regressed in surviving mice and no multinucleate hepatocytes were observed. The authors concluded that the hepatic lesions in TCDD-treated mice differed substantially from those observed in the rat after TCDD administration and suggested that the fatty liver in the mouse could be the result of decreased food uptake. Eight days after the compound was given, the liver was larger and paler than controls and a progressive necrotic centrilobular lesion developed. 504. Jones, K. G., and Sweeney, G. D. (1980) Dependence of the porphyrogenic effect of 2,3,7,8- tetrachlorodibenzo-p-dioxin upon inheritance of aryl Toxicol. Appl. Pharmacol. 53:42-49. The induction of several hepatic enzymes and the porphyrogenic effects of TCDD was observed in hybrid mice that were backcrossed from genetically AHH (aryl hydrocarbon hydroxylase) responsive and non-responsive mice. Doses of 25 ug/kg TCDD in 1,4-dioxane were administered weekly to groups of 6-9 C57BL/6J (AHH responsive Ah/Ah) mice, (non-AHH responsive, ah/ah) DBA/2J mice and D2 (B6 D2) F,/J (backcrossed) mice. Urinary porphyrins and hepatic cytochrome P-450 levels, AHH, uroporphyrinogen decarboxylase (UD) and aminolevulinic acid (ALA) synthetase activities were assayed. After 6 weeks of treatment, C57BL mice excreted 6 times as much porphyrin (comprised primarily of uroporphyrin) as DBA mice, which excreted primarily coproporphyrin. UD activity was inhibited only in C57BL mice. The genotype of backcrossed mice (from a cross of DBA mice with C57BL X DBA offspring) were categorized as AHH responsive (Ah/ah) or AHH non-responsive (ah/ah) from zoxazolamine paralysis times after beta-naphthoflavone pretreatment. Half of each group was administered TCDD weekly for 11 weeks. Only 174 �mice with the Ah gene that controls AHH responsiveness showed elevated porphyrin excretion. Only these groups had significantly decreased UD levels, by 80% in Ah/Ah (C57BL) mice and 33% in Ah/ah mice. AHH activity and microsomal cytochrome P-450 levels were induced by TCDD in all groups and ALA-synthetase activity was unchanged in all groups. The authors concluded that porphyria susceptibility genetically segregates with the gene controlling AHH responsiveness and these phenotypes are controlled by the same gene or are closely linked. The levels of porphyrin excreted by Ah/Ah mice and Ah/ah mice after TCDD treatment were indistinguishable, which contrasts with the theory of Poland and Glover (1975) that the heterozygous mice have a lower affinity TCDD receptor than homozygous mice. 505. Jones, K. G., and Sweeney, G. D. (1979) Iron deficiency prevents liver toxicity of 2,3,7,8- tetrachlorodibenzo-p-dioxin. Fed. Proc. Fed. Am. Soc. Exp. Biol. 38(3):536. [Abstract, only.] 506. Jorgenson, T. A., Rushbrook, C. J., and Newell, G. W. (1976) In vivo mutagenesis investigations of ten commercial pesticides. Toxicol. Appl. Pharmacol. 37(1):109. [Abstract, only.] 507. JRB Associates, Inc. (1980) EPA 2,4,5-T/Silvex cancellation hearing: Review of direct testimony and supporting documents on Seveso Accident. Final report of EPA contract no. 68-01-6280: Assignment 1. 60 p. [Background material.] 508. Jung, H. D., and Wolf, F. (1977) Contact eczema due to Selest 100 herbicide in forestry. Dtsch. Gesundh.-Wesen 32(31);1464-1467. [Not available.] 509. Juzwiak et al. (1973) A study of the extent of the pesticide threat to the health of agricultural workers in Olesnicki County. PTL-MS 28(ll):419-422. The authors measured concentrations of 2,4-D sodium salt in air at a pesticide manufacturing plant and during spraying of agricultural fields. Five 40-60 minute air samples were collected from various locations in a 2,4-D warehouse. Methods of sample analysis were not specified. Concentrations in air averaged 0.0012 - 0.0036 mg/1 with a range of 0.0005 - 0.0065 mg/1. The average concentrations of 2,4-D in air exceeded the maximum allowable concentration of 2,4-D for both the Soviet Union and the USA. Air samples collected during agricultural 175 �spraying also exceeded recommended standards. Concentrations of 2,4-D and MCPA measured on tractors ranged from 0.003 - 0.026 mg/1 while measurements taken 20-120 m from the field ranged from 0.0016 - 0.010 mg/1. The authors concluded that the exposure of warehouse and agricultural employees to harmful effects of this herbicide is high. 176 �510. Kalra, S. K., and Chahal, K. S. (1979) Degradation of diuron (3-(3,4,-dichlorophenyl)-l,1,-dimethylurea) in cows. Ecotox. Environ. Safety 3(4):362-368. Excretion and metabolism of diuron was studied in the cow. Four Holstein dairy cows were administered diets containing 5, 10, 25, or 50 ppm diuron for 33 days and milk, urine, and feces were collected daily for the first 5 days and weekly thereafter. Blood samples were collected 15 minutes, 1 hour, and 12 hours after the first feeding, daily for 3 more days and weekly thereafter. Diuron levels were determined in all biological fluids by a colorimetric method. Recovery of diuron was 79-89% and data was corrected accordingly. Metabolites were separated by thin layer chromatography. Toxic effects were seen after the highest 2 dosages and included pyrexia, reduced milk yield, and herauresis. For all cows, from 33-43% of the doses of diuron were recovered in urine, 6-7% in feces and none in milk. The authors reported that "The major portion of the herbicide was excreted in urine, followed by feces, and the least in blood" and presented in a table entitled "Mean Percentage Excretion of Diuron Residues and Metabolites" values of 2-5% for blood for the 4 cows, but did not explain which blood samples were analyzed for these values. Two metabolites, 3-(3,4~dichlorophenyl)-lraethylurea and 3(3,4-dichlorophenyl) urea were detected in urine and accounted for 21-23% and 6-8% of the dose. Diuron was stated to be present in smaller amounts than the metabolites, although data presented in the table did not agree with this statement. About 35% of the dose of diuron was not accounted for in the samples analyzed. The authors concluded that diuron was metabolized by 2 N-demethylation steps, analogous to reactions in higher plants, and could be degraded by gut flora. The inconsistencies in the presentation of data obscure the findings of these experiments. 511. Kanezaki, H., Sera, T., Inoue, Y., and Takahashi, T. (1973) On the health disturbance of the inhabitants around a pesticide factory in Araki area in Kerume City. (Abstract). J. Jap. Ass. Rural Med. 22(3):198-199. [Not available.] 512. Karickhoff, S.W., Brown, D.S., and Scott, T.A. (1979) Sorption of hydrophobic pollutants on natural sediments. Water Research 13:241-248. [Background material.] 513. Kaufman, D. D., and Kearney, P. C. Microbial transformations in the soil. In Herbicides: Physiology, Biochemistry, Ecology. Vol. 2. ed., L. J. Audus"i (New York: Academic Press, 1976) pp. 29-64. [Review article.] 177 �514. Kay, J. H., Palazzolo, R. J., and Calandra, J. C. (1965) Subacute dermal toxicity of 2,4-D. Arch. Environ. Health 11:648-651. The subchronic dermal toxicity of 3 commercial formulations of 2,4-D were studied in rabbits. The dimethylamine salt, isooctyl ester or butyl ester of 2,4-D (other components of the formulations were not given) was applied to shaved and abraded or intact skin and covered for 7 hours per day with plastic. At the end of each daily application the compound was washed from the skin with soap and water. The compounds were applied as 0.6 to 3.1% 2,4-D acid equivalents in water or furnace oil and were administered 3 days per week for 3 weeks. Vehicle controls received oil or water only. Body weights, incidence of mortality, blood chemistries and hematology, behavioral changes, and local skin reactions were monitored. At the end of the treatment period, the animals were killed and organs were weighed and examined grossly and microscopically. Of 112 rabbits used in the study, 12 died during the experiment, but none of the deaths were atributed to 2,4-D treatment. All rabbits that received oil, including vehicle controls, experienced pain from the applications and local severe inflammatory reactions. The reaction produced by water or aqueous 2,4-D solutions was milder. No alterations were observed in treated rabbits compared to their controls in body weights, hematologic and clinical blood chemistry results, or weights or microscopic or gross pathology of any tissue (including peripheral and central nervous system tissues) except the skin. Inflammation of the skin was similar in incidence and degree for all water-treated groups, regardless of whether 2,4-D was administered. Inflammation in the oil-treated groups was more severe in the presence of 2,4-D. The authors concluded that exposure to 2 to 5 times the anticipated amount of 2,4-D expected from home or field use produced no significant adverse effects attributed to 2,4-D except local inflammation that was produced by the vehicles, alone. 515. Kay K. (1975) Conference on toxicology- epidemiology-health effects of pesticides. Clin. Toxicol. 8(3):289-300. [Review article.] 516. Kaye, Scholer, Fireman, Hayes, and Handler (Hearing Attorneys for the • Dow Chemical Co.). In re: 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T). Dow Prehearing Memorandum No. 2. FIFRA Docket No. 295 et al. U.S. Environmental Protection Agency; Before the Administration. January 18, 1974. 25 pp. [Not available.] 178 �517. Kaye, Scholar, Fireman, Hayes, and Handler (Hearing Attorneys for .the Dow Chemical Co.). In re: 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Dow Prehearing Memorandum No. 3. FIFRA Docket No. 295 et al. U.S. Environmental Protection Agency; Before the Administration. February 22, 1974. 25 pp. [Not available.] 518. Kearney, P. C., Woolson, E. A., and Ellington, C. P., Jr. (1972) Persistence and metabolism of chlorodioxins in soils. Environ. Sci. Technol. 6(12):1017-1019. The authors studied persistence and metabolism of TCDD in two soil types, Hagerstown silty clay loam and Lakeland loamy sand. The major differences between the two soil types are that Hagerstown soil is high in organic matter and supports a high microbial population, and the Lakeland soil is low in organic matter soil and microbial activity. TCDD was added to 100 g portions of each soil at 1, 10, and 100 ppm. Both soils were analyzed at days 20 40, 80, 160, and 350 for TCDD by gas chromatography. In addition, C-TCDD was applied to soil samples at 1.78, 3.56 and 17.8 ppm for a metabolism study. Between 50-70% of the TCDD applied to soil samples persisted for 350 days. Persistence of TCDD did not seem to vary with soil type. Concentration of TCDD had no effect on persistence in Lakeland soil, but increasing TCDD concentration seemed to result in larger residues in Hagerstown soil. No TCDD metabolites were found in treated soil after 1 year. The authors concluded that TCDD is relatively persistent in soils. However, the concentrations used in this study were 10 -10 higher than what would occur in an actual field situation. Extrapolation of these data to such a situation may not be possible. 519. Kearney, P. C., Woolson, E. A., Isensee, A. R., and Helling, C. S. (1973) Tetrachlorodibenzodioxin in the environment: Sources, fate, and decontamination. Environ. Health Perspec. 5:273-277. [Review article.] 520. Kehrer, J. P., Haschek, W. M., and Witschi, H. (1979) The influence of hyperoxia on the acute toxicity of paraquat and diquat. Drug Chem. Toxicol. 2(4):397-408. The acute toxicity of diquat was determined in rats in the presence of oxygen concentrations of 20-100%. Male Fisher rats were administered a dose of 5-80 mg/kg diquat in saline intravenously and immediately transferred to chambers ventilated with 100% oxygen. Other groups of rats were administered 20 mg/kg diquat intravenously and then were exposed to chambers with 40, 60 or 80% oxygen. Mortality and LT-0 values (time until half of the group died) were recorded. Distribution of [ C]-diquat was determined in rats administered 20 mg/kg diquat intravenously by removing tissues from 1-10 hours after the injection 179 �and counting radioactivity. Lungs removed from rats administered 10 mg/kg diquat were inflated, embedded, stained and examined by light microscopy. The LTc« values for rats in 100% oxygen decreased from 3041 rain, with a 5 mg/kg dose to 87 min. for an 80 mg/kg dose. The LTcn values for a control group administered the same doses of diquat and then placed in room air was not reported. Death resulted from respiratory failure. The LTc0 values for a dose of 20 mg/kg diquat decreased from 1454 min. in 40% oxygen to 809 min. in 100% oxygen. Diquat levels in plasma, lung, liver, and kidney were the same for rats exposed to room air as for rats exposed to 100% oxygen. At each time point, the lung and plasma had the lowest diquat concentrations of the 4 tissues and the kidney had the highest concentration. Perivascular edema was present in lungs from diquat-treated rats in high oxygen, but not in vehicle controls exposed to high oxygen or in diquat-treated rats exposed to room air. The effects of paraquat were also described. The authors concluded that a toxic interaction between diquat and oxygen occurred, the mechanism of which was unclear. 521. Keil, J. E., Caldwell, S. T., and Loadholt, C. B. (1977) Pesticide usage survey of agricultural, governmental, and industrial sectors in the United States, 1974. U. S. Environmental Protection Agency Report No. 540/9-78-007. 67 pp. [Review article.] 522. Kenaga, E. E. (1975) The evaluation of the safety of 2,4,5-T to birds in areas treated for vegetation control. Residue Review 59:1-19. [Not available.] 523. Kenaga, E. E. (1974a) 2,4,5-T and derivatives: Toxicity and stability in the aquatic environment. Down to Earth 30(3):19-25. [Review article.] 524. Kenaga, E. E. (1974b) Toxicological and residue data useful in the environmental safety evaluation of dalapon. Residue Rev. 53:109-151. [Review article.] 525. Khan, S. U., (1973) Interaction of humic acid with chlorinated phenoxyacetic and benzoic acids. Environ. Letters 4(2);141-148. The authors studied the interaction of humic acid and 2,4-D or picloram, Humic acid is present in nearly all material soil and water systems. A finely ground sample of humic acid (Ig) was mixed with 200 ml of a water solution containing 500 (u) mole 2,4-D or 300 (u) moles picloram. After 3 days of mixing, the humic acid was centrifuged and 180 �the supernatant removed. This was followed by washing with 25 ml portions and one 400 ml portion of water until all the unbound herbicide was removed. The humic acid was then dried in an oven at 30°C. The amount of herbicide adsorbed was determined by subtracting the concentration of herbicide recovered in the solution from the initial concentration. Humic acid adsorbed 30 (u) moles/g 2,4-D and 18 (u) moles picloram/g. The authors concluded that these data suggest that herbicides applied to soils or water form complexes with humic substances, which would affect the activity, bioavailability, and persistence of the herbicides in soil and water. 526. 14 Khanna, S. C., and Fang, S. C. (1966) Metabolism of C-labeled 2,4-dichlorophenoxyacetic acid in rats. J. Agric. Food Chem. 14(5):500-503. The tissue distribution and excretion of 2,4-D were studied in the rat. A single dose of 1-100 mg of [ C]-2,4-D per rat was administered by stomach tube to Wistar rats (weighing 225-400 g). Radioactivity was determined in tissues and in feces, expired air and urine collected up to 144 hours after treatment. Tissue samples were also fractionated into subcellular components and the radioactivity associated,with each fraction was determined. No radioactivity was detected as 00_ or other metabolites in expired air and 93-96% of low doses (1-10 mg) of 2,4-D were excreted in the urine iri 24 hours. At higher doses, less of the dose was recovered in the urine and maximum urinary excretion occurred between 24 and 48 hours. Of 12 tissues examined for radioactivity, the half-lives were below 1 hour for a 1 mg dose for the blood, liver, kidney, heart, and spleen and 3 hours for an 80 mg dose. One metabolite of 2,4-D was separated in tissue and urine extracts by a counter-current separation technique, but was not identified. The liver contained the highest level of metabolite. From 57% (in liver) and 86% (in lung) of the tissue radioactivity was in the soluble fraction, with less in the nuclear fraction and below 10% in mitochondrial and microsomal fractions. The radioactivity in the soluble fraction was ether extractable. The authors concluded that larger doses of 2,4-D were absorbed and excreted more slowly than smaller doses and little biotransformat ion or macromolecular binding of 2,4-D occurred. 527. Khera, K. S. (1976a) Distribution, metabolism and perinatal toxicity of pesticides with reference to food safety evaluation: A review of selected literature. Advan. Mod. Toxicol. Pt. 1:369-420. [Review article.] 528. Khera, K. S. (1976b) Significance of metabolic patterns in teratogenic testing for food safety. Clin. Toxicol. 9(5):773-790. [Review article.] 181 �529. Khera, K. S. and McKinley, W. P. (1972) Pre- and postnatal studies on 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid and their derivatives in rats. Toxieol. Appl. Pharmacol. 22:14-28. The effects of 2,4,5-T, 2,4-D, and several of their derivatives on the teratology and postnatal development of the rat were studied. Pregnant Wistar rats were administered test compounds perorally in 0.5% aqueous gelatin or corn oil on days 6 to 15 of gestation. On day 22 of gestation, fetuses were weighed and examined for skeletal and visceral anomalies. Other litters were delivered and the offspring were monitored for body weight and gross defects until they reached 12 weeks of age. 2,4,5-T and its butyl ester contained less than 0.5 mg/kg TCDD. Maternal toxicity was observed only with the highest dose of 2,4,5-T, 150 mg/kg. No visceral defects were observed in fetuses of herbicidetreated groups. The four parameters measured, including number of viable fetuses, number of dead fetuses, average fetal weight, and percent skeletal malformations, were not altered by doses of 25 or 50 mg/kg of either herbicide. A statistically significant dose-related response was observed in these parameters at doses of 100 and 150 mg/kg. The moderate effect of each parameter at 100 mg/kg was statistically significant compared to controls except for the effect of 2,4,5-T on the number of viable fetuses. The highest dose, 150 mg/kg, produced maternal toxicity. The derivatives of 2,4-D tested included the isooctyl ester, the butyl ester, the butoxyethynol and dimethylaraine salts. In general these derivatives produced significant decreases in fetal weight and percentage of malformed fetuses. 2,4,5-T butyl ester caused no deleterious effects of any parameter of either 50 or 150 mg/kg doses. The most common skeletal defects observed in affected animals were wavy ribs, additional ribs, and sternal malformations. Dilated renal pelvis was observed in 7-45% of fetuses from all treated groups and 20-35% of controls. The incidence of this defect did not correlate with any dose or compound treatment. No decrease in post-natal survival was observed in offspring of 2,4-D or 2,4,5-T treated rats. When offspring of 2,4-D and 2,4,5-T groups given up to 100 mg/kg dose were mated with other rats of the same generation and treatment group, no decrease in fertility was observed from data on numbers of conceptions and viable and dead fetuses. No data or description of methods was provided for this part of the study. The authors concluded that the teratologic effects observed in fetuses was not incompatible with life, since no compromise in survival, growth, or reproductive capacity were observed in treated offspring. 530. Khera, K. S., Huston, B. L., and McKinley, W. P. (1971) Pre- and post natal studies on 2,4,5-T, 2,4-D, and derivatives in Wistar rats. Toxieol. Appl. Pharmacol. 19:369-370. [Abstract, only.] 182 �531. Khera, K. S., Whalen, C., Trivett, G,, and Angers, G, (1979) Teratogenicity studies on pesticidal formulations of dimethoate, diuron and lindane in rats. Bull. Environ. Contain. Toxicol. 22(4-5):522-529. The teratologic effects of diuron were studied in the rat. From 125 to 500 mg/kg of a commercial formulation containing 80% diuron (the identities of other ingredients were not known) were administered to pregnant Wistar rats from days 6-15 of gestation. On day 22, corpora lutea were counted and fetuses were weighed, examined for external malformations, and then stained with alizarin red and examined for skeletal malformations or fixed in Bouin's fluid and examined for visceral anomalies. The numbers of corpora lutea and live fetuses per pregnancy and the percentage of dead or resorbed fetuses were the same for the three diuron-treated groups as the values for the control group. A decrease in fetal weight of about 12% observed in the highest dosage group compared to the control group was statistically significant. A significant increase in the incidence of malformations over control levels (from 5% in controls to 10% with 250 mg diuron) was observed in the group given the middle dose of diuron but not in the group given the highest dose. The incidence of wavy ribs was also increased in the middle dose group. Maternal toxicity, measured as a decrease in maternal weight gain, was observed in the 500 mg/kg treatment group. The authors concluded that 250 mg/kg produced teratologic effects and 500 mg/kg caused maternal toxicity, while a single anomaly of delayed ossification of the calvarium produced by the lowest dose, only, may indicate that the low dose of 125 mg/kg may also be a toxic dose. This assessment of the-lowest dose seems inappropriate, since the effect was not seen at higher effects and therefore was probably a random occurrence unrelated to treatment. 532. Khera, K. S., and Whitta, L. L. (1968) Embryopathic effects of diquat and paraquat in the rat. Ind. Med. Surg. 37:553. [Abstract, only,] 533. Khubutiya, R. A., Ugulava, N. A. (1973) Cytogenetic effect of herbicides. Tr. Nauchno-Issled. Inst. Zashch. Rast. (Tislis) 25:97-99. [Not available.] 534. Kimbrough, R. D. (1980*) 2,3,7,8-tetrachlorodibenzodioxin (TCDD) Toxicity in animals, relevance to human health with notes on 2,4,5-T, picloram, cacodylic acid and 2,4-D. Presented at the 2d Continuing Education Conference on Herbicide Orange, Washington DC, May 28-30. [Review article.] 183 �535. Kimbrough, R. D. (1979) The carcinogenic and other chronic effects of persistent halogenated organic compounds. Ann. N.Y. Acad. Sci. 320:415-418. [Review article.] 536. Kimbrough, R. D. (1974) The toxicity of polychlorinated polycyclic compounds and related chemicals. CRC Critical Reviews in Toxicology 2:445-498. [Review article.] 537. Kimbrough, R. D. (1972) Toxicity of chlorinated hydrocarbons and related compounds. Arch. Environ. Health 25;125-131. [Review article.] 538. Kimbrough, R. D., Carter, C. D., Liddle, J. A., Cline, R. E., and Phillips, P. E. (1977) Epidemiology and pathology of a tetrachlorodibenzodioxin poisoning episode. Arch. Environ. Health 32(2);77-86. The health effects in animals exposed to soil sprayed with TCDDcontaminated salvage oil are described. Tissues from seven horses and four cats were examined histopathologically, and findings included vascular lesions, fibrosis of the liver, bile duct proliferation, hyperkeratosis, gastric ulcers, degenerative changes of the kidney, and edema and hemorrhage of the lungs. In addition to the presence of TCDD and trichlorophenol in the soil, reported in an earlier publication (Carter et al. 1975), polychlorinated biphenyls were found in soil sample analyses. The authors also briefly described the health effects in exposed humans, which were published elsewhere. The authors theorized that the lack of serious illness in humans, compared to the incidence in animals, reflected less absorption or lower dermal sensitivity in humans. 539. Kimmig, J., and Schulz, K. H. (1957a) Occupational acne due to chlorinated aromatic cyclic esters. Dermatologica 115:540. The authors described the clinical symptoms of workers involved in the manufacture of 2,4,5-trichlorophenol and 2,4,5-T and experiments in rabbits designed to identify the acnegenic agent in the manufacturing process. The workers were classified on the basis of the severity of skin changes as severe (9 cases), moderate (14) or mild (8). The clinical course of chloracne, which appeared in the summer and fall of 1954 in the subjects, started as facial dermatitis and developed into acneiform eruptions on the face and neck and then, in severe cases, on the trunk, arms and legs, and genitals. Other symptoms observed in the affected workers were chronic blepharo-conjunctivitis, fatigue, and weakness of the legs. Evidence of liver damage was revealed in 3 cases 184 �from clinical examination. No incidences of systemic symptoms or types of clinical tests used were presented. Skin symptoms remained 20 months after exposure to the toxic compounds was terminated. An additional instance of chlbracne in a laboratory worker involved in the animal experiments (described below) was also mentioned. One hundred compounds involved in the synthesis of 2,4,5-T were tested for acnegenie potency. Each compound was dissolved in polglycol and painted on the inside of one rabbit ear daily while the other ear served as the vehicle control. Active compounds produced inflammation in 2-4 weeks and several days later caused follicular swelling, hyperkeratoses, and cysts. Large cysts formed at later stages. Histologically, inflammatory infiltrations and edema of the cutis were observed and dilated follicles filled with keratinous material. Chlorinated dibenzofurans were potent hepatotoxins and acnegens but could not be isolated as byproducts of the manufacturing process. TCDD applied 3-4 times at 3-4 day intervals at doses of 0.005-0.01% was acnegenic and a single oral dose of 0.05-0.1 mg/kg was hepatotoxic and lethal to most rabbits in 20 days. TCDD was isolated as a byproduct of 2,4,5-T synthesis. Technical 2,4,5-trichlorophenol, but not pure trichlorophenol, was acnegenic. The authors concluded that TCDD was the acnegenic contaminant of trichlorophenol responsible for chloracne in exposed workers. 540. Kimmig, J., and Schulz, K. H. (1957b) Chlorinated aromatic cyclic ethers as the cause of so-called chloracne. Naturwissenschaften 44:337-338. Experiments that identified the acnegenic properties of TCDD are summarized. The identity of chloracne and hepatic toxicity in workers involved in the synthesis of trichlorophenol from tetrachlorobenzene led the authors to investigate the acnegenic potencies of various chemical precursors and byproducts formed in the process in the rabbit. These compounds were synthesized for the study and one laboratory technician involved in the synthesis developed severe chloracne. Compounds were applied to the rabbit ear and inflammatory changes in 2 to 4 weeks and symptoms resembling acne were recorded. (The number of rabbits tested, the method of application, or a list of test substances were not proyided.) TCDD was also administered in single oral doses of 0.05 to 0.5 mg/kg to rabbits and mortality and autopsy findings were reported. TCDD was the most potent acnegen, producing acne at concentrations of 0.002-0.01%; larger dermal doses caused hepatic necrosis and premature death. Other active acnegenic compounds were trichlorodibenzofuran and tetrachlorodibenzofuran. Inactive compounds included 2,4,5-trichlorophenol, pentachlorophenol, 2,3,4,-tetrachlorobenzene and various chlorinated diphenylethers and unsubstituted and monochlorinated dibenzofurans. Oral doses of TCDD produced death in all animals within 2 weeks. At autopsy, necrosis and fatty degeneration of the liver were observed. TCDD was successfully isolated from the byproducts of the trichlorophenol process. The authors concluded that byproducts of the manufacturing process were responsible for causing chloracne in the factory workers. 185 �541. King, C. T. G., Horigan, E. A., and Wilk, A. L. (1971) Screening of the herbicides 2,4,5-T and 2,4-D for cleft palate production. No reference: 233. [Abstract, only.] 542. King, M. E., and Roesler, A. R. (1974) Subacute intubation study on rats with the compound 2,3,7,8-tetrachlorodioxin. IIT Research Inst. Report to EPA No. IITRI-L6073-12. 60 pp. The subacute oral toxicity of TCDD in the rat is described. SpragueDawley rats (70 per group) were administered doses of 1 and 0.1 ug/kg/ week of TCDD in acetone-corn oil (1:9) by oral intubation. TCDD was administered 2 times weekly on Tuesday and Thursday for 28 weeks, followed by a 12-week recovery period. Animals were weighed weekly and rats (6 per group) were killed after 2-28 weeks and necropsies were performed. The body weights of treated animals remained below the control rats during the treatment and recovery periods; for the higher dosage group, this difference reached statistical significance. The only organ weights, expressed relative to body weights, that were statistically different from controls were increased liver weights in all groups. Hepatic changes were observed histopathologically starting at 28 weeks of treatment (no animals were killed between 16 and 28 weeks). More males than females were affected by the lower dose and by the end of the recovery period, fewer animals were affected than at the end of the treatment period or after the first 4 weeks of the recovery period. Changes included increased numbers of lipid vacuoles in centrilobular hepatocytes and electron microscopic suggestions of smooth endoplasmic reticular proliferation (which was difficult to discern because the tissue fixation techniques used were not appropriate for electron microscopy). Mild hepatocellular necrosis, multinucleated hepatocytes, enlarged hepatocytes at the peripheny of the lobule increased numbers of hemosiderin-containing macrophages no other organs were affected. The authors concluded that TCDD produced delayed, reversible hepatic toxicity in rats and the histopathologic lesion was suggestive of impairment in hepatic lipid transport or metabolism. 543. Kinoshita, F. K., and DuBois- K. P. (1970) Induction of hepatic microsomal enzymes by Herban , diuron and other substituted area herbicides. Toxicol. Appl. Pharmacol. 17(2):406-417. The effects of subacute administration of monuron and diuron on induction of several rat hepatic microsomal enzymes is described. Female 53-day-old Holtzman rats (3 per group) were administered 10-200 mg/kg/ day of diuron in carboxymethyl cellulose orally for 5 days. On the sixth day, rats were killed and whole liver homogenates were prepared and assayed for activity of the phosphorothioate detoxification (PTD) system and 0-demethylase activities. Significant increases of 144% and 192% occurred in the PTD activity and of 254% and 335% in 0-demethylase 186 �activity at the 2 highest doses of diuron, 100 and 300 mg/kg, respectively. Male and female 30-day-old Holtzman rats (3 per group) were administered diets that contained 100-2000 ppm diuron for 1-13 weeks and then were killed and liver homogenates were assayed for various enzyme activities. Significant, sustained increases occurred in PTD activity of females fed 1000 and 2000 ppm diets and in 0-methylase activity for all groups compared to the control group (whose treatment was not described). N-methylase activity was increased in all male groups and in females fed 500-2000 ppm at 1-3 weeks, but returned to control levels in all groups by week 6. Female 30- and 53-day-old rats (5-6 per group) were fed diets that contained 1000 ppm diuron for 1 week and then hepatic enzyme activities were determined. 0-methylase activity was increased 135% in the younger group of rats only, and N-demethylase and PTD activities were unaltered in either age group. The induction potencies of other substituted urea herbicides were also studied. The authors concluded that diuron produced weak enzyme induction (relative to DDT and other pesticides not investigated in this study) and this induction was only observed within the first few weeks of the feeding regimen, and sex-related differences in the inductive response were evident. 544. Kirkland, G. L., Jr. (1978) Population and community responses of small mammals to 2,4,5-T. Research Note, USDA Forestry Service, Pacific Northwest Forest Range Experimental Station Report No. PNW 314. 6 pp. [Not available.] 545. Klein, R. E., and Harrigan, E. T. (1969) Comparison of Defoliants Vol JL Directorate of Test and Evaluation, Armament Development and Test Center, Air Force Systems Command, Eglin AFB, Fla. Report No. ADTC-TR-69-30. [Background material.] 546. Klingman, D. L., Gordon, C. H., Yip, G., and Burchfield, H, R. (1966) Residues in the forage and in milk from cows grazing forage treated with esters of 2,4-D. Weeds 14(2):164-167. The levels of 2,4-D in milk from cows that grazed 2,4-D sprayed pastures and 2,4-D levels in forage are reported. One pasture was sprayed with 2 Ib per acre of 2,4-D isopropyl ester and another with 2 Ib per acre of 2.,4-D-2-ethylhexyl ester (twice the recommended agricultural rate). For each pasture, two cows were grazed for 6 days prior to spraying and for several weeks after spraying. The experimental protocol was repeated 3 years later and 1 additional cow per pasture was added 2 days after the spraying and another 4 days after the spraying. Milk collected from the exposed cows and forage samples from the fields were analyzed for 2,4-D by gas chromatography. The highest 187 �levels of 2,4-D were obtained in milk samples collected in the afternoon of the first exposure day and on the subsequent day with peak levels of 0.05-0.06 ppm 2,4-D. No 2,4-D was detected on the third day after exposure to the isopropyl ester or 14 days after exposure to the 2-ethylhexylester (no samples were collected between the 3rd and 14th day). For the second experiment conducted 3 years after the first, peak levels were 0.03 ppm 1 day after spraying and for animals from both fields 0.01 ppm 2,4-D was detected in all samples for the 7 days after spraying and in milk of cows introduced to the fields 2 days after spraying. No 2,4-D was detected in milk from cows introduced 4 days after spraying (detection unit, 0.01 ppm). Forage levels decreased rapidly, from 58 ppm of 2,4-D (acid) immediately after the butyl ester was sprayed to 5 ppm after 1 week and from 37 to 14 ppm for the 2-ethylhexyl ester in 1 week. The levels of the esters in forage were substantially below the acid levels and only the acid form was detected in milk. The authors concluded that only low levels of 2,4-D would be expected in milk of cows grazing on 2,4-D-sprayed fields. 547. Knutson, J. C., and Poland, A. (1980) 2,3,7,8-tetrachlorodibenzo-pdioxin: failure to demonstrate toxicity in twenty-three cultured cell types. Toxicol. Appl. Pharmacol. 54:377-383. The toxicity of TCDD was assessed in 23 different cell types, in vitro. Cell types that were tested included tissues and species that exhibit sensitivity to TCDD toxicity in vivo. Cells were exposed to TCDD for usually at least 1 week. -Concentrations of TCDD of 10- M were used, and in some cultures, 10- and 10M concentrations were also tested. The effect of TCDD on cell morphology, cell viability, growth rate, and aryl hydrocarbon hydroxylase activity. Fourteen of 22 cell lines tested, showed no detectable enzyme induction 3 showed 2-3 fold increases in activity, 2 showed 7-9 fold increases and 3 showed 40-650 fold increases. TCDD produced no changes in cell morphology, viability, or growth rate in any of the 23 cell lines. The authors concluded that their results were not surprising, as cytosol receptor binding was necessary, but no necessarily sufficient for TCDD, in order to elicit a toxic response. Alterations introduced by the culture conditions or missing cell-cell interactions were suggested to explain the differences of in vivo and in vitro experiments. 548. Kociba, R. J., Keeler, P. A., Park, C. N., and Gehring, P. J. (1976) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 35:553-574. The subacute, oral toxicity of TCDD in the rat is described SpragueDawley rats (12<male and 12 female per group) were administered 0.001, 0.01, 0.1 or 1.0 ug/kg of TCDD in acetone-corn by oral gavage, 5 days per week for 13 weeks. Five rats of each sex was killed in each group and the remainder were observed for a 13-week post-treatment period before they were killed. Body weights, food consumption, general appearance, hematological parameters, urinalysis and blood chemistry analyses were described for the exposure and post-exposure periods and 188 �complete pathological examinations were performed at necropsy. Treatment-related deaths occurred in only the highest dosage group; 4 females died during treatment, and 2 males and 2 females died after treatment ended. Aortic thrombosis and adrenal hemorrhage each occurred in 1 of these rats, but not in the rats in this group that were killed. Reduced body weights and food consumption occurred in the 2 highest dosage groups, and icterus and reduced activity were observed in the highest dosage group. Sex-dependent hematologic changes were observed in the highest dosage group and included decreased red blood cells, packed cell volume, and red blood cells in males. Increased urinary excretion of porphyrins and delta amino-levulinic acid and decreased creatinine excretion occurred from the highest dose. Blood chemistry changes from this dose included increased bilirubin, blood urea nitrogen, and alkaline phosphatase levels. Liver weights relative to body weights were elevated in all but the lowest dosage group and relative thymic weights were decreased from the 2 highest doses. Histopathologic changes occurred in the 2 highest dosage groups and included liver degeneration, lymphoid depletion of the thymus and morphological changes of reproductive organs suggestive of functional suppression. Hepatic levels of TCDD, measured by gas chromatographymass spectrometry, were 0.3, 0.035, and 0.003 ug per gm liver at the end of 13 week exposures to 1.0, 0.1, and 0.01 ug/kg doses, respectively. The authors concluded that no discernable ill effects occurred from doses of 0.01 ug/kg/day of TCDD administered for 13 weeks to rats and that no additional effects were likely from higher doses, beyond those already described, because steady state body burdens were probably reached by 13 weeks. 549. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Gehring, P. J. (1979a) Long-term toxicologic studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in laboratory animals. Ann. N.Y. Acad. Sci. 320:397-404. This paper summarizes the data presented in Kociba et al. (1978). new information was reported. 550. No Kociba, R. J., Keyes, D.G., Lisowe, R. W., Kalnins, R. P., Dittenber, D. D., Wade, C. E., Gorzinski, S. J., Mahle, N. H., Schwetz, B. A. (1979b) Results of a two-year chronic toxicity and oncogenic study of rats ingesting diets containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Fd. Cosmet. Toxicol. 17(3):205-221, 1979. In a two-phase study, the author investigated the long-term toxicity and oncogenicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in rats. Groups of 7- to 8-week-old Spraque-Dawley rats were fed in their diet either 99 percent pure 2,4,5-T in acetone at doses of 3, 10, or 30 mg/kg (comparable to approximately 85, 230, and 677 ppm), or .lust acetone. Test groups consisted of 60 males and 60 females each, while the control group consisted of 96 males and 96 females. No TCDD was found in the 2,4,5-T as determined by gas chromatography-mass spectrophotometry (limit of detection was 0.33 ug/kg). The primary phase of 189 �the study lasted 2 years, but 10 rats of each sex from each group were killed for a interim of the phase study at 118-119 days. This subchronic group was weighed once a week for the first 3 months from the start of the experiment; observations of general health and possible toxicological responses were made at these times. After the first 3 months body weights was recorded monthly, but other observations continued on the weekly schedule. Food consumption was recorded twice weekly. In the chronic group, 20 rats of each sex from test and control groups were observed and weighed on the same initial schedule; weekly observations continued to the 6th month. Pood consumption was measured twice weekly for the first 4 months and thereafter for 1 week each month. In both subchronic and chronic phases, routeine urinalysis (for specific gravity, pH, glucose, protein, ketones, bilirubin, and occult blood) and blood samples from the tail vein (for total erythrocyte count, total and differential leucocyte count, thrombocyte and reticulocyte count, packed cell volumes, and hemoglobin) were done for the subchronic phase on days 82 and 83, and for the chronic phase on days 89-90, 364-365, and 726-726. More in-depth urinalysis were performed on five rats of each sex from each of the subchronic groups three times before day 106 and just before the interim kill; and for the chronic phase, on four or five rats of each sex from each group after days 95-97, 188-190, 361-363, 564-566, and 698-700. This analysis measured creatinine, coproporplyrin, and uroporphyrin with or without -aminolevulinic acid. Serum samples from 10 rats of each sex per group were analyzed for urea nitrogen; glutamic-pyruvic transaminase (S6PT) and alkaline phosphatase activities (SAP); total, direct, and indirect bilirubin; and total protein, albumin, and globulin. Eyes were examined both by glass slides placed against the cornea under fluorescent light, and by preservation of eyes from five rats of each sex and group in Zenker's fixative. In addition, a complete gross pathological examination was made of representative sections of all major organs and tissues from all animals. Any grossly observed lesions or suggestion of tumor formation were preserved, and weights were recorded for liver, kidneys; brain, heart, thymus, spleen, and testes or ovaries/uterus. Portions of fat, liver, and kidney were saved from five rats of each sex and group for possible 2,4,5-T analysis. For the chronic phase, representative parts of the skull (including nasal turbinates and ear canal) were also examined. Peripheral blood and femoral bone marrow smears were also made for this group. Sections of tissues embedded in paraffin were stained with hemaboxylin and eosin and examined histologically. Tissues from lowdose rats in the subchronic phase were not examined, as there was no evidence of treatment-related effects in the high-dose group. For this subchronic phase, there were no toxicologically significant deviations from controls for body weights, food consumption, mortality, palpable masses and general toxicological analyses, hematological, and urinary parameters, BON, SGPT, SAP, bilirubin, protein, albumin, or globulin. There was a statistically signficant increase in relative kidney weights for high-dose males. For the chronic phase, no toxicogically significant changes were found in body weights, food consumption, mortality in the lower dose groups, palpable masses and toxicological observations, hematological parameters, BUN, SGPT, SAP, bilirubin, protein, albumin, or globulin. In the high-dose group, males showed a 190 �decrease in mortality in the latter part of the study, and median-dose females showed increases in the incidence of minimal degenrative inflammatory changes including focal renal tubular atrophy and renal aggregation of lymphoid cells; these changes were considered related to less severe chronic renal disease in these rats. These rats also showed an increase in total urine volume and in the excretion of coproporphyin and uroporphyrin. Males receiveing 10 mg/kg/day showed an increase in coproporphyrin excretion in the early part of the study. In the high-dose females coproporphyrin was shown to be increased in most samples; uroporphyrin decreased after 119 days but increased again after 566 days. In the lowest dose males and in the 3- and lOmg/gk females no significant differences were noted. There was a decrease in absolute heart weights of high-dose males, but this was of questionable significance due to an absence of any gross of histopathological changes. High and median-dose females showed both gross and microscopic evidence of increased mineralized deposits in the renal papillae or pelvis, on occasion accompanied by a localized reaction of the adjacent renal-pelvis epithelium. Females also showed evidenced of increased pigment content in the cytoplasm of the proximal convoluted tubular epithelial cells. Males particularly showed a decrease in the severity of spontaneous chronic nephropathy and a secondary decrease in mineralization of pulmonary alveoli, myocardium, myocardial blood vesels, and gastric mucosa and muscularis. The increased renal pigment noted gave a higher positive reaction with Malloy's stain for iron. Lung changes evidenced in high-dose males may or may not have been treatment-related. There was some increase in focal pulmonary interstitial inflammation, focal accumulations of alveolar microphages and cholesterol clefts; females in this group showed focal accumulation of secreted material in the alveoli. Cardiovascular changes occurring in high-dose females included dilated, flaccid ventricle; the opposite change occurred in males of this group. A variety of tumor types occurred in both control and test rats at an incidence level not considered toxicologically significant. Total numbers of tumors per group, average number per rats, and latency, were all comparable to control rats. With one exception, the incidence of individual tumor types was comparable between treated and control groups; however, the incidence of interfollicular C-cell adenoma was higher in females given the 3 mg/kg/day done (10 tumors vs. none in controls). This increase was not believed to be treatment related since the incidence of C-cell adenoma in control rats was swollen than what would have been historically expected in this strain of rat (up to 17%). 551. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., et. al. (1978) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46:279-303. The authors studied the effects of TCDD in a 2-year feeding study in rats. Groups of 6-7 week old male and female Sprague-Dawley Spartan substrain rats (50 males and 50 females per group) were fed 0, 0.1, 0.01, or 0.001 ug TCDD/kg/day. TCDD (99 percent purity) in acetone was added to standard lab feed. A negative control group (86 males, 86 191 �females) received standard diet. New stocks of feed with TCDD were prepared 6 times during the study. Periodically, feed samples were taken to analytically verify the TCDD content of the food. All rats were palpated and weighed on a monthly basis. Blood and urine samples were collected from 7-8 rats/sex/group at 3, 12, and 23 months. Urinary excretion of creatine, coproporphyrin, uroporphyrin and S-amino-levulinic acid were measured in 4-5 rats/sex/group at 3-4, 12, and 23 months. Serum samples were analyzed from 7 rats/sex/group for urea nitrogen, glutamic pyruvic transaminase, bilirubin, cholesterol, and triglycerides. Gross pathological examinations were conducted on all rats. Weight of major organs were measured and organ-to-body weight ratios calculated. Histological examinations were conducted on tissues from all rats in the control and high dose groups and select tissues from the other groups. Liver tissue was examined by electronmicroscopy. Statistical analyses of the data included analysis of variance, Fischer exact test, and the Mantel-Haenszel test. Multiple toxicological effects were observed in animals ingesting diets containing 2200 ppt TCDD (0.1 ug TCDD/kg/day). These included increased mortality, decreased body weight gain, slight depression of some hematologic parameters, increased urinary excretion of porphyrins, and S-amino levulinic acid, increased serum alkaline phosphase, glutamic pyruvic transaminase, and gamma-glutamyl transferase, and morphological changes in hepatic lymphoid, respiratory, and vascular tissues. A statistically significant (Fisher's exact test) increase in the incidence of hepatocellular carcinomas was observed only in females at this dose. Squamous cell carcinomas of the lung, hard palate/nasal turbinate, or tongue were also observed and were statistically significant (Fisher's exact test). Ingestion of 210 ppt TCDD in the diet (0.01 ug/kg/day) induced less toxicity than the higher dose. Observed toxicological effects included increased urinary excretion of porphyrins in females, liver toxicity, increased hepatocellular nodules and increased focal hyperplasia in the lungs. At the lowest dose tested, 22 ppt TCDD in the diet (0.001 ug/kg/day), no effects related to TCDD administration were observed. 552. Kohli, J. D., Khanna, R. N., Gupta, B. N., Dhar, M. M., Tandon, J. S., and Sircar, K. P. (1974a) Absorption and excretion of 2,4-dichlorophenoxyacetic acid in man. Xenobiotica 4(2);97-100. Plasma and urinary levels of 2,4-D and metabolites were analyzed in human subjects administered 2,4-D orally. Six 22-30 year old male volunteers were administered 5 mg/kg 2,4-D (99% purity) in a gelatin capsule with water. Blood and urine samples collected over the subsequent 7 days were analyzed by gas chromatography for 2,4-D and its metabolites. No adverse effects were reported by any of the subjects or detected by clinical laboratory tests. A mean of over 20 ug/ml of 2,4-D was detected in plasma 1 hour after administration; peak levels were reached in 7-24 hours. Within 96 hours, 75% of the dose had been excreted and no metabolites were detected in any samples. From kinetic analysis of the plasma data using a model with first order rates of absorption and clearance, constants were determined for the computergenerated curve that best fit the data. Rates for absorption and 192 �clearance were 27 and life was 33 hours and The authors concluded rapidly by the kidney 553. -2 2 x 10 per hour, respectively; the plasma'halfthe volume of distribution was 10 x 10 I/kg. that 2,4-D was absorbed rapidly and excreted and was not biotransformed. Kohli, J. D., Khanna, R. N., Gupta, B. N., Dhar, M. M., Tandon, J. S., and Sircar, K. P. (1974b) Absorption and excretion of 2,4,5-trichlorophenoxy acetic acid in man. Arch. Int. Pharmacodyn. 210:250-255. The kinetics of absorption and excretion of 2,4,5-T by humans was studied. Eight male volunteers ingested from 2 to 5 mg/kg 2,4,5-T. No clinical symptoms of toxicity were detected by the subjects. Urine samples collected over the subsequent 4 days and blood samples collected over the subsequent 7 days were analyzed for 2,4,5-T. The maximum blood levels were reached 7-24 hours after ingestion, although after 1 hour 2,4,5-T was detected in the blood. After 96 hours, urinary 2,4,5-T accounted for 63% of the 5 mg/kg dose, 79% of the 3 mg/kg dose, and 73% of the 2 mg/kg dose. The half-life of plasma clearance, calculated assuming that the rates of absorption and excretion followed first order kinetics, was reported as 18.8 hours and the volume of distribution was calculated as 15.8 liters/kg. Based on kinetic data presented in the present paper and by other investigators, the author predicted that tissue accumulation would be unlikely after repeated exposure of 2,4,5-T, 554. Kolberg, J., Helgoland, K., and Jonsen, J. (1972) The herbicide 2,4-D. II. Triglyceride accumulation in L cells. Acta. Pharmacol. Toxicol. 31(5-7):481-487. Endogenous biosynthesis of fatty acids and uptake of fatty acids were evaluated in cultured fibroblasts exposed to 2,4-D. Mouse L929 fibroblasts were cultured in the presence of 500 ug 2,4-D (99+% purity) per ml culture medium for 24 hr., Fibroblasts were cultured in the presence of [ H]-palmitic acid and [ C]-acetate (together) for 24 hours. Lipids were extracted from whole cells and cell homogenates after lipid particles were separated by centrifugation. Lipid extracts were analyzed by thin-layer chromatography and triglycerides, cholesterol, and phosphorous by chemical procedures. The amount of lipid particles was maximum after 24 hr. of 2,4-D treatment, increasing to two times the pretreatment amount represented. Most of the additional lipid was triglycerides. Incorporation of palmitate and of acetate into lipid fraction increased threefold. Incorporation of only acetate into nonlipid fractions.occurred. Both precursors were incorporated into triglyceride and C was,incorporated into cholesterol, as well. The specific activity of H in triglycerides did not change and decreased for C in triglycerides and for both isotopes in phospholipids. The authors concluded that increased incorporation of both fatty acid precursors may have reflected an effect of 2,4-D on plasma membrane permeability. 193 �555. Kolberg, J., Helgoland, K. , Jonsen, J., and Tjeltveit, 0. (1971) The herbicide 2,4-dichlorophenoxyacetic acid. I: Effects on L cells. Act a. Pharmacol. Toxicol. 29:81-86. The effects of 2,4-D on cell growth of L929 cells were studied in vitro. Strain L929 mouse fibroblasts were grown in monolayer cultures for 24 hours and then the medium was replaced with medium containing from 50 to 500 ug/ml 2,4-D (as the sodium and potassuim salts). Culture media were renewed every 3 days and for some experiments cell layers were washed several times and control medium was used to replace 2,4-D treatment media. Cells were trypsinized and counted or analyzed for lipids cytochemically. 2,4-D produced a dose-related inhibition of cell growth without causing cytopathogenic changes. The effect on cell growth was reversible when 2,4-D-containing medium was replaced with fresh medium on day 3 or 12 of culture. Uniformly-sized lipid-staining vacuoles appeared in the cytoplasm after 3 hours of exposure to 350-500 ug/ml 2,4-D; increased in number during the next 21 hours; decreased subsequently and disappeared by day 4-5 of culture. Lower doses caused fewer or no vacuoles. The formation of vacuoles and absence of cell division by 2,4-D treatment was observed with time-lapse cinematography. The authors suggested that the presence of lipid vacuoles was not necessarily a sign of degeneration and did not attempt to relate their observations to 2,4-D toxicity in vivo. 556. Kolmodin-Hedman, B., and Erne, K. (1980) Estimation of occupational exposure to phenoxy acids (2,4-D and 2,4,5-T). Arch. Toxicol. Suppl. 4:318-321. Concentrations of 2,4-D and 2,4,5-T in air from the breathing zone of workers applying these herbicides and urine and plasma levels for workers after exposure are presented. Four workers engaged in spraying a 27, emulsion of the 2 phenoxy acids in kerosene made up the study group. All workers were male, all were smokers, previous exposure to herbicides varied from 2-14 years for the group, and the mean age was 39 years. Two additional staff members served as the control group and had low, indirect exposure to the herbicides. No other information on the exposure levels or on other characteristics of this group were given. Air was sampled from the workers' breathing zone for 60 minute periods. The effective spraying time was 2-4 hours/day and urine and blood samples were collected before the start and at the end of the first work day of the week, at the end of a week of exposure and up to 36 hours after the exposure week ended. Levels of phenoxy acids in body fluids were determined by gas chromatography and in air by UV-spectrophotometry and thin-layer chromatography. The lowest level of detection was 0.05 ug/ml in urine and 0.02 ug/ml in plasma. Levels of phenoxy acids were 0.1-0.2 mg/m in air samples. Plasma levels were all below 0.2 ug/ml. Plasma levels were variable, did not show a rising trend during the exposure week and fell to the limit of detection 1 day after exposure ended. The mean peak urine levels were 10 ug/ml of 2,4-D and 3.5 ug/ml of 2,4,5-T and occurred in the afternoon of the day of exposure. Subsequent urine levels were substantially lower in 3 of 4 cases. No phenoxy acids were detected in urine from 194 �the controls. The only symptom reported was slight eye irritation by 1 man from direct contact with the liquid. The authors concluded that the extent of occupational exposure to these phenoxy acids would be monitored most accurately from urine samples collected on the afternoon of the exposure day. 557. Kolmodin-Hedman, B., Erne, K., Hakansson, M., Engqvist, A. (1979) Occupational exposure to phenoxy acids (2,4-D and 2,4,5-T). Arbete. och. Halsa ( ) 2. [Abstract, only.] 558. Konstantinova, T. K. (1974) Experiments on the effects of 2,4,5-T butyl ester on pregnant animals and on the development of their offspring. Gig. Sanit. 39(8):101-102. The teratogenic effects of 2,4,5-T to offspring following maternal treatment and maternal toxicity are described. Albino rats (12-13 per group) were administered doses of 4.2, 0.42, 0.1 or 0.01 mg/kg 2,4,5-T butyl ester (purity and dioxin level not reported). The herbicide was administered orally, in water, throughout gestation. Maternal body weights, peripheral blood composition, histopathology, and central nervous depression (measured as a summation threshold index; no other explanation of the parameter was provided), and organ weights were determined. Fetal mortality, body weights, visceral malformations (determined by a microanatomical technique that was not described further) and skeletal malformation, visualized by alizarin stain, were determined. All doses, except the lowest dose, produced nervous system inhibition, and increased organ weight coefficients (calculations used to obtain this coefficient were not described); the organs with increased coefficients varied at different dose levels and included the liver at 4,2 and 0.1 mg/kg, brain and kidney at 4.2 and 0.42 mg/kg, the lung at 0.42 mg/kg and the heart at 0.1 mg/kg. The highest dose also produced hemodynamic disorders and dystrophic changes in maternal organs. Fetal changes from the highest dose included high fetal death, reduced body weights, malformations (hydrocephaly in 6 percent of fetuses, liver abnormalities in 19 percent and abdominal hemorrhage in 11 percent), plethora of internal organs and granular dystrophy in the liver and kidneys. At 0.42 mg/kg, 2,4,5-T produced hydrocephaly (4.2 percent), increased limb bone length and plethora and granular dystrophy. At 0.1 mg/kg increased fetal weight and limb bones and decreased litter sizes were observed and at 0.01 mg/kg, only increased weight and bone length occurred. The author concluded that 0.01 mg/kg was the threshold dosage for the toxic maternal and fetal effects of 2,4,5-T. The author's failure to analyze the dioxin content of 2,4,5-T or to describe methods or results adequately to compare them to other studies, the lack of dose-related effects or effects seen in rats by other investigators renders this paper useless for evaluating teratology of 2,4,5-T. 195 �559. Konstantinova, T. K. (1967) Toxicology and some problems of the embryotropic action of the butyl ester of 2,4-D. In Proceedings of a Conference on Problems on Hygiene and Toxicology of Pesticides, Bygodchikov, ed., Moscow Meditsina. [Not available.] 560. Ronscantinova, T. K., Efimenko, L. P., Antonenko, T. H., Nechkina, M. A., and Shilov, V. N. (1978) Data for the hygienic standardization of 2,4-D herbicides in environmental substances. Gigiena i sanitariya. 9-13. Reproductive effects of 2,4-D, 2,4-D sodium and amine salts, and 2,4-D butyl ester were studied in the rat. Groups of rats were administered 0.5-1.0 mg/kg 2,4-D, 2,4-D sodium or amine salt or 2,4-D butyl ester orally and the embryotropic, cytogenetic, and gonadotropic effects were studied. The explanations of the methods used to measure these parameters were given. Parameters were reported in a table as being altered or not altered by a dose of 0.5-1.0 mg/kg 2,4-D or by a dose between 0.1 to 0.01 mg/kg. The results were undecipherable. For example, under the heading "embryotropic effect" in the table of results were four subheadings, "mother," "fetus," "permeability of the placenta," and "progeny," which may have referred to toxicity in the mother, which is not an embryotropic effect. Under "cytogenetic effects in males" were subheadings of "one-time," "5 days," "45 days," "5-6 mo." but these time intervals were not explained and no alterations were noted from 2,4-D treatment (the only compound tested for these effects). All of the compounds studied produced an embryotropic effect in the fetus but not in the progeny and only 2,4-D amine salt and 2,4-D butyl ester caused an embryotropic effect in the mother,'2,4-D crossed the placenta and transport was not determined for other compounds. The authors concluded that regulation of occupational exposure to 2,4-D should be based on the levels that produce embryotropic effects. 561. Kontek, M,, Jasinski, K., Marcinkowska, B., Tokarz, F., Pietraszek, Z., and Handschuh, R. (1973) Electroencephalographic study of farm workers exposed to derivatives of arylalcanocarboxylic acids. Polski Tygodnik Lekarski. 28(25):937-939. The authors conducted electroncephalographic (EEC) examinations on 17 farmworkers, aged 19 to 42 years, who sprayed fields with various herbicides (including 2,4-D). EEC's were conducted on these individuals before and after the spraying season. Time intervals between the EEC examinations ranged from 4 to 28 days. A group of 41 farmworkers not involved with herbicide spraying was used as controls. Of the 17 sprayers examined prior to spraying activities, 7 exhibited slightly perturbed EEC activities in the form of irregular alpha waves and numerous, slow theta waves originating from all areas of both cerebral hemespheres. Seven individuals from the control group also exhibited irregular EEC patterns, but not to the same extent as the test group. EEC readings obtained from the 17 sprayers after the spraying season 196 �had ended revealed that 5 of them exhibited distinct abnormalities, in alpha wave frequencies and theta wave amphitudes. Four of these five were the same persons that displayed irregularities in their EEC's taken prior to the spraying season. The irregularities had apparently intensified in these individuals. An additional 8 persons from the study group also exhibited some irregular bioelectric activity following the period of spraying. Additional laboratory tests such as blood morphology, urinalysis and enzyme reactivity were conducted and no abnormalities in these parameters were evident. The authors concluded that EEC patterns were effected in persons that contacted arylalcanocarboxylic acid herbicides, concluding 2,4-D. The authors briefly correlated their findings with results of animal studies that demonstrated abnormal cerebral electrical activity caused by administration of 2,4-D. 562. Kopaczyk-Locke, K. (1973) Effects of paraquat and diquat on rat liver mitochondria. Fed. Proc. Fed. Am. Soc. Exp. Biol. 32(3):250. [Abstract, only.] 563. Koschier, F. J., and Acara, M. (1979) Transport of 2,4,5-trichlorophenoxyacetate in the isolated, perfused rat kidney. Pharmacol. Exp. Ther. 208:287-293. J_-_ Characteristics of 2,4,5-T excretion by perfused rat kidneys are studied. Urine was collected from a urethral catheter of anesthetized male Sprague-Dawley rats and perfusate fluid which contained various test compounds was circulated through the kidneys in situ. The levels of radioactivity in the urine and blood were analyzed after radiolabeled compounds were introduced to determine the rate of renal clearance for each compound. The ratio of 2,4,5-T clearance to inulin clearance was below 1, with 99.5% of the 2,4,5-T in the perfusate bound to bovine serum albumin. This ratio increased to 6 after dextran, which bound less than 30% of the perfusate 2,4,5-T, was used instead of albumin. The presence of probenecid and of para-aminohippurate each decreased the clearance ratio and increased tubular reabsorption. The kinetics for clearance of 2,4,5-T from perfusate fluid involved 2 phases and the half lives and rate constants were calculated for perfusate fluids which contained albumin and dextran, and inhibitors. At 1 mM, 2,4,5-T inhibited the clearances of PAH and tetraethylammonium and an initial diuresis, followed by a progressive fall in glomerular filtration rate. This concentration of 2,4,5-T did not alter oxygen consumption, Na,k-ATPase, Mg-ATPase, or water distribution studied in renal slices but did alter electrolyte balance. The authors concluded that proximal tubular secretion of 2,4,5-T is limited by plasma protein binding of 2,4,5-T; 2,4,5-T was also reabsorbed to some extent and caused nephrotoxicity. 1Q7 �564. Koschier, F. J., and Berndt, W. 0. (1977) Relationship between 2,4,5-trichlorophenoxyacetate and the renal organic base transport system. Biochem. Pharmacol. 26:1709-1713. The interaction of 2,4,5-T with the renal transport of an organic cation, tetraethylammonium (TEA) was studied in rat renal cortical slices. Kidneys,were removed from male Sprague-Dawley rats, sliced and incubated with C-labeled organic ions including 2,4,5-T, TEA, N'-methyl-nicotinamide (NMN) and para-arainohippurate (PAH). Radioactivity in the tissue homogenates and.media were determined as a measure of tissue uptake. Efflux of C-organic ions preloaded in tissue slices was determined by transferring the tissue through a series of seven sequential one minute baths and measuring the radioactivity in each wash chamber. Oxygen consumption by renal slices was measured by means of an oxygen electrode. At 10 M, 2,4,5-T did not alter oxygen consumption by renal slices in the presence or absence of lactate. The rate constants for TEA efflux were calculated from the loss of radioactivity from kidney slices preloaded with C-TEA and transferred through seven chambers of control medium and then through eight chambers with 2,4,5-T present in the medium. 2,4,5-T increased TEA efflux but not NMN efflux, which was studied in the same way. Cyanine, a competitive inhibitor of the base transport system, phenoxybenzamine, an irreversible inhibitor of this system and mepiperphenidol, a competitive inhibitor of the cation system, all failed to alter 2,4,5-T transport. The authors concluded that 2,4,5-T interacts with specific intracellular TEA binding sites to block TEA accumulation, rather than-with a carrier-mediated aspect of base transport and proposed a two step kinetic model to explain this conclusion further. The high degree of 2,4,5-T renal tissue binding was considered to be related to its interference with TEA transport. 565. Koschier, F. J., and Berndt, W. 0. (1976a) Specificity of 2,4,5-trichlorophenoxyacetate on tetraethylammonium transport. Toxicol. Appl. Pharmacol 38:297-306. The effect of 2,4,5-T or the uptake of organic anions and cations by rat kidney slices was evaluated. Male Sprague-Dawley rats were administered 90 mg/kg of 2,4,5-T (with less than 0.05 ppm TCDD contaminant) subcutaneously in 70% ethanol, 24 hours before the kidneys were removed. Control rats received ethanol only. Renal cortical slices were incubated in modified Krebs-Ringer phosphate,buffer which contained the organic cation or anion labeled with [ C], After a 5 min. incubation, to study initial uptake, or a 60 min. incubation, isotope in the media and in the tissue was counted by liquid scintillation methods. Compounds were also added to the incubation media to assess their effect on the radioactive anions or cations. Data were usually presented as double reciprocal plots. Uptake of 2,4-D was inhibited in 2,4,5-T pretreated rat kidneys and showed competitive Michaelio-Menten kinetics. By increasing substrate concentration from 0.4 to 22 ug/ml, inhibition by 2,4,5-T fell from 28 to 2.5%. Unlabeled 2,4,5-T competed for TEA steady-state uptake by 2,4,5-T- pretreated kidney slices, but did not compete for the initial influx of TEA. TEA only minimally 198 �inhibited 2,4,5-T uptake and hexamethonium had no effect on 2,4,5-T uptake. The presence of 2,4,5-T in the medium inhibited N'-methylnicotinamide (NMN) uptake minimally (by 6%). The presence of 10 M acetate in the medium augmented the effect of 2,4,5-T on NMN uptake. The authors concluded that their results were consistent with a twocompartment model system, in which the initial step of transmembrane influx of TEA is not susceptible to 2,4,5-T competition while the second step of accumulation of TEA in the proximal tubular cell is where 2,4,5-T competes. 2,4-D uptake also showed a biphasic double reciprocal plot, consistent with a two-compartment model. 566. Koschier, F. J., and Berndt, W. 0. (1976b) The nature of the inhibition of renal transport caused by the acute administration of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Tox. Appl. Pharmacol. 37:169. [Abstract, only.] 567. Koschier, F. J., and Berndt, W. 0. (1976c) In vitro uptake of organic ions by renal cortical tissue of rats treated acutely with 2,4,5trichlorophenoxyacetic acid. Toxicol. Appl. Pharmacol. 35:355-364. The renal excretion of 2,4,5-T and the effect of 2,4,5-T pretreatment on transport of organic ions were studied in the rat. Male SpragueDawley rats were administered a dose of 45-135 mg/kg 2,4,5-T in 70% ethanol, subcutaneously. After 24 hours, the kidneys were removed, sliced and incubated with a radioactively labeled organic ion for 3 hours. Radioactivity of the/tissue and media were then counted. In some rats, daily doses of [ C]-2,4,5-T were administered and radioactivity excreted daily in the urine was determined. The daily rate of urinary excretion was 80% of the administered dose (20 mg/kg) for the first 6 days and 100% of the dose on the seventh day. By day 18, only 1.2% of the total daily dose remained in the rat. After 6 daily doses of 90 mg/kg, 35% remained in the rat, but by day 17, only 2.4% of the daily dose remained. Weight loss, seen during the first week, was reversed during the subsequent days. One day after an acute dose of 2,4,5-T, the renal concentration and urinary excretion of 2,4,5-T were determined to be dependent on the amount administered, with a lower proportion excreted and higher tissue levels after large doses. After 2,4,5-T pretreatment (45-90 mg/kg) uptake of 2,4-D, of 2,4,5-T and tetraethylammonium chloride were inhibited, but uptake of paraaminohippurate and alpha-aminoisobutyric acid were not altered. PAH (1200 rag/kg) pretreatment did not affect in vitro renal transport of any of the organic ions. At higher pretreatment doses of 2,4,5-T and PAH, sporadic alterations in organic ion transport were observed. The authors concluded that high doses of 2,4,5-T may prolong the half-life of the herbicide by blocking its renal uptake and the almost complete excretion of daily doses of 2,4,5-T explains its low chronic toxicity. 199 �568. Koschier, I. J., Girard, P. R., and Hong, S. K. (1978) Transport of 2,4-dichlorophenoxyacetate by rat renal cortical slices. Toxicol. Appl. Pharmacol. 45:883-894. The kinetic characteristics of the efflux of 2,4-D from rat renal slices was determined in the presence of various metabolic inhibitors. Renal slices were prepared from kidneys of male,Sprague-Dawley rats and were preloaded by incubation with 7.65 ug of [ C]-2,4-D in 3 ml buffer for 1 hour at 25°C. The slices were then transferred through a series of 18 sequential 1 minute washes and the radioactivity transferred into each wash solution and the amount that remained in the tissue was counted. The rate constant for efflux was then calculated. Tissue extracts were analyzed by paper electrophoresis for radioactive metabolites of 2,4-D. The kinetics of efflux was comprised of a fast and a slow component. Compounds that significantly increased the slow phase when added to the efflux medium were probenecid, dinitrophenol, stilbene derivative, iodoacetamide, succinate; succinate and lactate both increased the rate for the fast phase. The rate of 2,4-D efflux was temperature-dependent, with faster rates for 15°C and 35°C than at 25°C. Efflux was significantly increased (slow phase only) in potassium-free buffer, but was unaltered in calcium-free buffer. Uptake of 2,4-D was linear for 7 hours and transported 2 times more 2,4-D into the kidney than was transported out by the efflux mechanism. At 20 uM, 2,4-D produced a 50% inhibition of para-aminohippurate (PAH) uptake by renal slices. No metabolites of 2,4-D were detected in renal tissue extracts. The authors compared the renal transport of 2,4-D with that of PAH.. They concluded that the in vitro influx of 2,4-D was rapid and 2,4-D was a potent inhibitor of PAH transport; although in vivo renal secretion of PAH was more rapid than for 2,4-D, other factors were proposed that could mediate this effect. 569. Koschier, F. J., Hong, S. K., and Berndt, W. 0. (1979) Serum protein and renal tissue binding of 2,4,5-trichlorophenoxyacetic acid. Toxicol. Appl. Pharmacol. 49:237-244. Binding of 2,4-D and 2,4,5-T to bovine serum albumin (BSA) was assessed as well as the capacity of 2,4,5-T to bind several sites.of rabbit and rat kidney. BSA and between 10-5 to 10-6M C-2.4-D or C-2,4,5-T were incubated at 25°C for 120 minutes and then centrifuged in an ultrafilter cone membrane. The radioactivity in the filtrate and retentate were determined to calculate the proportion of bound radioactivity. At 0.5 mg/ml BSA, 20% of 2,4-D and 40% of 2,4,5-T were bound and at 5.0 and 50 mg/ml BSA, over 80% of each herbicide was bound to BSA. When rat kidney cortex homogenates were used instead of BSA, about 30% of the 2,4,5-T and 25% of 2,4-D were bound to the tissue. Binding of 2,4,5-T to microsomes of rabbit renal cortex was complete in 15 seconds, and was high, with a binding capacity of 17.5 umol/g protein and an association constant of 2.4 x 10 M . About 25% of 2,4-D and 2,4,5-T bound to rat and rabbit cytosol fractions of the renal cortex. These values were substantially higher than binding of the other organic ions studied. Two 2,4,5-T binding sites were identified in the 200 �4 -1 rabbit cytosol fraction, with association constants of 1.5 x 10 M and 0.04 x 10 M and corresponding binding capacities of 4.5 and 630 umol/g protein, respectively. 2,4,5-T inhibited tetraethylammonium binding to the cortex cytosol but not N -methylnicotinamide binding. The authors concluded that the ability of 2,4,5-T to bind to tissue components at least partially accounts for its renal accumulation after acute and chronic administration, and affects its nephrotoxicity by concentrating 2,4,5-T in proximal tubular cells or protecting the cell by lowering the free 2,4,5-T concentration within the cell. 570. Kouri, R. E., Ratrie, H., Atlas, S. A., Niwa, A., Nebert, D. W. (1975) Aryl hydrocarbon hydroxylase induction in human lymphocyte cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Life Sciences 15(9):1585-1595. The induction of aryl hydrocarbon hydroxylase (AHH) activity by TCDD was determined in cultured human lymphocytes. Lymphocytes were isolated from 19 healthy volunteers and cultured in the presence of mitogens. TCDD, added to the culture medium at a concentration of 33 ng per ml, produced a 1.7-2.4 fold increase in AHH activities of cells from 6 individuals. At 10 ng/ml, TCDD induced AHH activity from 1.7-2.7 times in cultures of cells from 13 individuals. Maximum induction, with a 2.9 fold increase in enzyme activity, was produced in 1 culture by TCDD at 100 ng/ml. Induction of AHH activity by 3-methylcholanthrene was maximal at 1,5 uM, compared with a maximally effective dose of TCDD of 30 nM, indicating that TCDD is about 50 times more potent than 3-MC in this culture system. Individuals whose cells showed low basal enzyme activity also showed the lowest increases in activity in the presence of inducers. The authors discuss the implications of their results as they relate to long-term risk potential of TCDD as a synergistic agent with other chemical carcinogens and they draw inferences related to the genetic mode of regulation of AHH induction, suggesting that several non-linked genetic loci may be involved. 571. Kouri, R. E., Rude, T. H., Joglekar, R., Dansette, P. M., Jerina, D. M., Atlas, S. A., Owens, I. S., and Nebert, D. W. (1978) 2,3,7,8Tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-methylcholanthrene-initiated subcutaneous tumors in mice genetically "nonresponsive" at Ah locus. Can. Res. 38(9):2777-2783. % The authors studied the effects of intraperitoneal and subcutaneous injections of TCDD on the induction of hepatic, skin, and subcutaneous tissue aryl hydrocarbon hydroxylase (AHH). They also studied the related effects of intraperitoneal and subcutaneous pretreatment with TCDD on the tumorigenesis of 3-methylcholanthrene (3-mc). Two strains of mice were tested: C57BL/6Cum(B6), which is genetically susceptible to 3-mc-induced tumors; and DBA/2Cum(D2), which is a resistant strain. Treatment began at 4 to 6 weeks of age. Mice received a single intraperitoneal dose of TCDD in p-dioxane, varying from .05 to 100 ug/kg. Three days after administration, hepatic AHH was measured. The enzyme 201 �assay was not described in this report. In the B6 strain, 1 ug TCDD/kg induced hepatic AHH to 70 percent of the maximum inducible level; in D2 no increase was noted. At levels of 60-100 ug TCDD/kg, AHH was induced in D2 to 80 percent of that in B6. Mice received TCDD subcutaneously in a single dose ofo either 1 or 100 ug/kg, and skin microsomal fractions were tested daily for AHH induction. Skin and subcutaneous tissue AHH was induced in B6 by the high dose at only a slightly higher level and more rapidly than in D2. At the low dose, AHH was induced in B6 at a. much higher rate than in D2. Skin and subcutaneous tissue AHH was increased more than 20- to 50- fold in both strains by subcutanebus administration of TCDD. Hepatic AHH was increased more than 8- to 10-fold in both strains by intraperitoneal administration. Skin and subcutaneous tissue AHH was not measured after intrapertoneal treatment. Other hepatic enzymes were also monitored after TCDD treatment, specifically epoxide hydrase, GSH S-transferase, and glucoronosyltransferase, but only AHH activity was found to increase at a significant level following TCDD treatment. Tumorigenesis was studied by pretreating mice intraperitoneally or subcutaneously with either 1 or 100 ug TCDD in p-dioxane per kg; pretreatment was done either 48 hours prior to or at the same time that application of 3-tnc was made. Mice were treated with 3-mc in 0.5 ml trioctanoin in subcutaneous doses of 150 mg. They were checked weekly by palpation for tumors. When a subcutaneous tumor grew to 1.0 cm in diameter, latency was calculated. After the eight month observation time, the carcinogenic index (CI) was calculated as the percentage of tumor incidence divided by the average latency in days, multiplied by 100. Several control groups were also used and consisted of mice treated with either 150 mg 3-MC, 0.05 ml trioctanoin, or TCDD (1 mg or lOOmg). No tumors developed from treatment with the trioctanoin vehicle nor from the maximum dose of TCDD. When either dose of TCDD was given i.p. 2 days before 3-mc initiation, neither D2 nor B6 mice showed any significant change in the level of sensitivity to 3-mc. When TCDD was given i.p. at the same time as 3-mc, B6 mice did not show any change at either dosage. D2 mice showed a significant increase in sensitivity to 3-mc only when both doses of TCDD were given s.c. simultaneously with 3-mc, or the high dose of TCDD was given s.c. simultaneously with 3-mc. The C.I. increased from 5 to 38 when the high dose of s.c. TCDD was used, and to about 14 when either the low dose of s.c. TCDD or high dose of i.p. TCDD was used. The authors concluded that the AHH inducibility of the B6 strain was already at a maximal level following 3-mc treatment, and that the change in sensitivity of the D2 mice is evidence that TCDD is a cocarcinogen. The TCDD cocarcinogenic activity thus appears to be highly dependent on species, dosage, dose route, and pretreatment time employed in animal testing. 572. Kouri, R. E., Salerno, R. A., and Whitmire, G. E. (1973) Relationships between aryl hydrocarbon hydroxylase inducibility and sensitivity to chemically induced subcutaneous sarcomas in various strains of mice. J. Natl. Cancer Inst. 50(2):363-368. The authors studied the relationship between susceptibility to 3-methylcholanthrene (3-mc) - induced sarcomas and hereditary hepatic 202 �inducibility of aryl hydrocarbon hydroxylase (AHH) activity in 14 strains of mice. A carcinogenic index (CI) was established for weanlings treated subcutaneously with 150 ug 3-mc and observed for 8 months; the CI applied accounted for both tumor incidence and latency. The most sensitive strains used were C3H/fMai (CI 100); Blo.Br/J(83); C57BL/6Cum(78); C3HHen(77); C58/J(77); BALB/cCr;(74); and C57BL/10ScSn(64). The least sensitive strains were SWR/J(31); Snell/ Mail(24); 129/J(25); SJL/J(24); DBA/2J924); AKR/J(12); and DBA/U(9). All the mice tested were female except BALB/cCr. At 4 weeks of age mice were inoculated intraperitoneally with 100 mg 3-mc/kg in a trioctanoin solution at .05 mg/dose. Twenty-four hours after inoculation, the hepatic levels of AHH in the mice were measured using the procedure of Nebert and Belboin (1969) as modified by Thomas et al. (1972). The authors determined AHH inducibility as the ratio of average AHH activity /g wet liver weight of 3-mc treated mice divided by the average in untreated mice. They also tested for group-specific (gs) antigen (primarily the gs-1 murine virus) in mose spleens, using microfilter complement fixation tests with pools of sera from Fischer rats with induced sarcomas from the Moloney murine virus. The author observed a direct relationship between 3-mc sensitivity and AHH inducibility. Mice with high CI for 3-mc sensitivity were inducible; those with a low CI were not. There was some indication of a link between virogene expression (measured by gs antigen expression) and low CI/AHH noninducibility. The authors suggest that this could be related to the fact that certain strains have a tendency to spontaneous tumors (associated with high virogene expression) while being simultaneously resistant to chemically induced tumors. One strain tested, however, C58/J, was sensitive to both spontaneous and 3-mc-induced tumors. It was the only strain tested that was both AHH inducible and consistently positive for virogene expression. Varying the dosage of 3-mc confirmed this correlation. Inducible mice receiving 37.5 ug 3-mc had CIs equivalent to those in noninducible mice receiving 300 ug 3-mc. The authors compared the effects of 3-mc with those produced in similar testing with 150 ug DMBA or BP, and did not obtain the same differential response between inducible and noninducible strains. They postulate that this was partially due to the fact that these two chemicals are less effective carcinogens when given subcutaneously at doses equivalent to that of the 3-mc administered. The C3H/fMai strain (males) showed extreme sensitivity to all three compounds, which could not be explained. The authors did indicate that this showed that AHH inducibility is not the only factor in determining absolute sensitivity in this strain. 573. Kramer, C. G. (1974) Health of employees exposed to 2,4,5-T. Findings of the DOW Chemical Company, Corporate Medical Department, 2030 DOW Center, Midland, Mich. 19 pp. Dow chemical performed a morbidity analysis of 126 workers exposed to 2,4,5-T. This population was divided into 3 non-exclusive cohort groups which were compared separately to a control population of 4,600. Data were obtained from personnel records to determine length and extent of exposure. Data on selected health indices were obtained from 203 �the company preventive health records. Information from health records included both clinical lab tests and questionnaire data. Data were analyzed in a 2 step process. First step analyses were the Students T-Tests on 50 clinical parameters and comparative analyses of observed and expected frequencies on questionnaire response assuming a Poisson approximation to the underlying binomial distribution. Second step analyses performed were linear correlation between the log of total career exposure to the base 10 and the log of total exposure days. Dichotomous health inventory measures between exposure and responses were determined by the Wilcoxon and unpaired Rank Test. Data showed no statistically significant difference between the exposed and control groups at the 0.5 level. 574. Krieger, R. I., Lee, P. W. , Black, A., and Fukuto, T. R. (1973) Inhibition of microsomal aldrin epoxidation by diquat and several related bipyridylium compounds. Bull. Environ. Contain. Toxicol. The capability of diquat to inhibit microsomal oxidation was studied in rat hepatic microsomes in vitro. Microsomes were prepared and suspended in buffer containing diquat and_aldrin epoxidation was analyzed. At a concentration of 6.6 x 10 M, 50% inhibition of aldrin epoxidation activity was attained. A higher dose (amount was not reported) inhibited enzyme activity completely. The inhibitory capacity of paraquat and of a series of agents with redox potentials between -0.18 and. -0.55 v were tested in this microsomal epoxidation system. The authors concluded that diquat inhibits microsomal oxidation by disrupting the microsomal electron transport system. The brevity of descriptions of experimental procedure and the indirect method of correlating the redox-potential of a compound with its ability to inhibit microsomal aldrin epoxidation provide weak support for this tenuous conclusion. 575. Kuhn, E., and Stein, W. (1966) Model myotonia toward 2,4-D. Calcium absorption of the vesicles, its sarcoplasmatic reticulum among 2,4-D. Klin. Wschr. 44:700-702. [Foreign language.] 576. Kuhn, E., and Stein, W. (1964) Experimental myotonia with 2,4-D administration in the rat. Klin. Wschr. 42:1215-1216. [Foreign language.] 204 �577. Kumaki, K., Jensen, N. M., Shire, J. G. M., and Nebert, D. W. (1977) Genetic differences in induction of cytosol reduced-NAD(P): Menadione oxidoreductase and microsomal aryl hydrocarbon hydroxylase in the mouse. J. Biol. Chem. 252(1):157-165. The induction of activities by TCDD was studied in two strains of mice. A dose of 80 ug/kg of TCDD in para-dioxane was given to C57BL/6 mice and DBA/2N mice. Ten days later, the mice were killed and liver enzyme activities were assayed in TCDD-treated and dioxane-treated mice. Reduced-NAD(P)rmendione oxidoreductase activity in the cytosol was assayed spectrophotometrically and aryl hydrocarbon (benzofalpyrene) hydroxylase activity in microsomes was assayed by measuring fluorescence of the hydroxylated product. An eightfold increase in hydroxylase activity was produced by TCDD in each strain as well as in hybrid mice from a cross of the two strains. Oxidoreductase activity was induced 375% in C57BL mice, 150% in DBA mice and 365% in hybrids. Oxidoreductase activity in TCDD-treated mice of both strains were inhibited by alpha-naphthoflavone and metyrapone to the same extent as activity of dioxane treated mice. Induction of both enzyme activities followed the same time course, with activities of both enzymes returned to normal within 70-90 days after TCDD treatment. A backcross of hybrid mice with DBA mice was made and responsive (heterozygous for the Ah allele) and nonresponsive (homozygous for the Ah allele from DBA mice). Of 40 backcross mice, 27 had oxidoreductase activity that was not indueible by TCDD. The responsive mice were more susceptible to the toxic effects of TCDD than were non-responsive mice. Data were presented for enzyme inductions by other agents in several species of mouse. The authors concluded that two genes were involved with induction of these enzymes and only one, hydroxylase induction, involved the Ah locus. 578. Kurisaki, E., and Sato, H. (1979) Tissue distribution of paraquat and diquat after oral administration in rats. Forensic Sci. Internet. 14(3):165-170. The tissue distribution and histopathology of diquat administered orally to rats is described. Male Wistar ratsxwere administered 231 mg(cation)/kg or 116 mg/kg diquat (as the herbicide formulation, Reglox, containing 30% diquat) in water, orally and after 2 hours to 9 days were killed and tissues were removed, homogenized in acid and analyzed for diquat spectrophotometrically after separation on ion exchange chromatography. Other tissue samples were prepared for light microscopy. Within 2 hours of treatment, distension of the stomach was observed. At later times, slight congestion of all organs was observed and hemorrhages of the lung and intestines were observed. Two hours after exposure, diquat concentrations were highest in the lung and heart. At 24 hours diquat levels had decreased in the liver, heart, lung, and brain and were the same as at 2 hours in the kidney. At 48 hours the only change in tissue diquat levels was an increase in kidney levels. At 5 and 9 days diquat levels were constant or decreased for all tissues. Paraquat retention was also studied. The authors concluded that diquat was more rapidly excreted than paraquat and did not accumulate or cause toxicity in the lung. 205 �579. Kutz, F. W., Murphy, R. S., and Strassman, S. C. (1978) Survey of pesticide residues and their metabolites in urine from the general population. Environ. Sci. Res. 12:363-369. The levels of 2,4-D and 2,4,5-T in human urine samples were analyzed. Preliminary data were presented from a study of pesticide residues in fat, urine and blood of 28,000 civilian Americans between 6 months and 74 years of age, selected scientifically to include groups with high risk of poor nutrition. The study was incomplete at the time of the report and the composition of the group from which the preliminary data was obtained was not given. 416-418 urine samples were analyzed for 2,4-D and 2,4,5-T. The specific analytic methods used for 2,4-D or 2,4,5-T analyses were not indicated. The limit of detection was between 5 and 30 ppb for both residues. No 2,4-D or 2,4,5-T was detected in any urine sample. The samples were also analyzed for 18 other pesticides. The authors emphasized that their results were preliminary and should not be assumed to represent the general population. 580. Kutz, F., and Strassman, S. (1979) Survey of pesticide residues and their metabolites in the general population of the United States. In: Commission of the European Communities Off., For. Off. Publ. POB1033 Luxembourg 1, Lux. 8 pp. The results of analyses of 267 human urine samples for 2,4,5-T is presented. Samples were collected through a human monitoring survey system which obtained patient samples from pathologists in hospitals and private practice and samples from normal volunteers with documented herbicide exposure (no other details about type of herbicide, extent of exposure or number exposed were given). Samples were collected from hospital patients who were treated on an out-patient basis or were admitted for less than 24 hours. Analyses of each sample were performed by a multi-phenol method that was not described further. Of the samples analyzed, 1.5% had detectable levels of 2,4,5-T and all samples had less than 0.1 ppm 2,4,5-T. The levels of other herbicides in human adipose tissue, milk and urine samples were also presented. The authors did not present any conclusions pertaining to the 2,4,5-T levels they reported. 581. Kuz'minskaya, U. H., and Bersan, L. V. (1975) The effects of the sodium salt of 2,4-dichlorophenoxy-acetic acid on the glycolysis, ATP-ase and transketolase activity of the erythrocytes. Farmakologiya i toksikologiya. 38(1):102-104. The effects of 2,4-D on enzymes involved in cell metabolism were studied in rats. Male rats were administered 1 g/kg 2,4-D sodium salt in water orally and blood was removed after 10 minutes, 1 hour, and 1, 5, and 15 days. Other groups of male rats were orally administered a dose of 20 mg/kg daily and blood was removed after 3 and 6 months. Erythrocytes were isolated and assayed for glycolysis activity as the 206 �rate of lactate production, and for transketolase and ATPase activities. Significantly increased glycolysis occurred 10 minutes, 1 hour, and 5 days after treatment, a 31% increase in transketolase occurred after 10 minutes but was not sustained at later times, and peak ATPase activity occurred at 5 days and was 40% above controls. Three months after chronic administration significantly decreased glycolysis and increased enzyme activities occurred and at 6 months these effects were abolished or reversed. At 3 months, toxicity was evidenced by clinical signs that included apathy, weight loss and hemuresis. Clinical signs improved by 6 months. The authors concluded that the pentose-phosphate pathway was the predominant source of erythrocyte energy production after acute 2,4-D exposure and effects observed after chronic exposure reflected the toxic state of the animals. 207 �582. Lam, H. F., Takezawa, J., and Van Stee, E. W. (1979a) The effects of paraquat and diquat on lung function measurements in rats. Ann. Rev. Resp. Pis. 119:327. [Not available.] 583. Lam, H. F., Takezawa, J., and Van Stee, E. W. (1979) Evaluation the effects of paraquat and diquat in rats using pulmonary function tests. Environ. Health Perspec. 33:338. [Abstract only.] 584. Lamb, J. C., Moore, J. A., and Marks, T. A. Evaluation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in C57BL/6 mice; Reproduction and fertility in treated male mice and evaluation of congenital malformations in their offspring. National Toxicology Program, Research Triangle Institute, Research Triangle Park, NC. Report No. NTP-80-44. 57 pp. Mixtures of compounds present in Agent Orange were administered to male mice and reproductive toxicity was assessed. Mice were treated for 8 weeks with daily diets containing 40 mg/kg 2,4-D plus 40 mg/kg 2,4,5-T plus 2.4 ug/kg TCDD (group II), or 20 mg/kg 2,4-D plus 20 mg/kg 2,4,5-T plus 1.2 ug/kg TCDD (group III), or 40 mg/kg 2,4-D plus 40 mg/kg 2,4,5-T plus 0.16 ug/kg TCDD (group IV). After the exposure period, treated males were mated with untreated females. No changes were reported in any reproductive parameters for treated groups compared to the control group given a diet supplemented with the corn oil vehicle. Parameters measured were mating frequency, average fertility, percent implantation and resorption sites, percent fetal malformations, germ cell toxicity, sperm concentration, sperm motility, percent sperm abnormalities, survival of offspring, and neonatal development. Toxicity of the liver and thymus and decreased body weights were observed in treated males. These effects were dose-related and were reversible after treatment ceased. 585. Langer, H. G., Brady, T. P., and Briggs, P. R. (1973) Formation of dibenzodioxins and other condensation products from chlorinated phenols and derivatives. Environ. Health Perspect. 5:3-7. [Background material.] 586. Laporte, J. R. (1978) Multinationals and health: Reflections on the Seveso catastrophe. Intern. J. Health Serv. 8(4)619-632. [Review article.] 208 �587. Laporte, J. R. (1977) Effects of dioxin exposure. Lancet: 82-83. [Editorial.] 588. Larsson, B., Oskarsson, A., and Tjalve, H. (1977) Binding of paraquat and diquat on melanin. Exp. Eye Res. 25(4):353-359. Binding of diquat to melanin in vivo in mice and in vitro to bovine melanin are described. Pigmented C57-B1 mice and albino NMR1 mice were administered 3.3 mg/kg [ C]-diquat in saline, intravenously. Mice were killed 5 min. to 4 days after treatment and were prepared for whole body autoradiography. Pigment granules/were separated from bovine uveal tissue and were combined with [ C]-diquat and maintained at room temperature for 45 min. Non-bound diquat was separated by centrifugation and counted. A substantial amount of radioactivity was associated with melanin-containing tissues of C57-B1 mice and was absent from the same structures in albino mice. A concentrationdependent uptake of diquat occurred in vitro, with at least 97% of diquat bound to melanin when doses of up to 1 uraol of diquat were added to 10 mg of pigment granule suspension. Binding of paraquat was also studied. The authors concluded that diquat was strongly bound to melanin and suggested the binding may be ionic and may potentiate the toxic effect of cataract formation in non-albino animals by diquat by sequestering the compound in the organ where the lesion occurs. 589. Lavy, T. L. (1978) Measurement of 2,4,5-T exposure of forest workers. Project Completion Report to the National Forest Products Association. The levels of 2,4,5-T exposure of forest workers were measured and body burdens were estimated. The subjects were workers in 4 work crews who applied 2,4,5-T propylene glycol butyl ether ester (Esteron 245) by mist blower or helicopter at the rate of 2 Ib. per acre or by backpack at the rate of 1.6 Ib. per acre. Workers were experienced in their particular tasks in the spray operations and were instructed not to alter their normal work habits during the study. Workers did not wear gloves or protective clothing. Data were collected for 2 exposures of each worker, which were separated by at least 6 days. Air samples collected by each worker with air monitors, urine samples collected for 6 days (beginning 1 day prior to exposure) from each worker and 10 x 10 cm gauze patches, attached to the workers' clothing (arms, chest and back) during the spraying operations (but not during the mixing operations) were all monitored for 2,4,5-T acid by gas chromatography. (Recovery by this method was 78% in air samples, 79-120% in patches and 64-99% in urine). The 2 patch samples and the urine sample highest in 2,4,5-T were also analyzed for TCDD content by gas chroraatography-mass spectrometry (50-100% recovery of TCDD by this method). Urinary creatine excretion rates were determined for the workers. Urinary excretion of 2,4,5-T ranged from 0.002 mg/kg body eight for helicopter crew members to 0.072 rag/kg for formulation mixers. Dermal exposures were calculated from the patch levels of 2,4,5-T, bare skin areas (estimated from photographs of workers) and body weights. Dermal 209 �exposures ranged from no detectable exposure of helicopter crewmember to 1.7 rag/kg for mist blower workers. Air residues ranged from undetectable (below 0.15 ug/liter air) for helicopter crewmembers to 0.007 mg/kg for one of the backpack sprayers and 0.007 mg/kg for other backpack sprayers. In general, dermal exposure values correlated poorly with urine excretion levels. The ratio of TCDD to 2,4,5-T in the urine and patch samples analyzed was below the 0.04 ppm level in the original formulation. The authors concluded that 2,4,5-T analyses in urine were the most reliable indicator of exposure and that exposure levels were highest for workers that mixed the herbicides, followed by backpack sprayers, lowest for helicopter flagmen, followed by supervisors. 590. Lavy, T. L., Shepard, J. S., and Bouchard, D. C. (1980) Field worker exposure and helicopter spray pattern of 2,4,5-T. Bull. Environ. Contam. Toxicol. 24(1):90-96. The amounts of 2,4,5-T deposited on workers applying the herbicide by several methods is reported. Six 100cm gauze patches were applied to specified areas of clothing of workers applying 2,4,5-T. These workers included a five-man helicopter team, a tractor driver spraying 2,4,5-T on rice levees, a four-man tracker mist-blower team applying 2,4,5-T in a forest, and 12 forest backpack spray workers. The 2,4,5-T preparations applied were Thompson Hayward's Bed Weed LV-6 and Dow's ESTERON 245 (the 2,4,5-T esters in these preparations and other ingredients were not specified). The rate of application was 2 pounds of active ingredients per acre and the herbicide was applied in water. All spray operations except on rice levees were conducted in the morning with wind velocity of less than 5 mph. The time for each operation was 116 min. for the helicopter team, 65 min. for the rice tractor driver, 245 min. for the forest mist blower team and 180 min. for the backpack workers. In addition to the workers, one person (with gauze patches fixed to his clothing) stood under the helicopter spray and another walked through the spray area 2 hours after ESTERON 245 was applied. Three sets of mylar sheets ( 9 x 9 in) were placed 80, 160, 320, 640, and 960 feet from the helicopter spray path and were analyzed for 2,4,5-T after the herbicide was applied, to determine the extent of drift. All 2,4,5-T analyses were done by gas chromatography with a lower limit of detection of 10 micrograms per gauze patch or sheet. The mean levels of 2,4,5-T on the patches of workers were 0.043, 0.12, 0.94, and 5.43 mg/100 cm for the helicopter crew, tractor driver, mist blower crew, and backpack sprayers, respectively. For 8 of the 22 workers, the patch on the thigh area contained over half of the total amount of 2,4,5-T recovered from the 6 patches. The patches on the individual under the helicopter spray contained from 0.01 to 6.48 mg and the total (maximum) potential exposure was calculated to be 0.86 mg/kg body weight. The authors further estimated that 0.034 mg/kg 2,4,5-T would be absorbed and excreted by an individual from this exposure. No 2,4,5-T was detected in patches from the individual exposed to the spray area 2 hours after the application was made. Over 84% of the 2,4,5-T sprayed by helicopter was deposited within 80 feet of the spray path and 99%, within 160 feet. Slight drift occurred, 210 �with trace levels of 2,4,5-T detected in 2 of 12 samples taken beyond 320 feet from the spray path. The authors concluded that workers who applied 2,4,5-T were unlikely to receive doses of 20 mg/kg or more of 2,4,5-T, the amounts reported by others to cause fetotoxicity in mice. This report is important in providing an estimate of human exposure potential from normal herbicide application, unfortunately several important points were not included in the report and include the composition of the herbicides and the ester applied, the amount of time the two people who were exposed to helicopter spray remained in the sprayed area, the recovery of 2,4,5-T from gauze pads for the analytical method used, and urine levels for the exposed workers. 591. Lawson, E. R. (1976) 2,4,5-T residues in storm runoff from small watersheds. J. Soil Water Conserv. 31:217-219. In this study, the author measures levels of 2,4,5-T in storm runoff from sprayed forest areas. Two watersheds were chosen for the study: one 1.5 acre area on which all woody vegetation had been removed, and another 1.5 acre area which contained about 60 square feet of pine. Both watersheds were sprayed on Sept. 1971, June 1972, and July 1973; the cleared watershed was sprayed with 4 Ib acid equivalent 2,4,5-T isooctyl ester/acre and the partially cut watershed with 21 Ibs/acre. A control watershed a similar size was not sprayed. Storm runoff samples were collected and analyzed by gas chromatography. Only runoff samples taken 3 weeks after the first application showed any detectable 2,4,5-T. Samples from the cleared watershed had an average of 2.1 ppm 2,4,5-T, while samples from the partially cut watershed averaged 1.0 ppm. Storm runoff 7 and 11 weeks after the first spraying, and after the second and third spraying did not yield detectable levels of 2,4,5-T. The author concuded that none of the concentrations of 2,4,5-T found in this study were a hazard to wildlife or man. 592. Leahey, J. P., and Hemingway, R. J. (1975) The metabolism of diquat on hens and residues in eggs and tissues. Environ. Qual. Saf. [Suppl]. 3:157-162. The tissue distribution, biotransformation, and excretion of diquat is described for the chicken^. One hen each was administered and five daily doses of 4-5 ug/g [ C]-diquat ion (99.7 + %. purity) in feed, and one hen was given 14 daily doses of 0.4-0.5 ug/g [ C]-diquat in feed. The feces, tissues, egg yolk, and egg albumin were analyzed for radioactivity. Metabolites were separated by paper chromatography. Three days after a single dose of diquat, 98.5% of the dose had been excreted in the feces and 5 days after five doses were administered, 94.5% was excreted in the feces. Eggs contained less than 0.1% of the dose. The yolk of eggs collected from the hen given 14 doses contained increasing amounts of diquat to day 9, and lower levels, subsequently. The pellets given to the hen were found to have less diquat available 9 days after they were prepared than fresh pellets given on day 1 of the experiment. Diquat (unmetabolized) accounted for 72-80% of the radioactivity in fecal samples collected on days 1-5 from the hen that 211 �was being administered five daily doses, and 18-22% on days 6 and 7. Two metabolites, comprising 4 and 2% of the fecal radioactivity from day 5 samples were identified as the 9-oxo-6,7-dihydro-8H-dipyrido [l,2-a:2',1'-c]-5-pyrazinium ion(l)and l-oxo-l,2,3,4-tetrahydro-2Hpyrido-[l,2-a]-pyrazinium ion (II), respectively. The radioactive composition of egg yolks from hens after subacute exposure identified by isotope dilution analysis, was 54-85% metabolite I, 26-39% diquat and 2-10% metabolite II. Seven days after 5 doses of diquat and 4 hours after 14 doses were given, hens were killed and kidney, liver, lung, muscle, fat, and blood were analyzed for diquat. Kidneys contained 0.004 ug/g diquat and all other tissue had less than 0.001 ug/g diquat. The authors concluded that the amount of diquat that remained in eggs after a dose 40 times larger than expected from normal commercial use was toxicologically insignificant, as were the tissue levels in hens after subacute exposure and levels of the two less toxic metabolites (based on oral LDcn values in rats). 593. Lee, C. (1979) Dioxin (Dilemma)? Med. J. Australia 602-603. [Editorial.] 594. Lee, I. P., and Dixon, R. L. (1978) Factors influencing reproduction and genetic toxic effects on male gonads. Environ. Health Perspec. 24:117-127. The authors review metabolic parameters that modulate the function of the male gonad. Data are presented substantiating the ability of TCDD to induce gonadal mixed function oxidase activity. Aryl hydrocarbon hydroxylase (AHH) activity of the rat testis was induced twofold by one PO treatment of 10 ug/kg TCDD while AHH activity of the rat prostate was induced 150 times by this dose. Testicular AHH activity returned to normal within 72 hours, while prostate activity remained elevated after 3 weeks. The authors concluded that TCDD, by inducing enzymes that activate other chemicals, can modulate mutagenicity of germ cells and oncogenicity of prostate glands. 595. Lehman, A. J., and Habermann, R. T. (1964) Pathological changes in rats fed 2,4-D for 2 yrs. Unpublished report: FDA. [Not available.] 596. Lehn, P. J., Pate, B. D., Kim, D. R., and Voight, R. C. Evaluation of the effects of defoliants on the animal communities of Test Area C-52A, Eglin Air Force Base, Florida. In Abstracts, 1972 Meeting of the Weed Science Society of America p. 90. [Not available.] 212 �597. Leistra, M., Smelt, J. H. , and Zandvoort, R. (1975) Persistence and mobility of bromacil in orchard soils. Weed Res. 15:243-247. The authors studied the movement of bromacil through 1 m of orchard soil. Two study sites were used which had received bromacil applications annually for 6 to 7 years prior to the experiment. One orchard contained sandy loam soil (Groesbeek), while the other had silty clay loam soil (Est). Bromacil was applied at 1.6 and 2.4 kg/ha at Groesbeek and at 1.2 and 2.4 kg/ha at Est. Soil samples from the top 150 cm of soil were collected and analyzed for bromacil by gas chromatography. There appeared to be differences in bromacil movement depending on the soil type. One year after application, the amounts of bromacil remaining in the Groesbeek soil were 0.91 and 1.75 kg/ha for the 1.6 and 2.4 kg/ha dose, respectively. At Est, the soil residues were 0.39 and 1.32 kg/ha for 1.2 and 2.4 kg/ha, respectively. The highest concentrations of bromacil occurred at 10-20 cm in Groesbeek soil and in the 5-10 cm and 10-20 cm layers for the Est samples. For both soils, the average residual bromacil was 54% of the applied herbicide. The authors calculated the half-life of bromacil to be approximately 8 months. The authors also calculated the amount of bromacil leaching from the top 1 m of soil as 1.8-2.8%, depending on dose and soil type. The effect of repeated applications to the test sites was not discussed by the authors. In light of the 8-month halflife of bromacil, it would be expected that some accumulation of bromacil would occur after repeated application. 598. Leng, M. L. Comparative metabolism of phenoxy herbicides in animals. In Fate of pesticides in large animals. G. W. Ivie and H. W. Borough, eds., (New York:Academic Press, 1977) pp 53-76. [Review article.] 599. Leng, M. L. (1978) 2,4,5-T RPAR - Review of EPA's rationale and their calculations for exposure to 2,4,5-T and TCDD. [Unpublished paper] DOW. 7 p. [Background material.] 600. Leonard, A., and Lauwerys, R. R. (1980) Carcinogenicity, teratogenicity, and mutagenicity of arsenic. Muta. Res. 75:49-62. [Review article.] 601. Levina, M. M. (1973) Industrial hygiene in monuron production. Tr. Prom. Sanit. Proizvod. Pestits. 178-192. [Not available.] 213 Gig. �602. Lewis, H. J. 177:745. (1972) Herbicide Study, [letter to the editor] Science [Editorial.] 603. Leyland, A. (1973) A case of 2,4,5-T poisoning in the dog. Vet. Rec. 92(6):149-150. The autopsy findings of a dog that ingested a lethal dose of 2,4,5-T is reported. Stomach contents contained 2,000 ppm 2,4,5-T and exposure was suspected to be from 2,4,5-T contaminated water or food from a farm that had been treated with a formulation of esters of 2,4,5-T. Hemorrhages were observed in the gastrointestinal tract, kidneys, and diaphragm. Severe gastritis and congestion of the renal cortex were also evident at autopsy. The liver was not examined because it had undergone extensive autolysis in the 2 days after death which preceded the examination. The authors concluded that death was caused by ingestion of a dose of 2,4,5-T approximately 3-4 times larger than the L V 604. Lindahl, R., Roper, M., and Deitrich, R. A. (1978) Rat liver aldehyde dehydrogenase - immunochemical identity of 2,3,7,8-tetrachlorodibenzo-p-dioxin inducible normal liver and 2-acetylaminofluorene inducible hepatoraa isozymes. Biochem. Pharmacol. 27:2463-2465. Rat liver aldehyde dehydrogenases, induced by different conditions including TCDD treatment, were differentiated from each other by immunochemical techniques. Three aldehyde dehydrogenase isozymes were prepared from rat liver cytosol. Tau isozyme was induced by TCDD administration to rats phi isozyme was induced by phenobarbital; alpha isozymes were induced by chemical carcinogens (2-acetylaminofluorene, for example) and are hepatoma-specific isozymes. Rabbit antiserum against alpha and tau isozymes as well as the 3 isozyme antigens were obtained from ethods described in other publications and were studied by Ouchterlony 2-dimensional immunodiffusion. Anti-alpha serum reacted with the tau isozyme forming a precipitin line. Anti-tau serum reacted with alpha isozyme but not with normal (uninduced) liver preparations. Neither serum reacted with phi isozyme. The authors described some previously published kinetic and physical properties of the enzymes and concluded that the TCDD-induced enzyme is immunochemically identical to the hepatoma isozymes, but distinct from the basal and phenobarbital induced isozymes. 605. Lindquist, N. G., and Ullberg, S. (1971) Distribution of the herbicides 2,3,5-T and 2,4-D in pregnant mice: accumulation in the yolk sac epithelium. Experientia 27:1439-1441. The uptake and distribution of [ C]-2,4-D and [ C]-2,4,5-T were followed in the pregnant mouse by autoradiography. NMRI mice were given a single intravenous injection of 10 ul of either compound in 50% 214 �ethyl alcohol. Mice were killed 5 minutes to 4 days after the injection and whole body autoradiography was performed. Radioactivity accumulated in the visceral yolk sac epithelium in mice given 2,4,5-T early (day 8-9) or late (day not indicated) in pregnancy. In early pregnancy no isotope was associated with the fetus, whereas at the later stage, the concentrations of isotope in the fetal blood, abdomen, pericardium, and ocular fluid reached that of the maternal blood. Radioactivity in C-2,4-D treated mice accumulated only slightly in the yolk sac, reached the fetus, and was eliminated from all tissues within 24 hours. Both compounds were evenly distributed in tissues other than the yolk sac. The data in this report were presented as a brief summary without any other details of the results provided. 606. Lisk, D. J., Gutenmann, W. H., Bache, C. A., Warner, R. G. , and Wagner, D. G. (1963) Elimination of 2,4-D in the urine of steers fed 4-(2,4-DB) or 2,4-D. J. Dairy Sci. 46:1435-1437. The amount of 2,4-D excreted in urine and feces after one dose was administered to steer was determined. One Holstein steer was fed a diet that contained 5 ppm 2,4-D for 1 day. On the subsequent 7 days, urine and feces were collected and analyzed for 2,4-D by gas chromatography. The limit of sensitivity of this method was 0.1 ppm and recovery was 82%. Urine collected on day 1 contained 85.3 mg (17.6 ppm), on day 2 contained 15.4 mg (1.0 ppm) and on day 3 contained 0.1 ppm. Of the total dose of 113.5 mg administered, 100.7 mg were recovered in urine within 2 days of administration. Another compound, 4-2,4-dichlorophenoxybutyric acid, administered to one steer was recovered in the urine as 2,4-D only. The authors concluded that 2,4-D was excreted unchanged primarily in the urine. 607. Litchfield, M. H., Daniel, J. W., and Longshaw, S. (1973) The tissue distribution of the bipyridylium herbicides diquat and paraquat in rats and mice. Toxicology 1(2):155-165. The distribution and accumulation of diquat into mouse tissues is presented. Male Alderley Park mice were administered 50 mg(cation)/kg [ C]-diquat in saline intravenously. After 10 min. to 72 hr., mice were killed and prepared for whole-body autoradiography. A group of 40 Alderley Park rats were administered a diet that contained 250 ppm diquat ion as diquat dibromate monohydrate for up to 8 weeks. Food intake and body weights were recorded weekly and at 2, 4, 8, and 9 weeks 10 treated rats and 5 control rats were killed and tissues were weighed, homogenized, extracted with trichloroacetic acid, and diquat was separated by ion-exchange chromatography and analyzed colorimetrically. Ten minutes after injection, diquat was distributed to all tissues and was concentrated in cartilage and gall bladder and low in the brain and spinal cord. After 1 hr., the levels and radioactivity were lowered in all tissues (especially in cartilage) than at 10 min. except the urine and intestinal epithelium, which had higher levels. At 24 hr. diquat was present in theintestine and bladder, and at 72 hr. in the gastrointestinal tract, only. In 7 days only 5.5% of a single 215 �dose of [ C]-diquat was excreted in the urine by rats and by 14 days only 5.8% was excreted in the urine. No alterations in food consumption or body or organ weights were observed for rats on a diet with 250 ppm diquat, compared to controls. Diquat concentrations were highest in the kidney and in large intestine at 1.2 ppm by 8 weeks in both tissues. No diquat was detected in blood or muscle and low levels were detected in the brain. One week after exposure ended, no diquat was detected in any tissues (lower limit of detection, 0.05 ug/g). The authors concluded that diquat was rapidly distributed and eliminated and was not accumulated. The authors suggested that secretion of diquat into the intestine occurred in the absence of biliary excretion. 608. Lloyd, J. W., Thomas, J. H., and Mawhinney, M. G. (1973) -2,4,5-T and the metabolism of testosterone -1, 2- H_ by mouse prostate glands. Arch. Environ. Health 26(4):217-220. The effects of 2,4,5-T on fructose levels, testosterone accumulation, and metabolite formation in the prostate gland and on weights of male sex organs were studied in mice. Male Swiss-Webster mice were administered a daily dose of 6.25 to 25 mg/kg of 2,4,5-T for 10 consecutive days. On the eleventh day, 10 ug/kg of [ H]-testosterone was administered intraperitoneally and 5 minutes later, the prostate glands were removed and radioactive testosterone and metabolites were extracted and analyzed by thin layer chromatography. In some groups of rats, no radioactivity was administered; instead, on the eleventh day, the prostate gland was removed and analyzed for fructose, or the liver was removed, sliced, or homogenized and incubated in the presence of [ H]-testosterone. Radioactive metabolites were analyzed chromatographically. In 2,4,5-T-treated rats, a 35% reduction in prostate uptake of testosterone was observed. The size of the effect was the same for all 3 doses of 2,4,5-T. The relative proportion of radioactivity associated with each metabolite in the prostate or in liver tissue was unaltered after 2,4,5-T treatment. Fructose levels were unaltered in treated mice and the weights of the seminal vesicles, prostate glands, and testes all were the same for 2,4,5-T treated mice as those of vehicle controls. The authors concluded that 2,4,5-T could have produced a direct effect on the prostate gland, interfering with uptake of testosterone, and suggested inhibition of androgen binding to target tissue as a possible mechanism. 609. Lock, E. A. in the rat. (1979) The effect of paraquat and diquat on renal function Toxicol. Appl. Pharmacol. 48(2):327-336. The effects of diquat on renal clearance of several compounds and of plasma and red cell volumes were studied in the rat. Male Alderley Park (Wistar derived) rats were administered 540-900 umol per kg of diquat perorally, followed in 24 hours by administration subcutaneously of [ C]- or [ H]-labeled inulin, para-amino hippurate (PAH), N'-methylnicotinamide (NMN), or diquat. Water equal to 5% body weight was then given intraperitoneally. Blood samples and urine samples were collected subsequently and analyzed for urea content and radioactivity. 216 �Plasma and cell volumes were determined in rats injected with Cr-tagged red cells.in plasma containing I-labeled human serum albumin. Cr and I radioactivities were determined in blood samples and in the excised kidneys and volumes were calculated from the extent of dilution of the administered isotopes. One day after diquat was administered, renal clearances of urea, inulin, PAH, NMN, and diquat were all significantly reduced to 17 to 55% of vehicle control levels. Urine flow was also reduced and the filtration fraction was double the values in saline-treated controls. Doses of 680 and 900 ug/kg of diquat produced a significant decrease of 14-20% in plasma volume, no change in red cell volume and a significant increase in hematocrit 24 hours after treatment. The higher dose also produced a significant decrease of 18% in renal plasma volume. The slightly higher clearance of diquat than inulin indicated that active secretion of inulin occurs. The renal toxicity of paraquat was also studied. The authors concluded that the renal changes produced by diquat occur secondary to hemodynamic changes which involve water redistribution to the lumen of the gastrointestinal tract and resultant decreases in plasma volume and renal blood flow. In man, the reduced renal excretion of diquat was proposed to enhance the systemic toxicity of diquat. 610. Lock, E. A., and Ishmael, J. (1979) The acute toxic effects of paraquat and diquat on the rat kidney. Toxicol. Appl. Pharmacol. 50(l):67-76. The effects of diquat on renal function were studied in the rat in vivo and in vitro. Male Alderley Park (Wistar-derived) rats were administered a single dose of 680 umol/kg or 900 umol/kg diquat in saline perorally. Some rats were also administered 50 uCi/kg [ C]- diquat. Urine and plasma biochemical analyses and cell counts were performed and urinary enzymes were assayed fluqrimetrically with 4-methylumbelliferyl substrates. Uptake of H-para-aminohippuric acid (PAH) and [ C]-N'-methylnicotinamide (NMN) from incubation medium., into renal cortical slices,was determined with diquat in the medium [1- C]glucose or [6- G]-glucose was included with diquat in the medium of some renal slices and [ ,£]~ carbon monoxide. C07 production was determined by trapping [ C]-CO- on potassium hydroxide saturated filters and then determining radioactivity of,the filters. Some renal slices were incubated in medium containing [ C]-acetate, then homogenized and the radioactivity in fatty acid extracts of tissue homogenates was determined. A dose of 680 ug/kg of diquat produced proteinuria and glucosuria during the next 24 hours. The urinary albumin to total protein ratio was not altered by this dose but an increased number of cells was excreted in the urine. After 24 hours, 7.5% of the oral dose of diquat was excreted in the urine. Mild focal hydropic degeneration was observed in the proximal tubules after diquat treatment. At ImM in the medium, diquat inhibited the accumulation of NMN (by 55%), but not of PAH into renal slices. Slices of kidneys removed from diquat-treated rats when in vivo clearance was reduced did not show reduced NMN or PAH,accumulation, compared to control kidney slices. Oxidation of [1- C]-glucose, but not of [6- C]-glucose was 217 �stimulated 30% by 0.1 and 55% by ImM diquat but oxygen consumption was unaltered by these doses. Incorporation of [ C]-acetate by fatty acids was increased by 40% by 0.1 or ImM diquat added to culture medium but not in renal slices from rats treated in vivo 24 hours earlier. The effects of paraquat and mercuric chloride on renal function were also studied. The authors concluded from their metabolic studies that orally administered diquat had not entered renal cells sufficiently within 24 hours to alter the redox state of the cell. From the PAH and NMN accumulation studies in vitro, they concluded that in vivo alterations in NMN and PAH clearances reported by others were the result of altered renal hemodynamics. 611. Loos, M. Phenoxyalkanoic acids. In Herbicides; Chemistry, degradation, and mode of action. Vol. 1, P. C. Kearney and D. D. Kuvman, eds., (New York: Marcel Dekker, Inc., 1975) pp 1-128. [Review article.] 612. Lucier, G. W., Lui, E. M. K., and Lamartiniere, C. A. (1979) Metabolic activation/deactivation reactions during perinatal development. Environ. Health Perspec. 29:7-16. [Review article.] 613. Lucier, G. W., McDaniel, 0. S., and Hook, G. E. R. (1975) Nature of the enhancement of hepatic uridine diphosphate glucuronyltransferase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Biochem. Pharmacol. 24:325-334. The characteristics of hepatic enzyme induction by TCDD were investigated in the rat. CD rats were administered a single oral dose of TCDD in acetone-corn oil (1:9) and actinomycin D was administered intraperitoneally immediately after TCDD to some rats. Hepatic microsomes were prepared from treated and vehicle-control rats. Glucuronyltransferase activity was solubilized from microsomes with deoxycholate, and then purified by ammonium sulfate precipitation, dialysis, and molecular sieve chromatography. Glucuronidation of 1-naphthol and paranitrophenol (PNP) were assayed colorimetrically and of testosterone and estrone were assayed using radioactive substrates. Product formation was quantified for all substrates by separation of radiolabeled substrates and products on ion exchange chromatography. The activity of beta-glucuronidase was determined by measuring hydrolysis of PNP-beta-D-glucuronide. Maximum (sixfold) induction of PNP glucuronidation was observed between 9 and 16 days after a dose 25 ug/kg of TCDD was given to male rats and the enzyme levels remained elevated (by 160%) after 73 days. A lower dose produced smaller increases in enzyme activity with the same time course for induction. Glucuronidation of 1-naphthol showed the same induction pattern by TCDD as PNP, while no induction of testosterone or estrone glucuronidations or beta-glucuronidase occurred. The identity and amount of each 218 �glucuronidaCion product was confirmed by the results of ion exchange chromatography. The magnitude of induction of PNP glucuronidation^was the same in rats aged 17, 38,_8jQ, and 335 days when TCDD was administered. Addition of 10 TCDD directly to the microsomal incubation mixture did not alter rates of glucuronidation for any of the 4 substrates tested. Addition of magnesium or detergent (triton X-100) stimulated enzyme activity from TCDD-treated rats and controls equally. Microsomal cholesterol and phospholipid contents and kinetic parameters for glucuronidation activity from TCDD-treated and control rats were the same, except for an increase in the maximum velocity of the reaction by enzyme from treated rats. Actinomycin D seemed to block the TCDD inductive effect, although it also decreased total microsomal protein, complicating the interpretation of this result. Purified glucuronidation enzymes from treated and control rats eluted in the same positions chromatographically, with far more activity associated with peaks from TCDD-treated preparation. The pH optimum of 7.2 and temperature optimum of 40"C for TCDD treated and control DNP glucuronidation was established. The authors concluded that TCDD exerts its effect on glucuronidation indirectly, probably by increasing synthesis or decreasing degradation of the enzyme. The conclusions are supported by well-designed experiments that considered many aspects of enzyme induction and used the most technically appropriate methodology to test each aspect. 614. Lucier, G. W., McDaniel, 0. S., Hook, G. E. R., Fowler, B. A., Sonawane, B. R., and Faeder, E. (1973) TCDD-induced changes in rat liver microsomal enzymes. Environ. Health Persp. 5:199-209. The effects of TCDD on hepatic microsomal enzyme activities and liver function was studied in the rat. The time-course of enzyme induction was studied in male Charles River rats (3 per group) who were administered a single oral dose of 5 or 25 ug/kg TCDD in acetone-corn oil and were killed 1-38 days later. Hepatic microsomes were prepared and analyzed for microsomal enzyme activities and serum ornithine transcarbamylase activities were measured. Dose-response relationships were examined in another series of experiments in which male and female rats were administered a single dose of 0.2, 1.0, 5.0 or 25 ug/kg TCDD and livers were removed 3 days after treatment. Hepatic microsomes and mitochondrian were isolated. Microsomal enzyme activities were assayed and mitochondrial oxidative phosphorylation rates were measured polarographically with an oxygen electrode. No rats died in any treatment group. In the time-course study, aniline hydroxylation activity increased by 100%, UDP glucuronytransferase activity by 600%, cytochrome P-450 and b_ levels by 60% and aminopyrine demethylation decreased by 30%. These changes were maximal 3-16 days after treatment and were still different from control values after 38 days. Liver function was unaltered, as were NADPH cytochrome c reductase and beta-glycuronidase activities, Microsomal protein contents increased significantly 28 days after treatment, by 60%. For the dose-response study, dose-related elevations in glucuronyltransferase (to 500%, maximum) and benzpyrene (to 1400%) were observed, with larger increases in female than male microsomes. Increased cytochrome P-450 and b_ 219 �levels and aniline hydroxylation activity of up to 100% occurred, while oxidative demethylation of aminopyrine, ethylmorphine and benzphetamine decreased and NADPH cytochrome C reductase activities were unchanged. The ratio of smooth to rough endoplasmic reticular enzyme and protein levels were decreased by TCDD. No alterations in mitochondrial state 3 or state 4 oxidative phosphorylation rates were observed after TCDD treatment. The authors concluded that hepatic microsomal enzymes are extremely sensitive to TCDD body burdens and recommended 8 areas for future research. 615. Lucier, G. W., Sonawane, B. R., McDaniel, 0. S., and Hook, G. E. R. (1975) Postnatal stimulation of hepatic microsomal enzymes following administration of TCDD to pregnant rats. Chem. Biol. Interact. 11:15-26. The effect of TCDD administration to pregnant rats on maternal, fetal and neonatal enzyme levels is described. Pregnant Charles River rats were administered 3 ug/kg TCDD in acetone-corn oil (1:9) on day 5, 10 or 16 of gestation. At various stages of gestation or after birth, maternal livers, livers of offspring, and placenta were removed and microsomes were prepared and assayed for benzypurene hydroxylase (BPH) and UDP gluouronyltransferase (UDPGT) activities. For 1 experiment newborn rats of mothers treated on day 5 of gestation were fostered by control mothers from birth to day 8 and control newborns were likewise fostered by TCDD-treated mothers. TCDD treatment did not alter fetal, placental or fetal liver weights, litter sizes, fetal microsomal protein contents or the incidence of gross malformations. Both maternal BPH and UDPGT (para-nitrophenol, substrate) activities were induced about 15 fold during pregnancy and remained elevated by 3 fold, 3 weeks after birth. Prenatal TCDD treatment reuslted in elevated UDPGT levels in 4-day old or older offspring. Treatment on day 5 with TCDD did not alter BPH levels of fetuses on day 20 of gestation, but caused elevated levels in noenates. Treatment on day 10 or 16 of gestation resulted in elevated fetal and neonatal BPH levels. Neonatal cytochrome P-450 and b- levels were also elevated following TCDD treatment on day 10 of gestation. Placental BPH and UDPGT levels were induced 20 and 3 fold respectively from TCDD treatment on day 16. Testosterone glucuronidation activity was not induced in any offspring by TCDD treatment during gestation. Newborn rats exposed to TCDD only by consumption of milk of treated foster mothers and newborn rats exposed only in utero exhibited elevated enzyme levels, comparable and intermediate between control and continuously exposed rats, respectively.' The authors concluded that multiple factors contribute to TCDD-induction of neonatal enzymes. 616. Luning, K. G., and Ryman, N. (1975) Comments on tests of dominant lethals in mice. Mutat. Res. 31:127-128. [Editorial.] 220 �617. Luster, M. I., Albro, P. W. , Faith, R. E., and Lawson, L. D. (1978) Inability of passive antibodies to reverse the effects of dioxin toxicity. Chemosphere 7(l);29-34. Immunization with antibodies raised against protein-bound TCDD was studied as a means of protection against the acute effects of TCDD in the mouse. l-Amino-3,7,8-trichlorodibenzo-p-dioxin (TriCDD-NH.) conjugated to bovine serum albumin or bovine thyroglobulin and 1-amino-TCDD diazo-linked to human serum albumin were injected into rabbits. Sera were assayed by immunodiffusion analysis for antibodies and complement-inactivated. Gamma globulin was then isolated by ion exchange chromatography, dialyzed, ultrafiltered, and administered intramuscularly to male C57B1 mice. A single dose of 250 ug/kg TCDD (99+% purity) in acetone-corn oil (1:6) by gavage. The immune rabbit globulin (IRG) administered had the capacity to bind 44% of the total TCDD administered and TCDD competed as well as triCDD-NH, for displacement of binding of radioactive antigen with antibody. Mortality rates and body weights were recorded for all mice. Both of these parameters were the same for mice treated with IRG 1 day prior to TCDD IRG 1 day after TCDD, saline plus TCDD, or normal rabbit globulin plus TCDD. Substantial weight loss and the same time course for lethal effects occurred in each group. Control mice treated with normal globulin plus corn oil survived and gained weight. The authors proposed specific technical difficulties as well as tight TCDD-receptor binding as explanations for the failure of immune globulins to protect against TCDD toxicity. Less than half of the dose of TCDD maximally could bind the administered antibody and inactivation by metabolism during the 24 hours between administration of antigen and antibody could have caused the negative effect, invalidating any conclusions regarding mechanisms of TCDD toxicity. 618. Luster, M. I., Clark, G., Lawson, L. D,, and Faith, R. E. (1979a) Effects of brief in vitro exposure to 2,3,7,8 tetrachlorodibenzo-pdioxin (TCDD) on mouse lymphocytes. J. Environ. Pathol. Toxicol. 2:965-977. The effects of in vitro TCDD exposure of mouse spleens on lymphocyte functions are described. Female B,C,F1 mouse spleens were submerged in a solution of TCDD (99+% purity), I C]-TCDD (99% purity), amino-3,7,8trichlorodibenzo-p-dioxin (Tri CDD-NH-) or 3,4,3', 4'-tetrachlorobiphenyl (TCB) in dimethylsulfoxide (DMSO) for 10 sec. Cell cultures were prepared from the treated tissue and cell viability was monitored by a trypan blue exclusion technique. Lymphocyte responses to mitogens (phytohemagglutinin, conconavalin A and lyopolysaccharide mitogen) were assessed by determining the treated to control ratio of radioactive precursor incorporation into RNA and protein. Tissue incorporation of radioactivity from [ C]-TCDD was determined in some spleens and binding of [ H]-conconavalin A (Con A) to TCDD-treated splenic lymphocytes was assessed in vitro. Total uptake of TCDD ranged from 0,29 ng per spleen after 5-10 sec. of immersion to 0.66 ng after 60 min. Compared to non-treated controls, DMSO treatment reduced thymidine, leucine and uridine incorporation in response to mitogens. Immersion of spleen in 221 �5-50 ng TCDD/ml DMSO reduced these rates of incorporation below vehicle controls for all 3 mitogen treatments. A decrease in cell viability was observed in TCDD-treated cultures after 48 hours, but the effect was not dose-dependent. Neither Tri CCD-NH« nor TCB produced statistically significant decreases in DNA synthesis of Con A-treated lymphocytes. No decrease in Con A binding or histological changes by light or electron microscopy were observed in TCDD-treated spleens. The authors concluded that DMSO-solubilized TCDD produced a direct effect on splenic lymphocyte proliferative response to mitogens, especially T-lymphocyte mitogens. 619. Luster, M. I., and Faith, R. E. (1979) Assessment of immunologic alterations caused by halogenated aromatic hydrocarbons. Ann. N.Y. Acad. Sci. 320:572-578. [Review article.] 620. Luster, M. I., Faith, R. E. , and Clark, G. (1979b) Laboratory studies on the immune effects of halogenated aromatics. Ann. N.Y. Acad. Sci. 320:473-486. [Review article.] 621. Lutz, H., and Lutz-Ostertag, Y. (1973) Pesticides, teratogenesis and survival in birds. Arch. Anat. Histol. Embryol. 56:65-78. The effects of spraying 2,4-D on fetal mortality and hatchability of several species of birds is described. Three preparations of 2,4-D (amino salt) were used in this study. Lot H (purity and other ingredients not reported) in water was sprayed at the rate of 880 g/hectare on red partridge and gray partiridge eggs before incubation and on pheasant and quail eggs before, or 3 or 7 days after incubation started. Lot W (purity and other ingredients not reported) in water was sprayed on group of quail eggs before incubaton at 2 rates, 880 and 110 g/hectare. A purified lot (99.1% purity) of 2,4-D dimethylamine salt in water was sprayed on pheasant and red partridge eggs and before incubation at the rate of 1200 g/hectare. Impurities in this preparation included other phenoxyalkanoic acids and 400 ppm 2,4dichlorophenol. Control quail and pheasant eggs were sprayed with water. Mortality prior to hatching and the proportions and types of extermal malformations in chicks at hatching were recorded. Treatment and control groups contained 100-350 eggs, except for 1 group of 632 pheasant eggs sprayed on day 0 with lot H. Lot H produced 74-75% mortality of pheasant eggs and 43-77% mortality of all other eggs prior to hatchng, compared to 6% for control quail eggs and 46% for control pheasant eggs. From 36-42% of the quail and pheasant eggs treated before incubation and 22-30% of eggs treated on day 3 or 7 with lot H hatched normally. Malformations included lordosis, shriveled feet, and paralyzed toes and occurred in 12-13% of all quail groups and up to 4% of the other groups treated with lot H. Mortality of quail embryos 222 �sprayed with the higher dose of lot W was 17% 2 days before hatching and 13% for the lower dose 3 days before hatching. The rate of hatching was 71-72% for both groups and cell hatched chicks were normal. The purified preparation resulted in 26-30% mortality before hatching. The authors presented a review of a previous study from their laboratory on 2,4,5-T and studies on other pesticides. The authors concluded that only lot H was teratogenic and did not attributed this toxicity to 2,4'-D because the other lots were unable to produce teratogenicity. 622. Lutz, J. F., Byers, G. E., and Sheets, T. J. (1973) The persistence and movement of picloram and 2,4,5-T in soils. J. Environ. Qual. 2(4):485-488. The authors describe the persistence and movement of 2,4,5-T and picloram in three soil types: clay loam (Fannin), fine sandy loam (Chandler), and loam (Chester). Experimental plots on each of the three soil types were sprayed with 2.24 kg/ha 2,4,5-T plus picloram. Four additional plots received 4,48 kg/ha herbicide. Plots were 0.04 acre in area, with an average slope of 27%, and vegetation consisted of orchard grass. Soil core samples (20 per plot) were collected immediately after spraying and 15, 50, and 100 days after spraying. Samples were analyzed by gas chromatography (detection limit 1 ppb picloram, 3 ppb 2,4,5-T). Approximately 90% of the 2,4,5-T and 60% of the picloram disappeared 15 days after application. By 100 days, 2,4,5-T had essentially disappeared, while about 10% picloram remained. Results were similar for both spraying levels. For all soils treated with 2.24 kg/ha, 68% or more of the total picloram remaining could be found in the top 7.5 cm of soil. Less than 10 ppb 2,4,5-T was detected at soil depths greater than 7.5 cm. At 4.48 kg/ha, more picloram but not more 2,4,5-T penetrated to the 15-45 cm depths than at 2.24 kg/ha. Both herbicides disappeared most rapidly from Fannin soil and least rapidly from Chandler soil. Little downslope movement of herbicide was observed, possibly, according to the authors, because of the high hydraulic conductivity of the soil resulting in very low runoff. The authors made no conclusion about the overall effects of these herbicides on the ecosystem under study. However, it appears that neither herbicide is very persistent or mobile in soil. 623. Lynn, G. E. (1965) A review of toxicological information on TORDON herbicides. Down to Earth 20(4);pp. 6-8. [Review article.] 223 �624. MacKay, D., and Leinonen, P. J. (1975) Rate of evaporation of low-solubility contaminants from water bodies to atmosphere. Environ. Sci. and Techno 1. 9:1178-1180. [Background material.] 625. Maddy, K. T., and Edmiston, S. (1976) Summary of occupational illnesses and injuries reported by physicians as due to exposure to pesticides of workers cleaning and/or repairing pesticide handling machinery. California Dept. of Food and Agriculture, Report No. ACF 59-300. 5 pp. [Background material.] 626. Maddy, K. T., Peoples, S. A., and Tochilin, S. J. (1976) Occupational illnesses due to exposure to pesticides or their residues as reported by physicians in California in 1975. California Dept. of Food and Agriculture, Report No. ACF 59-202. 17 pp. [Background material.] 627. Madge, D. S. (1977) Effects of trichlorophenoxyacetic acid and chlorodioxins on small intestinal function. General Pharmacology 8(5-6):319-324. Absorption of D-glucose was studied in everted gut sacs of mice treated with TCDD or 2,4,5-T in vivo. CD-I mice were administered a single dose of 2,4,5-T methyl ester (99.6% pur.ity) or TCDD in corn oil by oral intubation or vehicle only. From 1-28 days later the small intestine was removed and everted sacs were prepared and incubated with test sugars or amino acids. Small intestines were examined histologically 7 days after treatment with TCDD, 2,4,5-T or vehicle. TCDD elicited slight histopathological damage while no other compounds produced any change in gut histology. 2,4,5-T at doses of 25-250 mg/kg and TCDD from 10-75 ug/kg produced dose-dependent decreases in D-glucose absorption. Changes in fluid transfer by all treatments were minor. Maximum decrease in glucose absorption occurred 14 days after 2,4,5-T treatment. Neither TCDD nor 2,4,5-T treatment altered galactose, L-histidine or L-arginine transfer 7 days later. The decreases in D-glucose transport were eliminated by adding an exogenous energy source, D-mannose to serosal fluid or by adding D-maltose which metabolizes glucose. D-glucose metabolism by the gut was high from the levels of accumulated D-glucose in treated intestine. The author concluded that 2,4,5-T or TCDD treatment enhanced D-glucose metabolism by the gut during transfer, without affecting the transfer mechanism, itself. 224 �628. Magnusson, J., Rarael, C., and Eriksson, A. C1977) Mutagenic effects of chlorinated phenoxy acids in Drosophila melanogaster. Hereditas 87:121-123. The authors investigated the induction of sex linked recessive lethals, nondisjunction, and chromosome loss with 2,4,5-T and 2,4-D. 2,4,5-T was tested in two different forms: purified 2,4,5-trichlorophenoxyacetic acid containing less than 0.1 ppm TCDD and 2,4,5-T butoxyethyl ester containing 1 ppm TCDD dissolved in petroleum ether and with an unspecified emulsifier. A purified preparation (unspecified) of 2,4-D was also tested. In all experiments, each compound was dissolved in ethanol (amount unspecified) and mixed in the usual corn agar growth medium. In the chromosome loss and nondisjunction tests, males and females were treated during their entire larval period with 250 ppm 2,4,5-T butoxyethyl ester or 100 ppm 2,4-D. No increases in chromosome loss or nondisjunction were induced by 2,4,5-T or 2,4-D. However, only 1 dose of each of these compounds was tested. For the recessive lethal test both the dose of each herbicide and type of 2,4,5-T were changed. Therefore, no comparison can be made between the two phases of the experiment. In the recessive lethal test adult wild type Karsnas 60 strain males were treated with 1,000 ppm 2,4-D or 2,4,5-T containing less than 0.1 ppm TCDD. After 2 weeks of treatment males were mated to Muller 5 strain females (age and number unspecified). No effects of these chemicals on successive broods were studied. A total of approximately 13,000 individuals in the combined F, and F« generations were observed per chemical tested; 10,000 individuals from the control group were scored. When F, and F results were combined both 2,4-D and 2,4,5-T induce significant increases (p less than 0.01) in recessive lethals. However, when data from each generation were tabulated separately only 2,4-D induced a statistically significant increase in recessive lethals in F-. This is an indication that 2,4-D gives rise to lethal mosaics. 629. Mahle, N. H., Higgins, H. S., and Getzendaner, M. E. (1977) Search for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin in bovine milk. Bull. Environ. Contam. Toxicol. 18(2):123-130. The authors analyzed milk samples from dairy and beef cows for TCDD contamination. Twenty-five milk samples were obtained from cows in Missouri, Arkansas, and Oklahoma grazing on pasture that was treated with 2,4,5-T for brush control. Sample collections occurred 5 days to 48 months after spraying had occurred. Exact spray formulations and quantities could not be documented. Milk was analyzed by GC-MS (detection limit 1 ppt). Control samples were obtained from a local supermarket. No TCDD was detected in any of the samples. 225 �630. Maiback, H. I., and Feldman, R. (1974) Appendix P: Systemic absorption of pesticides through the skin of man. In: Occupational Exposures to Pesticides, A Report to the Federal Working Group on Pest Management from the Task Group on Occupational Exposure to Pesticides, Washington, DC. pp. 120-127. The extent of absorption of 2^4-D and diquat were studied in human male volunteers. C-2,4-D and C-diquat were applied to the forearms of groups of six men and absorption was calculated from the urinary excretion of radioactivity over the 5 day period following exposure. The extent of urinary excretion of the compounds, estimated from excretion of intravenously administered C-2,4-D and C-diquat, was also used to calculate absorption. (The urinary excretion rates after intravenous., administrat ion of the. compounds were not reported). A mean of 5.8% of C-2,4-0 and 0.4% of C-diquat was absorbed. Occlusion of the exposed skin for 24 hours, increased penetration of C-2,4-D to 14.7% of the dose and of C-diquat to 1.4% of the dose, based on 5 day urinary excretion rates. Skin damaged by removing the stratum corneum with cellophane,tape absorbed 33.8% of the dose of C-2.4-D and 3.8% of the dose of C-diquat. Penetration of C-2,4-D was decreased by washing the forearm for 2 minutes with soap and water to 0.5% of the dose when washed one minute after 2,4-D was administered and 3.7% when washed after 4 hours. Absorption of 10 other pesticides was also studied under various experimental conditions. 631. Mailman, R. B., and Hodgson, E. (1972) The cytochrome P-45Q substrate optical difference spectra of pesticides with mouse hepatic microsomes. Bull. Environ. Contam. Toxicol. 8: (3):186-192. The types of difference spectra produced from binding of pesticides with microsomal enzymes was identified. Hepatic microsomes from inbred mice (from the North Carolina Department of Health) were prepared and incubated with each herbicide. The optical difference spectra of the pesticide-exposed microsomes relative to a control sample of microsomes without pesticide added was recorded and characterized as type I with the absorption minimum at 416-420 nm and maximum at 385-390 nm or type II, with the minimum at 390-410 nm and maximum at 425-435 nm. The spectral size reported for each pesticide was calculated as the difference in optical density between the peak and trough of the spectrum, and was reported as a proportion of the P-450 optical density. Diuron, bromacil, picloram, and 2,4-D all produced type I difference spectra with relative spectral sizes of .126, .004, .016, and .024, respectively. The absorbance of diquat (or a metabolite) obscured its binding with microsomal enzymes. The binding of a total of 51 compounds were characterized in this system. The authors concluded that aromatic compounds generally cause type I binding while compounds with an unhindered nitrogen atom produce type II binding. 226 �632. Majuradar, S. K., and Golia, J. K. (1974) Mutation test of 2,4,5trichlorophenoxyacetic acid on Drosophila melanogaster. Can. J. Genet. Cytol. 16:465-466. The authors studied the ability of 2,4,5-T to induce recessive lethals in Drosophila melanogaster. Recessive lethals were detected by the Muller-5 technique (BASC-method) in which two-day-old Oregon R male flies were fed 250 or 1,000 ppm 2,4,5-T for 15 days. Negative control flies received only their usual diet while positive controls received 250 ppm ethyltnethane sulfonate. The stock of 2,4,5-T used in this study contained no detectable dioxin (detection limits not specified). After treatment males were mated with BASC females and kept in vials containing medium without 2,4,5-T. Three 4-day matings were conducted to determine the effects of 2,4,5-T on the spermatic cycle of the males. At the end of each 4-day mating period, males were placed in bottles containing new females. The absence of wild type males in the F2 generation of flies was used as the end point for induction of recessive lethals. The authors reported that 2,4,5-T induced a statistically significant increase in recessive lethals at 1,000 ppm but not at 250 ppm, according to the significance test of Kastenbaum and Bowman. Results seemed to be pooled for all three matings, so that no conclusions can be drawn on the sensitivity to 2,4,5-T of various portions of the spermatic cycle in Drosophila. 633. Majumdar, S. K., and Hall, R. C. (1973) Cytogenetic effects of 2,4,5-T on i" vivo bone marrow cells of Mongolian gerbils. J. Hered. 64:213-216. The authors studied the effects of 2,4,5-T on chromosomes of Mongolian gerbil bone marrow cells. Male and female 50 to 80-day-old animals received 5 daily intraperitoneal iniections of 10, 30, 50, 70, or 100 mg/kg 2,4,5-T dissolved in dimethyl sulfoxide, a total dose of 50, 150, 350, and 500 mg/kg respectively. The authors reported that the 2,4,5-T used in these experiments contained no measurable amount of TCDD but did not report the detection limits of their chemical analysis. Ten animals were treated at each dose level except at 100 mg/kg where 17 animals were treated because of low mitotic index. Water and DMSO negative control groups were included in the study, but no positive control group was reported. Twenty four hours after the last injection and 2 hours after an intraperitoneal injection of 0.2 ml of a 0.4 mg/ml solution of Colcemid, the animals were killed and chromosome spreads prepared from bone marrow cells. Metaphases (500 per treatment group) were scored for chromatid gaps, chromatid breaks, and fragments. Percent abnormal metaphases were analyzed statistically by the t-test. The authors did not state whether scoring took place by a double-blind technique to minimize bias on scoring. No increases in chromosomal aberrations were observed up to 150 mg/kg dose level. At 250 mg/kg, chromosome damage (chromatid gaps and breaks) increased significantly. No significant differences in fragments were observed. No comparison of the 500 mg/kg dose and controls was reported. The authors also statistically analyzed using the t-test the total number of abnormal metaphases per dose group. Up to 150 mg/kg, no significant differences 227 �were observed between control and treated groups. Groups receiving 250 mg/kg, 350 mg/kg, or 500 mg/kg showed statistically significant increases in abnormal metaphases. No analysis of aberrations based on the sex of the animals was presented. 634. Malcolm, G. N. (1977) 2,4,5-T and human birth defects. New Zealand Institute of Chemistry, Bulletin No. 11, Sept. 29, 1977. [Editorial.] 635. Malizia, E., Andreucci, G. , Chiavarelli, M. , Amato, A., and Gagliardi, L. (1979) A follow up of 20 months in Seveso, an environmental calamity. Vet. Hum. Toxicol. 21(Supplement):139-140. [Review article.] 636. Mailing, H. V., and Wassom, J. S. Action of Mutagenic Agents. In Handbook of Teratology, Wilson, J. G., Fraser, P., eds. (New York: Plenum Press, 1977) pp. 99-152. [Review article.] 637. Manis, J., and Apap, R. (1979) Intestinal organic anion transport, glutathione transferase and aryl hydrocarbon hydroxylase activity: Effect of dioxin. Life Sciences 24(15):1373-1380. The effects of TCDD on intestinal transport of iron penicillin, and para-amino-hippurate and hepatic and intestinal glutathione-Stransferase (GSH) and aryl hydrocarbon hydroxylase activities were studied in the rat. Male Sprague-Dawley rats were administered 17 ug/kg TCDD in dioxane orally 2 days before sacrifice or intraperitoneally 1-14 days before sacrifice. Everted gut sacs were prepared from the duodenum to measure iron transport and from, the mid-intestine to.measure penicillin transport. ^FeSO, or f C]-benzyl penicillin or [ C]-aminohippurate transport was evaluated by introducing the radiolabeled chemical to one side of the gut and measuring the radioactivity on each side after a 2 hour incubation period at 37°C. GSH-T activity was measured in supernatants from 100,000 xg centrifugation of tissue homogenates. AHH activity was measured in crude liver homogenates with benzo(a)pyrene as substrate. Serosal transfer of iron was increased by 38% in gut sacs from rats given TCDD orally, compared to vehicle controls, while mucosal transport of iron, transport of penicillin, and intestinal GHS-T activity remained unchanged. Hepatic GSH-T activity increased by 70% and AHH activity by 20-fold from TCDD treatment. Intestinal AHH activity increased 100-fold after oral and intraperitoneal treatments with TCDD. No change in penicillin or para-aminohippurate uptake, or GSH-T activity were observed in intestines of rats pretreated with TCDD 1-14 days previously. Hepatic GSH-T levels were elevated 1.5-2.0-fold 228 �4 and 11 days after pretreatment but no significant elevation in activity was observed 1 or 14 days after pretreatment. The authors concluded that TCDD selectively inhibited 2 intestinal mechanisms (iron transport and AHH activity), without altering other systems, as GSH-T activity which is sensitive to stimulation by other inducers. 638. Manis, J., arid Kim, G. (1979a) Stimulation of iron absorption by various polyhalogenated aromatic hydrocarbon environmental contaminants. Bioch. Pharmacol. 28(18) :2841-2843. The effect of 2,4,5-T on in vitro intestinal iron transport was studied in the rat. The report also included results of 14 other polyhalogenated aromatic hydrocarbons on in vitro and in vivo iron transport. Male Sprague-Dawley rats were administered 100 mg/kg 2,4,5-T in dioxane by gastric intubation 1 day prior to excising the intestine. -Everted duodenal sacs were prepared and incubated in medium containing FeSO,. The radioactivity inside and outside of the sac and associated with gut tissue were determined at the end of the incubation period and net serosal transfer and mucosal transfer of iron was calculated. Mucosal uptake of iron was increased significantly in gut sacs from 2,4,5-T treated rats compared to transfer in sacs from vehicle treated controls, while no alteration in serosal transfer was observed. The authors suggested that stimulated iron transport from exposure to certain polyhalogenated hydrocarbons, including 2,4,5-T, could pose health hazards related to changes in iron absorption and metabolism. 639. Manis, J., and Kim, G. (1979b) Stimulation of iron absorption by polychlorinated aromatic hydrocarbons. Am. J. Physiol. 236(6):E763-E768. The effects of TCDD on intestinal transport of iron, calcium, galactose and proline and hepatic and intestinal aryl hydrocarbon hydroxylase (AHH) were studied in the rat and mouse. Male Sprague-Dawley rats were pretreated with TCDD in dioxane by intraperitoneal injection or gastric intubation. Pretreated rats showed normal weight gain (compared to controls) and none of the pretreated rats died from TCDD treatment. Iron absorption was studied in vivo by anesthetizing the rat and tying off a loop of the duodenum. The loop was filled with medium containing FeSO, and after 30 minutes the radioactivity in the luminal fluid gut and in the gut wall was determined. Transfer of iron from the lumen into the mucosa (mucosal uptake) was calculated as the amount of isotope lost from the lumen during the 30 minute experimental period and the amount of iron transferred to the bloodstream was calculated as the difference between mucosal uptake and the isotope in the excised gut wall. Everted gut sacs were also prepared from sections of duodenum and were used to study in vitro iron transport. AHH activity was assayed in liver and intestine from rats and mice. Transfer of iron to the blood was increased 41%-67% by pretreatment of rats with 33 ug/kg of TCDD by either route. Mucosal uptake was affected minimally. Serosal transport was increased 100-108% by everted guts of rats 229 �pretreated 2 days previously with 22-42 ug/kg TCDD by either route, while mucosal transport was nonaffected. The effect was diminished when pretreatment occurred longer than 2 days prior to removing the gut. Serosal transfer of calcium was decreased 26% and mucosal uptake by 11% in TCDD in everted sacs from pre-treated rats, while galactose and proline transport were not effected. Everted gut sacs from TCDDpretreated mice showed 78-156% increases in iron transport over control levels. AHH activity was induced 20-fold in mouse liver, and 100-1000-fold in mouse intestine by doses of 31-124 ug/kg TCDD. In the rat a 200-fold increase in intestinal AHH levels occurred at 10 ug/kg TCDD, a dose that failed to alter iron transport. The authors concluded that TCDD selectively affects discrete intestinal mechanisms for transport of specific compounds and is not a general metabolic effect. The gut transport effect of TCDD is not mediated by enzyme induction solely because these 2 effects occur at different doses. 640. Manis, J., and Kim, G. (1979c) Introduction of iron transport by a potent inducer of aryl hydrocarbon hydroxylase, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch. Environ. Health 34(3):141-145. The effects of TCDD on intestinal iron transport and aryl hydrocarbon hydroxylase (AHH) activity were studied in the rat. Male SpragueDawley rats were given 67 ug/kg of TCDD in dioxine by gastric intubation 2-4 days prior to excising various sections of the gut. Everted gut sacs were incubated at 37°C in the presence of Fe-iron sulfate and the transfer of radioactivity into or out of the sacs were determined by liquid scintillation counting of the bath medium and fluid within the sac. The AHH activity of the mucosal layer was measured using benzo(a)pyrene as substrate. In everted duodenal sacs from TCDD-pretreated rats, 50% more iron was transferred serosally than by sacs from either non-treated controls or vehicle controls. This TCDD effect did not occur in gut sacs prepared from more distal segments of the intestine. AHH activity was significantly elevated in the duodenum as well as the adjacent distal section of TCDD-treated rats compared with the vehicle controls. After a dose of 17 ug/kg of TCDD, the increase in serosal transfer of iron fell to 35% above controls 2 days after TCDD treatment and was not significantly increased statistically after 4 days. Hepatic GSH-transferase levels were significantly elevated to 70% and 102%, 2 and 4 days after 17 ug/kg of TCDD was given orally, compared to vehicle controls. Two to 4 days after an intraperitoneal dose of 17 ug/kg TCDD was given, no changes in iron transport occurred, while hepatic GSH-transferase remained elevated to 80% above control levels for 2 weeks after TCDD was injected. The authors concluded that the locus for iron transport stimulation is more accessible to TCDD from the mucosal surface than the serosal surface of intestinal cells. 230 �641. Mantovani, A., Vecchi, A., Luini, W., Sironi, M., Candioni, G. P., Spreafico, and Garratini, S. (1979) Effect of 2,3,7,8 tetrachlorodibenzo-p-dioxin on macrophage and natural killer cell mediated cytotoxicity in mice. Institute di Ricerche Farmacologiche "mario negri" via Enitrea 62-2, Milano, Italy. The effects of TCDD on cellular immunity was studied in mice. Target cells were prelabled with H-thymidine and the ability of peritoneal macrophages from TCDD-treated mice to lyse these cells was determined. The effect of TCDD treatment on the cytostatic and cytocidal activity of macrophages against tumor cells after endotoxin stimulation was also measured. TCDD stimulation of peritoneal exudate cells was determined by measuring the number of nucleated cells and the proportion of polymorphs in this cell population over 47 days after treatment. The effect of spleen natural killer cells in producing lymphoid cytotoxicity was evaluated in vitro. The effect of TCDD on H-thymidine uptake of lymphoma TCDD did not affect the functional status of macrophages or natural killer spleen cells in any of these studies. In TCDD-treated mice, the total numbers of spleen cells and macrophages recovered were diminished. The authors suggested that TCDD effects on cells that are important in in vivo resistance against infection and neoplasia may lead to impairment in these functions. 642. Manzo, L., Gregotti, C., Di Nucci, A., and Richelmi, P. (1979) Toxicology of paraquat and related bipyridyls: biochemical, clinical and therapeutic aspects. Vet. Hum. Toxicol. 21(6) :404-410. [Review article.] 643. Marriage, P. B., Saidak, W. J., and Von Stryk, F. G. (1975) Residues of atrazine, simazine, linuron, and diuron after repeated annual applications in a peach orchard. Weed Res. 15(6):373-379. [Not available.] 644. Martin, J. V. (1979) 2,4,5-T. Lancet p. 1243. [Editorial.] 645. Martinez-Cabruja, R. (1976) Liver response and other effects of tetrachlorodibenzo-p-dioxins. Arch. Farmacol. Toxicol. 2(3):211-234. [Not available.I 231 �646. Mason, R. W. (1975) Binding of some phenoxyalkanoic acids to bovine serum albumin in vitro. Pharmacology 13(2):177-186. ( A high affinity hydrophobic binding site on bovine serum albumin and several sites with lower affinity for phenoxy-acids are described. Bovine serum albumin (BSA) was combined with 2,4-D or 2,4,5-T in vitro at 37°C or 23°C and the extent of binding was assessed by ultrafiltration and presented as Scatchard plots. Affinity constants were calculated from the Scatchard plots. Absorption peaks were determined for difference spectra of mixtures of 2,4-D or 2,4,5-T with cetrimide or BSA. The differential spectral constant for 2,4,5-T was greater than for 2,4-D. The binding properties of other phenoxy acids were studied. The author concluded that BSA contained a hydrophobic binding site with high affinity for phenoxy acids and compared this site to a binding site on human serum albumin implicated in drug binding. 647. Matsumura, A. (1970) The fate of 2,4,5-trichlorophenoxyacetic acid in man. Jap. J. Environ. Health 12:20-25. The pharmacokinetics of 2,4,5-T was described. Three male volunteers ingested 100-150 mg of 2,4,5-T and either blood or urine was collected from each subject periodically during the subsequent 72 hours. Levels of 2,4,5-T in urine of workers of a chemical manure factory and in the workplace were assayed. Plasma levels of 2,4,5-T, plotted as a function of time, showed a biphasic pattern and each of the 2 components, which reflected absorption and excretion, showed first order kinetics. Absorption was rapid, since the maximum plasma concentration was reached 4 hrs. after a single oral administration. The half-life for plasma clearance was 11 hr. About 80% of the dose ingested by volunteers was excreted in the urine in 72 hrs. No metabolites were detected in gas chromatograms of urine samples. Workers excreted up'to 3.6 mg of 2,4,5-T in urine daily; the breathing zone in the factory contained 0.2-0.7 mg/m 2,4,5-T, while components of the factory apparatus contained up to 15 mg/m of 2,4,5-T. The volume of distribution for 2,4,5-T, 8% of the body weight, approximated the total blood volume. The authors concluded that intake of 2,4,5-T by humans can be established by urinalysis, because almost all of the dose is gradually excreted into the urine unchanged. 648. Matsumura, F., and Benezet, H. J. (1973) Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Environ. Health Perspec. 5:253-258. The authors measured the bioaccumulation of TCDD and three other pesticides in three model aquatic ecosystems and its microbial degradation in 100 species of microorganisms known to degrade persistent pesticides. Bioaccumulation of ring-labeled C-TCDD was measured in Ostracoda (algae) species, brine shrimp, mosquito larvae, and northern brook silverside fish. Numbers of organisms used were not specified. In the first model ecosystem 5-10 pmole TCDD was added directly to water, food, and primary organisms, and this mixture was 232 �then added to the aquarium containing invertebrate test organisms. Final concentrations in the test organisms of 1,592, 1,956, and 7,069 ppb TGDD, were observed in daphnids, brine shrimp, and algae respectively. In the second model ecosystem, TCDD (20 pmole ppb) was applied to the inner surface of a container containing the test organism food source. Twenty-four hours later the primary food organisms were transferred to an aquarium containing the test invertebrates. Final concentrations of 279 and 879 ppb TCDD were found in algae and daphnids, respectively. Flaws in these two models are extensive: the compounds were used above the limit of water solubility, and the extent of direct pick-up from partitioning and food intake is uncertain. To correct for the solubility problem, a third model ecosystem was used. TCDD (1.62 (u)g) was added to Ig sand; the solvent was evaporated, and the sand was added to a test aquarium containing invertebrates and fish. Final concentrations of TCDD were 2, 157, and 4,150 ppb in fish, brine shrimp, and mosquito larvae, respectively. Tn a two-step bioaccumulation experiment, mosquito larvae were exposed to TCDD (1.62 (u)g) and were then added to an aquarium containing fish. The TCDD concentration in fish increased to 708 ppb but in the mosquito larvae TCDD remained nearly the same as the previous experiment. The length of time of the study was not reported. In additional testing, TCDD was microbially degraded by only 5 of the 100 species tested, and TCDD (0.1 (u)mole) translocation from sand to organic soil or water was observed to be nearly nonexistent in a 10 x 1.5 cm glass column. 649. Matsunaka, S., and Kuwatsuka, S. (1975) Environmental problems related to herbicidal use in Japan. Environ. Qual. Saf. 4:149-159. [Review article.] 650. Matsushita, T., Arimatsu, Y., Misumi, J. , Tomio, T., and Nomura, S. (1975) Skin disorders caused by herbicides sodium chlorate and sodium 2,2-dichloropropionate. Kumamota Med. J. 28(4):164-169. One case report and a field study of 76 workers exposed to the herbicide 2,2-dichloropropionate are described. A 42-year-old man had been engaged in spraying sodium chlorate and phenoxy herbicides for 5 years when he began to spray the herbicide, sodium 2,2-dichloropropionate. Within a few days he developed contact dermatitis on the face and hands and nausea. A patch test of 0.1% sodium 2,2-dichloropropionate showed mild erythema after 24 hours. A moderately high blood sugar level was the only notable clinical finding (but other tests that were conducted were not reported). A group of 76 forest workers (mean age, 40.8 years) who sprayed herbicides about 10-20 days per year and were employed in this occupation for 6-9 years (mode of work period of the group) were compared with 16 control workers from the same area. The number of men in the group who were exposed to sodium 2,2-dichloropropionate was not specified, although this herbicide and sodium chlorate were used by at least some workers in the group. Clinical blood chemistry tests, hematology tests, urinalyses, and dermatologic tests were .performed and reports of other symptoms 233 �were obtained by questionnaire and by interview. Nausea, skin lesions, and copious sxtfeating were reported significantly more frequently by exposed workers than controls. The symptoms that were reported to be associated with sodium 2,2-dichloropropionate exposure were nausea, skin lesions, anorexia, and pain in the throat; the size of the group or incidences of these symptoms were not given. No differences in the results of patch tests for 2,2-dichloropropionate or in clinical findings were observed in the exposed group, compared to the control group. Contact dermatitis was diagnosed in 6 cases out of the group of 76 workers and symptoms were described as mild. A case of leucomelanoderma in a worker exposed to sodium chlorate was also described. The authors concluded that an allergic reaction to 2,2-dichloropropionate occurred only in the single case report presented, and warranted precautions in handling this herbicide. 651. Matthews, H. B., and Kato, S. (1979) The metabolism and disposition of halogenated aromatics. Ann. N.Y. Acad. Sci. 320:131-137. [Review article. ] 652. May, G. (1973) Chloracne from the accidental production of tetrachlorodibenzodioxin. Br. J. Ind. Med. 30:276-283. This report describes the health effects in workers at a factory in Derbyshire, England, that manufactured 2,4,5-T from trichlorophenol. In 1968, the building was closed after an exothermic reaction caused an explosion which killed one worker. Eleven of the 14 workers in the building at the time of the explosion were affected, as evidenced by abnormal liver function tests or abnormal white cell counts. Within 10 days these test results were within normal limits, and the factory was reopened, with the damaged areas sealed off. During the next 8 months, 79 cases of chloracne developed, primarily in maintenance workers, who did not use gloves to handle factory equipment. TCDD was identified as the causative agent. The most severe cases started as malar erythema. All cases involved the face; the extremities and trunk were also involved in some cases. Conjunctivitis accompanied the early stages of the condition. Most cases resolved within 4 to 6 months of treatment with steam, ultraviolet light, and lotions. No systemic symptoms developed in these workers. The building was finally closed, and heavy equipment was dismantled and buried or repeatedly cleaned with steam. An incident in Germany, involving dioxin exposure and an explosion, was also described. Many of the exposed workers developed chloracne and severe liver damage. 653. Mazarean, H. H., Dux, L., and Cuba, F. (1979a) Changes of metabolism during experimentally induced myotonia of rats I. Alterations in lactate and malate dehydrogenase isoenzyme activities. Biochem. Med. 22(3):350-358. Alterations in several enzymes involved in energy production were measured in several muscles of rats after 2,4-D administration. Doses 234 �of 100 mg/kg of 2,4-D in aqueous sodium bicarbonate solution were administered intraperitoneally to rats daily for 14 davs. Controls were given sodium acetate. Myocardium, soleus (red muscle), and semimembranosus (white muscle) samples were removed, homogenized, and the supernatant after centrifugation (8,000 g for 20 minutes) was assayed spectrophotometrically for lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities. Isoenzymes were separated by polyacrylamide gel electrophoresis and the stained bands were quantitated by densitometry. The total LDH activity was not altered in any muscles by 2,4-D treatment. The density of the isoenzyme band, LDH 5 was elevated significantly in the semimembranosus of treated rats while LDH 4 was decreased. The LDH 5 band for the m. soleus muscle was increased in density while the LDH 1, 3, and 4 bands were decreased after treatment. Myocardial LDH 4 and 5 decreased while LDH 1 increased after treatment. The ratio of H subunits, calculated from the proportions of each LDH isoenzyme were significantly elevated in myocardium and decreased in the other two muscles after 2,4-D treatment. MDH activity decreased in all three myotonic muscles, with the largest change in the myocardium. The proportion of MDH associated with cytoplasm of the soleus and semimembranosus muscles was significantly decreased. The authors concluded that the elevation of LDH 5 isoenzyme during myotonia in muscles that favor anaerobic glycolysis represents an alternate pathway that is not active when the oxygen supply is normal. The decrease of H-related isoenzymes was less in soleus than in semimembranosus because the red muscle has a higher oxidative capacity from its extensive capillarization. The increased LDH 1 activity was suggested to be a secondary effect from the high circulating lactate level. The decreased MDH levels were attributed to the myotonic, the limitations of mitochondrial oxidative processes during hypoxia. The actual activities of isoenzymes have not been measured in these studies and are assumed to correlate with densitometry results from polyacrylamide gels to enable the authors to draw their conclusions that a shift to anaerobic energy production occurs in muscles after 2,4-D treatment. 654. Mazarean, H. H., Dux, L., and Cuba, F. (1979b) Changes of metabolism during experimentally induced myotonia of rats II. Alterations in creatine kinase and acid phosphatase activities: a possible mechanism in the development of muscle damage. Biochem. Med. 22(3):359-364. Enzyme changes in several types of muscles after 2,4-D administration were measured. Myotonia was induced in rats by 2,4-D administration (described in another report, Mazarean; 1979a). Muscle homogenates of m. soleus, m. semimembranosus, and myocardium were assayed for acid phosphatase and creatin kinase activities. After 2 weeks of treatment, the acid phosphatase activity was increased substantially in all tissues and the creatine kinase activity was about one-half of the original values for all three muscles. The authors concluded that the decrease in creatine kinase was proportional to the loss of muscle tissue through cellular damage and the increase in acid phosphatase reflects an increase in lysosomal damage accompanying induced myotonia. 235 �The authors proposed that prolonged muscle contraction and hypoxia of skeletal muscles damage the oxidative energy-producing system which stimulates the rate of anaerobic glycolysis. 655. Mazzocchi, P. H., and Rao, M. P. (1972) Photolysis of 3-(p-chlorophenyl )• 1,1-dimethylurea (monuron) and 3-phenyl-l,1-dimethylurea (fenuron). J. Agric. Food Chem. 20(5):957-959. The authors report on the photolysis of monuron under anaerobic conditions. A solution of 9 g monuron in 450 ml ethanol was irradiated for 84 hours in a bank of GE-G15 low pressure mercury lamps (2537A). Formation of photolysis products was measured by gas-liquid chromatography and thin layer chromatography. The following products were identified: 3-phenyl-l,1 dimethyl urea and methyl p-chlorophenyl carbamate. The anaerobic conditions of the experiment prevents extrapolation of these results to a field situation. 656. McCarthy, F. (1980) Statement of the Agent Orange Victims International before the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants, Sept. 22, 1980. 3 pp. [Testimony.] 657. McCollister, D, D., and Leng, M. L. (1969) Toxicology of picloram and safety evaluation of TORDON herbicides. Down to Earth 25(2):5-10. [Review article.] 658. McConnell, E. E., and McKinney, J. D. (1978) Exquisite toxicity in the guinea pig to structurally similar halogenated dioxins, furans, biphenyls, and naphthalenes. Toxicol. Appl. Pharmacol. 45:298. [Abstract, only.] 659. McConnell, E. E., and Moore, J. A. (1977) The toxicopathology of TCDD. Manuscript provided at Workshop TCDD, Milan, Oct. 23-24. [Not available.] 660. McConnell, E. E., Moore, J. A., and Dalgard, D. W. (1978a) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicol. Appl. Pharmacol. 43(1):175-87. The acute toxicity of TCDD was studied in monkeys. Female juvenile Rhesus monkeys (three per group) were administered by oral gavage a single dose of 70(u)g/kg or 350(u)g/kg TCDD (99+ percent purity) in 236 �corn oil or vehicle only. Body weights, blood chemistries, hematology and general appearance, appetite and behavior were monitored until death or until animals became moribund and were killed. Histological examination of an extensive number of tissues was performed, and raaior organ weights were recorded. Hepatic porphyrin levels were assessed quantitatively and qualitatively (by examining tissue for fluorescence under ultraviolet light). Monkeys in the high dose group died or were killed between days 28 and 34, and in the low dose group on days 14, 42, and 47. Monkeys in both treatment groups showed the same pattern of toxicity. TCDD-treated monkeys lost from 13 to 38 percent of their body weight. This loss began by day 3, recovered slightly (days 7-9), then fell continuously until death. Appetite was variable throughout the experiment, and water consumption fell at the end of the study. Blepharitis, loss of fingernails and eyelashes, and facial alopecia with acneiform eruptions progressed in treated monkeys. Hetnatologic changes included a trend toward increased neutrophils, and in the high dose group, lymphopenia and decreased platelet counts. Blood chemistry tests showed progressively decreased cholesterol levels to half of control values for the high dose group, decreased glucose concentrations starting on day 14, and decreased albumin levels by day 30. Elevated glutamic oxaloacetic transaminase, aldolase, and terminal blood urea nitrogen values were reported. The organ to body-weight ratios of the liver, adrenals, and kidneys, and the absolute heart and spleen weights were significantly elevated in both treatment groups. The thyraus weights, both absolute and relative, were lower in treated groups than in controls. The treated monkeys had no body fat, the bile ducts were distended with hyperplasia of the mucosal lining, and congestion of the intestine and depletion of lymphoid tissue, especially of the thymus, were observed. Multinucleated hepatocytes were observed in the liver of one monkey. No elevation of hepatic porphyrins was detected. Half of the monkeys showed epithelial hyperplasia of the renal pelvis. Gastrointestinal lesions, degranulation of the exocrine pancreas, and a mild loss of cellularity and increase in myeloid to erythroid cell ratio in sternal bone marrow were observed. The authors concluded that death was caused by starvation, which resulted from an inability to use consumed nutrients. No other lesions appeared to be severe enough to be lethal. 661. McConnell, E. E., Moore, J. A., Haseman, J. K., and Harris, M. W. (1978b) The comparative toxicity of chlorinated dibenzo-p-dioxins in mice and guinea pigs. Toxicol. Appl. Pharmacol. 44(2):335-356. The acute toxicities of various dioxins, including 2,3,7,8-TCDD were studied in the mouse and guinea pig. TCDD (98% purity) was administered by oral intubation in corn oil to male C57B1/6 mice and male Hartley guinea pigs. Controls were given corn oil. Treatment groups were comprised of 8 mice or 6 guinea pigs. After one animal died in each group, the remaining animals were killed after they became moribund. Serum proteins were fractionated by cellulose acetate electrophoresis. Histopathologic examination was performed on a number of tissues and the weights of major organs were determined. Porphyrins 237 �were detected by fluorescence of liver, skull, and teeth under ultraviolet light. The symptoms produced by the various dioxins were described together because all compounds produced the same effects in the same species and only the doses needed to elicit effects were different for the various dioxins. The LD50_3_ for 2,3,7,8-TCDD was 2 ug/kg in the guinea pig and 284 ug/kg in the mouse. A dose-related decrease in weight gain or weight loss occurred in both species. In guinea pigs, failure of individual weight stabilization within 7-10 days of treatment was associated with a poor prognosis. Dehydration and, near death, decreased food consumption were described in guinea pigs and death occurred within 17-20 days and by 12 days from higher doses. Exopthalmu's and distension of the abdomen occurred in mice and death occurred within 22-25 days and by 18 days from higher doses. Decreased thymus, testicle, and heart weights were reported, while the liver weights were higher after treatment in both species. Significantly decreased serum alpha-globulin levels occurred in the mouse only and fluorescence was observed in mouse tissues only. Hemolysis and hyperproteinemia were present in samples from moribund animals of both species. Histological changes in guinea pigs included a reduction of fat depots and muscle mass, sternal bone marrow hypocellularity, hypoplasia of the renal pelvis, and occasionally of the mucosa of the urinary bladder, adrenal hemorrhages and lesions, loss of seminiferous components of the testicles. In mice, changes included ascites and subcutaneous edema, hydrothorax, loss of the body fat, retro-orbital hemorrhage, and hepatic and testicular lesions. Intestinal hemorrhage and microcysts and thymic and splenic lesions occurred in both species. Depletion of lymphoid tissue was apparent in several tissues. Death was attributed to depletion of energy reserves and the reason why the usual metabolic sources did not replace the energy reserved was not known. 662. McCorkle, F. M., Chambers, J. E., and Yarbrough, J. D. (1977) Acute toxicities of selected herbicides to fingerling channel catfish, Ictalurua punctatus. Bull. Environ. Contain. Toxicol. 18(3)267-270. This paper describes the acute toxicity of 2,4-D and 2,4-D dimethylamine salt, dalapon, diuron, monuron, and 2,4,5-T to one-year-old channel catfish fingerlings. Static bioassays were conducted in 76 liter tanks containing 5 fish (average weight 14g). Each compound (purity and solvent unspecified) was tested for 48 hours at 1 and 10 ppm. However, no analysis was made to verify herbicide concentrations in the treated water. All of the above herbicides produced less than 10 percent toxicity in 48 hours. No controls were reported. The authors concluded that these herbicides were not toxic to catfish at 10 ppm and could be used around ponds without causing harm to the catfish. 663. McCormack, K. M., Gibson, J. E., and Hook, J. B. (1976) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on renal function in rats in vivo. Toxicol. Appl. Pharmacol. 37(1):177. [Abstract, only.] 238 �664. McKinney, J. D. (1978) Analysis of 2,3,7,8-tetrachlorodibenzo-paradioxia in environmental samples. In: Chlorinated Phenoxy Acids and Their Dioxins. ed. G. Ramel, (Ecol. Bull. no. 27 Scolkholm: Swedish Natural Science Research Council, pp. 53-66. [Background material.] 665. McLennan, M. W. 9(2):88. (1974) 2,4-D toxicity in dairy cattle. " Pestic. Abstr. [Abstract, only.] 666. McLeod, J. G. (1971) Peripheral neuropathy caused by drugs and toxic substances. Aust. N.Z. J. Med. 3:268-269. [Review article.] 667. McNulty, W. P. (1977) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin for Rhesus monkeys: Brief report. Bull. Environ. Contain. Toxicol. 18(1):108-109. The toxicity of subchronically administered TCDD was evaluated in two Rhesus monkeys. Male monkeys, 1 year of age, were fed diets containing either 2 or 20 ppb TCDD. Histological examination of tissues was performed when death occurred. The monkey given the higher dosage showed a loss of appetite and decreased activity. Vomiting occurred and on the twelfth day when death occurred, the animal lost 30% of its body weight. The monkey given the low dosage diet retained its appetite. The eyelids became swollen and the face was edematous after 9 weeks, when the monkey underwent surgery to remove a biopsy sample of the stomach, and was given a control diet subsequently. Two weeks later when the animal died, the body weight loss was 332. Mucous metaplasia of the gastric mucosa was observed in biopsy sections and at autopsy of both animals. Other changes observed in both monkeys were cachexia, hyperplasia, and ulceration of the gastric mucous, squamous metaplasia of sebaceous glands and thymic atrophy. Total intake was estimated to be well below 10 ug/kg TCDD. The author concluded that young male Rhesus monkeys show high susceptibility to TCDD toxicity, compared to other laboratory animals. 668. McNutt, W. S., and Robins, H. I. (1974) The temperature sensitive adenylylation of tRNA in carcodylate and other buffers. Bioch. Biophy. Acta. 366(4):411-423. 239 �669. McQueen, E. G., Veale, A M . 0., Alexander, W. S., and Bates, M. N. (1977) 2,4,5-T and human birth defects. Report prepared by Dept. of Health, New Zealand. 41 pp. The causal relationship between 2,4,5-T exposure and 3 separate instances of clusters of neural tube defects that occurred in New Zealand is evaluated. For each of the 3 clusters of 4-8 neural tube defects maternal exposure to pesticides and drugs during early pregnancy, (when the neurospores close) family history of neural tube defects, illnesses during pregnancy, date of birth of afflicted child, and occupation of the father and other relevant information are described. The doses of 2,4,5-T and of TCDD required to cause defects were extracted from animal studies and maximum maternal exposure levels from agricultural use of these chemicals was estimated. The first cluster of cases occurred in Northland. One case of spina bifida occurred in 1975; in 1976, 2 cases of anencephaly and 4 of spina bifida were reported. This incidence was not considered statistically different from the expected incidence of 4-5 cases. Two cases had family histories of spina bifida and 2,4,5-T exposure in early pregnancy could not be confirmed in any of the 7 cases. Clomiphene was administered to 1 of the mothers just before conception. One case of spina bifida and 3 of anencephaly occurred in Taranaki between 1974 and 1977. The probability of the occurrence of this incidence by chance alone was calculated to be 1 in 80. The mother of one of the cases of anencephaly and the father of another case were siblings. The mother a third case had another child with spina bifida. Exposure of 1 mother to 2,4,5-T sprayed in the vicinity of her house 2 weeks after the date of her last menstruation was documented; water sources included both roof and main water supplies in this case. Pregamal (folic acid and ferrous sulphate) was the only drug consumed by more than 1 mother in this group. In a 4-month period (December, 1975 to January, 1976) 7 cases of spina bifida and 1 of anencephaly were born in Waikato. The probability of this incidence of occurring by chance was calculated at 1 in 140. Two of the involved families had histories of neural tube defects, and a third family history included other congenital deformities. Two cases involved 2,4,5-T exposure of the fathers, but the temporal relationship of exposure to conception could not be established. One mother had influenza in early pregnancy and one mother took iron tablets. The authors determined that a woman would have to drink 42,840 liters of roof water contaminated with 2,4,5-T from the spraying rates and TCDD contamination used in New Zealand agriculture to approach a teratogenic dose of 2,4,5-T or TCDD. The committee concluded that no evidence of an association between herbicide exposure and neural tube defects could be established. 670. Meselson, M., O'Keefe, P., and Baughman, R. (1978) The evaluation of possible health hazards from TCDD in the environment. Presentation for Symposium on the Use of Herbicides in Forestry, Arlington, Va. Feb. 21-22, 1978. 11 pp. [Background material.] 240 �671. Midwest Research Institute and Criteria and Evaluation Division, OPP, EPA. (1975a) Substitute chemical program—initial scientific and minieconomic review of bromacil. Criteria and Evaluation Division, OPP, EPA. PB-241 SOI. 79 pp. [Review article.] 672. Midwest Research Institute. (1975b) Substitute chemical program initial scientific and minieconomic review of monuron. Environmental Protection Agency. PB248 110. 120 pp. [Review article.] 673. Midwest Research Institute. (1975c) Substitute chemical program. Initial scientific review of cacodylic acid. Prepared for Environmental Protection Agency, PB-251 541. 130 pp. [Review article.] 674. Military operations: herbicide operations. MACV Dir. 525-1. (1969) U.S. Military Assistance Command, Vietnam. DTIC No. AD 779794. 130 pp. [Background material.] 675. Mill, T. (1980) Data need to predict the environmental fate of organic chemicals. In Dynamics, exposure and hazard assessment of toxic chemicals. Bizuanual Haque, e d . ( A n n A r b o r : A n n Arbor Science), pp. 297-322. [Background material.] 676. Miller, A. S. 21(5):2-4. (1979) 2,4,5-T: Chemical time bomb. Environment 677. Miller, R. A., Norris, L. A., and Hawkes, C. L. (1973) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic organisms. Environ. Health Persp. 5:177-186. The authors describe the toxicity of TCDD to fish (guppies, cohp salmon, and rainbow trout) and aquatic invertebrates (snail, oligochaete worm, and mosquito larvae). The effects of 24-96h exposure of young salmon to greater than 23 ng/g body weight results in death in 10-80 days. Fish exposed to toxic levels of TCDD had a declining interest in food after 5 (guppies) to 8 (salmon) days. Skin discoloration and fin necrosis appeared in 15 days in guppies and in 30 days in salmon. Erosion of the upper jaw was also observed in guppies 241 �surviving 30-60 days. Exposure of salmon to TCDD levels of 0.054 ng/g for 24 hours or longer resulted in 12 percent mortality in 60 days, to 5.4 ng/g resulted in 55 percent mortality during 60 days and to 54 ng/g resulted in 100 percent mortality in 40 days. In all cases, duration of exposure seemed to be less important than levels of exposure. Tn both salmon and guppies, larger fish survived longer periods of TCDD exposure than smaller fish. Tn rainbow trout, fed diets containing 2.3 ppm TCDD for 4 weeks, no deaths or signs of toxicity were observed at 6.3 pg/tank/week or 6.3 ng/tank/week. At 6.3 ug/tank, decreased appetite in fish was observed at 10 days, and fin necrosis at 14 days. The authors indicated that deaths began occurring at 33 days, but no numerical data were presented. TCDD at 0.2 ppb had no effect on pupation of mosquito larvae during a 30 day test period. However, the same dose of TCDD for 36 days inhibited reproduction but no juvenile or adult survival, in pulmonate snails. Exposure of adult oligochaete worms to 2 ppb TCDD in water for 55 days resulted in a decrease in reproduction of worms. 678. Mitchell, J. W. Hodgson, R. E., and Gaetiens, C. F. (1946) Tolerance of farm animals to feed containing 2,4-D. J. Animal Sci. 5:226-232. The toxicity of 2,4-D sprayed on pastures was assessed in sheep and cows that grazed on the treated pasture. An 0.15% aqueous solution of purified 2,4-D was sprayed at the rate of 215 gallons per acre and starting on the subsequent day 2 cows and 2 sheep grazed on the exposed field for 48 and 12 days, respectively. Autopsies were performed on the cows at the end of the exposure period. A lactating Holstein cow was fed 5.5 gms of 2,4-D daily for 106 days. During the last 38 days of exposure, milk was collected and fed to a calf. 2,4-D levels were estimated by a bioassay; the sera of the cow and calf and in cow tissues removed at autopsy at the end of the exposure period were analyzed for 2,4-D. Levels were determined by measuring growth stimulatory effects. Aqueous extracts of the tissues were applied to bean seedlings and internodal elongation was measured after 24 hours. No adverse effects were observed in sheep and cows that grazed on sprayed fields or a cow fed 2,4-D or calf exposed to 2,4-D in milk, although no controls were studied to compare the data for milk production, food consumption, or body weights in the lactating cow. Milk and cow serum, but no tissues, gave positive reactions in the bioassay used to measure 2,4-D. The authors concluded that purified 2,4-D was not toxic. The methods used in this study are outdated and recent studies provide more information on the extent of 2,4-D toxicity. 679. Miura, H., Omori, A., and Shibue, M. (1975) The effect of chlorophenols on the excretion of porphyrins in urine. Pestic. Abstr. 5(7):456-457. [Abstract, only.] 242 �680. Moilanen, K. W., and Crosby, D. G. (1974) The photodecomposition of bromacil. Arch. Environ. Contain. Toxicol. 2Cl):3-8. The authors studied the photodecomposition of bromacil under simulated sunlight conditions. An aerated 10 ppm aqueous solution of bromacil was irradiated using a sunlight simulator for 6 days. An identical solution was aerated in the dark as a control. Both solutions were analyzed by gas-liquid chromatography and thin layer chromatography to identify photodecomposition products. Only 1 photoproduct was formed, 5-bromo-6-methyluracil. Decomposition of the herbicide proceeded slowly; 96% of the herbicide remained after 6 days irradiation. The authors concluded that bromacil is stable in sunlight and that photo decomposition probably contributes only a minor role to the environmental disappearance of bromacil. 681. Monarca, G., and Di Vito, G. (1961) Folia Med. 44:480-485. A case report of accidental inhalation exposure to a toxic dose of 2,4-D is described. A 57-year-old man sprayed a 40% aqueous solution of 2,4-D by a manual pump under windy conditions. During the evening and day after the work was performed, the farmer experienced generalized asthenia, profuse perspiration, vomiting and oliguria and was admitted to the hospital. Central and peripheral effects that were experienced in the early phase of illness included dizziness, change of refexes and uncertain walking. The only alterations in routine laboratory test results was a transitory albuminuria. The clincal condition of the patient gradually improved, until 18 days after the accident, when sytnptons related to the nervous system worsened and a serious form of hemorrhagic enterocolitis developed. Neural effects included uncertain walking, sluggish palellar and achilles tendon reflexes, positive Romberg sign and dysmetria. Laboratory tests did not provide additional information at this stage. By 5 months, all svmptons except hyporeflexia had receded. The authors suggested that the acid form of 2,4-D produced the early effects and the phenol metabolite elicited enterocloitis by an irritative-caustic action on the mucous membranes of the gastrointestinal tract. 682. Montgomery, M. L., and Norris, L. A. (1972) A preliminary evaluation of the hazards of 2,4,5-T in the forest environment. Ind. Veg. Manage. 4(l):19-22. [Review article.] 683. Moore, J. A. (1980) Statement at the Meeting of the Interagency Work Group to Study the Possible Long-Term Health E f f e c t s of Phenoxy Herbicides and Contaminants, Sept. 22, 1980. 7 pp. [Testimony.] 243 �684. Moore, J. A. (1978) Toxicity of 2,3,7,8-tetrachlorodibenzo-paradioxin. In Chlorinated Phenoxy Acids and Their Dioxins. ed. 0. Ratnel (Ecol. Bull, No. 27, Stolkholm: Swedish Natural Science Research Council, 1978) pp. 133-144. [Review article.] 685. Moore, J. A., Gupta, B. N., Zinkl, J. G., and Vos, J. G. (1973) Postnatal effects of maternal exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). Environ. Health Perspec. 5:81-85. The effect of TCDD on the development of the kidney in fetal and neonatal mice is studied. C57B1/6 strain mice were administered TCDD orally in acetone-corn oil or the vehicle alone. On day 18 of gestation fetuses were removed and tissues were examined histologically. When 3 ug/kg of TCDD was administered on days 10 through 13 of gestation, a mean of 55.4% of the fetuses of each litter had cleft palate, 95.1% had kidney anomalies, and 83.1% had bilateral kidney anomalies. At 1 ug/kg, TCDD administered on days 10-13 caused 1.9% cleft palate, 58.9% kidney anomalies, and 36.3% bilateral kidney anomalies. The same dose administered on day 10 only caused no cleft palates, 34.3% and 8.8% kidney anomalies, and bilateral kidney anomalies, respectively. None of these malformations were observed in vehicle controls. The renal anomaly observed in treated animals was hydronephrosis, characterized by a reduced or absent renal papilla in an enlarged renal pelvis. A reciprocal cross-fostering study was conducted in which mice exposed to TCDD in utero nursed untreated mothers and mice born to control mothers nursed treated mothers. The highest incidence of hydronephrosis, 34%, occurred in mice exposed to TCDD before and after parturition. The incidences of hydronephrosis were 2-2.1% for those exposed in utero, 8.5% for those exposed after birth, and 1.7% for unexposed mice. When mothers were treated on the day of parturition with 3 or 10 ug/kg TCDD, 71-75% of the pups per litter had hydronephrosis, while none of the controls and 12% of the 1 ug/kg dose group (given at parturition) were afflicted. A dose-related decrease in thymic weight of pups in the 3 and 10 ug/kg dose groups was mentioned. The authors concluded that the renal disorder was progressive hydronephrosis which involved the right kidney in 90% of the unilateral cases. Complete atrophy of the right kidney was observed in 55-day-old mice from the highest treatment group. The dose and length of exposure to TCDD determine the severity of the condition, although the authors postulated that pathogenesis of lesions developed in utero or postnatally were the same. The strain and species-specific nature of this striking defect should be kept in mind in extrapolating these results to human exposures. 686. Moore, J. A., Harris, M. W., and Albro, P. W. (1976) Tissue distribution of [ C]tetrachlorodibenzo-p-dioxin in pregnant and neonatal rats. Toxicol. Appl. Pharmacol. 37(1):146-147. [Abstract, only.] 244 �687. "More on Agent Orange". (1980) Congressional Record - U.S. Senate, Sept. 30. S 14160-S 14162. [Testimony.] 688. Morgan, D. P. (1976) Recognition and management of pesticide poisonings. U. S. Environmental Protection Agency Report No. EPA-540/9-011. pp.13-30. [Review article.] 689. Morton, H. L., Moffett, J. 0., and Martin, R. D. (1974) Influence of water treated artificially with herbicides on honey bee colonies. Environ. Ent. 3(5):808-812. In this paper, the authors study the effects of 2,4,5-T ingestion on honey bees. Ten colonies of bees were placed in 2 apiaries at least 2 miles from the nearest water source and at least 3 miles from areas treated with pesticides. One apiary was used as a control and received untreated water, the other apiary had water available that contained 1,000 pptn 2,4,5-T triethylamine salt (purity unspecified). The experiment was initiated in the dry season when the only available water was that which was supplied to.the bees. Treatment continued for 12 weeks; water was changed every 2 days. Numbers of dead bees and brood production was measured in each colony. Adult worker bees, honey, and wax were also removed at intervals over a 24-month period and analyzed for 2,4,5-T content by gas chromatography. The number of bees dying in traps on colonies treated with 2,4,5-T did not differ significantly from controls at the end of the 12-week treatment period. However, a large number of bees were drowned in the 2,4,5-T-treated water, possibly, according to the authors, because of the change in surface tension for the treated water. Treated colonies examined for brood development had less capped brood than controls 1 month after the study began. There was little brood production during the second month of treatment. One month after the rainy season started the bees had access to an untreated water source, and brood production increased. No differences in brood production were seen during the second year of the study. The concentration of 2,4,5-T in adult worker bees reached a peak of about 150 ppm during treatment. When treatment was terminated, 2,4,5-T levels decreased to about 5 ppm. Detectable levels of 2,4,5-T were found in bees during most of the 2-year study. In honeyj 2,4,5-T reached a peak of 50 ppm. Upon removal of treatment, 2,4,5-T levels in honey decreased to about 10 ppm. 2,4,5-T was last detected in honey 480 days after intitation of the experiment. The compound was detected in wax as long as 650 days after initiation of the experiment. The authors concluded that 2,4,5-T could be transferred to honeybee colonies, honey, and wax if the bees were exposed to contaminated water sources. This can potentially be another source of human exposure to 2,4,5-T. The effects of paraquat on honey bees was also discussed in this paper. 245 �690. Morton, W. E., Crawford, E. D., Maricle, R. A., Douglas, D. 0., and Freed, V. H. (1975) Hypertension in Oregon pesticide-formulating workers. J. Occ. Med. 17(3):182-185. The incidence of hypertension was determined for chlorophenoxy herbicide workers, other pesticide workers and non-pesticide-exposed workers. Workers at 28 plants who were engaged in the formulation of pesticides were requested to complete a questionnaire indicating clinical symptoms, family history of hypertension, and hazardous product exposure. Blood pressures were measured (number of examiners taking blood pressure was not reported). Of 3 sets of readings, the set of readings with the lowest systolic value was used for the study. The control group was comprised of workers from a wood products plant, as well as employees of the State health department and from the medical school. All chlorophenoxy herbicides workers were employed at the same plant. Compliance for the herbicide, pesticide and control groups were 85%, 85%, and 10%, respectively and the sizes of the study groups ranged from 69-84. The groups were similiar in terms of sex ratio, age range and body size. Tobacco use was highest and alcohol use lowest for herbicide workers. Exposure to phenoxy-herbicides was indicated by 68% of the herbicide workers and to phenoxy herbicide intermediates, by 78% Over half of the herbicide group was exposed occupationally to pesticides for more than 10 years, while 2/3 of the pesticide workers were exposed for less than 10 years. Herbicide workers indicated experiencing higher incidences of symptoms than the other groups (specific symptoms for each organ system or statistical analyses of any results were not reported). The mean systolic and diastolic blood pressures were the same for all 3 groups, although 39% of the herbicide groups, 29% of the pesticide workers and 30% of the control group were diagnosed with hypertension (systolic pressure, at least 150 and/or diastolic, at least 90). Family histories of hypertension were most frequent among the herbicide group and age-standardization of results did not alter the trends. The authors concluded that the modest excess in the prevalence of hypertension among herbicide workers was probably attributed to higher predisposition, evidenced by data on family histories. The authors indicated that the size and selection bias of their control group was unsatisfactory and that exposure to corrosive chemicals and solvents by both experimental groups was probably a significant confounding factor in the study. 691. Moses, M. (1979) Effects of TCDD on human health. Testimony before the House Subcommittee on Oversight and Investigations, June 26, 1979. 6 pp. [Testimony.] 246 �692. Hosier, A. R., and Guenzi, W. D. (1973) Picloram photolytic decomposition. J. Agric. Food Chem. 21(5):835-837. The authors report on the photolytic decomposition of picloram under laboratory conditions. An aqueous solution of 2.08 x 10-'M picloram sodium salt was irradiated with 300-380 nm ultraviolet light. Within 72 hours, picloram was 99% degraded. After 34 hours of irradiation, 11 degradation products were observed by thin layer chromatography. Attempts to characterize these compounds were unsuccessful. The authors also discussed the possible mechanisms of picloram degradation. 693. Muller, R. 0. (1980) Statement of Executive Director of Vietnam Veterans of America before the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants, Sept. 22, 1980. 11 pp. [Testimony.] 694. Mullison, W. R. (1980) 2,4,5-T update — January 1980. Presented at the 29th Conference of the Texas Agricultural Aviation Association, January 15-17, San Antonio, Texas. [Editorial.] 695. Mullison, W. R. (1980) Some public concerns about 2,4,5-T. Presentation to the 33rd meeting, Western Society of Weed Science, Salt Lake City, Utah, March 18-20, 1980. [Editorial.] 696. Murai, Y., and Kuroiwa, Y. (1971) Peripheral neuropathy in chlorobyphenyl poisoning. Neurology 21:1173-1176. Results of clinical examinations and nerve conduction velocity tests in patients that had been exposed to chlorobiphenyl (kanechlor 400) are described. Twenty-one patients, ranging from 7 to 60 years of age, were examined. Neurologic symptoms in most of the patients included numbness, muscular aches, hypoesthesia and areflexia. No symptoms were found that suggested the involvement of the central nervous system. Nerve conduction velocities were measured in both motor (tibial nerve) and sensory nerves (radial and sural nerves). Results of these tests demonstrated that conduction velocities in motor nerves were within the normal range while conduction velocities in the sensory nerves were reduced in about half of the patients. There was one exception; conduction velocities in the motor nerve were slowed in one patient that exhibited areflexia in all four limbs. The authors concluded that accidental ingestion of chlorobiphenyl caused sensory neuropathy, and that this could be an early manifestation of mixed (sensory and motor) polyneuropathy. ' 247 �697. Murakami, M., and Fukami, J. (1978) Persistent pesticides and environmental chemicals are taken up to a greater extent than non-persistent compounds by cultured human cells. Bull. Environ. Contain. Toxicol. 19(4) :423-427. Uptake of 2,4-D and 2,4,5-T by human embryonic lung cells is described. Confluent monolayer cultures of human embryonic lung diploid cells were cultured in the presence of 4 x 10 M F C]-2,4-D or [ Cl-2,4,5-T for 4, 24, and 48 hours. The cell layer was digested with sodium hydroxide and counted for radioactivity. Total cell protein was assayed to evaluate cell growth. For 2,4-D, uptake varied between 2.6 and 5 pmol/mg cell protein for the 3 time points and cell growth was 79% of control cell growth. For 2,4,5-T, uptake varied between 6.6 and 12 pmol/mg protein and cell growth was 83% of control growth. Uptake and cell growth were also tested for DDT, dieldrin, aldrin, PCS, HCB, carbaryl, malathion, parathion, and chlordimeform. Uptake of 2,4-D and 2,4,5-T were less than for the other compounds, but they were more toxic in terms of cell growth. The authors concluded that the test compounds that are persistent in the environment were taken up to a greater extent by cultured cells but cytotoxicity was not the result of greater uptake of the test compound. 698. Muranyi-Kovacs, I., Rudali, G., Imbert, J. (1977) Study on the carcinogenicity of 2,4,5-T in mice. (Meeting Abstract). Presented at the 4th Meeting of the European Association for Cancer Research, Lvon, France, Sept. 13-15. p. 64. [Not available.] 699. Muranyi-Kovacs, I., Rudali, G. , and Imbert, J. (1976) Bioassay of 2,4,5-trichlorophenoxyacetic acid for carcinogenicity in mice. Br. J. Cancer 33(6):626-633. The authors evaluated the carcinogenicity of 2,4,5-T in a feeding study in mice. Male and female (6 weeks old) XVII/G and C3Hf mice were given water containing 2,4,5-T (100 mg/1) for 2 months after which the animals received 80 ppm 2,4,5-T in their diets up to 28 months or until death. The 2,4,5-T preparation used in this study had less than 0.05 dioxins by gas chromatographic analysis. The authors did not report any analysis of food or water treated with 2,4,5-T to confirm their formulations. In addition, they did not specify the length of time or temperature at which a particular water or diet formulation was stored. The authors also did not report the number of animals initially used in the experiment. However, 40-45 control animals per group and 20-25 treated animals per group were alive during the llth to 12th month of the experiment. Tumors developing in the test animals were placed in one of two classes: 1) incidental tumors which were discovered at necropsy of an animal which died from other causes and 2) non-incidental tumors which were diagnosed during the animal's lifetime or caused the death of the animal. In XVII/G animals, there was no significant difference between the number of tumor bearing mice observed and the 248 �number expected by Peto's method of statistical analysis. However, in C3Hf mice the differences between observed and expected non-incidental tumors were significant in both male and female mice. When incidental and non-incidental tumors were group together, a significant difference was observed in only females. Incidence of incidental tumors alone was not significantly different in either sex. When males and females were pooled, incidence of total tumors and non-incidental tumors was statistically different between control and treated animals. From this information, the authors concluded that additional data are required to make an assessment of the carcinogenic potential of 2,4,5-D. 700. Murphy, J. M., Murfin, G. D., Jamieson, N. L., Rambo, A. T., Glenn, J. A., Jones, L. P., and Leighton, A. H. (1974) The effects of herbicides in South Vietnam. Part B. Working papers: Beliefs, Attitudes, and Behavior of Lowland Vietnamese. National Academy of Sciences-National Research Council. AD -779 030. [Background material.] 701. Murray, F. J., Smith, F.F., Nitschke, K. D., Humiston, C. G. , Kociba, R. J., and Schwetz, B. A. (1979) Three generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50(2):241-252. Reproductive toxicity was demonstrated in rats treated with TCDD in a three generation study. Rats were administered 0, 0.001, 0.01, or 0.1 ug TCDD/kg/day orally by monitoring consumption and adjusting dietary TCDD accordingly. For the fQ parental generation, administration of the TCDD diet was initiated 90 days prior to mating (at about 7 weeks of age) and was continued for the duration of the experiment. The f_ generation produced two litters, the (f. ) and second ^ f i i ) generations. The f,, generation was mated to produce an f- generation and the f_ generation was mated to produce an f3 generation. All animals, except the control group, were fed diets containing TCDD throughout the experiment. Litter size and body weights were recorded at parturition and 1, 7, 14, and 21 days after birth. Gross and histopathological examinations were performed on weanlings not selected for mating. Signs of toxicity were seen in only one f/v adult in the high dosage group. Of the 4 litters delivered from fQ females in the high dosage group, only one litter had viable pups (4/5) and no subsequent matings were made. For the other two treatment groups, the fertility indices, body weights, number of days from cohabitation to delivery, and postnatal survival were adversely affected in the f. and f^ generations of the intermediate dosage group, compared to the control group, as were the sizes and gestational survival indices of their litters. The reproductive capacity of the f,. rats of the middle dosage group and all of the low dosage groups resembled the controls. Dilated renal pelvis was observed in rats from the high and low dosage groups. A crossmating study was performed on rats from the f.. geneation which had received the TCDD diet for 12 months. Male or female rats from the f« control group or the fQ group given 0.1 ug TCDD/kg/day were mated with 249 �younger, untreated rats. Females were killed 20-21 after sperm was detected in vaginal smears and numbers of viable, dead, and resorbed fetuses were recorded. Incidences of pregnancy from matings of f-. males of the high dosage group with untreated females and of f~ control males with untreated females were both 58-60% compared to 5% for matings of untreated males and f_ control females and 15% for matings of untreated males and f~ females from the highest dosage group. The 70% incidence of resorptions for the last group was significantly higher than for its control (15%). The decreased fertility of all f rats was attributed to the age of the females when the cross-mating study was conducted. 702. Myers, P. W. (1981) Status of Herbicide Orange Studies. Presentation before the Veterans Affairs Committee, House of the Representatives. (Subcommittee on Oversight and Investigations) released by the Committee on Veterans Affairs, House of Representatives, 39 pp. [Testimony.] 250 �703. Nagy, Z. S., Mile, I., and Antoni, F. (1975) The mutagenic effect of pesticides on Escherichia coli WP2 try. (1975) Acta Microbiol. Acad. Sci. Hung. 22:309-314. The authors evaluated the mutagenicity of 2,4-D and 29 other pesticides in Escherichia coli WP2 try. Two bacterial strains which revert from tryptophan requiring to non-requiring were used. One strain (her ) was repair deficient in addition to requiring tryptophan; the other strain (her ) was repair proficient; 2,4-D was tested in a spot test without metabolic activation either as 1-3 mg crystals or 20-25 ul liquid. Both positive and negative controls were included in the assay. Numerical values were not reported. According to the authors, 2,4-D was negative in this assay. 704. Nash, R. G. and Beall, Jr., M. L. (1980) Distribution of Silvex, 2,4-D and TCDD applied to turf in chambers and field plots. J. Agric. Food Chem. 28:614-623. [Background material.] 705. Nash, R. G., and Beall, M. L., Jr. (1978) Environmental distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) applied with Silvex to turf in microagroecosystem. Final Report EPA-IAG-D6-0054, Agricultural Environmental Quality Institute, U.S. Department of Agriculture, Beltsville, Maryland. [Background material.1 706. Nash, R. G., Kearney, P. C., Fertig, S. N., et al. (1981) Agricultural exposure to 2,4-D. [Abstract of paper presented at 181st Am. Chem. Soc. National Meeting, Atlanta, Ga., April 1981.] [Abstract, only.] 707. Nash, R. G., Kearney, P. C., Maitlen, J. C., Sell, C. R., and Fertig, S. N. (1981) Agricultural applicator exposure to 2,4-D. (in press) Urinary excretion data for 43 workers who applied 2,4-D were used to estimate 2,4-D exposure levels. A group of 17 aerial applicators supplied 24-hour urine samples on alternate days of a 2-week period of peak 2,4-D spraying. A group of 26 ground applicators provided six consecutive 24-hour samples following a single 2,4-D application. Urine samples were analyzed for 2,4-D by gas-liquid chromatography. An average of 0.4 ppm 2,4-D was detected in urine collected prior to the experimental period from both groups. The mean level of 2,4-D excretion for aerial applicators over the 2 week period was 0.11 mg/kg body weight per day. The highest levels were excreted by mixer/loaders (0.02 mg/kg/day) and the lowest by pilots (0.006 mg/kg/day). Ground applicators were divided into three groups by occupation. Applicators 251 �excreted a total of 0.005 mg/kg of 2,4-D; mixer/loaders excreted 0.007 mg/kg; and mixer/loader/applicators, 0.018 mg/kg. The urinary levels from each group were not associated with age, weight, or type of clothing, and were associated with the type of .iob, length of time of exposure, and amount of 2,4-D applied. The half-lives for excretion of 2,4-D by ground applicators were between 35-48 hours. The average total exposure was estimated to be 12.7 ug/kg for all ground applicators. The authors concluded that exposure of an 80 kg man to 2,4-D during spraying 30 days per year for 30 years could result in absorption and excretion in an estimated 0.9 g of 2,4-D total, which is far below 2g, a dose that produced no adverse effects when administered intravenously to one man (reported elsewhere). 708. National Center for Toxicological Research. Study. (1975) 2,4,5-T Teratology [Not available.] 709. National Forest Products Association. (1980a) Forest Industry Statement before the Interagency Work Group on Phenoxy Herbicides Public Meeting Sept. 22, 1980: Proposed Outline. Sept. 11, 1980, 1 p. [Testimony.] 710. National Forest Products Association. (1980b) Statement of the National Forest Products Association before the Interagency Working Group on Phenoxy Herbicides on Health Effects Information, Sept. 22, 1980. 19 pp. [Testimony.] 711. National Research Council. (1974) The Effects of Herbicides in South Vietnam: Part A. Summary and Conclusions. National Academy of Sciences, Washington, D.C. AD-774-749. [Review article.] 712. National Institute for Occupational Safety and Health. O978) Criteria for a recommended standard; Occupational exposure during the manufacture and formulation of pesticides.DHEW (NIOSH) Publ. No. 78-174. [Background material.] 713. National Veterans Law Center. (1980) Statement of the National Veterans Law Center before the Senate Veterans Affairs Committee, United States Senate, Sept. 10, 1980. 31 pp. [Testimony.] 252 �714. Neal, R. A., Beatty, P. W., and Gasiewicz, T. A. (1979) Studies of the mechanisms of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ann. N. Y. Acad. Sci. 320:204-213. The authors proposed a series of mechanisms of action to account for the acute toxicity of TCDD and tested each hypothesis, experimentally. The hypothesis that TCDD inhibits mitosis was tested by examining the effgct of TCDD on the growth of five mammalian cell lines in vitro. At 10 M, TCDD had no effect on any cells tested. The similarity of TCDD toxicity to the hypothyroid state and other reasons led to an experiment that tested the ability of triiodothyronine (T-) administration to protect mice from TCDD toxicity. The survival time of these mice was prolonged, but T, did not decrease the mortality. The authors tested the effect of TCDD on Rana catesbeiana tadpole metamorphosis, a T»-dependent process and found no alteration, nor any toxicity in the tadpoles or adults of the species. The effect of TCDD on the oxidation-reduction state of the cell and on the ATP levels was examined. Rats were treated with 50 ug/kg TCDD intraperitoneally and 14 later the livers were excised. The ATP content of the liver and the ratio of the levels of oxidized to reduced pyridine nucleotides (NAD/NADH) were the same in TCDD treated and ad libitum-fed control rats. The total liver content of the several pyridine nucleotides were measured in another experiment and only the NAD levels of TCDD-treated rats were significantly lower than control values. Next, the hypothesis that TCDD produced toxicity by mimicking glucocorticoid compounds or stimulating their biosynthesis (through enzyme induction) was studied. At 7 and 14 days after 50 ug/kg of TCDD was administered to rats, plasma corticosterone levels were 2.5 times the levels of pair-fed controls. All adrenalectomized rats given 10, 20, 40, or 80 ug/kg TCDD died within 6 days, while the 20 day LD^,. in the rat including sham-operated and vehicle controls was 50 ug/kg. Even at a 200 fold molar excess, TCDD was unable to compete with [ H]-dexamethansone for the rat cytosol glucocorticoid receptor. TCDD was also unable to interfere with a peripheral action of glucocorticoids, the induction of rat liver tyrosine aminotransferase activity by dexamethasone. Although TCDD markedly affects lipid metabolism, the fatty acid compositions of the livers, plasma, and adipose tissue of guinea pigs were unaltered by TCDD,treatment. Absorption of nutrients was not impaired by TCDD, as [ C]-glucose, f C]-alanine and.! C]-oleate administered orally to guinea pigs produced as much [ C]-CO_ by TCDD-treated animals as pair-fed controls. Other parameters that were studied but did not reveal further information on the mechanism of TCDD toxicity were increases in blood NH~ increased superoxide anion levels, changes in cyclic nucleotide metabolism and inhibition of riboflavin coenzyme activity. The authors concluded that TCDD produces stress an acute stress in rats that is combated by increased glucocorticoid output, TCDD does not interfere with thyroid hormone binding nor causes a pathologic alteration in the cellular energy state in the rat. The details of the experimental protocols were not provided, but the rationale and interpretation of results were adequately described and successful identification of the mode of action of TCDD would greatly enhance understanding TCDD toxicity. 253 �715. Nebert, D. W., and Jensen, N. M. (1979) The Ah locus: genetic regulation of the metabolism of carcinogens, drugs, and other environmental chemicals by cytochrome p-450-mediated monooxygenases. CRC Crt. Rev. Biochem. 6:401-430. [Review.] 716. Nebert, D. W., Robinson, J. R., and Poland, A. P. (1973) Genetic expression of several drug-metabolizing enzyme activities inducible in the mouse by aromatic hydrocarbons. Genetics 74:S193. [Abstract, only.] 717. Neely, W. B., Branson, D. R., and Blau, G. E. (1974) Partition coefficient to measure bioconcentration potential of organic chemicals in fish. Environ. Sci. and Technol. 8(13):1113-1115. [Background material.] 718. Neilands, J. B. (1973) Survey of chemical and related weapons of war. Naturwissenschaften 60:177-183. [Review article.! 719. Nelson, B. (1969) Herbicides: Order on 2,4,5-T issued at unusually high level. Science 166: 977-979. [Editorial.] 720. Nelson, C. J., and Holson, J. F. (1978) Statistical analysis of teratologic data: problems and advancements. J. Environ. Pathol. Toxicol. 2:187-199. [Review article.] 721. Nelson, C. J., Holson, J. F., Green, H. G., and Gaylor, D. W. (1979) Retrospective study of the relationship between agricultural use of 2,4,5,-T and cleft palate occurrence in Arkansas. Teratology 19:377-384. The author performed a retrospective study to determine a possible causal relationship between 2,4,5-T exposure and the teratogenic effect of cleft palate in Arkansas. The ecological model employed estimated the herbicide exposure level by the number of rice fields in a given area. Data were obtained from both the vital records and the files of the State Crippled Children's Services for the years 1943 to 1974 inclusive. Data were apportioned in six-year intervals with the 254 �exception of the 2 final intervals which had seven years. Regression analyses were performed separately on cleft lip with and without cleft palate and cleft palate only, prevalence rates versus years by expdsure group (high, medium or low) race and sex. A significant linear trend with time for cleft lip with or without cleft palate was found for high exposure black females and high and low exposure white males. Black females exhibited increased rates with increased exposure. For cleft palate only a significant linear trend with time was found for high exposure black females, low exposure black and white males and high and low exposure white females. The author concluded that since data were not available to compare individual exposures and the difficulty of determining if cases in the assigned groups actually received a high or low exposure that no inferences of cause and effect should be drawn. 722. Neubert, D., and Dillmann, I. (1972) Embryotoxic effects in mice treated with 2,4,5-trichlorophenoxy-acetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch. Pharmacol. 272(17):243-264. An extensive study designed to separate the teratogenic effects of 2,4,5-T from those of TCDD was conducted with NMRI mice. Three preparations of 2,4,5-T containing less than 0.02 ppm dioxin (A), 0.05 +_ 0.02 ppm dioxin (B), and (C) an unknown amount of dioxin, but with physical properties suggestive of higher dioxin contamination than (B). The butyl ester of 2,4,5-T, 2,4,5-trichlorophenol, and pure TCDD were also tested. Compounds were administered in rape seed oil by stomach tube to pregnant mice on various schedules. On day 18 of gestation the number of viable fetuses, resorbed fetuses and implantation sites were counted, placentae and fetuses were weighed, and the incidence of cleft palate was determined. A total of 700 pregnant mice and 7,000 fetuses were used in these experiments. The LDen for non-pregnant mice given 10 daily oral doses was 130 mg/kg 2,4,5-T. Non-pregnant mice given 120 mg/kg 2,4,5-T on this regimen gained significantly less weight than controls but no fatalities occurred. Pregnant mice given doses of 2,4,5-T above 30 mg/kg, or TCDD above 2 ug/kg showed decreased weight gains. At doses above 30 mg/kg, 2,4,5-T or 2,4,5-T butyl ester administered from day 6 through day 15 of gestation produced cleft palates in a dose-dependent manner. The purest preparation was significantly less potent than the other preparations. Administration of a single dose of 300 mg/kg 2,4,5-T was most effective in producing cleft palate when administered on day 12 compare to any other day between days 6 and 15. Doses higher than 45 mg/kg 2,4,5-T given on days 6 through 15 significantly increased the total number of resorptions and this increase occurred sporadically among litters. All doses of 2,4,5-T caused a reduction in fetal weight which was not dose-dependent and was not accompanied by a decrease in placental weights. TCDD administered on days 6-15 at doses above 2 ug/kg produced cleft palate and embryolethal effects. Single doses of TCDD were most effective in producing cleft palate when administered on day 8 or day 11 of gestation. Although potentiation of the effect of 2,4,5-T in producing cleft palate occurred when TCDD was administered simultaneously, contamination of less than 1 ppm TCDD would not lead to potentiation of the effect of 30-60 mg/kg 2,4,5-T in producing cleft 255 �palate. Trichlorophenol at doses up to 9 mg/kg was not teratogenic but produced a slight increase in embryo mortality. The authors concluded that neither TCDD nor trichlorophenol were likely to be responsible for causing or potentiating the embryotoxicity produced by impure preparations of 2,4,5-T, although other unidentified contaminants may be active in this regard. The doses of 2,4,5-T shown to be active in causing embryotoxic effects were considered to be low compared to the application rates of 10 to 300 mg/m used for the herbicide. 723. Neubert, D., Zens, P., Rothenwallner, A., and Merker, H. J. survey of embryotoxic effects of TCDD in mammalian species. Health Persp. 5:67-79. (1973) A Environ. The embryotoxic effects of pure preparations of TCDD and of 2,4,5-T alone and in combination were studied in the mouse and data from other laboratories on the embryotoxicity of these compounds were reviewed. Pregnant NMRI mice were administered TCDD (98.6% pure) or 2,4,5-T (less than 0.02 ppm contaminant) or both in rape seed oil by stomach tube. On day 18 of gestation, fetuses were removed and dead fetuses, including resorptions, were counted. Probit plots of the incidences of cleft palate and of resorptions from daily doses of 2-10 ug/kg TCDD given during days 6-15 of pregnancy overlapped. The probit plot of cleft palate incidence in mice given 10-50 ug/kg TCDD on day 13 only paralleled the data from doses given on days 6-15, but no increase in resorptions occurred from any dose given on day 13, only. From 6 to 8 fetuses per litter had cleft palates after 9 ug/kg TCDD was administered on days 6-15 or on days 9-13. Mortality was increased only from treatment on days 6-15, with 9 fetuses per litter affected, compared to one dead fetus per litter in controls and in the group treated on days 9-13. The maximum incidence of cleft palates resulted from administration of TCDD on day 11, with half of the maximum incidence produced from administration on day 10 or day 12. Maximum incidence of cleft palate occurred from administration on day 12, with 60% of maximum incidence from treatment on day 13 and about 15% of maximum from day 11 treatment. (The doses used were not indicated.) The probit doseresponse curves were compared for TCDD, 2,4,5-T, 6-aminonicotinamide (6-AN), endoxan, and dexamethasone with cleft palate incidence as the response. TCDD was the most potent and 2,4,5-T the least potent compound; 2,4,5-T showed the most shallow response and 6-AN showed the steepest response. A combination of 60 mg/kg 2,4,5-T and a non-teratogenic dose of 2 ug/kg TCDD or 0.2 ug/kg TCDD showed potentiation of the incidence of cleft palate. This combination responds to contamination of 2,4,5-T with 3.3 ppm TCDD. Nonteratogenic levels of 2,4,5-T of 30 mg/kg TCDD at more than 20 ppm can cause potentiation. TCDD also potentiated the incidences of cleft palate produced by 6-AN and by dexamethasone and by combinations of these compounds and 2,4,5-T. The authors concluded that the degree of potentiation by TCDD was related to the steepness of the dose-response curve of the other teratogen, assuming that TCDD did not alter the metabolism of the teratogen. This study presents a good review of the embryotoxicity of TCDD and pertinent data on the potentiation of combinations of relevant compounds which is essential to understand other studies that employed TCDD-contaminated preparations of 2,4,5-T. 256 �724. Newmeyer, J. (1973) Herbicides. Th e E n c ydopedia of Chemistry 3 d e d, Hatnpel, C. A., and Hawley, G. G. eds. (Re'inhold"," NY: Van Nostrand) 526-529. [Not available.! 725. Newton, M., and Snyder, S. P. (1978) Exposure of forest herbivores to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in areas sprayed with 2,4,5-T. Bull. Environ. Contam. Toxicol. 20(6):743-750. The authors describe the analysis of livers of mountain beavers for TCDD contamination. Ten beavers were captured 45-60 days after aerial spraying of 2,4-D<2.2 kg/ha) and 2,4,5-T (2.2 kg/ha). "Animal livers were analyzed by gas chromatography-mass spectrometry (detection limit 3-17 ppt). No TCDD contamination was detected in either the exposed animals or a control animal captured in an untreated area. The authors concluded that forest herbivores are unlikely to accumulate TCDD in their tissues at detectable levels. 726. Nicholson, S. A., and Clerman, R. J. (1974) Toxicity of diquat to the crustacean amphipod Hyalella from Chautauqua Lake. Environ. Letters 7(3)-.215-227. The authors report on the toxicity of diquat to the crustacean Hyalella. Organisms were collected from Lake Chatauqua and tested in lake water. A commercial preparation of diquat (.3 - 4.02 ppm cation) was added to tanks containing 10 animals. Test animals were examined at 24, 48, 72, and 108 hours. The median 24 hour tolerance limit (TL ) °^ Hyalella was determined to be 1.26 ppm (0.67 ppm cation). The authors concluded that Hyalella is killed by diquat levels commonly used for weed control. 727. Nielsen, K., Kaempe, B., and Jensen-Holm, J. (1965) Fatal poisoning in man by 2,4-dichlorophenoxyacetic acid (2,4-D): Determination of the agent in forensic materials. Acta Pharmacol. Toxicol. 22:224-234. A case of suicide by ingest ion of 80 mg/kg of the dimethylamine salt of 2,4-D by a 23-year-old man was reported. The formulation ingested also included dimethylamine which was considered to be of low toxicity. Tissue samples were analyzed for 2,4-D contents and were examined for histological changes. All organs showed acute congestion. Severe, degenerative changes were observed in the ganglion cells and acute pulmonary emphysema was noted in the lungs. The kidneys, liver, spleen and adrenals showed acute congestion, but no degenerative changes. The pancreas, prostate, and testes appeared normal on histological examination. All organs analyzed contained 2,4-D. The highest concentrations were detected in the stomach contents followed by the blood. The brain, on the other hand, had almost negligible amounts suggesting extreme sensitivity of the ganglion cells to 2,4-D or anoxia as the cause of the observed histopathology. The authors were unable to 257 �identify the mechanism of 2,4-D poisoning, but a diabetogenic effect on the pancreas (suggested by others) was considered unlikely since no histological changes were observed in this organ. 728. Nienstedt, W., Parkki, M., Uotila, P., and Aitio, A. (1979) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the hepatic metabolism of testosterone in the rat. Toxicology 13:233-236. The metabolism of testosterone was studied in homogenates of livers from rats after pretreatment with TCDD. Adult male Wistar rats were administered 20 ug/kg of TCDD in acetone-corn oil (1:99) intragastrically 1 week before,they were killed. Hepatic tissue homogenates were incubated with [4- C]-testosterone for 2 min. No cofactors for conjugation reactions were added to the mixture. Radioactive metabolites were separated by thin-layer chromatography and quantified by liquid scintillation counting. Only non-conjugated steroids were identified in the digestion medium. The production of polar compounds was reduced significantly from 78% of the radioactivity by liver homogenates in vehicle controls to 56% by the homogenates from TCDD-treated rats. Increases in the proportions of the radioactivity associated with testosterone and with dihydroxysteriod intermediates occurred with TCDD treatment. The authors concluded that TCDD exerted its effect by inhibiting the cytochrome P-450 dependent hydroxylation reaction that produces the polar metabolites of testosterone, without altering enzymatic reduction reactions that produce the radioactive intermediates isolated in this study. The importance of this alteration of testosterone metabolism on reproductive function in the male has yet to be demonstrated. 729. Nikolaev, A. Z., Subkhankulova, F, B., Geller, I. S. (1970) Immune reactions in methylmercaptos, phosphamide, aldrin and monuron poisoning. Farmakol. Toksikol. 33(6):737-741. [Foreign language.] 730. Nisbet, I. C. T (1980) Direct testimony before the U. S. Environmental Protection Agency, FIFRA Docket Nos. 415, et al. [Testimony.] 731. Nitro Workers' Deaths, Dioxin Link Not Found. October 10, 1980. [Editorial.] 258 The Charleston Gazette, �732. Niwa, A., Kumaki, K., and Nebert, D. W. (1975) Induction of aryl hydrocarbon hydroxylase activity in various cell cultures by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Molec. Pharmac. 11:399-408. The characteristics of TCDD induction of aryl hydrocarbon hydroxylase (AHH) activity were compared to 3-methylcholanthrene (3-MC) induction in cultures of various types of cells. AHH activity was assayed in cultures of 10 established cell lines, of fetal primary cultures from 5 animal species, and of human lymphocytes. For each of four cell lines, the lag time from introduction of the inducer into cultures until induction occurred, the length of rapid increase in enzyme activity and the maximal specific activities for TCDD and 3-MC were similar; the magnitudes of the maximum responses varied for different cell lines. Induction by either agent was blocked by actinomycin D and by cycloheximide and maximally effective doses of both agents did not produce a greater induction than either compound administered alone. The cytotoxic effect of TCDD (in the range of 0.7-1.0 uM TCDD) was not related to its AHH-inducing activity. The dose of TCDD producing a half maximal inductive response ( D ) varied from 0.1 nM for C58BL/6N E ^ mouse cells to 8.0 nM for human lymphocytes and over 200 nM for some established cell lines. The ED for fetal cells from chick, hamster, rat, rabbit, and two mouse strains and for six established animal cell lines were below 2 nM, while the ED,.,, of 6.4 nM for one mouse strain (AKR/N) and 11 for a human liver established cell line approached the value for human lymphocytes. TCDD was consistently more effective than 3-MC as an inducer, ranging from 250-900 times more potent, depending on the cell line. . The authors recommended the use of H-4-II-E cell cultures for assaying TCDD at levels of 10 moles/ml culture medium by monitoring AHH induction. The authors also proposed a difference in affinity or number of TCDD-"receptor" sites to account for the differences in responsiveness to TCDD of different cell lines. 733. Nolan, R. J., Smith, F. A., and Hefner, J. G. (1979) Elimination and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female guinea pigs following a single oral dose. Toxicol. Appl. Pharmacol. 48(l)Pt. 2:A162. [Abstract, only.] 734. Norback, D. H., and Allen, J. R. (1973) Biological responses of the nonhuman primate, chicken, and rat to chlorinated dibenzo-p-dioxin ingestion. Environ. Health Persp. 5:233-240. [Review article.] 735. Norman, R. L., Johnson, E. F., and Muller-Eberhard, U. (1978) Identification of the major cytochrome P-450 form transplacentally induced in neonatal rabbits by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fed. Proc., Fed. Am. Soc. Exp. Biol. 37(6):1720. [Abstract, only.] 259 �736. Norris, L. A. (1966) Degradation of 2,4-D and 2,4,5-T in forest litter. J. Forestry 64:475-476. [Background material.] 737. Norris, L. A., Montgomery, M. L., and Johnson, E. R. (1977) The persistence of 2,4,5-T in a Pacific Northwest forest. Weed Sci. 25(5):417-422. The authors report on a study of the persistence of 2,4,5-T in forest vegetation and soil after aerial applications of the herbicide. The study area consisted of 87 ha in a highly productive Douglas fir forest. 2,4,5-T isooctyl ester (2.24 kg/ha) in diesel oil was sprayed by helicopter over the area in March 1971. A second application on the same mixture was applied to 67 ha in March 1972. Samples of the current season's vegetation (vine maple, blackberry vines, composites of several grass species, 15 cm terminal branch segments of Douglas fir), forest floor material, and forest soil were collected within 2 hours of application and 1,3,6, and 12 months after treatment. Samples were analyzed by gas chromatography (detection limit 0.01 ppm). In forest vegetation, maximum concentrations of 2,4,5-T occurred immediately after application and ranged from 10.6 ppm in vine maple to 114.5 ppm in grass. Concentrations dropped sharply during the first 3 months. One year after spraying, 2,4,5-T levels ranged from 0.48 ppm in vine maple to 0.03 ppm in blackberry foliage. Therefore, months after application only vine maple contained detectable levels of 2,4,5-T, In the area which received 2 herbicide sprayings, vegetation concentrations were also highest immediately after spraying and declined rapidly. After 1 year, only vine maple had detectable levels of 2,4,5-T (0.02 ppm). Levels of 2,4,5-T in forest floors increased during the first month after herbicide spraying and then declined rapidly. About 0.3% of the original application remained after 24 months. Levels of residues were higher after the second application than after the first application probably as a result of reduced vegetation due to the first spraying. One year after the second application, 2,4,5-T residue levels were identical to levels remaining 1 year after the first application. 2,4,5-T residue levels in soil were very low compared to forest floor and vegetation samples. After the first spraying, 2,4,5-T residues in the 0-15 cm layer of soil peaked at 0.08 ppm 3 months after spraying. No 2,4,5-T was detected in the top layer (0-15 cm) of soil after 12 months. In addition, no 2,4,5-T was detected deeper than 0.15 cm. After the second spraying, residues were detected in the top layer immediately after spraying but not at any other time. The authors concluded that no accumulation of 2,4,5-T occurred in the forest sample tested and that the short persistence of this compound should not adversely affect the forest environment. 260 �738. Norris, L. A., Montgomery, M. L., Webb, W. L., Schroeder, H. J. Jr., and Gross, F. (1976) Distribution of 2,4-D and picloram applied by a mist blower. Bull. Environ. Contain. Toxicol. 16(6): 631-639 . [Background material.] 739. Norris, L. A., and Moore, D. G. (1970) The entry and fate of forest chemicals in streams. In Proc., Symp., Forest Land Uses and Streams Environment. fCorvallis, Oregon: Oregon State University PC:138-158, [Background material.] 261 �740. O'Connor, G, A., and Anderson, J. U. (1974) Soil factors affecting the adsorption of 2,4,5-T, Soil Sci. Soc. Am. Pro. 38(3).-433-436. The authors describe soil factors that affect the adsorption of 2,4,5-T. Four soil types were studied, three agricultural soils (Glendale, Palouse, Ephrata) and one forest soil (Ordnance). Each soil was handled in four different ways: l) untreated; 2) organic matter removed, calcium saturated; 3) organic matter and oxide coatings removed, calcium saturated; 4) calcium saturated. C-2,4,5-T labeled at the carboxyl carbon position (specific activity 4.93 c/mg) was added to soil samples, and adsorption was determined from the.specific activity of the original solution and the activity of C-2,4,5-T remaining. The amount of herbicide adsorbed tended to increase with increasing organic matter. However, herbicide adsorbed was not in proportion to the amount of organic matter. Adsorption of 2,4,5-T in the four soils ranged from 14-290 (u)g/g soil. Removal of organic matter reduced the adsorption of 2,4,5-T, while removal of oxides of Fe and Al had little effect on three of the four soils studied. No differences in 2,4,5-T adsorption were observed when calcium-saturated soil was compared to untreated soils. The authors concluded that organic matter may be the single most important factor influencing the adsorption of 2,4,5-T by soil. 741. Okey, A. B., Bondy, G. P., Mason, M. 1., et al. (1978) Regulatory gene product of the AL locus. J. Biol. Chem. 254(22):11636-11648. The isolation and characterization of the cytosol receptor product of the Ah_ locus is described, the receptor was isolated from liver cytosol from 5 strains of mice and Sprague-Dawley rats, using sucrose density gradient analysis. The isolated receptor was characterized as to its affinity, capacity, specificity, thermolability and protease susceptibility. The isolated receptor showed high-affinity, lowcapacity binding to TCDD. The receptor bound polycyclic aromatic inducers of cytochrome P,-450 was thermolabile, and was primarily protein. The receptor was isolated in mouse strains that are responsive to enzyme inducers and not in non-responsive strains. The liver contained 60 fmol of receptor per mg of cytosolic protein, equivalent to 5,500 receptors per cell. Kidney and lung tissue contained receptor in the same concentration range as in liver. [ H]-TCDDreceptor complex was ioslated from the livers of responsive strains of mice after in vivo TCDD treatment. The authors concluded that cytochrome P,-450 induction and other effects elicited under Ah locus control, required that the presence of the receptor and its translocation to the mucleus, bound to the inducer. 742. Okey, A. B., Bondy, G. P., Mason, M. E., et al. (1980) 1979 Temperature-dependent cytosol-to-nucleus translocation of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in continuous cell culture likes. J. Biol. Chem. 255(23):11415-11422. The effect of TCDD on aryl hydrocarbon hydroxylase activity and characteristics of the TCDD-Ah receptor complex in various cell lines 262 �are described. Doses that produced half-maximal stimulation of aryl hydrocarbon hydroxylase activity were determined in Hepa-1, H-4-II-E, HTC and VERO continuous cell lines. The effect of temperature, and of proteases on the [ H]-TCDD-receptor complex and the time required for the complex to move from the cytosol to the nucleus were determined in vitro. About 1% TCDD taken up by the Hepa-1, H-4-II-E and HTC cell lines was bound to the receptor and a concentration of 0.28 nM TCDD produced half-saturation of nuclear binding sites in Hepa-1 cells. The receptor was more thermolabile in cytosol than in the nucleus and was susceptible to proteases, but not nucleases. The receptor was estimated to be 65, from its movement in sucrose density gradients. The half-maximal TCDD doses for enzyme induction were 0.23, 0.45, 110 and greater than 200 nM for H-4-II-E, Hepa-1, VERO and HTC cells, respectively. Hepa-1 cells had twice as many receptors as H-4-II-E cells and four times as many as HTC cells, while VERO cells had no detectable receptors. The authors concluded that enzyme induction does not necessarily result in cell lines (such as HTC cells) in which cytosol receptors could be demonstrated and translocation to the nucleus takes place. 743. Okonek, S., and Hofmann, A. (1975) On the question of extra corporeal hemodialysis in diquat intoxication. Arch. Toxicol. 33(3):251-257. A case report of diquat poisoning from ingestion in a suicide attempt is described. A 43-year-old woman ingested an unknown quantity of commercial diquat herbicide preparation (Reglone; 200 g/1 diquat ion). After 2 days of decreased appetite and vitality, an unknown amount of the same preparation was ingested again. The patient went into shock, was anuric, and had hemorrhagic mucosal necrosis of the mouth, throat, and esophagus within 1 day. Forced diuresis and hemodialysis were instituted and blood samples were collected and analyzed for 2,4-D colorimetrically after separation of 2,4-D by ion-exchange chromatography. At the end of the 6.5 hr. hemodialysis period, the serum diquat level had fallen to 30% of the initial level. A second hemodialysis was started 10.5 hr. after the first period ended and lasted 5 hr. The serum 2,4-D level was reduced by 30% by the second hemodialysis. The patient died in protracted cardiovascular collapse 19 hours after the second hemodialysis. The authors concluded that hemodialysis was unable to remove a toxicologically significant amount of diquat from the circulation 1 day after ingestion. 744. Olds, K. L., (1976). Monitoring of pesticide disposal practices, Iowa Army Ammunition Plant, Burlington Iowa, Sept 74 - Jan 75. USNTIS Publication No. AD030862.18 pp. 745. Oliver, R. M. (1975) Toxic effects of 2,3,7,8- tetrachlorodibenzo 1,4-dioxin in laboratory workers. Br. J. Ind. Med. 32:49-53. Three cases involving scientists who synthesized TCDD in a laboratory and exhibited symptoms of dioxin toxicity are described. Two scientists 263 �prepared TCDD in a fume hood wearing gloves and an overalls. Eight weeks later, one man developed chloracne on the face and neck, which subsided in 18 months. This scientist, who had no other clinical changes, had severe acne as an adolescent. The second scientist was involved in two syntheses, one week apart. Six weeks after the first synthesis, he noticed excessive oiliness of the skin and chloracne developed in two waves, which simulated the two exposure incidents. A follicular rash that developed on his hands and arms when the chloracne appeared cleared rapidly. No other abnormal findings were evident at the time, and the chloracne healed within one year. Two years after the exposure, the second scientist developed abdominal pains, flatulence, headaches, excessive fatigue, decreased concentration, irritability, hirsutism, blurred vision, and decreased muscular coordination. All of these symptoms, except the abdominal pains, subsided in 6 months. The third scientist worked with the TCDD preparation synthesized by the other scientists. Three years after his exposure, he developed an inability to concentrate, indigestion, diarrhea, decreased sense of taste, visual problems, insomnia, thigh pains, excessive oiliness of the skin, and hirsutism. All symptoms except hirsutism subsided in 6 months. A blood examination 3 years after the synthesis revealed hypercholesterolemia in all three men and hyperlipoproteinemia in the latter two men. None of the patients showed evidence of acquired porphyria. The authors concluded that TCDD exposure was the likely cause of all of the symptoms described. 746. Olson, J. R, , Holscher, M. A., and Neal, R. A. (1980) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the golden Syrian hamster. 'Toxicol. Appl. Pharmacol. 55:67-78. The acute toxicity of TCDD in the hamster is described. TCDD was administered orally or intraperitoneally to groups of male and female golden Syrian Hamsters and the mortality rate was determined 50 days after a single dose was administered. Body weight gain and chemical and enzyme analyses of blood were determined during the 50 day period after exposure. Histopathology and body weight gains were determined at autopsy. The 50 day intraperitoneal LD,-n was above 3,000 ug/kg of TCDD and the oral LD,-n was calculated at 1157 ug/kg. Weight gains were reduced after TCDD was administered by either route. Thymic atrophy was the most severe lesion noted at autopsy, while severe ileitus and peritonitis were observed in orally-treated animals. Chemical changes in treated animals included decreased albumin, chloride, urea nitrogen, and triglyceride levels and increased alkaline phosphatase, bilirubin, protein, iron and cholesterol levels. The authors concluded that the hamster is the most sensitive mammalian species to TCDD toxicity and that higher toxicity after oral, compared to peritoneal exposure was caused by ileitus and peritonitis, seen only after oral dosing. 264 �747. Olson, J. R. , Gasiewicz, T. A.., and Neal, R. A. (1980) Tissue distribution excretion, and metabolism of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) in the golden Syrian hamster. Toxicol. Appl. Pharmacol. 56:78-85. The distribution, excretion, and metabolism of TCDD in the hamster is described; A single oral or intraperitoneal dose of 650 ug/kg of [ H]or [ C]-TCDD was administered to groups of male golden Syrian hamsters. The tissue distribution and urinary, fecal and biliary excretion of radio-activity were determined during the subsequent 35 days. Excretory rates were analyzed pharmacokinetically. The presence of metabolites were assessed in various tissues and isolation of biliary metabolites was carried out by high pressure liquid chromatography. The liver, adipose tissue, and adrenals contained the highest concentrations of radioactivity which was present as unmetabolized TCDD. Elimination was probably first-order for both isotopes, with half-times of 11-12 days for intraperitoneal doses and of 15 days for oral doses. The urine and feces contained 35 and 50%, respectively, of the doses administered in 35 days. On day 7 after treatment, the 24-hour bile sample contained 1% of the dose, which was recovered as 1 major and several minor metabolites, but not as unmetabolized TCDD. No enterohepatic cycling was observed. The authors concluded that once TCDD metabolites were formed, they were rapidly excreted in bile and the enhanced rate of TCDD biotransformation partially explains the low sensitivity of this species to TCDD toxicity. 748. Olson, R. J. , Trunible, T. E., and Gamble, W. (1974) Alterations in cholesterol and fatty acid biosynthesis in rat liver homogenates by aryloxy acids. Biochem. J. 142(2):445-448. The effects of 2,4-D and 2,4,5-T on fatty acid biosynthesis by rat liver homogenates in vitro was studied. Radioactive substrates of fatty acid synthesis were incubated with rat liver homogenates (whose preparation was not described) in the presence of inhibitors and cofactors. Non-saponifiable lipids were extracted and the radioactivity in the extracts as well as in the fatty acid fraction and cholesterol fraction were determined. Thin layer chromatography was used to separate and identify some radioactive melabolites. From 23-31% of the incorporated radioactivity from [2- C]-mevalonate was recovered in the non-saponifiable lipid fraction, with 89% in cholesterol, 2% in squalene and 4% in farnesol. Both 2,4-D and 2,4,5-T (less than 1 ppm dioxin,contaminant) produced dose-related decreases in the incorporation of [ C]-nevalonate into non-saponifiable lipids of 77% maximum inhibition by 2,4-D and 65% maximum by 2,4,5-T. The proportion of non-saponifiable lipid radioactivity recovered as cholesterol decreased to 37% with 9mM 2,4-D and to 5% with 4.5mM 2,4,5-T, while the squalene fraction comprised 35% of the radioactivity in 2,4-D treated incubates and 54% with 2,4,5-T present, and 20% of the radioactivity was in^armene for 2,4-D and, 2,4,5-T homogenates. Incorporation of [1- C]-nevalonate into CO,, was unaltered by phenoxyacid treatment and incorporation of [1- C] isopentenyl pyrophosphate into cholesterol resembled nevalonate incorporation in 265 �treated and control incubations, 2,4-D produced 100% inhibition of [2- C]-acetate into non-saponifiable lipids and 2,4,5-T produced./3% inhibition. At low concentrations, both compounds stimulated [2- C] acetate incorporation into fatty acids and inhibited incorporation at the highest concentrations (4.5-9.OmM). The authors concluded that both compounds inhibited precursor incorporation into lipids but were not able to identify which step(s) were blocked and indicated that no implications related to acute toxicity were found from the data. 749. Orberg, J. (1980) Effects of low protein consumption on the renal clearance of 2,4-dichlorophenoxyacetic acid (2,4-D) in goats, Acta Pharmacol. Toxicol. 46(2):138-140. The rate of 2,4-D clearance was determined in goats that were fed a low protein diet. Four female goats, including one 6-month-old kid, were administered a normal diet of 6.3% digestible protein for 3 of 5 periods of 4-6 weeks and a low protein diet of 1.4% digestible protein for the second and fourth period. At the end of each feeding period, urea, [ C]-2,4-D and [ H]-inulin were administered by intravenous infusion. The resultant plamsa level for 2,4-D was 10 ng/ml. Both blood and urine samples were collected during the infusion period and analyzed for urea and for radioactivity. The urea and inulin clearances were 3 and 1.7 times lower during the periods of low protein consumption than during control periods; the 2,4-D clearance was about 1.7 times lower during the low protein than control period. The authors concluded that the small decrease in 2,4-D clearance by about 50% would not be likely to significantly enhance the toxicity of the compound in animals with low protein consumption. 750. Orberg, J. (1980) Observations on the 2,4-dichlorophenoxyacetic acid (2,4-D) excretion in the goat. Acta Pharmacol. Toxicol. 46(1):78-80. The rate of renal clearance of 2,4-D and the proportion of 2,4-D bound to plasma proteins was determined in goats. Three female goats were administered 2,4-D by continuous venous infusion at the rate of 0.5-32 mg [ C]-2,4-D per minute. [ H]-inulin was also infused and urine and blood samples, collected for six consecutive 10 minute periods, were counted by radioactivity. Protein binding was determined in blood samples by equilibrium dialysis from the amount of radioactivity accessible to both sides of a cellulose membrane. Urinary metabolites were analyzed by thin-layer chromatography. About 97% of the plasma 2,4-D was bound to protein for 2,4-D plasma concentrations less than 20 ug/ml and gradually decreased at higher 2,4-D plasma concentrations. The ratio of 2,4-D clearance to inulin clearance and the glomerular filtration rates were constant for plasma concentrations below 40 ug/ml of 2,4-D; both parameters steadily decreased at higher plasma concentrations. 2,4-D was excreted much more rapidly than inulin at low 2,4-D plasma levels, despite high protein binding; at high levels, inulin is transported more rapidly than 2,4-D. The authors concluded that 2,4-D was transported by tubular secretion and 2,4-D altered renal blood flow and/or renal blood pressure (because it decreased the rate of glomerular filtration). 266 �751. Oreopoulos, D. G. , and McEvoy, J. Med. J. 45(527):635-637. (1969) Diquat poisoning. Postgrad. A case report of diquat poisoning after accidental ingestion is presented. An 18-year-old man accidentally ingested diquat and attempted to spit it out. During the next 10 hours, he had diarrhea. Ten hours after the ingestion he was hospitalized and treated by gastric lavage. After 56 hours no symptoms were apparent and forced diuresis was initiated as a prophylactic means. A good diuretic response ensued and treatment continued for 11 days because diquat metabolites were detected qualitatively for 9 days. All clinical test results were normal during and after treatment and no clinical symptoms emerged. The authors concluded that acute poisoning by diquat followed a different course than paraquat poisoning and differences in tissue distribution and elimination by man and by other species may exist. 752. Orians, G. H., and Pfeiffer, E. W. (1970) Ecological effects of the war in Vietnam, Science 168:544-554. [Review article.] 753. Ott, M. G., Holder, B. B., and Olson, R. D. (1980) A mortality analysis of employees engaged in the manufacture of 2,4,5-trichlorophenoxyacetic acid. J. Occup. Med. 22(l):47-49. Mortality data is reported for workers that were occupationally exposed to 2,4,5-T during its manufacture at Dow Chemical Co. A group of 204 people were occupationally exposed to 2,4,5-T for at least 1 month between 1950 and 1971. Only 3 of these workers could not be traced for this study. Eleven deaths occurred in the total group. .Observed deaths for the group of 11 workers, for the group of 6 workers with less than 1 year of exposure, and for the group of 5 workers with more than 1 year of exposure were lower than expected for all categories, except external causes (accidents and suicides). The categories were malignant neoplasms, diseases of cardiovasuclar system and all other causes (no deaths fell into this last category). Six deaths, including 5 in the low exposure group, were from external causes, including 2 suicides (that occurred more than 10 years after exposure), 3 car accidents and 1 fire. The expected incidence was 3.7. The authors concluded that no adverse effects detectable by mortality-statistics were observed in workers exposed to 2,4,5-T during its manufacture. The small number of deaths that have occurred in the exposure group indicate that is premature to evaluate the effects of 2,4,5-T exposure of these workers by mortality statistics. 754. Ott, M. G., Holder, B. B., and Olson, R. D. (1979) A longevity survey of employees exposed to 2,4,5-trichlorophenoxyacetic acid. Unpublished report of the Medical Department, Dow Chemical Company, Midland, Mich. 15 pp. [Not available.] 267 �755. Paggiaro, P. L., Martino, E., and Mariotti, S. (1974) A case of 2,4-dichlorophenoxyacetic acid (2,4-D) poisoning. Med. Lavoro 65(3-4):128-135. A case of accidental poisoning by 2,4-D is reported. The subject, a 63-year-old man, inhaled fumes of a solution comprised of 44% 2,4-D isopropyl ester. Within 2-3 hours the subject lost consciousness and within 2 days cephalea and fever appeared, followed by myalgia in the neck and lower limbs, anorexia, muscular hypertonia, tachyarrhythmia, urinary incontinence and constipation. Sepsis of the urinary tract was identified by urine culture analysis. No compromise in thyroid function was observed. Therapy with hydroquinidine corrected the intermittent nodal tachycardia in 5 days and muscular hypertonia in 4 days. The authors reviewed the symptoms produced from acute and chronic 2,4-D exposures in humans and observed that cephalea, constipation and tachyarrhythmia were symptoms that were observed only in the current case. The authors concluded that 2,4-D poisoning caused the nodal tachycardia of the patient and that a large amount of 2,4-D was inhaled (the amount was not estimated) and suggested that 2,4-D interfered with membrane polarization of striated muscle fibers. 756. Palm, C. E. (1968) Original arsenical herbicides. In Classification ahd chemistry of herbicides, chapter 10, Principles of Plant and Animal Pest Control, Volume 2. No. 1597 [Washington, D.C.~; National Academy of Sciences] pp. 167-169. [Review article.] 757. Palmer, J. S. (1963) Chronic toxicity of 2,4-D alkanolamine salts to cattle. J.A.V.M.A. 143(4):398-399. The toxic effect of 2,4-D was described in cattle. Yearling steer (1 per dosage level) were administered daily doses of 50-250 mg/kg of 2,4-D alkanolamine salt (comprising 65% of a commercial formulation) for up to 112 days. Body weights and signs of intoxication were recorded during the exposure period and gross pathology was assessed at necropsy. A total of 112 treatments of 50 mg/kg doses caused no intoxication. Doses of 100, 200, and 250 mg/kg produced signs of toxicity after 86, 34, and 15 treatments, respectively. These signs included cracked muzzles, ulcerated mucous membranes, epistaxis upon restraint, nasal discharge, apathy, and depression. The steer that received 20 treatments of 250 mg/kg doses had tympany, severe hemorrhages of the large intestine, and pulmonary congestion and the steer given 44 treatments of 200 mg/kg showed hind leg muscle weakness and a staggering gait. Death (in unspecified animals) was attributed to kidney inflammation and a pneumonic condition. The authors concluded that 2,4-D had a low order of toxicity and probably did not accumulate in bovine tissues. 268 �758. Palmer, J. S., and Radeleff, R. D. (1964) The toxicologic effects of certain fungicides and herbicides on sheep and cattle. Ann. N. Y. Acad. Sci. 111:729-736. The toxic effects of orally administered 2,4-D and 2,4,5-T to sheep and cattle were described. Sheep, primarily Delaine breed, and cattle, primarily dairy breeds, were orally administered commercial preparations of 2,4-D alkanolamine salt, 2,4-D propylene glycol butyl ether esters, 2,4,5-T propylene glycol butyl ether esters, or 2,4,5-T triethylamine salt and dalapon in gelatin capsules daily or five times per week. One animal was treated with each test dosage of each compound. Clinical signs of toxicity and necropsy findings were reported. Sheep given 481 doses of 100 rag/kg of either form of 2,4-D were unaffected by treatment; 7 doses of 500 mg/kg of 2,4-D salt were lethal to another sheep and 9 doses of 250 mg/kg of the ester was lethal. The tolerated and lethal doses of 2,4-D in cattle were 50 mg/kg for 112 doses and 200 mg/kg for 44 days respectively. Poisoned animals showed anorexia, depression, weight loss, muscle weakness, ataxia, and ulceration of bovine nasal mucous membranes. In sheep, 481 doses of 100 mg/kg of the 2,4,5-T salt was tolerated, and 369 doses of 100 mg/kg of the 2,4,5-T ester or 7 doses of 250 mg/kg were lethal. In the cow, 7 doses of 250 mg/kg of the 2,4,5-T ester was also lethal. Anorexia and weight loss occurred in 2,4,5-T poisoned animals. Necropsy findings for these animals included degeneration of the kidneys and liver, cardiac hemorrhages, congestion of visceral blood vessels, and rumen atony. Dalapon was tolerated at doses of 100 mg/kg for 481 days or 500 mg/kg for 10 days to sheep or 500 mg/kg for 8 days to cattle. The authors concluded that accidental ingest ion of any of these compounds is unlikely to present a significant health hazard to sheep or cattle. 759. Park, J., Darrien, I., and Prescott, L. F. (1977) Pharmacokinetic studies in severe intoxication with 2,4-D and Mecoprop. Clin. Toxicol. 18:154-155. The pharmacokinetics of 2,4-D is described from data from a male subject who ingested 70 ml (estimated) of an herbicide comprised of 10% 2,4-D and 20% 4-chloro-2-methylphenoxypropionic acid (Mecoprop), as the amine salts. Clinical symptoms 3 hours after ingestion included neuropathy, myopathy, hyperventilation, pyrexia, and coma. Alkaline diuresis, which was initiated to force excretion of the herbicides reversed the coma, pyrexia, and hyperventilation. The levels of 2,4-D and Mecoprop in blood and urine were analyzed by gas liquid chromatography and the plasma half-lives and renal clearances were calculated before and during diuresis. Urine samples were acid-hydrolyzed to determine the proportion of each conjugate that was excreted as acidlabile conjugates. The plasma half-lives of 219 hours for 2,4-D and 39 hours for Mecoprop were reduced to 5 and 14 hours, respectively during alkaline diuresis, with concomitant increases in the clearances of both compounds during diuresis. About 10% of total 2,4-D and 40% of Mecoprop were metabolized to acid-labile conjugates. The authors concluded that renal clearance at alkaline urine pH and the higher efficacy of alkaline diuresis for 2,4-D than Mecoprop reflected the 269 �higher pKa value for 2,4-D and more limited metabolism compared with Mecoprop. 760. Pasi, A., and Embree, J. W., Jr. (1976) Further comments on the assessment of the mutagenic properties of diquat and paraquat in the murine dominant lethal test. Mutat. Res. 31:123-125. [Editorial.] 761. Pasi, A., Embree, J. W. Jr., Eisenlord, G. H., and Hine, C. H. (1974) Assessment of the mutagenic properties of diquat and paraquat in the murine dominant lethal test. Mutat. Res. 26:171-175. The authors tested the mutagenic effects of diquat in a dominant lethal test in Swiss-Webster mice. Five males 8-10 weeks old were given a single intraperitoneal injection of 6 nmole/kg body weight diquat dibromide monohydrate which corresponded to the LD,. for this compound. A control group of 10 males received a single intraperitoneal injection of physiological saline (10 ml/kg). No positive control was included in the study. Two hours after treatment each male was caged with 3 untreated females, 8-10 weeks old. After one week, males were removed and placed with another group of 3 untreated females. This was repeated for eight consecutive weeks, in order to assess tile effects of diquat on the entire spermatogenic cycle of the mouse. Fifteen days after being caged with the male, the females were killed and uteri scored for pregnancy, total number of implants, early fetal deaths and late fetal deaths. The following statistical analyses were performed on the results: analysis of variance, chi-square, and student's t-test. No significant differences in total implants or early fetal deaths were observed indicating no mutagenic effect. However, the pregnancy rate between diquat treated weeks 1 through 5 (p less than 0.05 week 1 and 4, p less than 0.01 than week 2,3,5) and untreated controls decreased significantly. This antifertility effect corresponds to effects on postmeiotic early and late spermatids and epididymal sperm for weeks 1, 2 and 3. For weeks 4 and 5 the antifertility effect corresponds to effects on premeiotic early and late spermatocytes. 762. Paynter, 0. E., Tusing, T. W., McCollister, D. D., and Rowe, V. K. (1960) Toxicology of dalapon sodium (2,2-dichloropropionic acid, sodium salt). J. Agric. Food Chem. 8(1):47-51. The acute, subacute, chronic, and reproductive toxicities, of dalapon were studied in various animal species and tissue levels of dalapon were determined in the rat and dog. Single doses of dalapon (83-85% purity) were administered by intubation to rabbits, guinea pigs, rats, mice, and chickens. The LD,-0 values were calculated and gross and histopathological examinations were performed. Dermal irritation of 10 daily applications undiluted, \7« and 10% aqueous solutions of dalapon was tested on intact and abraded rabbit skin. Eye irritation produced 270 �by powdered dalapon or 1% or 10% aqueous solutions was tested in the rabbit. A heifer and a calf were administered 1 g/kg dalapon by oral intubation orally for 10 successive days. Two dogs were orally administered dalapon 5 days per week for 80 days. Doses were increased from 50 mg/kg per day initially to 1000 mg/kg per day. Wistar-Dow rats (10 per group) were fed diets with from 0.0115 to 1.15% dalapon for 97 days. Dogs (3 per group) were administered daily doses of 15, 50 or 100 mg/kg dalapon in capsules, 5 days per week for 52 weeks. Carworth Farm rats (44 per group) were fed diets with 100, 300, or 1000 ppm dalapon for 2 years. A 3-generation reproductive study was performed on rats fed diets of 0.03 to 0.3% (3000 ppm) dalapon. The LD5Q values ranged from 3.86 g/kg for the rabbit and guinea pig to 9.3 g/kg for the male rat. Death occurred rwithin 1 day and the only autopsy finding was the presence of a large amount of fluid and gas in the gastrointestinal tract. Undiluted dalapon produced hyperemia and dermal necrosis on both intact and abraded skin, 10% solutions were moderately irritating and 1% solutions were ineffective. Powdered dalapon produced severe conjunctivitis and corneal injury, while aqueous solutions were less potent eye irritants. Dalapon produced anorexia, lassitude, and diarrhea in the heifer, but no clinical effects in the calf or gross or histopathological changes except mild renal lesions in the calf. No subacute effects were observed in dogs that received 161-200 grams total of dalapon except vomiting. Biochemical and hematological analyses, urinalyses, and gross and histologic pathology exams revealed no adverse effects in dogs or rats after subacute or chronic treatment. An increase in kidney weights occurred in both species after chronic treatment. No reproductive effects were detected in any generation of the rat study. Levels of dalapon in dog and rat tissues and milk after chronic exposure were low relative to the administered doses, with maximum levels of 28 ppm in the rat kidney and 78 ppm in the dog kidney. The authors concluded that dalapon has a low order of toxicity, but protection against eye exposure should be used when handling the compound and dermally applied dalapon should be washed off with water. 763. Pazderova, J., Lukas, E., Nemcova, M., Spacilova, M., Jirasek, L., Kalensky, J., John, J., Jirasek, A., and Pickova, J. (1974) Chronic intoxication by chlorinated hydrocarbons formed during the production of sodium 2,4,5-trichlorophenoxyacetate. Prac. Lek. 26(9):332-339. The health of 55 workers from a 2,4,5-T factory in Czechoslovakia is described. All of the information was presented in two previous reports; see Jirasek et al. (1973) and Jirasek et al. (1974). 764. Pazderova-Vejlupkova, J., Lukas, E., Nemcova, M., Pickova, J., and Jirasek, L. (1980) Chronic poisoning by 2,3,7,8-tetrachlordibenzop-dioxin. Pracov. Lek. 32:204-209. The health of 55 workers involved in the manufacture of 2,4,5-T was observed over 5 years. The contaminant, TCDD, was detected on the walls and furnishings of the factory, as well as in the 2,4,5-T. Each 271 �worker had an average of five medical examinations. Chloracne was observed in 95 percent of the workers, and increased elimination of uroporphyrins occurred in 41 percent (half of these workers also showed dermal symptoms of porphyria cutanea tarda). Alterations in lipid metabolism occurred frequently: hyperlipemia was seen in 67 percent of the workers, hyperchloesterolemia in 56 percent, and hyperphospholipemia in 42 percent. These lipid-metabolic parameters improved over the 5-year observation period. Polyneuropathy was present in one-third of the workers at the end of the observation period. No renal, cardiovascular, hematologic, or optic problems were encountered in the workers. At the time of publication, the only deaths among the exposed workers were: two in traffic accidents, one from cirrhosis of the liver and infectious hepatitis (age 40), one from arteriosclerosis (age 57), and two from bronchogenic carcinoma (ages 47 and 50). The authors indicated that the number of observed cases of bronchogenic carcinoma was too small to imply a causal relationship with exposure. Two of the workers' wives each reported having one miscarriage, and 18 children were born during the period in which the exposed workers remained ill. The authors indicated that this incidence of 2 abortions in 18 pregnancies was far below the worldwide incidence of 15-20 percent for spontaneous abortions. The authors did not indicate that they used a systematic method to collect information on reproduction or that they considered psychological factors which would increase voluntary birth control. The authors suggested that polyneuropathy was a direct response of nerve tissue to TCDD and potentiated negative effects on fat and carbohydrate metabolism. The authors attributed some of the psychological disturbances they observed to the fear of death, disfigurement, and disability. This TCDD exposure is significant, but the authors do not present the time of the appearance of symptoms or their duration, or thoroughly describe the incidence or extent of each symptom. 765. Pegg, D. G., Hewitt, W. R., McCormack, K. M., and Hook, J. B. (1976) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on renal function in the rat. J. Toxicol. Environ. Health 2:55-65. Renal function of rats treated with TCDD was studied in vivo and in vitro. Male Sprague-Dawley rats were administered a single dose of 10-50 ug/kg TCDD intraperitoneally or 1 or 5 ug intragastrically. Some rats were then given drinking water containing ammonium chloride until the kidneys were removed to establish acidosis. After 3 to 7 days, the kidneys were removed and accumulation of organic molecules was measured in kidney slices incubated in medium containing [ C]- labeled compounds. Glucose and ammonia production.during a 1 hour incubation period were analyzed in renal slices. [ C]-paraaminohippurate (PAH) and [ H]-inulin clearances were measured in vivo under a saline load and during control periods sleeping times were recorded for rats administered 115 mg/kg hexobarbital. Growth rates of rats treated with 10 or 25 ug/kg TCDD were significantly below control rats after 3 and 7 days and hexobarbital sleeping times were increased substantially by 25 ug/kg of TCDD. PAH uptake was decreased 7 days after 1 or 5 ug of TCDD 272 �was given orally or 25 ug/kg TCDD was injected but was unaltered 3 or 7 days after 10 ug/kg TCDD was given. A dose of 25 ug/kg TCDD did not alter 2-deoxyglucose uptake 7 days later. Ammonia and glucose pro=duction were unaltered by TCDD in non-acidotic and acidic rats. Glomerular filtration rate, measured as inulin clearance and effective renal plasma flow (PAH clearance) were decreased 7 days after 25 or 50 ug/kg of TCDD was given, although their ratio was not altered by TCDD. Urine flow rates following volume expansion were lower in TCDD-treated rats than in control rats. Fractional sodium excretion was affected minimally by TCDD treatment. The authors concluded that TCDD-induced impairment of renal function reflected the systemic toxicity of TCDD and not a specific effect on a particular aspect of renal function. 766. Peoples, S. A. (1975) Review of arsenical pesticides. In Arsenical Pesticides, ACS Symposium Series Vol. 7. [Washington, D.C.1American Chemical Society] pp. 1-12. [Review article.] 767. Peoples, S. A., Maddy, K. T., Peifer, W. R., and Edmiston, S. (1979) Occupational exposures to pesticides containing organoarsenicals in California. Vet. Hum. Toxicol. 21(6):417-421. The authors tabulated occupational accidents involving cacodylic acid, methanearsenic acid, and their salts which occurred in California from 1975 to 1977. At'least 22 of the 34 incidents involved herbicides containing cacodylic acid or its sodium salt (or both). Nine of the 34 incidents involved systemic symptoms, including vomiting, diarrhea, and abdominal pain. Of the remaining cases, 13 involved eye irritation and 12 involved skin injuries, including contact dermatitis and allergic rash. All cases responded well to treatment. Most accidents resulted from equipment failure or lack of use of protective gear. 7.68. "Perspectives on Chlorinated Dibenzodioxins and Dibenzofurans." (1973) Lee, D. H. K.; Falk, H. L.; Dixon, R. L.; Fishbein, L. G. R.; Mailing, H.; Moore, J. A.; Hoel, D.; Hart, L., eds. Environmental Health Perspectives Vol. 5. (Research Triangle Park, North Carolina: DHEW.) 313 pp. [Review article.] 769. Peterson, J. (1978) Seveso: The event. Ambio. 7(5-6):232-233. [Editorial.] 273 �770. Peterson, R. E., Modhukar, B. V., Yang, K. H., and Matsuraura, F. (1979) Depression of adenosine triphosphate activities in isolated liver surface membranes of 2,3,7,8-tetrachlorodibenzo-p-dioxin treated rats: Correlation with effects of ouabain biliary excretion and bile flow. J. Pharmacol. Exp. Ther. 210(2):275-282. The effects of TCDD on liver surface membrane adenosine triphosphatase (ATPase) levels and on biliary excretion are described. Male Holtzman rats were administered a single oral dose of-10 or 25 ug/kg TCDD in acetone-corn oil (1:19). After 2-40 days, [ H]-ouabain was administered intravenously; bile and blood samples were collected over the next 60 minutes and biliary excretion.of ovabain was calculated. In other animals, biliary excretion of [ C]-erythritol was determined. Liver surface membranes (LSM) were isolated from some TCDD-treated rats, examined by electron microscopy to confirm purity of the preparation and Na+, K+-ATPase and Mg++ATPase were both assayed. [ H]-TCDD was administered to a group of rats and the radioactivity associated with the isolated LSM fraction was determined. The effect of TCDD added to LSM preparations from control rats on ATPase activity was also reported. Both types of ATPase levels from TCDD-treated rats were depressed over the 40 day period, compared to vehicle controls and to pair-fed controls. Bile flow, bile salt excretion and ouabain and erythritol biliary excretions were all depressed by TCDD, with signs of recovery evident by the last time point. Blood clearance of ouabain was also decreased. Ten days after [ H]-TCDD treatment, the LSM fraction contained 0.0136 picomol TCDD per ug LSM.groteiniq The equivalent of this concentration in vitro was 10 to 10 M; even concentrations of 10 M TCDD in vitro failed to alter ATPase activity. The authors concluded that the LSM was a site of TCDD action and the effect of TCDD on LSM ATPase activity and biliary excretion of ouabain correlated (but were not necessarily causally related). 771. Pfeiffer, E. W., and Orians, G. H. (1972) "Chapter 3: The Military Uses of Herbicides in Vietnam," In Harvest of Death. Neilands, J. B., ed., (Free Press) p. 117-176. [Background material.] 772. The Phenoxy Herbicides - Second Edition. (1978) Council for Agricultural Science and Technology, Report No. 77. 28 pp. [Review article.! 773. Pilinskaya, M. A. (1974) Cytogenetic effect of the herbicide 2,4-D on human and animal chromosomes. Tsitologiya i Genetika 8(3):202-206. The author describes the cytogenetic effects of 2,4-D on human lymphocytes in vitro and in mouse bone marrow cells in vivo. Human lymphocyte cultures were established from two healthy donors with no known exposure to mutagens. 2,4-D was added to cultures at 0.002, 274 �0.02, 0.2, 2.0, 20 or 50 ug/tnl. Chromosome spreads were prepared according to the author's laboratory methods, which were unspecified. Metaphases (500 per dose level) were examined and scored for chromatid fragments and exchanges and chromosome fragments, dicentrics, exchanges, and rings. Results were analyzed by the X method. No increase in chromosome damage was observed at the lowest dose of 2,4-D, 0.002 ug/tnl. At 0.02 and 0.2 ug/ml chromosomal aberrations increased with increasing dose and were significantly different from the controls. At doses of 2.0 ug/ml, high cytotoxicity was observed. Chromosome damage did not increase above the levels observed at 0.2 ug/ml. The most frequent type of chromatid damage observed was single acentric fragments. Chromosome aberrations were mainly paired acentric fragments; 81.4% of the chromosome breaks occurred in the long arms. The authors concluded that 2,4-D was a potential genetic hazard to humans. The cytogenetic effects of 2,4-D in mice were also examined. Male outbred white mice (18-200g; 6 mice per dose level) received a single oral dose of 10, 50, 100 or 300 mg 2,4-D/kg body weight. Ten control mice were also included in the study. Mice were killed 20 hr after administration of the herbicide. Chromosome spreads were prepared and 200 metaphases per animal were scored. Increased chromosome aberrations were observed only at the toxic doses of 100 and 300 mg/kg. Single acentric fragments were the most common form of chromosome damage; paired fragments occurred much less frequently; and translocations were rare. From these results the author concluded that 2,4-D is a weak mutagen in mice. 774. Piper, W. N., Rose, J. Q., and Gehring, P. J. (1973b) Excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Environ. Health Persp. 5:241-244. The experiments published in this report were presented in a previous publication, see Piper, W.N., Rose, J.Q., and Gehring, P.J. (1973a). 775. Piper, W. N., Rose, J. Q., and Gehring, P. J. (1973a) Excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Symposium on chlorodioxins, origin and fate, Washington, DC. Advances in Chemistry Series 120:85-91. The levels of TCDD excreted and distributed in various rat tissues up to 7 days after administration was,determined. Male Sprague^Dawley rats were administered 50 ug/kg [ C]-TCDD (specific activity, 2.8 uCi/mg; 93-95% purity) in acetone-corn oil (1:9) by oral intubation. The CO- of expired air was trapped and urine and feces were collected; all samples were analyzed for radioactivity as well as tissue samples. About 30% of the dose was excreted in feces in 48 hours, 53% was excreted in feces in 21 days, 13% in urine and 3% in expired air. Beyond the first 2 days (after unabsorbed TCDD had been excreted in the feces), clearance of radioactivity from the body showed first order kinetics, with a half-life of 17 days. After 3, 7, and 21 days the liver contained 3.2, 4.5, and 1.3% of the dose of TCDD per gram and fat contained 2.6, 3.2, and 0.4%, respectively. Tissue levels were lower 275 �for 12 other tissues reported at the same time periods. The carcass was analyzed for radioactivity (result was not reported) and total recovery was calculated as 96.8% of the dose. The authors concluded that some of the dose was not absorbed and the liver and fat are the primary sites for TCDD localization; some metabolism of TCDD occurred because C0_ was recovered in expired air. The authors noted that the administered dose was toxic to the animals, and TCDD may have been excreted differently by animals in better physical condition. v 776. Piper, W. N., Rose, J.Q., Gehring, P. J. (1971) Paper presented at the meeting of the American Chemical Society, Washington, DC. [Not available.] 777. Piper, W. N., Rose, J. Q., Leng, M. L., and Gehring, P. J. (1973) The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral administration to rats and dogs. Toxicol. Appl. Pharmacol. 26(3):339-351. The pharmacokinetics of 2,4,5-T plasma clearance and urinary excretion were studied in the rat and the dog. Single doses of 5 to 200 mg/kg [ C]-2,4,5-T (98+% purity) in acetone-corn oil (1:9) were administered by intubation to Sprague-Dawley rats (6 per dosage group) and a 5 mg/kg dose was administered by the same procedure to 4 beagle dogs. Blood, urine, feces, and expired air were collected and analyzed for radioactivity. Plasma protein binding of [ C]-2,4,5-T was determined in vitro as the amount of isotope that exchanged freely between plasma retained in dialysis tubing and the surrounding 2,4,5-T solution. Urinary metabolites were extracted and separated by thin-layer chromatography. Data for clearance of 2,4,5-T in the rat was simulated by a one-compartment model with apparent first-order rates of absorption and clearance. Rate constants and corresponding half-lives were from the computerized best-fit. The values that provided the closest simulation of the actual data when used in the equations derived from the kinetics model. The half-lives and volumes of distribution both increased with higher doses. Doses of 5 and 200 mg/kg resulted in half-lives of 4.7 and 25.2 hours, respectively, and 14.4 and 43.7 liters/kg, respectively. The rate of excretion decreased with higher doses and was comprised primarily of urinary excretion. The proportion of the dose excreted in feces increased at higher doses and less than 1% was recovered in expired air. Urinary metabolites were separated in urine from the highest doses groups and comprised up to 5% of the urinary radioactivity. Total recovery of radioactivity, (including radioactivity in the carcass) was 91.1%. The same kinetic pattern was seen for 2,4,5-T clearance in the dog although the half-life of 77 hours and volume of distribution were considerably larger than for the rat. After 9 days 42% of the dose was recovered from the urine and 20% from feces. (Cross-contamination of their samples was suspected). Three urinary metabolites accounted for 10% of the total urinary radioactivity, the remainder recovered as 2,4,5-T. No sex-related differences in results were observed. When 2,4,5-T was present at 276 �concentrations of 0.04 to 425 mg/1; 89.7% of 2,4,5-T bound reversibly to plasma protein. The authors concluded that detoxification mechanisms were altered at high doses of 2,4,5-T, decreasing the rate of clearance and that the slower rate of 2,4,5-T excretion of dogs compared to rats could account for the higher toxicity in dogs. 778. Pirie, A., and Rees, J. R. Eye Res. 9(2):198-203. (1970) Diquat cataract in the rat. Exp. The pathological changes associated with diquat-induced cataracts and the distribution of diquat to the eye are described for the rat. Wistar rats were fed a diet that contained 0.05 or 0.075% diquat dibromide for at least 1 year. The rats were killed and the eyes were removed and prepared for histology or analyzed for ascorbic acid, enzymes, and GSH Ievels1(reducing groups). Four male rats were administered 0.5-1.0 mg of [ C]-diquat intraperitoneally and 1-3 hours later ocular tissues, intraperitoneal fluid, and blood were analyzed for radioactivity. Gross examination of treated rats within 4-8 months revealed the development of a thin sheet of opacity which formed under the posterior capsule of the lens and took on an irregular pattern. This stage was followed by formation of a clearly defined nuclear cataract and then shrinkage and complete opacity ensued. At the earliest stage a thin plaque of ribonuclease-sensitive, basophilic material was observed histologically. No consistent decrease in mitosis occurred in,lens epithelia cells of treated rats. The concentration of [ C]-diquat remained below plasma concentrations 1-3 hours after treatment. Ascorbic acid levels in the ocular fluids decreased gradually with age and decreased more rapidly in diquattreated rats. The decrease in ascorbic acid levels were evident in rats with posterior opacity only. In rats with severe cataracts, lens weight was increased, total protein was diminished, and GSH levels remained unaffected. The authors concluded that very little of the administered dose of diquat reached the lung, and, therefore, diquat probably acted catatytically to produce cataracts that had different properties than cataracts caused by other agents. 779. Pirie, A., Rees, J. R., and Holmberg, N. J. (1970) Diquat cataract: formation of the free radical and its reaction with constituents of the eye. Exper. Eye Res. 9(2):204-218. Reactions of diquat with various components of the eye were studied in vitro and distribution of diquat to the eye was studied in the rabbit. Diquat was added to extracts of bovine lens, aqueous humor and vitreous humor and the effects of added chemicals and sources of light on the formation of diquat radicals was monitored as a decrease in ascorbate concentration in the incubation mixture. At 0.2 mM diquat concentration, catalysis of diquat radical formation in extracts of aqueous humor was accelerated in the presence of sunlight, was independent of metals, and produced hydrogen peroxide at a slower rate than the rate of ascorbate loss. In extracts of vitreous humor, in addition to ascorbate utilization hyaluronic acid depolymerization occurred in the 277 �presence of 0.05 mM diquat. At 0.5 mM, diquat also caused a 33% reduction in ascorbic acid of rat lens in vitro. Dutch rabbits (2) were administered 30 mg/kg diquat dichloride intraperitoneally. Controls were given saline. One hour after treatment, aqueous and vitreous humors were removed, exposed to ultraviolet light, and analyzed for ascorbic acid. Ascorbic acid levels were from 10% to 75% of control levels. Reduction of glutathione by glutathione reductase in the presence of diquat in aqueous humor or bovine lens extracts was demonstrated by an increase in the production of reduced glutathione end product measured after exposure to sunlight. Diquat had no effect on reduced glutathione levels in the absence of ascorbic acid. The authors concluded that diquat radical formation could lead to the formation of cataracts and to an alteration in ascorbic acid levels without a change in reduced glutathione levels. / 780. Pirie, A., Rees, J. R., Holmberg, N. J. rat. Biochem. J. 114(4): 89 pp. (1969) Diquat cataract in the [Abstract, only,] 781. Pocchiari, F. 2,3,7-Tetrachlorodibenzo-para-dioxin decontamination. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stolkholm: Swedish Natural Science Research Council, 1978a) pp. 67-70. [Background material.] 782. Pocchiari, P., Silano, V., Zampieri, A. (1979) Human health effects from accidental release of tetrachlorodibenzo-p~dioxin (TCDD) at Seveso, Italy. Ann. N. Y. Acad. Sci. 320:311-320. The areas southeast of a chemical plant in Meda, Italy, are categorized according to the degree of TCDD contamination in the soil, and a summary of results to date of a study of the health status of residents in this area is presented. An explosion at the Meda chemical plant that was synthesizing trichlorophenol resulted in TCDD contamination of the surrounding area. Zone A, with an area of 110 ha received about 2 kg of TCDD, and the 733 residents were evacuated 2 weeks after the explosion. Zone B, 270 ha, and zone R, 1,430 ha, each received 20 g of TCDD. The 4,800 residents of zone B and 22,000 in zone R were prohibited from cultivating or consuming local crops or raising poultry or other animals. At the time of publication, 50 cases of chloracne were detected in residents of zone A, two-thirds of them in children under 12 years old. In the other two zones, 0.5 percent to 1.2 percent of the residents exhibited skin lesions, compared to 0.3 percent observed in other towns in northern Italy. Neurological screenings in 1977 and 1978 revealed 7 percent and 12 percent respectively of the group from zone A with idiopathic clinical neural damage, compared to 1 percent of those from zones B and R identified in 1977. Subclinical damage, including reduced nerve conduction velocity, was seen in less than 5 278 �percent of any group. Of approximately 200 factory workers from the Meda plant and another factory in zone A, 4 percent showed clinical damage in peripheral nerve fibers and 3 workers showed polyneuropathy of the legs. No decrease in immune response was detected in 45 children from zone A when laboratory tests performed triannually from 1976 were compared with those from a control group of 45 unexposed children of the same age. Of 34 aborted fetuses examined (including 4 spontaneous and 30 therapeutic abortions), none had any alterations that were attributed to TCDD exposures. The frequency of malformations in newborn infants in the contaminated areas matched the expected frequency. The difficulties in obtaining reliable reproductive data were discussed. Of a group of 1,654 subjects from all three zones who were examined, 32 percent had evidence of hepatomegaly. Transient rises were observed in the percentage of abnormal liver function test results in 1976, An increased incidence of aberrations in peripheral lymphocytes was observed in factory workers and zone A inhabitants, compared to control groups. An increased incidence of childhood infectious diseases was attributed to the increased medical surveillance. The authors chose to postpone their conclusions on the effects of TCDD on human health until more data from their ongoing studies could be compiled and reviewed. 783. Pohl, R. J., Philpot, R. M., and Fouts, J. R. (1976) Cytochrome P-450 content and mixed function oxidase activity in microsomes isolated from mouse skin. Drug Metab. Dispos. 4(5):442-450. Induction of skin microsomal enzyme activities by dermally applied TCDD in the mouse is described. Swiss-Webster mice were pretreated with 0.3 (u)g of TCDD applied in acetone to about 30 cm of shaved skin. Controls were treated with acetone only. Microsomes prepared from the skin and the liver were assayed for aryl hydrocarbon hydroxylase activity, 7-ethoxy-coumarin deethylase activity and cytochrome P-450 content. Hepatic enzyme levels were induced three-fold 24 hours after TCDD treatment and five-fold after 72 hours. Skin microsomal enzymes were induced four to eight-fold after 24 hours and six to 30-fold after 72 hours. The P-450 content of both tissues was elevated. A spectral shift of the peak wavelength to 447 nm occurred for TCDD-induced cytochrome P-450 from skin. The authors concluded that mixed function oxidase activity in the skin is potentially capable of detoxifying topically applied chemical's or metabolizing them locally to irritants. 784. Poiger, H., and Schlatter, Ch. in rats. Nature 281:706-707. (1979) Biological degradation of TCDD Isolation of biliary metabolites of TCDD in the rat is described. Female Sprague-Dawley rats were orally administered 20 uC of [ H]-TCDD (41 Ci per mmol; 98.7% purity of radiochemical), several days after their bile ducts were connulated. Bile was collected for the subsequent 4 days and then the animals were killed and the livers were removed. Total radioactivity of the bile and liver were determined and the chemical nature of the biliary radioactivity was indentified by 279 �dichloromethane extractability, dialyzability (exclusion limit or type of dialysis tubing was not stated), enzyme susceptibility, and removal by lyophylzation and by thin layer and gas liquid chromatographies. About 1% of the dose was excreted daily in bile and 8.5-12% was recovered in liver. Two metabolites were isolated from bile, as water soluble conjugates. The authors concluded that TCDD was excreted in the bile in 2 distinct metabolic forms, which may have resulted from cleavage of the ether group. 785. Poland, A., Greenlee, W. F., and Kende, A. S. (1979) Studies on the mechanism of action of the chlorinated dibenzo-p-dioxins and related compounds. Ann. N. Y. Acad.^Sci. 320:214-230. [Review article.] 786. Poland, A. and Glover, E. 2,3,7,8-Tetrachlorodibenzo-para-dioxin and enzyme induction. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No 27,(Stolkholm:Swedish Natural Science Research Council, 1978) p 145-148, [Review article.] 787. Poland, A., and Glover, E. (1975) Genetic expression of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin: Evidence for a receptor mutation in genetically non-responsive mice. Mol. Pharmacol. 11:389-398. The potency of TCDD in inducing hepatic aryl hydrocarbon hydroxylase (AHH) activity is described for 15 strains of mice. Female mice, 4-9 weeks of age, were administered TCDD in para-dioxane, intraperitoneally or methylcholanthrene in corn oil. 10-16 hours later, they were killed and hepatic microsomal AHH toxicity was determined. The dose of TCDD that produced half of the maximal level of induction was found to be about 1 nmole/kg for 3 strains of mice that also show an AHH-inductive response to MC and about 10 nmoles/kg for 3 strains that are nonresponsible to MC. A single dose of 3 or 30 nmoles/kg TCDD or 0.3 m moles/kg MC were administered to 3 treatment groups for each of 14 strains of mice. For 7 strains, only the higher dose of TCDD induced AHH activity and for the remainder all 3 treatment groups showed induced AHH activities. Heterozygous offspring of a cross between a non-responsive strain and a responsive strain had sensitivity to TCDD that was intermediate between the 2 parental strains. The authors concluded that nonresponsive strains of mice were the consequence of a genetic mutation that produced an induction receptor with diminished affinity for the enzyme inducers, including TCDD and MC. 280 �788. Poland, A., and Glover, E. (1974) Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methyl-cholanthrane. Mol. Pharroacol. 10:349-359. The potency, time-course and species specificity of TCDD induction of aryl hydrocarbon hydroxylase (AHH) was compared to these parameters for 3-methylchloranthrene induction of AHH. Male Sprague-Dawley rats were administered TCDD in paradioxane or methylcholanthrene (MC) in corn oil or sodium pentobarbital in saline, intraperitoneally. One day later, livers were removed and microsomes were isolated and assayed for AHH activity. At 0.22 ug/kg TCDD produced half-maximal induction and was estimated to be 28,640 times as potent as MC on a molar basis. Both induces produced the same level of maximum stimulation and this level was not exceeded when the 2 compounds were tested in combination. Some rats were maintained for up to 35 days after an inducer had been administered and then AHH activity was assayed. MC-induced AHH activity and carbon monoxide-binding cytochrome peaked 4 days after treatment, then dropped sharply, whereas TCDD-induced AHH activity and CO-binding cytochrome remained elevated for 21 days, and showed a slight decrease by 35 days. Shifts in spectral maximas and changes in the ethyl isocyanide difference spectra induced by each agent followed the same time course as shown for AHH activity. Both agents induced AHH activity in rat kidney, intestine and lung, but not in the testicle. TCDD and MC produced 30-50% increases in aminopyrine N-demethylase and NADPH-cytochrome C reductase activities, compared to 4-5 fold increases induced by phenobarbital. Data on species differences in TCDD potency from other reports were discussed. The authors concluded that TCDD induction resembled MC induction in all respects studied except for a higher potency of TCDD; higher apparent affinity for an "induction receptor" and resistence to biotransformation were presented as possible explanation for TCDD's higher potency. 789. Poland, A., and Glover, E. (1973a) 2,3,7,8-Tetrachlorodibenzo-pdioxin: A potent inducer of delta-aminolevulinic acid synthetase. Science 179:476-477. TCDD was shown to inhibit 8-aminolevulinic acid (ALA) synthetase activity in chick embryos. At 4.7 x 10 moles per egg, TCDD injected into the air sac of eggs on day 17 of gestation, caused a 2-fold increase in ALA synthetase activity and at 1.6 x 10 mole/egg caused a 35-fold increase in activity 2 days after treatment. The TCDD effect was blocked by actinomycin D and by cycloheximide after 5 days, treated eggs maintained 70% of the maximally induced enzyme activity. Other dibenzo-para-dioxins which were reported (elsewhere) to be lethal and acnegenic at low doses also inhibited ALA synthetase. The ability of TCDD to inhibit ALA synthetase was considered relevant to its presumed ability to cause porphyria cutanea tarda in exposed workers. (This data is published in more detail in Poland and Glover, 1973b). 281 �790. Poland, A., and Glover, E. (1973b) Studies on the mechanism of toxicity of the chlorinated dibenzo-para-dioxins. Environ. Health Perspec. 5:245-251. The effect of TCDD on chick embryo and rat-aminolevulinic acid (ALA) synthetase and aryl hydrocarbon hydroxylase activities was studied, TCDD, dissolved in para-dioxane, was injected into chick embryos and.„ after 8 hours, hepatic enzyme activities were assayed. At 4.7 x 10 mole per egg, TCDD caused a 2-fold increase in ALA synthetase activity and at 1.6 x 10 mole per egg, TCDD caused a 35-fold increase compared to activity of control,eggs that were injected with para-dioxane only. Similarly at 1.6 x 10 moles per egg, TCDD^doubled AHH activity, and maximal induction was produced by 1.6 x 10 mole per egg. Tnduction of AHH by TCDD was maximum 18 hours after treatment was initiated, and persisted for 5 days before declining. The inductive effects in both enzymes were produced by other dibenzo-para-dioxins with carbons at positions 2, 3, 7, and 8 substituted with halogens in 3 positions with the 4th position free. TCDD produced a 5 fold increase in rat liver AHH activity at 3.1 x 10 mole/kg and was 3 x 10 times more potent than 3-methylcholanthrene (3-MC) in its capacity to induce AHH activity. Although both TCDD and 3-MC showed the same level of maximum response, induction by TCDD persisted for over 1 month, while AHH activity of 3-MC treated rats returned to control values in 8 days. Spectral characteristics of the cytochrome P-450 enzyme system indicated that both TCDD and 3-MC induced formation of the same enzyme system. The authors discussed their results in terms of proposed mechanisms of toxicity of TCDD. 791. Poland, A., and Glover, E. (1980) 2,3,7,8-tetrachlorodibenzo-p-dioxin: Segregation of toxicity with the Ah locus. Molec. Pharmacol. 17:86-94. The potency of TCDD in eliciting thymic atrophy and, in fetuses, cleft palate were determined in responsive, non-responsive and hybrid strains of mice. C57BL/6J, DBA/2J and hybrid mice of these 2 strains were administered a single intraperitoneal dose of TCDD. (Prior to TCDD treatment, hybrid mice were phenotyped by their zoxazolamine paralysis times after beta-naphthoflavone induction.) Five to six days after TCDD treatment, the animals were killed and the thymuses were removed and weighed. Aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin0-deethylase activities were assayed in the liver and thymus. \ Hj-TCDD binding was determined in hepatic and thymic cytosol. Cleft palates and fetal deaths were determined on day 18 of gestation, in litters of mice that were administered a single subcutaneous dose of TCDD on day 10 of gestation. C57BL/6J mice showed 10 fold higher sensitivity to thymic involution, high incidence of cleft palate, high level of enzyme induction and a high level of specific cytosol binding TCDD, compared to the responses by DBA/2J mice. Hybrid mice showed intermediate responses and were correlated with their phenotype. The potencies of other compounds as enzyme inducers correlated with their potencies in producing thymic atrophy. Four of 5 strains of responsive mice with high affinity cytosol receptors showed high incidences of cleft palate, 282 �after TCDD treatment while 5 non-responsive strains showed low incidences at the same dose. The authors concluded that TCDD binding to the cytosol receptor was a necessary step for toxic responses to be elicited by TCDD, because toxic responses segregated with the Ah locus, which determines the receptor. 792. Poland, A., Glover, E., and Kende, A. S. (1976) Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. Biol. Chetn. 251(16):4936-4946. The characteristics of a cytosal "receptor" species from mouse liver that binds TCDD is described for methyl cholanthrene (MC) responsive and non-responsive strains of mice. C57BL/6J mice and DBA/2J mice were administered a single intraperitoneal dose of f C]-TCDD and killed from 3 hours to 14 days later. Livers were removed and fractionated into cytosol, nuclear, mitochondrial and microsomal fractions. Radioactivity associated with each fraction was determined. The extent of high-affinity binding was determined in the cytosol fraction by subtracting the total cytosol-bound radioactivity and the radioactivity bound nonspecifically to low affinity sites (i.e. the amount of radioactivity that was not displaced from the binding site when a 200-fold excess of 2,3,7,8-tetrachlorodibenzofuran was added to the sample). Hepatic uptake of [ C]-TCDD, expressed as a percentage of the total dose, was about 2-3 times higher in MC-responsive (C57BL/6J) mice than in nonresponsive mice; hepatic uptake of radioactivity remained elevated for 14 days in both species, and uptake increased in DBA mice at higher doses of TCDD and decreased in C57BL mice at higher TCDD doses. Hepatic uptake of radioactivity for 2 hybrid strains of mice from crosses of DBA mice with 2 responsive strains) were intermediate between the parental values. Each of the 4 subcellular fractions retained 20-40% of the total hepatic radioactivity and only the level in the supernatant fraction was reduced in mice that were pretreated with a large excess of TCDD. A small pool of high-affinity binding sites were identified in C57BL mouse cytosol; an equilbrium dissociation constant of 0.27 nM and a total of 8.4 pmol of binding sites per g liver were calculated for this pool. The high-affinity binding pool was absent in DBA cytosol. The binding affinities of various structural analogues of TCDD and other arylhydrocarbon hydroxylase inducers for the high-affinity receptor (in C57BL mice) correlated highly with their potencies in inducing hydro1ase activity in vivo. About 60% of the high specific binding of TCDD to cytosol was lost in the presence of trypsin while no loss occurred when RNase and DNase were tested. The authors concluded that a single locus codes for the induction reception protein that has a high affinity for TCDD and activates AHH induction as a consequence of TCDD binding; nonresponsive mice have an altered receptor with lowered affinity for inducers. 283 �793. Poland, A. P., Glover, E., Robinson, J. R., and Nebert, D. W. (1974) Genetic expression of aryl hydrocarbon hydroxylase activity. J. Biol. Chem., 249(17).-5599-5606. Microsomal enzyme induction by TCDD was studied in several inbred strains of mice, including strains that are responsive to 3-methylcholanthrene (3MC) enzyme induction and strains that are non-responsive to 3MC. Responsive strains included C57BL/6N, C57BL/6J, C3H/HeN, BALB/cAnN and CBA/HN strains and the nonsponsive strains studied were DBA/ZN, DBA/2J, AKR/N, NZW/BLN, and NZB/BLN strains. Mice were administered 40 ug/kg TCDD in dioxane intraperitoneally or 4 ug (in 4 applications over 2 days) dermally. Enzyme activities were assayed in microsomal preparations 48 hours after TCDD administration. Benzo(a)pyrene hydroxylase, 7-ethoxycoumarin Ortho-deethylase, paranitroanisole 0-demethylase, and 3 methyl-4-methyl aminoazobenzene N-demethylase activities were assayed. The total cytochrome P-450 content of microsomes was determined spectrophotometrically from the carbon monoxide and ethyl isocyanide difference spectra. TCDD (ip) induced hydroxylase activity to the same extent in 3MC-responsive and 3MC-non-responsive mice. Microsomal enzymes from the liver, bowel, lung, and kidney responded to TCDD treatment. Dermal application of TCDD induced hydroxylase activity in the skin and liver of both responsive and non-responsive strains. Formation of new hepatic cytochrome P-450 was demonstrated in non-responsive mice after TCDD administration spectrophotometrically and by changes in the EPR spectra at low temperatures. TCDD-induced enzymes were sensitive to alphanaphthoflavone inhibition but not to metyrapone inhibition. Spectral changes characteristic of induction of new cytochrome P-450 also occurred in lung, bowel, kidney, and skin microsomal preparation from 3MC-non-responsive mice after TCDD treatment. The authors concluded that non-responsive mice probably do not recognize most polycyclic hydrocarbon inducers (i.e. 3MC) while they possess the regulatory and structure genes required for new cytochrome P-450 formation. They proposed that defect in inducer recognition involved a mutation yielding a defective receptor with diminished ability to bind inducers. TCDD was proposed to interact with the same receptor that other inducers bind. These conclusions are well founded, based on the experiments reported here, which represent a thorough consideration of induction of various enzymes in various tissues and strains of mouse in which appropriate and specific technical assays were applied. 794. Poland, A., and Kende, A. (1976) 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Environmental contaminant and molecular probe. Federation Proceedings. 35(12):2404-2411. [Review article.] 284 �795. Poland, A. P., Smith, D., Matter, G., and Possick, P. (1971) A health survey of workers in a 2,4-D and 2,4,5-T plant. With special attention to chloracne, porphyria cutanea tarda, and psychologic parameters. Arch. Environ. Health .22(3) -.316-327 . Results are reported of physical examinations of 73 male employees in a factory that manufactures 2,4-D and 2,4,5-T. Some of the workers in the factory had been included in a study conducted 6 years previously (Bleiberg et al. 1964). Medical histories were obtained and physical examinations were performed, with special emphasis on neurologic and dermatologic conditions. Each worker also responded to the Minnesota Multiphasic Personality Inventory (MMPI). [Routine automated blood analyses were performed on serum samples from workers, and urine was analyzed for porphyrins and porphyrin precursors.] Acne was present in 66 percent of the workers and was considered severe in 18 percent. The presence of scarring, hyperpigraentation, hirsutism, and complaints of eye irritation each correlated with the severity of acne, while the length of employment or the employee's location within the plant was not related to severity. Only three workers had conjunctivitis, although hyperemia of the buccal and nasal mucosa was present in 11 percent and 32 percent, respectively, of the employees. No cases of porphyria cutanea tarda were identified. Maintenance men had statistically higher coproporphyrin excretion levels than workers in administrative positions, but both sets of values were within the normal range, No hyperferremia was observed. Blood analyses revealed minimal liver dysfunction. About 30 percent of the employees had gastrointestinal complaints. The employees had the expected frequencies of abnormal conditions related to the cardiovascular, pulmonary, metabolic, and hematologic systems. The MMPI correlated only hypomania with severity of chloracne. The authors concluded that uroporphyrinuria identified in workers in a previous study and chloracne can occur independently and have different clinical courses, even though they may potentially have a common etiology. The etiology of both conditions was not known, but the authors suggested dichlorophenol, a precursor of 2,4-D as the etiologic agent of porphyria cutanea tarda, and TCDD as a potentially acnegenic compound. 796. Possible Long-Range Adverse Health Effects on Individuals that were Exposed to The Herbicide Agent Orange. Letter to Ralph H. Metcalf, U.S. House of Representatives. (1978) U.S. General Accounting Office, Washington, DC, 20 pp. [Editorial.] 797. Pratt, I. S., Keeling, P. L., and Smith, L. L. (1980) The effect of high concentrations of oxygen on paraquat and diquat toxicity in rats. Arch. Toxicol. [Suppl] 4:415-418. The effects of diquat on mortality and lung pathology were evaluated in the rat in an atmosphere of 85% oxygen. Wistar rats were administered 10 or 20 mg/kg diquat subcutaneously and then placed in air or in an 285 �exposure chamber with 85% oxygen. Deaths were recorded daily and some rats were killed between 2 and 24 hours after dosing and lung tissue was examined by electron microscopy. After the higher dose was administered, 50% of the rats exposed to air died, with a mean time to death of 48 hours. Slight damage of type I, II, and epithelial cells was observed. No rats given the lower dose and exposed to air died and no lung damage was observed. All of the rats given the higher dose and exposed to high oxygen died, with a mean time to death of 17 hours. Severe lung edema and moderate damage to type II cells occurred. Half of the rats given the lower dose and high oxygen died (in an average of 41 hours), but no tissue damage occurred. Rats exposed to 85% oxygen alone showed no tissue damage or mortality. Paraquat toxicity was potentiated 10 fold by high oxygen, compared to the 2 fold increase in diquat toxicity. The authors proposed the hypothesis that bipyridyls exert their toxicity by generating superoxide anions and suggested that this anion generation is likely to be facilitated by high oxygen concentrations. They did not indicate whether their results supported this hypothesis. 798. A preliminary assessment of herbicides and defoliation. Environ. Sci. Technol. 2(3):176-181. (1968) [Review article.] 799. Preliminary dioxin implementation plan. U.S. Environmental Protection Agency - Office of Pesticide Programs, Criteria and Evaluation Division, Washington, DC, 18 pp. [Background material.] 800. Prescott, L. F., Park, J, and Darrien, I. (1979) Treatment of severe 2,4-D and mecoprop intoxication with alkaline diuresis. Br. J. Clin. Pharmac. 7:111-116. A case involving intentional ingestion of a weedkiller comprised of 10% 2,4-D and 20% 4-chloro-2-methylphenoxypropionic acid (mecoprop) is described. The ingestion induced vomiting. The patient became aggressive, then confused, and finally lost consciousness for 4 days. The patient remained confused for 4 days after he regained consciousness. Pyrexia and hyperventilation both responded to alkaline diuresis, which was initiated 42 hours after the ingestion. Myotonia persisted for several days after the patient regained consciousness. Plasma enzyme levels indicated severe muscle injury and electromyography, which was performed 6 days after the ingestion, indicated mild hyopathy. Weakness of the legs was noted 2 months after the incident. The plasma concentration was 400 ug/ml for 2,4-D and remained constant until the pH of the urine rose above pH7 after initiating alkaline diuretic therapy. The clearance half-life then fell to 3.7 h. Consciousness was regained when the blood levels of 2,4-D and mecroprop each were 100 ug/ml. 286 �801. Pritchard, J. B. (1980) Accumulation of anionic pesticides by rabbit choroid plexus in vitro. J. Pharmacol. Exper. Therap. 212(2):354-359. Uptake of 2,4-D by rabbit choroid plexus and the effects of 2,4-D on uptake of other organic ions, including neurotransmitter metabolites were studied in the rabbit. Lateral plexi from male New Zealand rabbits were excised and incubated in medium that contained the C-labeled anion to be tested. After incubation, radioactivity in the tissue and medium were counted. The tissue-to-medium ratio of radioactivity was 40-60 at 2,4-D concentrations up to luM. Above this 2,4-D concentration, the transport mechanism was saturated. Uptake was inhibited by 90% when oxygen was replaced by nitrogen or by ImM cyanide. DDA (a polar DDT metabolite) and chlorophenol red each produced doserelated inhibition of 2,4-D uptake which could be partially overcome with higher doses of 2,4-D. Likewise, 2,4-D produced a dose-dependent inhibition in DDA uptake and in uptake of the serotonin metabolite, 5-hydroxy-3-indoleacetic acid. The authors concluded that 2,4-D are actively transported into the choroid plexus by the organic anion system and then may also be bound within the plexus; 2,4-D accumulation in the plexus was thought to be similar to transport by the kidney and by the liver, described by other investigators and a. potential for neurotoxicity of 2,4-D was suggested. 802. Pushkar, M. S. (1969) Morphological changes in the organism under the effect of the herbicide reglone (diquat). Vrach. Delo. 9:92-96. [Foreign language.] 287 �803. Radosevich, S. R., and Winterlin, W. L. (1977) Persistence of 2,4-D and 2,4,5-T in chaparral vegetation and soil. Weed Sci. 25(5):423-425. The authors studied the distribution, persistence, and vertical movement of 2,4-D and 2,4,5-T in soil afer herbicide application to chaparral. Four replicate plots (186 m ) were sprayed with 4.5 kg/ha 2,4-D (butoxy propyl ester) or 2,4,5-T (propylene glycol butyl ester). Samples of soil surface litter and of the terminal 10-15 cm of chamise foliage, understory grass, and forbs were collected before and immediately after spraying and 30, 60, 90, 180, and 360 days after herbicide application. Soil samples were collected at the same times after herbicide application at 0-5, 10-15, 25-30, and 55-60 cm depths. Determination of 2,4-D and 2,4,5-T in the samples collected was by gas-liquid chomatography. After application, soil surface litter contained over 50% of the 2,4-D or 2,4,5-T applied, followed by understory grass and forbs (25.2% 2,4-D; 31.2% 2,4,5-T), and chamise (20.7% 2,4-D; 18% 2,4,5-T). Residues of both herbicides decreased up to 93% on foliage and litter and within 30 days. In the soil, residues of only 0.1% 2,4-D and 0.07% 2,4,5-T were detected immediately after application. No detectable herbicide was found below 5 cm. Soil residues of both herbicides remained constant for 90 days after application. However, by day 180 for 2,4-D and 360 for 2,4,5-T, only 0.01% of the orginal application could be detected in the soil. This experiment took place during the dry season, therefore, the effects of surface runoff and percolation through the soil could not be measured. The authors also studied the loss of herbicide from paper disks in a greenhouse bioassay under conditions similar to the chaparral. In seven days, nearly all the 2,4-D and 2,4,5-T had disappeared. The authors felt that either volatilization or decomposition was responsible for herbicide disappearance. From the work of other scientists, they eliminated photodecomposition as a possibility and concluded that volatilization was responsible for rapid herbicide disappearance. In addition, the authors demonstrated that 2,4-D and 2,4,5-T does not accumulate in soils and vegetation and is an unlikely contaminant of water supplies. 804. Raju, K. S., and Rangaswami, G. (1972) Studies on microbial degradation of herbicides in soil. Biochetn. J. 128(1) :40. [Abstract, only.] 805. Ramel, C. Genetic effects of phenoxyacetic acids in animals. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978a) p. 182-185. [Review article.] 288 �806. Ramel, C. Editor's preface, Conclusions and recommendations, Chemistry and Summary. In Chlorinated Phenoxy Acid and Their Dioxins, C. Ramel, ed. (Ecol. Bull No. 27, Stockholm: Swedish Natural Science Research Council, 1978b) p. 9-27. [Review article.] 807. Ramsey, J. C., Hefner, J. G., Karbowski, R. J., et al. (1979) The in vivo biotransformation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDDT~in the rat. Toxicol. Appl. Pharmacol. 48(l)Pt. 2:A162. 808. Ramsey, J. C., Lavy, T. L., and Braun, W. H. Exposure of forest workers to 2,4,5-T: calculated dose levels. Report from the toxicology laboratory, Dow Chemical Company, Midland, Mich. Pharmacokinetic analysis of data for 2,4,5-T urine levels of workers is described. Exposure conditions and urinary 2,4,5-T data were described in another report (see Lavy, T.L., 1978 report to the National Forest Products Association). From previous animal and human studies, the excretion of 2,4,5-T was established as a first order process that is independent of the route of administration; a 1 compartment pharmacokinetic model which incorporated these characteristics was used to estimate absorption levels by 3 methods. For 12 of the 41 exposures, the absorbed doses and rate constants were estimated as the values providing the best fit of the urinary excretion data by the kinetic model using the human excretion rate from a previous study. The second method involved mathematically estimating the proportion of the initial dose that was excreted on 7 successive days from the established rate constants; cumulative urine levels were then divided by this proportion to estimate absorbed doses for 39 of the 41 exposures. The third method, applied to all 41 exposures, used an equation that estimated exposures from data for urinary excretion over a limited interval, independent of the total quantity of urine collected. All 3 methods yielded similar results, with maximum exposure of 0.132 mg/kg and 0.156 mg/kg for a backsprayer and mixer, respectively, and lowest exposure of 0.001 mg/kg for helicopter flagmen. The average dose for all workers was 0.063 mg/kg. The authors estimated body burdens from their model. The authors concluded that the close fit of observed and calculated urinary 2,4,5-T values attested to the validity of the proposed kinetic model and that method 3 was the method of choice. Worker exposure levels were concluded to be far below the no-effect level of 20 mg/kg (for teratogenic and fetotoxic effects) put forth by the EPA, indicating that 2,4,5-T exposure presents a negligible toxic hazard to workers. 809. Ranish, N. A., Dettbarn, W. D., and Iyer, V. (1977) The influence of nerve stump length on (2,4-dichlorophenoxy)acetic acid-induced myotomia. Exp. Neurol. 54:393-396. The influence of nerve stump length on 2J4-D induced myotonia in the rat was investigated. Adult rats were subjected to unilateral sciatic 289 �nerve transections. Denervation was performed to either the level of the spinal cord or at the distal end of the nerves. This resulted in nerve stumps of two lengths in the different rats. 2,4-D (200 rag/kg) was administered intraperitoneally at 6 to 8 hour intervals and the myotonia evaluated 2 hours after each administration. After the first 48 hours, the 2,4-D was given daily for 10 to 12 days. Myotonia was measured by electromyographic examination of the gastroenemius muscle. After 12 days of examination the animals were sacrificed and the nerve stump lengths measured. The times of appearance of recorded changes in myotonia, the loss of myotonic activity and the onset of fibrillation were measured and compared to the length of the nerve stump. Longer time intervals between denervation and these myotonic parameters were associated with longer nerve stump lengths. The muscle retained its capacity to become myotonic, and the length of this period was found to be related to the nerve stump length. By one week after denervation, however, the muscle was no longer able to become myotonic in response to 2,4-D. The authors compared their results to results of other experiments that induced myotonia with 20, 25-diazacholesterol. The authors made no attempt to relate their study with studies of others that have induced myotonia with 2,4-D. The experiment lacked control animals and the purity and preparation of the 2,4-D compound was not described. 810. Rao, K. S., and Dad, N. K. (1979) Studies of herbicide toxicity in some freshwater fishes and ectoprocta. J. Fish Biol. 14:517-522. The authors studied the acute toxicity of 2,4-D to ectoprocta and dalapon to fish. According to the authors, dalapon toxicity was measured in Het eropneutes fossilis, Saratherodan mossambicus, and Puntius ticto caught locally in India. Concentrations of dalapon tested and results of the acute toxicity test were not presented. The authors reported that fish survived for 8 days in concentrations ranging from 1-2500 mg/1. 2,4-D butyl ester was tested in three species of ectoprocta, Plumatella casmiana, Lophopodella carteri, and Hyalinella punctata, at 3 and 4 mg/1. No toxicity was observed in L. carteri or H. punctata 3 mg/1 for up to 84 hours. P_. casmiana was more sensitive to 3 mg/1 2,4-D; 50 percent of the organisms were dead at 72 hours. Sluggishness was observed as early as 24 hours. In all three species, 4.0 mg/1 2,4-D produced toxicity; all 7_. casmiana and E_. punctata were dead at 48 hours while all L^. carteri were dead at 36 hours. In addition germination of JP. casmiana leptoblasts was not affected by 0.01 - 1.5 rag/1 2,4-D. 811. Rappe, C. (1979) Dioxiner och dibensofuraner tva substansgrupper i blickpunkten. Lakartidningen. 76:(1-2):21-24. [Not available.] 290 �812. Rasmuson, B., and Svahlin, H. Mutagenicity tests of 2,4-dichlorophenoxyacetic acid and 2,4,5,-trichlorophenoxyacetic acid in genetically stable and unstable strains of Drosophila melanogaster. In Chlorinated Phenpxy Acids and Their Pioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stolkholm: Swedish Natural Science Research Council, 1978) p. 190-192. The author tested 2,4-D and 2,4,5-T in a mutation assay in Drosophila melanogaster. The test involves use of a sex-linked genetically unstable system in Drosophila. The instability is caused by the insertion of foreign DNA into the regulatory part of the structural gene of the white locus. Mutations induced within the regulatory part of the gene influence control of the white locus somatically which results in different pigment synthesis. Mutagenic frequency is measured by the number of hatched males with pigmented eye sectors after larval exposure to a mutagen. In addition the authors also used a genetically stable strain to compare strain differences in mutation frequencies. Larvae of each strain were kept until eclosion on food containing 25 ppm 2,4-D or 100 ppm 2,4,5-T. Ethylmethane sulfohate (500 ppm) was used as a positive control. 2,4-D induced an increased mutation frequency in the unstable strain, but not in the stable strain. 2,4,5-T did.not cause increases in mutation frequencies in either strain. These results differ from recessive lethal studies in which both 2,4-D and 2,4,5-T were mutagenic. 813. Rawls, R. L. (1980) Reproductive hazards in the workplace. Chem. Eng. News 58(6):28-31. Part I. [Editorial.] 814. Rawls, R. L. (1979) DOW finds support, doubt for dioxin ideas. Eng. News 57(7):23-29. [Not available.] 815. Rawls, R. L., and O'Sullivan, D. A. (1976) Italy seeks answers following toxic release. Chem. Eng. News Aug 23:27-28,33-35. [Review article.] 816. Recommended Classification of Pesticides by Hazard. (1975) WHO Chronicle 29:397-401. [Review article.] 291 Chem. �817. Reggiani, G. (1980) Acute human exposure to TCDD in Seveso, Italy. J A Toxicol. Environ. Health 6:27-43. The health status 'of 17 people who were exposed to TCDD from a factory explosion in Seveso, Italy, in 1976 were reported. The subjects who were examined had been referred to the judge who held the inquest on the accident and included 13 children below 15 years of age. The environmental levels of TCDD in the vicinity of the factory were reported, and the tissue levels in one woman from the contaminated area who died 6 months after the accident were reported. Clinical and neurological examinations, which included electroencephalography, electromyography, and nerve conduction velocity measurements, were performed. Circulating complement, serum immunoglobulin levels, and lymphocytic response to nonspecific mitogens were determined. Chromosomal aberrations were analyzed, and laboratory tests were performed to assess hepatic function, metabolism of protein, lipids, and carbohydrates, porphyrin excretion, hematology, and urine content. Burnlike lesions of the skin developed in almost all cases. Within 3-7 days after the accident, exposed areas of the skin showed signs of acute inflammation, which subsided in 2-3 months. Hyperkeratosis, vermicular atrophy, and vesicles and pustules occurred in some cases. Chloracne developed in 12 cases, usually 2 weeks to 2 months after exposure, and involved the formation of blackheads and cysts on the face and in some cases also on the neck, shoulders, chest, lower trunk, and limbs. The duration of chloracne was 8 to 26 months, and residual scars remained on the faces of two of the most severely affected and youngest patients. None of the cases was classified as severe by the grading system recommended by an expert panel for evaluating the severity of chloracne. The only other changes identified in the patients were gastrointestinal disturbances within a few days of the onset of skin lesions and transient increases in serum enzyme levels, which were not considered severe enough to indicate hepatic injury. The authors concluded that visceral lesions had not occurred in the subjects of this study and attributed acute dermatitis to chemicals other than TCDD, which were released during the accident. Varied exposure to TCDD and varied individual susceptibility were presented as possible reasons why some people developed dermatitis only, and others developed chloracne, as well. 818. Reggiani, G. (1979) Estimation of the TCDD toxic potential in the light of the Seveso accident. Arch. Toxicol. 2:291-302. The results of a health survey of residents near an Italian factory that released TCDD are described. Chloracne observed in periodic screening of thousands of residents during a two-year period after the accident showed a trend to healing with few new cases reported. The expected incidence of adolescent acne is 0.1-0.4 percent; the observed incidence of chloracne was 0.6-1.2 percent. Clinical chemistry test results revealed no increase in hematologic or renal problems. Some abnormal results of hepatic function tests were reported briefly, as well as abnormal porphyrin patterns in an undisclosed proportion of examined people. Hepatomegaly was found in 8 percent of the factory 292 �workers. No adverse health effects were reported in a group of Swiss workers involved in the decontamination of the most contaminated area (zone A). The only neurologic effects attributed to TCDD contamination were subclinical, involving a reduction in sensory and motor conduction velocity of 1-2 nerves. The incidences of abortions and of congenital malformations were considered to be below the expected values. No fetal malformations in 34 aborted fetuses (including 4 spontaneous abortions) examined were attributed to the action of exogenous agents. A "representative group" of children born after the accident was examined, and no developmental abnormalities have been observed. Cytologic tests revealed no increase in chromosomal aberrations in blood cells from exposed individuals nor a decrease in immune capability in children. Tissue samples of a woman from Zone A who died of carcinoma of the pancreas 7 months after the accident were analyzed for TCDD. A total body burden of 40 (u)g was estimated, with tissue levels from 0.04 ppb TCDD in the kidney to 1.84 ppb in the fat. The author concluded that humans have a higher tolerance to TCDD than would be expected from animal data. The significance of these findings cannot be evaluated, because the data do not include details on the methods used to evaluate each health parameter, or the specific groups studied, or the controls. 819. Reggiani, G. (1979) Abstracts: Eighteenth annual meeting; 361. TCDD contamination in Itlay: The risk assessment of low level exposure, cumulative effect and long term consequences. Toxicology and Applied Pharmacology 48(1).:A180. [Abstract, only.] 820. Reggiani, G. (1978) Medical problems raised by the TCDD contamination. Arch. Toxicol. 40:161-188. [Review article.] 821. Rehder, H., Sanchioni, F., Cefes, F., and Gropp, A. (1978) Pathologicalembryological investigations in cases of abortion related to the Seveso accident. J. Swiss Med. 108(42):1617-1625. Results are presented of examinations of 34 aborted fetuses from mothers who were exposed to TCDD from the industrial accident in Seveso, Italy in July, 1976. An estimated 150 women in the affected area were in their first trimester of pregnancy when the accident occurred. A total of 125 women in the affected and bordering areas had decided by October, 1976, to terminate their pregnancies and 30 cases were approved. Fetuses from these elected abortions and from 4 spontaneous abortions that occurred concurrently, in this region were examined. Of the group of mothers who elected abortions, 3 were from zone A of highest contamination, 5 from zone B of moderate contamination and 13 from zone R of low contamination; 7 of the remaining mothers resided outside of these zones, but worked, travelled, visited 293 �or consumed food from the contaminated zones and 2 abortions were elected for psychological reasons. Fetuses ranged from a few millimeters (5 weeks, estimated gestational age) to 17.5 cm from crown to heel (20 weeks of age). Most fetuses were 10-12 weeks of age. Twenty-five fetuses, between 5 and 12 weeks of age, were removed by instrument curettage and many were mutilated, some with only a few organs remaining for examination. Five fetuses, 13-17.5 cm in length, were removed after prostaglandins were administered, sometimes by extraction which also damaged the fetal skin and sometimes internal organs, as well. The 4 spontaneous abortions occurred in zone A and R in week 11-24 of pregnancy; 3 fetuses were 11-19 cm in length, the fourth was resorbed. Fetal organs were weighed, fixed in Bouin's fluid, and examined by light microscopy. In 23 of the fetuses, no abnormalities in organ size, structures or developmental stage (compared to fetal age) were observed. One of these mothers showed mild symptoms of chloracne. Six fetuses showed changes from normal development and included a secondary obliterated duodenum (a potentially temporary condition), a fetus with a subepidermal cyst and a defective ventricle septum (considered an artifact from manipulation), 2 cases of prolapse of the retina and 1 of displaced liver and intestine (all considered to be artifacts), and 1 case of slightly retarded renal and tracheal development. The remaining 5 fetuses, including 4 from spontaneous abortions and 1 which showed signs of intrauterine death prior to the elected abortion, were described in detail and were attributed to heterogeneous mixture of alleged reasons for fetal death or abortion. The authors concluded that no evidence of a fetotoxic or teratogenic effect of human exposure to TCDD was documented by their results, although the study was limited by incomplete fetal material and limited examination techniques. 822. Rehwoldt, R. E., Kelley, E., and Mahoney, M. (1977) Investigations into the acute toxicity and some chronic effects of selected herbicides and pesticides on several fresh water fish species. Bull. Environ. Contam. Toxicol. 18(3):361-365. The authors briefly describe the acute and chronic toxicity of 2,4-D and 2,4,5-T in seven species of fish. In the acute toxicity experiments, striped bass, banded killyfish, pumpkinseeds, white perch, American eels, carp, and guppies were exposed to unspecified concentrations of the two herbicides for 24, 48, and 96 hours. Mean threshold limit values (Tl ) were then calculated. At 24 hours, Tl values for 2,4,5-T varied from 27.3-73.2 mg/1 depending on species. 2,4-D was less toxic than 2,4,5-T. Tl values for 2,4-D at 24 hours varied from 55.5 - 427.2 mg/1 depending on species. Ages and sizes of fish used in the experiment were not reported. In a long term 10 month experiment fish were exposed to aquaria concentrations of 0.1 ppm 2,4-D or 2,4,5-T. Numbers and kinds of fish used were not specified by the authors. No toxic effects were seen in exposed fish after 10 months. However, no histological examinations were performed. In a breeding experiment using 4 female and an unspecified number of male guppies, an unspecified concentration of 2,4-D or 2,4,5-T had no effect on offspring compared to controls. However, the lack of details presented in 294 �this paper prevents any form of complete evaluation. From the data, it appears that 2,4-D is less toxic than 2,4,5-T and species sensitivity to the herbicides seems to vary. 823. Renner, H. W. (1979) Monitoring of genetic environmental risk with new mutagenicity tests. Ecol. Environ. Safety 3:122-125. [Not available.] 824. Report on 2,4,5-T - A Report of the Panel on Herbicides of the President's Science Advisory Committee. By Dr. Colin M. MacLeod, Chairman. Washington, DC: Government Printing O f f i c e . (1971) 68 pp. [Background material.] 825. Riimaki, V., Asp., S., Seppalainen, A. M., and Heinberg, S. (1978) Symptotnology, morbidity, and mortality experience of chlorinated phenoxy herbicide (2,4-D and 2,4,5-T) sprayers in Finland. A clinical and epidemiological study. Working paper for an IARC working group meeting on "Coordination of Epidemiological Studies on the Long Term Hazards of Chlorinated Dibenzodioxins and Chlorinated Dibenzofurans." Lyon, France, 10-11 January. [Not available.] 826. Rip, J. W., and Cherry, J. H. (1976) Liver enlargement induced by the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). • J. Agric. Food Chem. 24(2)-.245-250. The effects of 2,4,5-T on rat liver weight, hepatic RNA and DNA syntheses, and enzyme induction were examined. One month old male Long-Evans rats were administered 10 mg of 2,4,5-T (less than 0.05 ppm TCDD contaminant) in 6 g of feed, daily for 1-11 days and were killed 18 hours after the final feeding or were fed the 2,4,5-T diet for 6 days and then a normal diet for 8 days before being killed. Liver, kidney, spleen, and body weights were recorded. Liver homogenates were fractionated into subcellular components and assayed for nucleic acids and protein. Hepatic glucose-6-phosphatase and acid phosphatase activities were assayed and the 2,4,5-T content of the liver was determined. Rats fed 2,4,5-T for 7 days showed a 28% increase in both wet and dry liver weights, but no changes in other organ wet or dry weights or body weight, compared to controls. The increase in absolute and in relative liver weight was 20% after 2 days and 150% after 6-7 days in another experiment. Total DNA content of the liver remained constant for 11 days of treatment and DNA per gram of liver decreased. RNA content per liver increased up to 20% in 2 days and by 26% at 6 days. Protein content per liver increased by 50% with 8 days of treatment and protein per gram of tissue remained constant. The increases in protein and RNA were comparable for each subcellular liver T95 �fraction and the crude homogenate. Liver weights were normal in rats fed a normal diet after treatment. Moderate inhibitions of both enzymes occurred. The ultraviolet absorption spectrum of 2,4,5-T extracted from treated rat livers showed the same profile as the 2,4,5-T standard and only 1.5% of the administered dose of 2,4,5-T remained in ,the liver after 24 hours. The authors concluded that the 2,4,5-T induced liver enlargement was caused by cellular hypertrophy, with minimal enlargement from herbicide hepatotoxicity and the increase in protein synthesis did not involve induction of 2,4,5-T metabolizing enzymes. 827. Roan, C. C., and Morgan, D. P. (1972) Alleged effects on human health of the use of herbicides in the area around Globe, Arizona. Arizona Community Pesticide Studies Project, 6 March. The authors reported on the analysis of human body fluids and tissues and water samples for 2,4-D, 2,4,5-T, and TCDD in 1969 and 1970. Four urine samples, three serum samples, and three adipose tissue samples were negative for all three compounds (detection limit 2 ppb, method of analysis not specified). From these few samples, the authors concluded that the general population in the Globe, Ariz., area did not have any chronic exposure to 2,4-D, 2,4,5-T, and TCDD. Twenty-two water samples over a one-year period were also collected and analyzed by gas chromatography (detection limit 0.01 ppb). All samples were negative for 2,4-D, 2,4,5-T, and TCDD. The authors concluded that there was no widespread environmental contamination of the area by 2,4-D, 2,4,5-T, and TCDD. 828. Robbins, A. (1979) Dioxin studies, 205(4413):1332. fletter to the editor! Science [Editorial.] 829. Robens, J. F. (1978) Tests for possible carcinogenicity of 20 pesticides in Osborne-Mendel rats and B6C3F1 mice. Toxicol. Appl. Pharmacol. 45(1):236. [Abstract, only.] 830. Robson, J. M. (1970) Testing drugs for teratogenicity and their effects on fertility. Brit. Med. Bull. 26(3):212-216. [Review article.] 296 �831. Rocchi, P., Perocco, P., Alberghini, W., Fini, A., and Prodi, G. (1980) Effect of pesticides on scheduled and unscheduled DNA synthesis of rat thymocytes and human lymphocytes. Arch. Tpxicol. 45:101-108. The authors studied the action of diquat and 16 other pesticides on scheduled and unscheduled DNA synthesis in rat thymocytes and human lymphocytes. Diquat (95% purity) in DMSO was added to rat thymocyte cell cultures to determine the concentration of diquat which gave 50-70% DNA synthesis inhibition. This dose was then used to determine the effect of diquat on DNA synthesis in human lymphocytes. Diquat (500 ug/ml) was added to cultures of human lymphocytes with and without hydroxyurea for 4 hours. During this culture period, the cells were labelled with tritiated thymidine to measure DNA synthesis. Cells treated with ultraviolet radiation were used to compare scheduled and unscheduled DNA synthesis. Diquat at 500 ug/ml produced approximately the same amount of inhibition of scheduled DNA synthesis in rat thymocytes as well as in human lymphocytes. In this system inhibition of scheduled DNA synthesis was comparable to inhibition of unscheduled DNA synthesis. The authors did not discuss the relevancy of these data. However, it appears from the data presented that diquat at the dose tested in human lymphocytes did not induce unscheduled DNA synthesis compared to controls. 832. Rodgers, C. A., and Stalling D. L. (1972) Dynamics of an ester of 2,4-D in organs of three fish species. Weed Sci. 20(1):101-105. [Background material.] 833. Roll, R. (1971) Studies of the teratogenic effect of 2,4,5-T in mice. Fd. Cosmet. Toxicol. 9:671. Incidences of fetal malformations and deaths in mice administered 2,4,5-T are described. NMRI mice were administered 20-130 mg/kg 2,4,5-T 99.6% purity; 0.05 ppm TCDD contaminant) in peanut oil, by oral intubation on days 6-15 of gestation. On day 18 of pregnancy (1 day prior to expected delivery) fetuses were removed, weighed, and examined for visceral malformations by Dawson's method) or for skeletal abnormalities in Alizarin red-stained specimens. The numbers of implantation sites, resorption sites and dead fetuses were noted. The LDeQ doses were calculated to be 674 mg/kg for adult NMRI male mice and 778 mg/kg for females. Maternal weight gains were reduced in rats administered 90 or 130 mg/kg doses. A small decreases in the numbers of full-term fetuses and small decreases in early and late resorption and dead fetuses occurred from treatment with 90 mg/kg doses and these changes were all substantial in the 130 mg/kg dosage group. A doserelated decrease in fetal weight and increase in cleft palate incidence occurred; these changes ranged from 1.23 g. of fetal weight for controls to 0.73 g. for the highest dosage group and 1.9% of controls with cleft palate to 48.8% for the highest dosage group. Dosedependent breast bone anomalies were considered to represent slow development, rather than lack of development that would produce 297 �permanent abnormal structures. The authors concluded that 2,4,5-T (and not the dioxin contaminant was responsible for the high incidence of cleft palate they observed and suggested that the mouse was more susceptible than the rat (studied by other investigators) to these teratogenic effects. 834. Rose, H. A., and Rose, S. P. refugees from South Vietnam. (1972) Chemical spraying as reported by Science 177(4050):710-712. The authors describe the findings they obtained from questionnaires they issued to 98 South Vietnamese refugees who had migrated to Hanoi. Eighty of the group were men, mostly between 25 and 45 years of age. Most of the 98 respondents (95%) were sprayed with herbicide at least 2 times, with 2 people reporting that they were sprayed 21-30 times. The remainder of the questions referred only to the last spraying mission they experienced. Chemicals were sprayed by air in 91% of the cases and the authors indicated that descriptions were compatible with spraying of Agents Orange, White, and Blue and of (ortho-chlorobenzylmalononitrile (CS). When asked about environmental effects, 95% indicated that trees were damaged or killed, 89% that crops were destroyed or became inedible, 48% that fish died, 39% that domestic animals died and 5% that cattle died. Abortions or monstrous births of animals were voluntarily mentioned by 5% of the group. Health effects reported were ocular problems, 79% of the cases; transient nausea and vomiting, 58%; skin burns and reddening, 56%, persistent skin effects including pustules, scabs and eczema, 1.62; fatigue or dizziness after spraying, 92%; prolonged fatigue, 17%. Respondents also reported health effects of others in their village, including long illnesses in the old and in children (observed by 11% of the respondents), deaths due to chemicals (8%), and human abortions (4%). The authors suggested that the reported eye defects could be attributed to exposure to CS and that fatigue was a common symptom of people exposed to 2,4-D or to organophosphorus insecticides. The authors concluded that the toxicology of individual agents could not be assessed because complex combinations of agents were sprayed in each episode and frequent episodes of spraying occurred. 835. Rose, J. Q., Ramsey, J. C., Wentzler, T. H., Hummel, R. A., and Gehring, P. J. (1976) The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36:209-226. The pharmacokinetics of TCDD distribution and excretion in the rat is described. Sprague-Dawley rats (6 per group) were administered a single oral dose of 1 ug/kg [ Cj-TCDD (99+% radiochemical purity) in acetone-corn oil (1:25) by oral gavage. Other rats (18 per group) were administered 0.01, 0.1 or 1.0 ug/kg/day of f Cl-TCDD, 5 days per week for 7 weeks. Urine, feces and expired air samples and tissue sample of rats killed after 1, 3, or 7 weeks of exposure were analyzed for radioactivity. Liver homogenates were extracted with organic solvents to determine extractable radioactivity and were analyzed by gas 298 �chromatography-mass spectrometry to determine the proportion of radioactivity present as non-metabolized TCDD. Toxic effects of TCDD in the rat were not mentioned. The body burden for 24 days following a single oral dose of TCDD was calculated by subtracting fecal excretion levels from the total dose administered. No radioactivity was detected in urine or expired air. Body burdens followed apparent first-order kinetics, with a half-life of 31 days for both sexes. Twenty-two days after a single do'se, fat and liver each contained 1.25% of the dose, and kidney, thymus and spleen each had less than 0.1%. When repeated doses were administered, radioactivity was detected in the urine of only the highest dosage group, accounting for 3% and 12.5% of the cumulative doses for males and females, respectively. Body burdens calculated from excretion data were close to the levels of radioactivity detected in carcasses at the end of exposure of 37% and 48% for the 1 and 0.1 ug/kg/day dosage groups, respectively. Body and burden data were fitted to a one compartment pharmacokinetic model and half-times and rate constants were calculated. About 75% of the 1 ug/kg doses were absorbed by male rats and about 90% by females; 90% of the 0.1 ug/kg doses were absorbed by both sexes. The half-life (from data for all groups) was 23.7 days. The steady state body burden was estimated to be 29 times the daily dose and 90% of the steady state value would be reached after 78.5 days of exposure. Radioactivity accumulated in the liver and fat and whole body at the same ratio; the concentration in the liver was about 5 times that of fat, and both levels plateaued with continuous administration. Other tissue levels of radioactivity were less than one-tenth of the hepatic levels. Recovery of .TCDD by chemical methods confirmed the radiochemical data, indicating that TCDD biotransformation was not detectable. The authors concluded that low, continuous exposure to TCDD is unlikely to result in tissue accumulation of TCDD; TCDD approached a constant concentration in the fat, liver, and whole body within 13 weeks of repeated administrations and the rate constant was independent of dose. 836. Rose, M. S., Crabtree, H. C., Fletcher, K. , andWyatt, I. (1974) Biochemical effects of diquat and paraquat. Disturbance of the control of corticosteroid synthesis in rat adrenal and subsequent effects on the control of liver glycogen utilization. Biochem. J. 138(3):437-443. The effects of diquat administration on glycogen depletion, and on adrenocorticotropic hormone (ACTH) levels, plasma corticosteroid levels and cyclic AMP content of the adrenal glands are described in normal, hypophysectomized and adrenalectomized rats. An intraperitoneal dose of 20 rag (cation/kg) of diquat dichloride was administered to male Alderley Park rats, including adrenalectomized and hypophysectomized rats. Vehicle controls received an injection of saline. Liver glycogen, blood glucose, plasma corticosteroids and ACTH, and adrenal cyclic AMP and catecholamine levels were determined at various times after administration. Rats administered diquat and starved for 24 hr. had a mean hepatic glycogen level of 27.7 mg/g liver (wet weight) compared to 0.8 mg/g in starved vehicle controls and 67.2 mg/g in fed vehicle controls. Rats administered diquat after a 28 hr. starvation period had levels of 11.5 mg/g of hepatic glycogen, compared to 4.6-4.8 299 �mg/g for the vehicle and untreated controls. In adrenalectomized rats, the liver glycogen level fell more rapidly in diquat-treated rats than in vehicle controls. The increase in blood glucose levels was maximum 1 hr. after diquat treatment at 110 mg of glucose per 100 ml blood and was less in hypophysectomized rats and minimal in adrenalectomized rats. Diquat administration did not cause a change in adrenal catecholamine levels or in clearance of corticosteroids from ACTHtreated, hypophysectomized rats. Diquat caused an increase in cyclic AMP and a sustained increase in plasma corticosteroids in starved rats, compared to a transient increase observed in vehicle controls and was ineffective in changing corticosteroid or cyclic AHP levels in hypophysectomized rats. Diquat caused an increase in ACTH levels (in intact rats) which disappeared by 24 hr. after diquat administration. Paraquat was also studied in these experiments. The authors concluded that diquat stimulated synthesis of adrenal corticosteroid synthesis possibly by increasing adrenal sensitivity to ACTH, and suggested that some toxic effects observed histologically after diquat and paraquat poisoning were related to increased corticosteroid synthesis. 837. Rose, M. S., Lock, E. A., Smith, L. L., and Wyatt, I. (1976) Paraquat accumulation: tissue and species specificity. Biochem. Pharmacol. 25(4) .-419-423. The uptake of diquat by rat tissues in vivo and in vitro is described. Male Alderley Park rats (3-4 per group) were administered 680 umoles/kg [ C]-diquat orally and tissues were removed after 2-30 hr. and radioactivity was determined. Tissues were removed fromaon-treated rats and were sliced and incubated in the presence of 10 M diquat. Radioactivity taken up by the tissues was determined at the end of the incubation period. The concentrations of diquat in lung tissue 2-30 hr. after in vivo treatment remained close to the plasma levels, whereas the kidney levels were 5-10 times the plasma levels, the levels for the liver and adrenals were 1-2 times plasma levels, and levels for brain and muscle were below plasma levels. Slices of renal cortex concentrated diquat to levels about double those in the medium and the effect was established within 1 hr. Tissues studied in vitro included those analyzed after in vivo exposure, as well as skin, heart, small intestine and spleen. No other tissue accumulated diquat after 1-2 hr. in culture. Uptake of paraquat was also studied. The authors concluded that only the kidney and possibly the adrenals and liver were capable of accumulating or retaining diquat. 838. Rose, M. S., and Smith, L. L. (1977) Minireview. Tissue uptake of paraquat and diquat. Gen. Pharmacol. 8:173-176. [Review article.] 300 �839. Rose, M. S., Smith, L. L. , and Wyatt, I. (1976) The relevance of pentose phosphate pathway stimulation in rat lung to the mechanism of paraquat toxicity. B iochem._ Pharmaco 1. 25(15): 1763-1767 . The effect of diquat on glucose metabolism by rat lung slices and diquat accumulation in the lung.were studied. Rat lung slices were incubated in the presence of [ C]-glucose and metabolism was determined by trapping and counted CO- generated. Diquat was introduced to the incubation medium at concentrations of 10 to 10 M or was administered intravenously (65 u moles/kg in saline) 0.5-18 hr. before the lungs were removed or orally (680 u moles/kg) 4-30 hr. before the lungs were removed, sliced, and incubated. Vehicle controls were used for the u.v. study. At 10 "M and 10 M, diquat caused twofold and fourfold stimulation, respectively, of [1- C] glucose metabolism. At all times after u.v. treatment and 30 hr. after ingestion, [1- _C]-metabolism was elevated about twofold. The effect of diquat at lO^M was not inhibited by impranine (10 M). The half-time for I C]-diquat to equilibrate with the volume of tissue available to [ Hl-water was 80 min. The metabolism of [6- Ol-glucose was not altered by diquat administered by any route. The effects of paraquat were also examined. The authors concluded that diquat and paraquat entered lung cells and stimulated the pentose phosphate pathway, generating free radicals; accumulation of paraquat was suggested as the reason why paraquat was toxic to the lung and diquat was not toxic. 840. Rose, M. S., Smith, L. L., and Wyatt, I. (1974) Evidence for energy-dependent accumulation of paraquat into rat lung. Nature 252(5481):314-315. Uptake of diquat by rat lung slices is described. Lung slices were prepared from male Alderley ?grk rats and were incubated in the presence of 10 M to 10 M [ C]-diquat for up to 2 hr. Radioactivity of the tissues was analyzed after the incubation period ended. The amount of diquat taken up by lung slices plateaued by 30 min. of incubation and the plateau level was dependent upon the concentration of diquat in the medium. The pattern of uptake of paraquat by lung slices was also described. The authors concluded that paraquat, but not diquat, was actively accumulated by liver slices by an energy-dependent process. 841. Rowe, V. K. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA Docket No. 415 et al., Nov 13. [Testimony.] 301 �842. Rowe, V. K., and Hymas, T. A. (1954) Summary of toxicological information of 2,4-D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. Amer. J. Vet. Res. 15:622-629. Acute and subacute toxicity of 2,4-D and 2,4,5-T is described for the dog, rabbit, guinea pig, rat and chicken. Various salts and esters of 2,4-D and 2,4,5-T were administered orally in water, olive oil, corn oil, or undiluted. For acute studies, mortality rates were determined 14 days after a single dose was administered. Brief descriptions of the toxic effects observed in treated animals were given. The LD values ranged from 300-1000 mg/kg for both compounds for all species except the dog, with an LD^Q of 100 mg/kg. The chicken was the least sensitive species. LD5Q values for the various forms of the phenoxy acids were comparable and commercial formulations had LD-. values that were proportional to the levels of active ingredients they contained. Neither compounds had cumulative effects, since repeated oral doses that were ineffective were only slightly lower than an ineffective single oral dose. For both compounds, symptoms in affected animals included muscular tenseness and weight loss. Irritation of the stomach and minor liver and kidney damage was also observed. The authors concluded that 2,4-D and 2,4,5-T used by recommended agricultural methods presented a negligible hazard to livestock and wildlife. 843. RPAR Assessment Team. (1979) Biologic and economic assessment of 2,4,5-T. A report of the USDA-STATES-EPA 2,4,5-T RPAR Assessment Team, Washington, DC, Feb 15. Exposure of 2,4,5-T and TCDD are estimated after assumptions regarding conditions of exposure are stated. Dermal absorption of 2,4,5-T, measured for four human volunteers in a laboratory experiment, and deposition and absorption determined for field workers were reported elsewhere (Newton, 1978; Lavy 1978a; and Lavy 1978b, respectively) were used to calculate exposure. Assumptions, including the concentration of 2,4,5-T used for spraying by workers with specific tasks, and the types of protective clothing worn, are stated for 10 different exposure situations. Maximum possible exposure levels were estimated from the rates of absorption, given in the previous laboratory reports, and were applied to the specific, assumed exposure conditions. Dosages were presgnted fbr 2,4,5-T and TCDD, based on a level of contamination of 3 X10 ppm TCDD in the 2,4,5-T mixture and an absorption rate for TCDD twice the rate of 2,4,5-T absorption. Maximum absorption was estimated for a backpack spray operator without protective clothing administering 40 Ib of acid equivalent per 100 gallons, with a predicted maximum absorption rate of 0.85 mg/kg/hr of 2,4,5-T and 2.1 x 10 ug/kg/hr of TCDD. The authors concluded that the levels of exposure observed after actual operational applications were substantially lower than those calculated from laboratory data with maximum exposure of 0.095 mg/kg/hr of 2,4,5-T calculated from data on workers under specific conditions of exposure. 302 �844. Rubenchik, B. L., Botsman, N. E., and Groban, G. P. (1970) The carcinogenic effect of the herbicide monuron. Vopr. Onkol. 16f10):51-53.• [Foreign language.] 845. Rubenchik, B. L., Botsman, N. E., and Shipko, G. P. (1969a) Study of carcinogenic properties of the herbicide monuron. Vopr. Ratsion Pitanii 5:85-86. [Not available.] 846. Rubenchik, B. L., Petrun, A. S., Pliss, M. B., and Shipko, G. P. (1969b) Monuron action on the liver when this herbicide is administered with food. Vopr. Pitan. 28(6)-.13-18. [Foreign language.] 303 �847. Safety of the herbicide 2,4,5-T. [Letters to the editor] 1977. Zea. Med. J. July 13, 1977:35-36. New [Editorial.] 848. Saint-Ruf, G. (1972) Formation of dioxin in the pyrolysis of sodium a-(2,3,7,8-trichlorophenoxy)-propionate. Naturwissen Schaften 59(12):648 [Background material.] 849. Saint-Ruf, G. , and Do-Phuoc Hien. (1975) Similarity of the biochemical effects of 2,3,7,8-tetrachloridibenzo-p-dioxin and 2,3,7,8tetrabromodibenzo-p-dioxin in the rat. C. R. Hebd. Seances Acad. Sci. Ser. D. 280(23):2709-2711. [Foreign language.] 850. Samraan, P. D., and Johnston, E. N. M. (1969) Nail damage associated with handling of paraquat and diquat. Br. Med. J. 1:818-819. Two cases of nail damage that were suspected to be caused by contact with Preeglone Extra are described. A 75-year-old man who diluted Preeglone Extra (which contains equal amounts of diquat and paraquat) concentrate with water frequently over a number of years, presented with damage to most of his fingernails. Damage included subacute parenychia, partial nail loss, discoloration, and hemorrhages below the nail. Two months later all nails were regrowing satisfactorily. A 50year-old man, who also diluted Preeglone Extra, frequently had nail damage on most fingers, which involved nail loss, discoloration, and infection. The toe nails were not involved in either case. A case of nail discoloration after handling paraquat was also reported. The authors concluded that local contact of the nails with the herbicide in concentrated form resulted in discoloration, nail softening and loss, accompanied by susceptibility to infection. 851. Sanders, H. 0. (1970) Toxicities of some herbicides to six species of freshwater crustaceans. J. Water Poll. Cont. Fed. 42:1544-. [Background material.] 852. Sanderson, C. A., and Rogers, L. J. (1981) 2,4,5-Trichlorophenoxyacetic acid causes behavioral effects in chickens at environmentally relevant doses. Science 211(4482):593-595. Alterations in behavioral development of chickens treated with 2,4,5-T in ovo are described. A single dose of 2,4,5-T (0.03 ppm TCDD contaminant) suspended in 0.5% gum tragacanth was injected into chicken 304 �eggs on day 8 or day 15 of incubation or was administered intraperitoneally to 2-day-old chicks. Doses of 7-53 mg/kg and 75-225 mg/kg were administered to eggs and chicks, respectively. Median lethal doses (L.DCQ), gross abnormalities and body weights were established. Behavioral activities measured included general activity (scored by an activity meter), ambulation (the frequency each chicken crossed into a new quadrant; the floor of the test box was divided into 4 quadrants), visual learning (the number of incorrect pecks at background pebbles by chickens seeking food) and jumping behavior (frequency of jumping). The LDcn doses were 53 mg/kg administered on day 15 of incubation and 200 mg/kg to 2-day-old chicks. In chicks given 27 mg/kg on day 15, 70% hatched. No dose-related gross malformations or weight loss from doses below 150 mg/kg on day 2 were observed. Jumping behavior increased significantly in chickens given 13 or 27 mg/kg on day 15 or 150 mg/kg after hatching. General activity increased significantly in chicks treated in ovo on day 15 with a 27 mg/kg dose and visual learning decreased in chicks treated in ovo on day 15 with 7 or 27 (but not 13) mg/kg TCDD. The authors concluded that doses of 22 mg/kg could reach the egg from recommended agriculture rates of 2,4,5-T spraying and these levels caused adverse behavioral effects in chickens. The relationship of the behavioral parameters measured to mammalian behavioral development are not obvious; the biological significance of the observed changes in measured frequencies and the possibility all of a 22 mg/kg dose of 2,4,5-T deposited on an eggshell of reaching the embryo remain unknown. 853. Sare, W. M. (1979) 2,4,5-T and the problems of toxicity. [letter to the editor] Med. J. Austr. 1(11):526. [Editorial.] 854. Sare, W. M. (1972) The weedicide 2,4-D as a cause of headaches and diplopia. [letter to the editor] New Zea. Med. J. 75(478):173-174. [Editorial.] 855. Sare, W. M., and Forbes, P. I. (1977) The herbicide 2,4,5-T and its possible dysmorphogenic effects. New Zea. Med. J. 85(588):439. [Editorial.] 856. Sare, W. M., and Forbes, P. I. (1972) Possible dysmorphogenic effects of an agricultural chemical: 2,4,5-T. New Zea. Med. J. 75(476):37-38. Two lethal cases of congenital deformities were reported. In the previous few years 2,4,5-T, which can be contaminated with dioxin, had been sprayed in the rural area of New Zealand where the births occurred. Both infants had gross myelo-meningocele and autopsy of one infant revealed other malformations (which were not described), in 305 �support of the potential causal relationship between TCDD exposure and birth defects. The authors indicated that the farms of both mothers had been sprayed during the first trimester of each pregnancy and rain water collected from the roof of each house was used as the domestic water supply. A neighbor in the sprayed area who did not collect roof water gave birth to a normal child. The farm of one of the affected families reported the lowest calving rate for pregnancy-tested cattle. The authors also calculated the potential exposure levels of TCDD from aerial spraying and an estimated fetotoxic dose of TCDD in humans extrapolated from animal data. 857. Sauerhoff, M. W., Braun, W. H., Blau, G. E., and Gehring, P. J. (1977) The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man. Toxicology 8:3-11. The pharmacokinetics of 2,4-D clearance from plasma was determined in five men. The men, aged 29-40 (and in good health, as evidenced by results in the normal range for all tests including physical exams and clinical blood and urine analyses) were administered 5 mg/kg analytical grade 2,4-D in milk (2) or as a powder followed by water. Urine and plasma samples were analyzed over the subsequent 144 hours by gas chromatography-mass spectrometry. Acid-labile urinary conjugates of 2,4-D were determined by acid-hydrolysis of urine samples prior to analysis. No signs of toxicity were detected by any subject. A two-compartment model was used to analyze data from the one subject, a one-compartment model was used for two additional subjects, and the data for the last two subjects were not analyzed kinetically. Neither model adequately described the data at the 95% confidence level. The half-lives for absorption were between 1.7 and 4.2 hours and for elimination from blood were 7-11 hours for the one-compartment model and 4 and 15 hours for the fast and slow components of the twocompartment model. The volumes of distribution were 283-294 ml/kg for the one-compartment model and 84 and 218 ml/kg for the central and slow exchange compartments of the two-compartment model. Urinary elimination was analyzed pharraacokinetically for all five subjects. From 48 to 97% of the dose was excreted in the urine, with a half-life of 10.2-28.4 hours. From 0 to 27% (mean 12.8%) of the dose of 2,4-D was excreted as urinary conjugates. The authors concluded that an oral dose of 2,4-D was well absorbed in man and was excreted from the body and cleared from plasma by apparent first-order rate processes. 858. Sauerhoff, M. W., Braun, W. H., Blau, G. E., and Gehring, P. J. (1976) The dose-dependent pharmacokinetic profile of 2,4,5-trichlorophenoxyacetic acid following intravenous administration to rats. Toxicol. Appl. Pharmacol. 36(3):491-501. The effect of the dosage of 2,4,5-T administered to rats on the pharmacokinetics of excretion were examined. Sprague-Dawley rats were administered 5 or 100 mg/kg of 2,4,5-T (99% purity) by intravenous infusion. Blood, urine, feces, and tissue samples were analyzed for radioactivity and urinary metabolites were extracted and separated by 306 �thin layer chromatography and gas chromatography-mass spectrometry. Plasma clearance of 2,4,5-T followed first order kinetics after the lower dose only. For the higher dose, the rate constant for the linear phase of plasma clearance (the faster component) was close to the value for the lower dose (0.16 per hour). The half-lives were 4.3 hours and 5.3 hours for the low and high doses respectively. The excretion of 2,4,5-T showed the same dose-dependent kinetics with the constants for the faster components from the high dose corresponding to the constants for excretion of the low dose. Less than 5% of either dose remained in the body after 48 hours. Tissue-to-plasma ratios of radioactivity were the same after the low and high doses, except for a higher kidney-toplasma ratio after the lower dose. Over 94% of the urinary radioactivity was associated with unchanged 2,4,5-T. The authors concluded that the higher dose of 2,4,5-T saturated the active transport process of renal excretion and that projections of toxicity of low doses of 2,4,5-T based on data from high doses are not justified because mechanisms of detoxification have been compromised at high doses. 859. Sauerhoff, M. W., Braun, W. H., Blau, J. E., and LeBeau, J. E. (no date) The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man. No reference pp. 136-137. [Abstract, only.] 860. Sauerhoff, M. W., Chenoweth, M. B., Karbowski, R. J., Braun, W. H., Ramsey, J. C., et al. (1977) Fate of silvex following oral administration to humans. J. Toxicol. Environ. Health 3:941-952. The pharmacokinetics of [2-(2,4,5-trichlorophenoxy) proprionic acid](silvex) was studied in human volunteers. Six men and one woman were each administered orally 1 mg/kg silvex which contained less than 0.01 ppm TCDD. Blood, urine, and fecal samples collected in the subsequent 168 hours were analyzed for silvex, by gas chromatographymass spectrometry, and urine and feces were analyzed for metabolites by hydrolysis, extraction, or derivitization of samples prior to the silvex analysis. No adverse effects were detected in the subjects after ingesting silvex and all clinical chemistry analyses on blood and urine were within normal ranges. Blood levels of silvex peaked 1-2 hours after ingestion. Blood clearance was biphasic, with half-lives of 4 and 16.5 hours for the two components. The best fit of the data was provided from a two-compartment model and the volume of distribution for each compartment was 115 ml/kg and 107 ml/kg. A mean of 80% of the dose of silvex was excreted in the urine in 144 hours, with 15-44% of the dose recovered as conjugates in the urine. 2,4,5-trichlorophenol was not detected in the urine. Urinary excretion was biphasic, with half-lives for each component of 5 and 26 hours. Fecal excretion of silvex accounted for 3.2% of the dose in one subject and below 1% for four other subjects. The authors noted that the rate for the slow phase of plasma clearance resembled the rate of clearance of 2,4,5-T described elsewhere and noted that the biliary route of excretion which predominates in the rat is not important in silvex excretion in man. 307 �861. Savidge, J. A. (1978) Wildlife in a herbicide-treated Jeffrey pine plantation in eastern California. J. For. 76(8):476-478. The author compares the populations of vegetation and wildlife on a 2,4,5-T sprayed area and an unsprayed area of a Jeffrey pine plantation. The study was conducted 6 years after an aerial spraying of 2,4,5-T (1.8 kg/acre acid equivalent). Each study area contained approximately 11 acres. The biggest difference between the two areas was the elimination of most of the Ceanothus velutinus (snowbrush) in the sprayed area. Live Ceanothus covered 30% of the unsprayed plot, but only 27, of the sprayed plot. According to the author, this difference was responsible for the changes seen in wildlife populations. Elimination of Ceanothus allowed a currant species (Ribes cereum) that was dormant at the time of spraying to thrive in the sprayed area. Other vegetation, such as the Jeffrey pine, were similar in dominance in both areas. The total number and number of species of birds on the unsprayed plot were approximately twice those of the sprayed plot. Most of the species eliminated on the sprayed plot used Ceanothus as a source of food or shelter. Nearly twice as many small mammals (mice, chipmunks, squirrels) were found in the sprayed plot. The author speculated that food and/or habitat changes might have been responsible for the increase. Deer usage also varied between the two plots. On the unsprayed areas, deer and deer trails were observed, while on the sprayed area neither were observed. The author did not make any conclusions on the significance of these changes. 862. Sawinsky, A., and Pasztor, G. (1977) Exposure tests in reglone spraying by aircraft. Z. Gesamte Hyg. 23(ll):845-846. [Not available.] 863. Scarpelli, D. G., Rao, M. S., Subbarao, V., et al. (1980) Activation of nitrosamines to mutagens by postmitochondrial fraction of hamster pancreas. Cancer Research 40:67-74. 864. Schantz, S. L., Barsott, D. A., and Allen, J. R. (1979) Toxicological effects produced in nonhuman primates chronically exposed to fifty parts per trillion 2,3,7,8-tetrachiorodibenzo-p-dioxin (TCDD). Toxicol. Appl. Pharmacol. 48(1):A180. [Abstract, only.] 865. Schardt, C., and Heathfield, C. (1979) "Agent Orange" - A Selected Bibliography. Prepared by the Veterans Administration. 13 pp. [Bibliography.] 308 �866. Schiller, C. M. (1979) Chemical exposure and intestinal function. Environ. Health Perspec. 33:91-100. [Review article.] 867. Schonborn, H., Schuster, H. P., and Kossling, F. K. (1971) Clinical and morphological findings in an acute oral intoxication with diquat (reglone). Arch. Toxicol. 27(3):204-216. [Foreign language.] 868. Schreiweis, D. 0., and Murray, G. J. (1976) Cardiovascular malformations in Oryzias latipes embryos treated with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Teratology 14(3);287-290. The teratogenic effects of 2,4,5-T were examined in developing fish eggs. j)ryzias latipes eggs were grown in the presence of of 10-50 ppra 2,4,5-T (2,4,5-T had less than 0.05 ppm dioxin contamination), which was introduced in ethanol (final volume of ethanol, 0.4%) at stage 10 (blastula stage) of development. Ethanol was added to control dishes of eggs. The rate of hatching and developmental features, including axis formation, initiation of heartbeat, and normal body pigmentation were followed. The rates of hatching were 100% in control and 10 ppm-treatment groups, 96% for the 14 ppm group, 47% for the 20 ppm group, 4% at 25 ppm and none at higher doses. Interruption of each developmental feature monitored showed a dose-related pattern. Cardiovascular anomalies also occurred at a frequency related to 2,4,5-T dose. These anomalies included enlarged veins, enlarged cardinals, and enlarged heart chambers, tube heart and hemorrhages. No other anomalies in other organ systems were discussed. The authors suggested that 2,4,5-T is selectively taken up by differentiating cardiovascular tissue and recommended rates of spraying for 2,4,5-T that would not interfere with reproduction in this fish species. 869. Schulz, K. H. (1968) Clinical picture and etiology of chloracne. Arbeitsmedizin-Sozialmedizin-Arbeitshygiene 3(2):25-29. [Review article.] 870. Schulz, K. H. (1957) Arch. Klin. Exp. Derm. 206:589-596. The clinical symptoms of workers in Hamburg, Germany involved in 2,4,5-T synthesis are described briefly and experimental results of efforts to elucidate the etiology of the observed pathology are presented. Thirty-one workers developed dermatological symptoms between the middle of 1954 and the spring of 1955. Comedones, pustules, furuncles and retention cysts appeared on the face, neck, back, chest, genitals, arms and legs. (The frequencies of these symptoms or temporal or clinical patterns were not described). Other 309 �symptoms reported were chronic conjunctivitis, blepharitis and complaints of nausea, debility and loss of appetite. Clinical tests did not reveal liver, kidney, central nervous or hemopoietic disorders (the tests used and the frequency of testing were not indicated). A chemical laboratory worker outside of Hamburg developed chloracne 10-14 days after working with a tetrachlorodibenzodioxin; no other details of this incident were provided. Various compounds used in the synthesis of 2,4,5-T or proposed as contaminants during the manufacturing process were tested for acnegenic activity in the rabbit ear; hepatotoxicity was tested with a bromosulfthalein method which was not described further. Technical and chemical grades of 2,4,5-trichlorophenol and its distillation residue, 1,2,4,5-tetrachlorobenzene, diphenyl ether, mono- to tetra-chlorinated diphenyl ethers, dibenzofuran, mono- to tetra-chlorinated dibenzofurans and a tetra-chlorinated dibenzo dioxin (chlorine positions were unknown) were each painted on rabbit ears and the development of chloracne at the site was monitored. Technical grade 2,4,5-trichlorophenol, its distillation residue, tri- and tetra-chlorinated dibenzofurans and tetrachlorodibenzo-dioxin each elicited an acnegenic response of inflammation in 5-7 days and keratotic-containing follicles several days later. These compounds all produced slight hepatotoxicity, except for the furans, which elicited a strong response and the dioxin, which was not tested for hepatotoxicity. Chlorinated diphenyl ethers also produced slight hepatotoxicity. The authors concluded that the tetrachlorodibenzo dioxin was likely to have been a contaminant in the distillation residue and was likely to be the etiologic agent in causing the observed dermatitis. 871. Schulze, J. A., Manigold, D. B., and Andrews, F. L. (1973) Pesticides in selected western streams 1968-71. Pest. Monit.'J. 7(1):73-84. The authors describe the occurrence of 2,4-D and 2,4,5-T in 20 western streams October 1968 through September 1971. Samples were analyzed by gas chromatography (detection limit 0.05 ug/1 2,4,5-T; 0.02 ug/1 2,4-D). The highest concentration of 2,4-D detected was 0.99 ug/1 while the highest concentration of 2,4,5-T was 0.40 ug/1. Of the total 109 2,4,5-T occurrence, 47% were detected at 2 stations. 2,4-D was detected 103 times. Amounts of herbicide applied to the watersheds under study was not presented. 872. Schultz, D. P., and Harmon, P. D. (1974) Residues of 2,4-D in pond waters, mud, and fish, 1971. Pestic. Monit. J. 8(3):173-179. The authors 1) measured residue levels and the rate of dissipation of 2,4-D in water, bottom mud and fish, 2) determined the effect of physical and chemical characteristics on uptake and dissipation and 3) assessed the efficacy of 2,4-D on waterhyacinth in Florida and Georgia ponds. Nine ponds of varying ecological and geographical types, located in Florida, Georgia and Missouri, were treated with DMA-2,4-D at 2, 4 or 8 Ib/acre in 1971. The formulation contained 4 Ib 2,4-b acid equivalent per gallon. Fish, mud and water samples were taken at intervals up to 147 days after application. Approximately 98% of the 310 �waterhyacinth was killed within 7 days. The highest residue level in Florida pond water was 0.345 mg/1 3 days after application of 8 Ib/acre; it then dissipated to less than or equal to 0.005 mg/1 14 days after treatment. The highest level in Georgia pond water was 0.692 mg/1 3 days after treatment with 8 Ib/acre 2,4-D. Levels decreased to less than 0.005 mg/1 28 days after treatment. In Missouri pond water, the highest level was 0.630 mg/1 14 days after application of 8 Ib/acre decreasing to less than detectable 56 days after application. After treatment, residues in mud were 0.046 mg/kg in Florida, 0.042 mg/kg in Georgia, and 0.170 mg/kg in Missouri, all treated at levels of 8 Ib/acre 2,4-D. Trace amounts of 2,4-D in mud were detected 112 days after application in the Florida and Georgia ponds and no residues were detected in the Missouri ponds 56 days after sampling. Residues in fish from Florida and Georgia ranged from 0.005 mg/kg to 1.075 mg/kg. In the Florida ponds, only one sample contained detectable levels after 14 days. In the Georgia ponds, only one sample showed detectable levels after 28 days. All fish samples from Missouri ponds showed no detectable amount of 2,4-D except 6 samples which showed trace amounts. The authors conclude that no difference in waterhyacinth kill was evident among the three treatment levels. Since the Missouri ponds had no waterhyacinth before treatment, the persistence of the herbicide in Missouri ponds is due to the lack of sufficient surface biomass to degrade 2,4-D. It is also mentioned that initial concentrations were twice as much in the Missouri pond due to a wrong assumption before spraying. 873. Schuth, C. K., Isensee, A. R., Woolson, E. A., and Kearney, P. C. (1974) Distribution of C and arsenic derived from [ C] cacodylic acid in an aquatic ecosystem. J. Agr. Fd. Chem. 22(6):999-1003. The authors studied the accumulation and distribution of cacodyli acid in model aquatic ecosystem using three soil types, Willacy sandy loam, Hidalgo clay loam, and Laredo silt loam. Duplicate soil samples were treated with 21 ppm C-cacodylic acid and layered on the bottom of glass aquarium tanks. Water was added to each of the tanks and allowed to equilibrate for weeks. Catfish, crayfish, daphnids, snails, filamentous algae, and duckweed were then added to each tank. Water samples were removed from each tank at 2-day intervals for 59 days for scintillation counting. In addition, water samples were removed for arsenite, arsenate, and total arsenic analysis by colorimetric and atomic absorption spectroraetry at 8, 22, 38, 50, and 59 days. Soil and organisms were also analyzed by the same methods. In the soil (average of all 3 types), after 59 days 13.5% of the C radioactivity and 40% of the As from the original cacodylic acid remained. According to the authors, the differences in C and as loss indicate that the C-As bond of cacodylic acid in the soil is being split, In water, the cacodylic acid increased nearly linearly for 30-50 days, leveled off, then decreased. Water analysis after 59 days did not show any cacodylic acid present. The authors concluded that ,C in the water was associated with volatile chemical forms such as *- -CO^ or organoarsenicals. Analysis of aquatic organisms for C and total arsenic revealed little litl bioaccumulation. Bioaccumulation ratios decreased as food chain 311 �position of the organism increased. According to the authors, the C data indicate degradation of cacodylic acid and uptake of C by the plants. 874. Schwetz, B. A., Norris, J. M., Sparschu, G. L., Rowe V. K., and Gehring, P. J. (1973) Toxicology of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5:87-99. The acute oral, parenteral, and dermal toxicities of TCDD, as well as the production of eye irritation, acne, and chick edema disease are summarized. TCDD was administered in corn oil: acetone (9:1) by gavage to Sherman rats, Swiss Webster mice, New Zealand albino rabbits, Hartley guinea pigs, and Beagle dogs, and the animals were observed for 2-8 weeks. Female rats were more sensitive than male rats, and rabbits were more sensitive than dogs to TCDD. The oral LD,-ns of the same TCDD preparation were 0.6 (u)g/kg in the guinea pig, 22-45 (u)g/kg in the rat, and 115 (u)/kg in the rabbit. Death was usually delayed, occurring several weeks after TCDD was administered. Weight loss was seen in all species after TCDD treatment, while other symptoms were species-specific. Histological lesions observed in most species included hepatic necrosis, fat atrophy, and periarteritis. Single doses of 32-500 (u)/kg TCDD in acetone were applied to the shorn abdominal skin of rabbits, and the site of administration was wrapped in cotton after the acetone evaporated. Rabbits were administered single doses of 32-500 (u)g/kg TCDD in corn oil suspension intraperitoneally and observed for 4 weeks. Lethality of TCDD in the rabbit was the same by all routes of administration. Eye irritation was evaluated in rabbits to which 2 mg (article does state 2 milligrams) of TCDD was instilled into the conjunctival sac of one eye. Delayed conjunctival chemosis was observed 13-22 days after TCDD was applied ocularly. Acnegenic activity was evaluated in rabbits that received 0.1 ml of the supernatant of a TCDD suspension in benzene applied to the inner surface of the ear 5 days per week for 4 weeks. Acne was produced by TCDD concentrations above 0.04 (u)g/ml, with the severity of the response dependent upon the concentration applied to the ear. White Leghorn chickens were administered 1 or 10 (u)g/kg TCDD daily in their diets and were observed for symptoms of chick edema syndrome. Daily doses of 1 or 10 (u)g/kg of TCDD produced chick edema syndrome, characterized by increased pericardial fluid volume, dyspnea, subcutaneous and pulmonary edema, and enlarged, mottled liver, and histologically identified atrophy of the germinal centers of the spleen, few bursal lymphocytes, and fatty degeneration. The toxicities of other chlorodibenzo-dioxins were also examined. The authors concluded that TCDD had a very high order of toxicity relative to the other dioxins tested, and the toxicological properties of the various chlorodibenzodioxins were different. The authors suggested that repeated contact with small amounts of TCDD would probably produce chloracne in man, but TCDD contact with the eyes would probably not impair vision seriously. 312 �875. Schwetz, B. A., Sparschu, G. L., and Gehring, P. J. (1971) The effect of 2j4-dichlorophenoxyacetic acid (2,4-D) and esters of 2,4-D on rat embryonal, foetal and neonatal growth and development. Fd. Cosmet. Toxicol. 9:801-817. The teratologic and reproductive effects of 2,4-D and two derivatives of 2,4-D were evaluated in rats. Pregnant rats were orally administered 12.5-87.5 mg/kg of 2,4-D daily from day 6-15 of gestation. Other rats received the molar equivalents of 2,4-D propylene glycol butyl ether (PGBE) or 2,4-D isooctyl ester (10) on days 6-15 or on days 5-8 or 10 on days 8-11 or days 12-15. All compounds were administered in corn oil and the control rats received only corn oil. On day 20 of gestatiop weights, and skeletal malformations at 21 days of age were recorded. No maternal toxicity, measured as body-weight gain, was observed with the doses administered. All 3 compounds caused a decrease in fetal body weight at doses at 75 and 87.5 mg/kg. Only PGBE produced a reduction in percent resorptions, but this effect was small and was not dose-related. The incidence of subcutaneous edema was increased by all 3 compounds administered at doses of 50 mg/kg or greater. Other skeletal defects included delayed ossification of sternebrae, wavy ribs, missing sternebrae (all significantly increased by high doses of 2,4-D or PGBE), sternebrae with split centers of ossification (significantly increased by high doses of 2,4-D or 10), lumbar ribs (caused by all 3 compounds) and delayed ossification of skull bones (significantly increased by 10). Administration of 87.5 mg/kg molar equivalents of PGBE or 10 on days 5-8 did not alter the numbers of implantations or of corpora lutea per litter. When administrated on days 8-11, this treatment produced a significant increase in resorption but did not produce a change in fetal size. The only alterations in post natal indices were decreases in viability and lactation indices in the 75 and 87.5 mg/kg (equivalent) groups treated with PGBE and 10. The delayed bone ossification was a temporary condition that was not observed in 3-week-old neonates. The authors concluded that 25 mg/kg or its molar equivalent was the no-effect dose for each of the 3 compounds and the toxicity increased in the order: PGBE, 2,4-D, 10. The authors also concluded that all 3 compounds were embryotoxic and fetotoxic, but were not teratogenic or detrimental to survival of offspring to 21 days of age. 876. Scientific Dispute Resolution Conference on 2,4,5-T. by The American Farm Bureau Federation, 101 p. (1979) Sponsored [Review article.] 877. Scifres, C. J., McCall, H. G., Maxey, R., and Tai, H. (1977) Residual properties of 2,4,5-T and picloram in sandy rangeland soils. J. Environ. Qual. 6(1):36-42. The authors studied the movement and persistence of 2,4,5-T and picloram in a sandy rangeland ecosystem. Three study sites were chosen: 1) Bastrop, consisting of S.lha of Coastal Bermuda grass in 313 �Edge-Tabor sandy clay load soil; 2) Caldwell, 3.8ha of Coastal Bermuda grass in Laverne-Tabor-Lakeland loamy fine sand, and 3) San Perlita which had coastal prairie climax vegtation. The Bastrop and Caldwell sites were sprayed with 0.56 Kg/ha 2,4,5-T and 0.56 kg/ha picloram using a ground sprayer in 1973. Bastrop was sprayed again in 1974. San Perlita received only one spraying in 1974. Twenty five vegation and soil, samples water samples were collected from each site and analyzed by gas-liquid chromatography (detection limit 10 ppb soil and 5 ppb water). Immediately after the first spraying at Bastrop, grass tissue contained 58,840 ppb 2,4,5-T and 109,351 ppb picloram. Levels of both herbicides decreased by about 90% during the 1st 7 days after application. No 2,4,5-T was detected at 112 days. Picloram persisted for 224, and detectable levels were measured at 365 days. Twice as much 2,4,5-T was detected on Bermuda grass after the 2nd spraying at Bastrop, the primary source of variation being a 50% decrease in the quantity of vegetation at application in 1974. Persistence of 2,4,5-T in 1974 was smilar to 1973. By 112 days, no 2,4,5-Twas detected. Picloram persisted for 112 days in 1974. At Caldwell, both herbicides persisted for longer periods. 112 days for 2,4,5-T and 224 days for picloram. In Bastrop soil samples, levels of 2,4,5-T increased from 0 to 7 days probably as a result of runoff. Only trace amounts ( lOppb) of 2,4,5-T were detected on days 28 and 56; no 2,4,5-T was detected at 112 days. For the most part, 2,4,5-T residues were restructed to the top 2.5 cm of soil. A similar pattern of 2,4,5-T residues was observed at Bastrop in 1974 and at Caldwell in 1973. At Bastrop in 1973, picloram residues were similar to the pattern five to 2,4,5-T. In 1974, picloram residues in soil did not peak until 28 days after spraying occurred in the top 18 cm of soil. Trace amounts of picloram were detected up the top 15 cm of soil up to 112 days after spraying. Except on day 56, no picloram residues were detected below 15 cm. At Caldwell, 2,5,5-T residue were present immediately after spraying. Only trace residues were detected at 7, 28, and 56 days and no residues were detected at 112 days. Except for trace A amounts of picloram at 2,5-15 cm on days 7, 28, and 56, no pilcoram residues were detected below the top 2.5 cm of soil at Caldwell. At San Perlita, neither 2,4,5-T nor piclorara residues were detected below the top 2.5 cm of soil. 2,4,5-T persisted for 34 days, while picloram was still being detected at 68 days. At Bastrop, residues of both herbicides were present in water runoff 27 days after application. At Caldwell, 2,4,5-T residues were detected in runoff at 27 days and picloram residues were present at 56 days. The authors made no conclusions regarding the effects of 2,4,5-T and picloram on the rangeland ecosystem. However, it appears that both 2,4,5-T are not persistent compounds in a rangeland environment, although persists slightly longer than 2,4,5-T. Neither compound appears to accumulate in vegetation or soil. 878. Seabury, J. H. (1963) Toxicity of 2,4-dichlorophenoxyacetic acid for man and dog. Arch. Environ. Health 7:202-209. The therapeutic efficacy of 2,4-D for fungal infections was evaluated in dogs and in man. Three dogs were infected with Histoplasma 314 �capsulatum and were treated with intravenous doses of 1.2-3.2 mg/kg of the sodium salt of 2,4-D for approximately one month (total doses for the three dogs were 277 mg, 389 mg and 464 mg). The dogs were autopsied from 85 to 202 days after the last injection. No gross or microscopic lesions were observed in the tissues examined except those attributed to the infection. No neurological tissues were examined and no comparison was made of the extent of infection of the 3 treated dogs compared to 3 control dogs that received only the fungus. Two patients with disseminated coccidioidomycosis (and poor prognoses) were treated with 2,4-D, The first patient was given intramuscular injections over 4 days of 40 mg (total) of the sodium salt of 2,4-D and 3.3 mg each of indole-3-propionic acid and indole-3-butyric acid. The patient died on the fifth day; no symptoms or autopsy findings were attributed to 2,4-D treatment. The second patient received a total of 12.7 g of 2,4-D over a period of 34 days (along with 369 mg indole-3-butyric acid and 38 mg of naphthaleneacetic acid) with no toxicity observed. An intravenous dose of 3600 mg of sodium 2,4-D 2 days later caused the patient to lapse into a deep stupor, accompanied by hyporeflexia and urinary incontinence. These symptoms were alleviated within 24 hours, although muscle weakness and lethargy persisted for an additional 24 hours. The patient died 2 weeks later and all lesions observed grossly and microscopically at autopsy were attributed to the fungal infection, including hepatic and renal congestion. Other tissues, including the treated muscle, heart, and pancreas appeared normal except for leptomeninges over the brain stem. Death was attributed to disseminated coccidioidomycosis. 879. Seefeld, M. D., Albrecht, R. M., and Peterson, R. E. (1979) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on indocyanine green blood clearance in Rhesus monkeys. Toxicology 14(3): 263-272. Hepatic function was assessed in monkeys treated with a single dose of TCDD. Adult male Rhesus monkeys were given TCDD in acetone-corn oil (0.5:9.5) by gastric intubation. One animal each received 5 (u)g/kg and 75 (u)g/kbitol dehydrogenase (SDH), and gamma glutamyl transpeptides (GTP) were also measured. Histopathological examination of the liver was performed.] Monkeys died or became moribund and were killed 4 weeks after the high dose, and 6-7 weeks after the middle dose. The monkey that received the low dose survived, and the only changes observed were elevated SDH and SGPT levels 2-5 weeks after treatment. The 2 monkeys given 25 (u)g/kg TCDD showed: elevated ICG clearance for 4 weeks after treatment, followed by a decrease until death occurred; weight losses of 27 percent and 42 percent of pretreatment weights; and elevated SDH and SGPT levels. The same changes occurred after the high dose, but the changes occurred sooner and were generally greater. Weight loss was 45 percent of pretreatment weight. Monkeys at the highest two doses had symptoms which included blepharitis, facial edema, changes in the skin and nails, and loss of hair. Only the last symptom was observed from the lowest dose. Lipid accumulation in the liver and occasional necrotic hepatocytes were observed. The authors concluded that serum SDH and SGPT levels were the most sensitive parameters of toxicity of those studied and suggested that the 315 �transient increase in ICG clearance observed after TCDD treatment reflected an adaptive response in increased hepatic blood flow. The authors suggested that the fall in ICG clearance may reflect hepatotoxicity or could be caused by the starvation state of the monkeys. 880. Seiler, J. P. (1979b) Phenoxyacids as inhibitors of testicular DNA synthesis in male mice. Bull. Environ. Contain. Toxicol. 21:89-92. The author tested 2,4-D and 2,4,5-T in a mouse testicular DNA synthesis inhibition test. According to the author this assay correlates well with the carcinogenicity of chemicals. One hour before treatment male mice (number unspecified) received 1 u C C-thymidine intraperitoneally. Herbicides were administered in a single oral dose: 200 mg/kg 2,4-D, 200 mg/kg 2,4,5-T acid, or 50, 100, 200, or 400 mg/kg 2,4,5-T isooctyl ester. The animals were given intraperitoneal injections of lOuCi HIjjhymidine 3-96^ours later. The ratios of H counts per ug DNA and of H counts per C counts were then compared to controls. Criteria for a positivie result was that thymidine uptake was depressed significantly (statistical test not specified). All three compounds significantly inhibited testicular DNA synthesis. The author concluded that more work needs to be done on the carcinogenicity of these compounds. 881. Seiler, J. P. (1978a) The genetic toxicology of phenoxy acids other than 2,4,5-T. Mutat. Res. 55:197-226. [Review article.] 882. Seiler, J. P. (1978b) Herbicidal phenylalkylureas as possible mutagens. I. Mutagenicity test with some urea herbicides. Mutat. Res. 58:353-359. The authors evaluated the mutagenicity of diuron, monuron and several other phenylalkylurea herbicides in the mouse testicular DNA synthesis inhibition (DSI) test, the Ames test, and the micronucleus test in mice. The herbicides used in the tests were either pure substances or commercial formulations. No specific designation was made for each compound. In the DSI test, each herbicide was administered in a single oral aqueous dose given by stomach tube to 4 male mice (age and strain unspecified) per dose level. Three^hours later each mouse received an intraperitoneal injection of lOu Ci [H]-thymidine. Thirty minutes later the mice were killed, testes removed and homogenized [ H]-TdR incorporation measured. Both monuron and diuron significantly inhibited (p less than 0.05) testicular DNA synthesis. Each compound was also screened for mutagenicity in the Ames test. Data for only one tester strain (Salmonella typhimurium TA1535) were reported. This strain reverts from histidine requiring to prototrophy by base pair substitution. Rat liver S9 was used as the metabolic activation system. Both compounds induced dose dependent increases in numbers of revertants in the plate assay and also in a number of tubes with 316 �microbial growth in the fluctuation assay. No positive or negative controls or numbers of replicate plates were reported. The authors also tested monuron and diuron in a micronucleus test in mice. Very few details were reported. Six mice per dose level (age, strain, sex unspecified) received doses of 1000 or 2000 mg/kg monuron or 1000 or 2000 mg/kg diuron. Six mice served as controls. Percentage of micronucleated polychromatic erythrocytes in bone marrow cells were scored. No differences between control and treated mice were observed. The testing presented in this paper cannot be viewed as reliable evidence of mutagenic effects primarily because the author fails to present methodology, description of animals used and controls, and criteria used in evaluating the results of his study. 883. Seiler, J. P. (1973) A survey on the mutagenicity of various pesticides. Experientia 15(5):622-623. The author tested 2,3,7,8-TCDD and 25 other pesticides in Salmonella typhimurium strains his G46, TA1530, TA1531, TA1532 and TA1534. In this system, the tester strain reverts from histidine dependence to histidine independence when treated with a mutagen. According to criteria set by the author, a strong mutagenic response was defined as a relative mutagenicity (number of revertants from treated plates per 10 bacteria/spontaneous reversions per 10 bacteria) of greater than 10. A medium mutagenic response was defined as a relative mutagenicity of 5-10, a weak response was a relative mutagenicity value of 3-5, a doubtful response was a relative mutagenicity of 1-2, and a negative response was defined as a relative mutagenicity of 1. 2,3,7,8-TCDD was a strong mutagen in strain TA1532, a doubtful mutagen in strains TA1531 and TA1534, strains which are reverted by frameshift mutagens. The compound was a direct acting mutagen, that is, it did not require exogenous metabolic activation for a mutagenic effect 2,3,7,8-TCDD was negative in G46 and TA1530, which are reverted through base pair substitutions. No numerical data for chemically induced-revertants or spontaneous revertants were presented by the authors. This prevents a proper evaluation of the test system in that laboratory. The author also did not include the numbers of replicate plates used in the experiment, the dose of TCDD used, or the number of times the experiment was repeated. Failure to include this information severely limits the value of the data since no quality of experimental design can be evaluated. 884. Shadoff, L. A., Hummel, R. A., Lamparski, L., and Davidson, J. H. (1977) A search for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an environment exposed annually to 2,4,5-trichlorophenoxyacetic acid ester (2,4,5-T) herbicides. Bull. Environ. Contam. Toxicol. 18(4):478-485. The authors measured TCDD residues in environmental samples taken from two areas where 2,4,5-T was extensively used as a herbicide. The first sample area was a Texas drainage impoundment that is part of a watershed in which large areas have been sprayed with 2,4,5-T for control of mesquite (0.5 Ib/acre, 2,4,5-T acid equivalent) and brush 317 �(3-4 Ibs/acre, 2,4,5-T). Samples of water, mud, catfish, and walleyed pike were collected from the impoundment. Human milk samples from mothers residing in the area were also collected. The second sampling area was a pond in Arkansas used to flood to rice fields that had been sprayed with 1.25 lb/acre/2,4,5-T 4-8 weeks previously. Application of 2,4,5-T had occurred for 18 years up to the sampling time. Water, mud, catfish, and walleyed pike samples were collected. Analysis of the samples for TCDD was performed by GC-MS; limits of detection for the system used in the study averaged less than 10 ppt. Samples sizes were 10 of fish and mud, 20 of human milk, and 500 ml water. No TCDD was detected under the conditions of the study. 885. Shafik, M. T., Sullivan, H. C., and Enos, H. F. (1971) A method for determination of low levels of exposure to 2,4-D and 2,4,5-T. Intern. J. Environ. Anal. Chem. 1:23-33. Levels of 2,4-D and 2,4,5-T in the urine of occupationally exposed workers and of rats after oral administration are presented. Twenty-two workers occupationally exposed to 2,4-D, 2,4,5-T or both were categorized by occupation and urine samples were analyzed for these herbicides by gas chromatography. No other details about the characteristics of the exposed group or the timing of urine collection were given. 2,4-D was detected in urine of farmers, spray operators (method of spraying was not given), and aircraft spray operators. Each group consisted of 2 people and urine levels in each of these workers ranged from 0.2 to 1.0 ppm. 2,4,5-T was detected in all samples from a group of 6 spray operators, 2 spray crew foremen and 2 aircraft spray operators; levels ranged from 0.05 to 3.6 ppm. No 2,4,-D was detected in urine from 2 herdsmen, 4 farm laborers and 2 project officers exposed to 2,4-D. 2,4,5-T was not detected in 2 farmers exposed to 2,4,5-T. Male Sprague-Dawley rats (two per group) were administered daily doses of 3.75 ug/kg to 37.5 mg/kg 2,4-D or 5 ug/kg to 50 mg/kg 2,4,5-T for 3 days. Urine samples were collected for 7 days after the start of exposure and were analyzed for 2,4-D and 2,4,5-T by gas chromatography. After the 7 days, from 28% of the highest dose to 75% of the lowest dose of 2,4-D had been recovered in urine and from 49% of the lowest dose of 2,4,5-T and all of the highest doses were excreted in urine. The authors described methods for detecting derivatives of these herbicides in biological samples and concluded that their methods were suitable for detecting low levels of exposure from analysis of urine collected within 24 hours of exposure. 886. Shapley, D. (1973) Herbicides: South Americans. No reference. Agent Orange stockpile may go to the [Editorial.] 318 �887. Sharma, R. P., and Gehring, P. J. (1979) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on splenic lymphocyte transformation in mice after single and repeated exposures. Ann. N. Y. Acad. Sci. 320:487-497. Cellular immunity is described from in vitro studies of splenic lymphocytes of mice treated with TCDD acutely or subacutely. Male CD-I mice were orally administered 0.01-10 ug/kg TCDD in acetone-corn oil (1:20), weekly for 8 weeks or 10 ug/kg, once. Animals were killed 2, 4 and 8 weeks after treatment started and splenic lymphocytes were isolated and cultured with phytohemagglutinin or pokeweed mitogen. Incorporation of [ H]-thymidine by lymphocytes from TCDD-treated mice and lymphocytes treated with TCDD in vitro was assessed. Total serum immunoglobulins were assayed by quantitative immunoelectrophoresis. Increased liver weights and hepatic necrosis were observed in mice administered 10 ug/kg/week of TCDD; spleen, thymus and kidney weights were not significantly altered by TCDD treatment. Lymphocyte transformation in the absence of mitogens was stimulated 2 and 4 weeks after TCDD treatments started; in the presence of mitogens, TCDD-treated lymphoytes showed less stimulation than non-TCDD-treated controls, after 2 and 4 weeks. The acute dose produced the same effects as the subacute dose although stimulation by the acute dose was.more transient than the subacute effect. In vitro doses of 10 to 10~ M TCDD produced a dose-related decrease in [ H]-thyraidine incorporation of lymphocytes and no change in the responses of these cells to mitogens. Immunoglobulin levels were increased in mice administered 0.01-0.1 ug/kg TCDD doses and decreased at higher doses. The authors concluded that TCDD was immunosuppressive in mice and the toxic effect on thymic tissue may be transient. 888. Sharp, C. W., Ottolenghi, A., and Posner, H. S. (1972) Correlation of paraquat toxicity with tissue concentrations and weight loss of the rat. Toxicol. Appl. Pharmacol. 22(2):241-251. The tissue distribution of diquat is described for the rat. Male Sprague-Dawley rats were administered 20 mg/kg of [ C]-diquat dichloride (containing diquat-dibromide carrier) intravenously. Other rats were administered 20 mg/kg [ C]-paraquat dichloride intravenously (both radioactive compounds 99+% pure). Animals were killed 1-10 days later and tissues were removed. Paraquat was analyzed colorimetrically in acid-extracts of lung, muscle, liver, and kidney homogenates. (Determination of diquat concentrations was not described.) The ratio of the concentrations of paraquat to diquat was reported. The ratios for the lung and muscle were about 10, ranging from 2-16 in muscle and 8-33 in lung. The ratios were below one for kidney and liver and were between 2 and 8 for heart, adrenal, spleen, stomach, ileum, testes, and thymus. The distribution and toxicity of paraquat were described further. The authors concluded that diquat, in contrast to paraquat, did not concentrate in the lung or cause pulmonary toxicity. 319 �889. Shavgulidze, M. M. , Nanobashvili, V. I., and Mirianashvili, M. N. (1976) On the toxicity of the herbicide 2,4-D. Veterinaria (4):103-104. The acute and chronic toxicities of 2,4-D were studied in sheep. Single doses of 200-900 rag/kg 2,4-D sodium salt were administered perorally to sheep. Other sheep were administered 120 daily doses of 18 mg/kg 2,4-D amine salt orally. Mortality and clinical signs from the acute doses recorded and after subacute exposure, clinical signs were recorded and hematological and biochemical blood tests were performed. A dose of 900 mg/kg killed all sheep. Clinical signs of intoxication occurred after doses of 500 mg/kg or greater and included general weakness, ataxia, immobility, muscular paralysis of the hind legs, difficulty in breathing, sensitivity to light, and lack of appetite. Death occurred 2-4 days after exposure. Fluctuations in both directions occurred during the first month for the erythrocyte count, leukocyte counts, hemoglobin levels, blood nitrogen levels, and "alkaline reserve" (no further explanation given for this parameter). No traces of 2,4-D, administered as a single dose of 300-400 mg/kg of the amine or sodium salt, were detected 12 days after exposure (no other methodology was reported for this experiment). The authors concluded that meat from sheep was safe for human consumption 12 days after sheep had ingested 2,4-D and that 2,4-D was a compound of moderate toxicity. 890. Shaw, B., and Hopke, P. K. (1975) The dynamics of diquat in a model eco-system. Environ. Letters 8(4):325-335. The authors report on the distribution and accumulation of diquat in a model aquatic ecosystem. An aquarium tank (20 gal.) containing Lake Chautauqua water and sediments and plants, Norlunge fingerlings, snails, and HyaleIla was treated with 64.5 mg diquat of which 13.5 mg was labeled with C. A control tank was also included in the experiment. Water samples were removed daily for 9 days and analyzed for C by scintillation counting. When radioactivity in the water reached background-levels, the experiment,was terminated. Plants, animals, and sediments were analyzed for C uptake C-diquat and not a metabolite or C02 was being taken up by the components of this model. The authors also performed a 96-hour acute toxicity study in muskelunge using diquat. However, more than 10% of the fish in the control tank died, which invalidates the experiment. 891. Shepard, B. (1980) Presentation at the 2d Continuing Education Conference on Agent Orange, Washington, DC, May 28-30. [Background material.] 320 �892. Sherman, H., and Kaplan, A. M. (1975) Toxicity studies with 5-bromo-3-sec-butyl-6-methyluracil. Toxicol. Appl. Pharmaeol. 34(2):189-196. A series of reproductive, acute, and chronic toxicity studies of 5~bromo-3-sec-butyl-6-methyluracil (bromacil) were performed in mammals, fish, and wildlife. An outline of the methods used for the studies and a brief summary of the results were presented. Acute oral toxicity was evaluated in male Charles River animals (presumably rats) after a 14 day evaluation period and in male mongrel dogs. Subchronic toxicity was studied in male Charles River rats given 10 doses of bromacil by gastric intubation over 2 weeks. Acute dermal toxicity was tested by applying a 70% aqueous paste of bromacil to the clipped skin of 3 rabbits and fitting each animal with a plastic collar to prevent ingest ion. After 24 hours, the compound was removed and the animals were observed for toxic effects for 2 weeks. Skin irritation and sensitization of 50% bromacil pastes were tested in guinea pigs given a single or 3 doses of bromacil. The dry powder and a 10% solution of bromacil in mineral oil were tested for ocular irritation in rabbits. Acute inhalation toxicity was tested in rats exposed to 2.1 mg/liter or 4.8 mg/liter bromacil in air for 4 hours. Acute toxicity of bromacil to fish and wildlife was tested by previously published methods that were not described. Pregnant rabbits were administered dietary levels of 50 or 250 ppm bromacil during days 8-16 of gestation. Fetuses were removed on day 28 or 29 and examined for gross malformations or stained with alizarin red to observe skeletal malformations. Resorptions were also counted. Other litters were delivered. A ninety day feeding study was performed on rats. The doses initially were 50, 500, and 250 ppm for 3 treatment groups. The highest dose was increased several times to a final dose of 7,500 ppm. Urine, hematology, and renal function tests were performed monthly and histopathology was conducted at the end of the feeding period. A 2-year feeding study was also performed in rats with groups administered 50, 250, and 1,250 ppm bromacil diet. In addition to parameters used for the 90-day study, food consumption, body weights, and tibia growth were monitored. A 2-year feeding study in dogs was performed using the same doses as for the rat study (except the highest dose was administered after a week of lower doses being administered). The same parameters were monitored as in the rat study, and respiration, pulse rate, and body temperatures were monitored twice weekly, as well. A 3-year reproduction study was performed in rats given 250 ppm dietary levels of bromacil. Offspring were maintained on the experimental (or control) diet for 110 days after weaning and then were mated with rats from the same group. The oral LD,-n was 5,200 mg/kg in the male rat. Doses of 100 mg/kg or greater produced etnesis in the dog, precluding establishing an l^cn for this species. No dermal toxicity, mild dermal and eye irritations, and no skin sensitization were observed. The LC,-~ for inhalation exposure was above 4.8 mg/liter and for fish was 70-165 ppm. For wildlife the dietary LCcn over 8 days was greater than 10,000 ppm. Subchronic toxicity was elicited as gastrointestinal and central nervous system disturbances from 10 daily doses of 1,035 mg/kg. Mortality of 1,500 mg/kg/day was 67% (4/6) and at 1,035 mg/kg was 17% (1/6). The highest dosage group in the 90-day study showed histological changes of the 321 �thyroid and liver and lowered body weight gain. No other groups showed any adverse effects. No malformations were identified in the teratology study. A decreased weight gain by the highest treatment group was the only deviation noted in both the chronic dog and chronic rat studies. No reproductive effects were observed. No carcinogenicity was observed in the studies with chronically treated animal groups. The authors concluded that bromacil has a very low order of toxicity. 893. Shirasu, Y., Moriya, M. , Kato, K., Furuhashi, A., and Kada, T. (1976) Mutagenicity screening of pesticides in the microbial system. Mytat. Res. 40:19-30. The authors studied the DNA damaging and mutagenic capabilities of bromacil, 2,4-D, diuron, monuron, 2,4,5-T, and 161 other pesticides using three bacterial assays, a rec assay in Bacillus subtilus and reverse mutation assays in Escherichia coli and Salmonella typhimurium. In the rec assay J3. subtilus strains HI7 Rec+ and M45 Rec- were utilized. Each chemical dissolved in DMSO was added in a 0.02 ml aliquot of a 1 rag/ml solution to a paper disc covering a streak culture of each of the two bacterial strains. After 24 h incubation, the length of inhibition of bacterial growth for each streak (rec+ and rec-) was measured. DNA damaging capability is measured by preferential inhibition of the rec-strain. However, criteria for a positive result was not reported by the authors. However, according to their results none of the above mentioned herbicides was positive in the assay. No metabolic activation was used and no positive controls were mentioned. In the reversion assay, E_. coli strains B/R try WP2 and WP2 try her were utilized. Both strains revert from tryptophan requiring to prototrophy when exposed to a mutagen that acts by base pair substitutions. Four strains of _S_. typhitnurium were also employed in the reversion assay, TA1535, TA1536", TA1537, and TA1538. These strains revert from histidine dependence to histidine independence by mutagens that induce base-pair substitutions (TA1535) or frameshift mutations (TA1536, TA1537, and TA1538). Each of the herbicides was dissolved in DMSO and added to a filter disc (0.02 ml of a 1 tng/ml solution) which was placed on an agar plate containing bacteria. Criteria for a positive result was not discussed by the authors. No positive controls were reported. In addition, metabolic activation was not included in the testing; therefore, only direct acting mutagens would show a positive result. All of the herbicides mentioned above were negative in both reversion assays. 894. Shirasu, Y. (1975) Mutagens and teratogens, 15(4):187-189. [Review article.] 322 Cong. Anom. �895. Shu, H., Talcott, R. E., Rice, S. A., and Wei, E. T. (1979) Lipid peroxidation and paraquat toxicity. Biochem. Pharmacol. 28(2):327-331. Distribution of diquat to the lung and production of lung edema was studied in the rat. A dose of 45 mg/kg [ C] diquat dibromide in saline was administered subcutaneously to rats and lung tissue was removed after 3 hr. from four rats or 6 hours from seven rats, perfused briefly to remove blood, digested, and counted for radioactivity. Ether anesthetized rats (6-11 per gr.) were administered [ Il-bovine serum albumin intravenously, immediately followed by a subcutaneously dose of 32 or 45 mg/kg diquat. Twenty-four hours after the higher dose and 48 hr. after the lower dose, the trachea was cannulated and saline was flushed into the lungs to remove recoverable radioactivity (recovery of radioactivity was estimated at 80% by this method). Blood was also collected and counted for radioactivity. Between 70 and 95% of the radioactivity from the lung lavage and plasma were precipitable with 10% trichloroacetic acid. Diquat was present in the lung at higher concentrations after 3 hr. than after 6 hrs. and the levels at both times were less than half of paraquat levels after equimolar doses had been administered. After 24 and 48 hours, diquat produced a 1.6 fold and 2.1 fold increase, respectively^ in alveolar albumin content and minimal effects in plasma levels of [ I]-radioactivity. Other effects of paraquat were described. The authors concluded that diquat produced less pulmonary edema and reached the lung in lower amounts than diquat and indicated that enhanced in vitro stimulation of lipid peroxidation of diquat over paraquat was not consistent with in vivo mechanisms of toxicity. 896. Siebert, D., and Lemperle, E. (1974) Genetic effects of herbicides: Induction of mitotic gene conversion in Saccharomyces cerevisiae. Mutat. Res. 22:111-120. The authors tested commercial preparation of dalapon, bromacil, diuron, diquat, 2,4,5-T amyl ester, and 2,4-D and 24 other herbicides in a mitotic gene conversion assay in Saccharomyce s cerevisiae. In the assay S_. cerevisiae strain D4 was employed. This strain is a diploid heteroallelic at two loci, ade 2 and trp 5 which makes the yeast adenine and tryptophan requiring. The induction of gene conversion by a genetically active compound results in cells that no longer require either adenine or tryptophan. Convertants are measured by use of selective medium. Yeast cells in the logarithmic growth phase were treated with 1,000 ppm of each of the herbicides in a liquid suspension culture at pH 4.5 or 7.0 for 16 hours after which cells reported by the authors. Purity of the commercial preparations tested was not reported. In addition criteria for a postive result were not presented by the authors. However, according to the authors, diquat and 2,4-D induced a significant increase in mitotic gene conversion compared to controls. Both compounds were tested at pH 4.5. 2,4-D increased convertants 5 fold at the ade 2 locus and 6 fold at the trp 5 locus, while diquat induced a 7 fold increase at the ade 2 locus and 4-5 fold at the try 5 locus. Bromacil (pH 4.5) and 2,4,5-T (pH 7,0) amyl ester increased 323 �convertants slightly but not significantly over controls while convertant frequencies induced by dalapon (pH 4.5) or diuron (pH 4.5) did not differ from controls. The rationale behind using 2 different pH values in the testing was not presented by the authors. 897. Sigmon, C. F. (1979a) Influence of 2,4-D and 2,4,5-T on life history characteristics of Chironomus (Diptera: Chironomidae). Bull. Environ. Contain. Toxicol. 21:596-599. The author studies the effects of 2,4-D and 2,4,5-T butoxyethanol esters on pupation, emergence, and mortality in Chironomus. Midges were collected from sewage oxidation ponds and transferred to culture dishes (10 animals/dish) of lake water in the laboratory. After overnight acclimation to the water, midges were exposed to 1 or 3 ppm 2,4-D or 2,4,5-T acid equivalent at 20, 25, and 30°C. Commercial preparations of herbicide were used; no purity data were available. Fresh herbicide solutions were added daily, and the mortality, pupation, and emerging of animals was recorded. Only 2,4-D at 30°C had any effect on Chironomus. At both 1 and 3 ppm, 2,4-D caused increased larval mortality and decreased pupation and emergence. The author stated that concentrations of herbicides in the experiment represented the highest values observed in waterways and runoff. Although effects from to 2,4-D were observed, the author concluded that these effects would not be important in nature. 898. Sigmon, C. (1979b) Oxygen consumption in Lepomis machrochirus exposed to 2,4-D or 2,4,5-T. Bull. Environ. Contain. Toxicol. The author determined the short term uptake of 2,4-D and 2,4,5-T bluegill sunfish and examined the metabolic response of the fish to the herbicides. Fish used in the experiment ranged from 0.28-23.30g and were randomly assigned testing tanks that housed either one fish greater than 2g or four fish less than 2g. Respiration was measured by the azide modification of the Winkler technique. Fish were exposed to water containing 3 ppm 2,4-D or 2,4,5-T, butoxyethanol esters. Respiration was determined at 20, 25, and 30C. The herbicides had no effect on respiration. Fish were also maintained for 8 days in water containing 3 ppm 2,4-D or 2,4,5-T at 20, 25, and 30C. Uptake of herbicides was measured by gas liquid chromatography. Both controls and 2,4-D exposed fish had uptake levels of less than 0,05 ppm while 2,4,5-T exposed fish had 0.06-0.12 ppm uptake. Uptake of 2,4,5-T was not related to temperature. The significance of this study was not discussed by the author. 899. Sigmon, C. F. (1979c) Physiological effects of 2,4-P and 2,4.5-T on selected aquatic organisms. A Report to the Office of Water Research and Technology, Washington, DC. USNTIS Publication No. PB294539/25T. 30 pp. [Not available.] 324 �900. Simmon, V. F., Mitchell, A. D., and Jorgenson, T. A. (1977) Evaluation of selected pesticides as chemical mutagens: in vitro and in vivo studies. U.S. Environmental Protection Agency, Research Triangle Park, N.C. Report No. EPA-600/1-77-028. 237 p. Twenty compounds, including bromacil, cacodylic acid, and monuron, were evaluated for mutagenic activity. Only bromacil was among ten compounds selected for assay in the mouse dominant lethal test. There was no time or dose-response effect in this assay associated with bromacil at 1250, 2500, and 5000 mg/kg of diet. Bromacil, cacodylic acid, and monuron were evaluated in the following test systems: unscheduled DNA synthesis in human fibroblast (WI-38 cells, reverse mutation in Salmonella Typhimurinum strain TA1535, TA1537, TA1538, and TA100 and in Escherichia coli WP2; mitotic recombination in Saccharomyces cerevisiae D3, and preferential toxicity assays in DNA repair-proficient and deficient strains of Escherichia coli (strain W3110 and M45, respectively) and Bacillus subti1 is (strains H17 and M45, respectively). These studies were reported in sufficient detail to support the author's conclusions and demonstrate that the study was well controled and apparently well conducted. All of the-results were negative with the exception that monuron (assayed at 10 , 1 0 , and 10 M) induced unscheduled DNA synthesis in the presence of metabolic activation enzymes. The authors concluded that this result may indicate that monuron is a procarcinogen and that this possibility should be evaluated using in vivo studies. 901. Simmon, S. F., Poole, D. C., Riccio, E. S., Robinson, D. E., Mitchell, A. D., and Waters, M. D. (1979) In vitro mutagenicity and genotoxicity assays of 38 pesticides. Environ. Mutagen. 1:142-143. [Abstract, only.] 902. Simpson, G. R., Higgins, V., and Chapman, J. (1978) Exposure of council and forestry workers to 2,4,5-T. Med. J. Austr. 2(11):536-537. [Not available.] 903. Simsiman, G. V., Daniel, T. C., and Chesters, G. (1976) Diquat and endothall: Their fates in the environment. Residue Rev. 62:131-174. [Review article.] 904. Sirons, G. J., Frank, R., and Dell, R. M. (1977) Picloram residues in sprayed Macdonald-Cartier Freeway right-of-way. Bull. Environ. Contam. Toxicol. 18(5):526-533. The authors describe the persistence and movement of picloram applied to the shoulders and median strip of a major highway. A commercial 325 �p formulation Tordon 101 containing 62.4 g (acid equivalent) picloram and 250.8 g (acid equivalent) 2,4-D present as triisopropanol amine salts, was applied at 350 g/ha picloram in alternate years beginning May 1968. Soil samples to 45 cm depth were collected at 7 sites along the Macdonald-Cartier Freeway beginning in May 1969 and continuing for 3 years. In 1971, grass samples were also collected at each site. Samples (50 g soil, 109 grass) were analyzed for picloram only by gas liquid chromatography (detection limit 0.01 ppm). No 2,4-D residue data were reported. The authors reported data for the study period 1968-1973. Samples were pooled in data presentation and no distinction was made between samples collected from median strips or shoulders. Picloram residues in the top 15 cm of soil were greatest one week after am accumulation in soils was presented. 905. Sjoden, P. 0., Archer, T., and Soderberg, U. (1977) Effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on radioiodine distribution in rats. Bull. Environ. Contam. Toxicol. 17(6):670-678. Alterations in tissue distribution of radioactive iodine were determined in rat after 2,4,5-T administration. Rats were orally administered 100 mg/kg of 2,4,5-T (with less than 1 ppm TCDD contaminant) in corn oil 1-15 days prior to sacrificed. A 10 ug dose of I was administered intramuscularly 1 day prior to sacrifice. The blood and several tissues were analyzed for radioactivity. Controls were administered corn oil without 2,4,5-T. No differences were observed among the results from male Sprague-Dawley rats or male or female Wistar rats and these results are discussed together. Data from control groups were not presented but all experimental data were presented as percentages of control group data. Serum radioactivity was depressed especially 1 day after 2,4,5-T treatment. Increased brain, liver, and female thyroid levels occurred 1-3 days after 2,4,5-T treatment. The changes in other tissues were moderate or low and the increases in tissue radioactivity were not adequate to account for the loss from the serum. The authors concluded that increased renal clearance of iodine accounted for the serum loss. Measurement of radioactivity in the urine would be a more appropriate way to evaluate iodine clearance after 2,4,5-T administration than the indirect method used which failed to recover the administered dose of isotope in tissues. 906. Sjoden, P. 0., and Soderberg, U. Phenoxyacetic acids: Sublethal effects. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978) p. 149-164. The sublethal effects of 2,4,5-T in rats were investigated in several different experiments. Various behavioral parameters were measured in adult and neonatal rats, including open-field activity, maze learning abilities and test-aversion discrimination. In addition, the effect of 2,4,5-T on food and water intake, thyroid regulation, electrolytic maintenance and amino acid metabolism in the basin were examined. For 326 �all experiments, the 2,4,5-T was mixed with corn oil and administered orally at a dose of 100 rag. Adult rats were given a single dose while pregnant rats received the dose on day 7, 8, or 9 of gestation. Neonatal rats that had been exposed to 2,4,5-T in this manner were behaviorally tested at 35, 60 and 90, 95 and 125 days of age. Results of these behavioral tests indicated that prenatal exposure to 2,4,5-T tended to increase the exploratory activity of 90-day old rats in the open-field test. Increased activity was also evident in 35 and 60-day old rats that had been cross-fostered with control dams. Older cross-fostered rats, aged 95 and 125 days, did not exhibit increased exploration in this test. Rats exposed prenatally to 2,4,5-T also exhibited disabilities in maze learning and electric shock-avoidance conditioning. Adult rats fed a combination of saccharin and 2,4,5-T associated saccharin with 2,4,5-T and developed a resistant longlasting taste aversion to the sweetner. Adult rats that received 100 mg of 2,4,5-T also displayed a reduction in food and water intake. Radioiodine distribution experiments revealed abnormal thyroid activity in adult rats that were given 2,4,5-T. These rats also exhibited electrolytic disturbances in the brain fluids. Reduction of amino acid synthesis, particularly tryptopham, in the brain of adult rats treated with 2,4,5-T was noted. Seratonin levels were also reduced in the brains of neonatal rats that had been exposed to 2,4,5-T prenatally. The authors concluded that acute exposure to 2,4,5-T, either prenatally or as an adult, produced significant effects on motor behavior, taste and learning abilities, food and water ingestion, thyroid activity and amino acid synthesis in the brain. They attempted to relate results of their taste-aversion experiments to animals in the wild that may be exposed to 2,4,5-T. A number of problems are associated with their investigation. The purity of the 2,4,5-T was not specified or even how the 2,4,5-T compound was prepared for administration was not described for any of the experiments. The ages, sex and number pf control rats used for some of the experiments were not reported. It is unclear * whether some of the radioiodine and amino acid studies were conducted using a separate group of rats or if rats in the other experimental group were examined. The authors also did not discuss other factors that may have influenced their results, particularly for the behavioral effects. 907. Sjoden, P. 0., and Soderberg, U. (1975) Long lasting effects of prenatal 2,4,5-trichlorophenoxyacetic acid on open field behavior in rats: pre- and postnatal mediation. Physiol. Psychol. 3(2):175-178. The behavioral development of rats treated with 2,4,5-T in utero or fostered by a 2,4,5-T treated mother were studied. Pregnant Wistar rats (12) were treated with one dose of 100 mg/kg of 2,4,5-T (less than 1 ppm TCDD contaminant) in vegetable oil by oral intubation on day 8, 9, or 10 of gestation. Ten pregnant control rats received vehicle only. Litters were reduced to 4 males and 4 females. Half of the litters were cross-fostered by dams of the other group and the remainder were fostered by their biological mothers. All offspring were weaned at 24 days of age and were tested on behavioral parameters at 35, 60, 95, and 125 days of age. Each rat was observed for 4 327 �minutes on 4 consecutive days for atnbulation activity (number of grids rat traverses), rearing (frequency rat rises on hind limbs), grooming (frequency rat washes face or body), and defecation. The colors used for the floor grid lines and fields were reversed on the fourth day. Offspring exposed prenatally or postnatally showed significantly more ambulation and rearing activities than controls on days 35 and 60. No changes were seen at 90 days of age; at 125 days of age, increased rearing and ambulation of prenatally exposed rats occurred only on day 1 and decreased activity of postnatally exposed rats also occurred on day 1 only. No sex-related differences were seen in any group. The authors concluded that 2,4,5-T exposure prenatally affects offspring during both prenatal and postnatal development, producing behavioral changes at doses that do not affect anatomical development. The authors speculated that altered thymus function may be responsible for observed behavioral modifications. The relationship of the parameters measured in this paper with any other behavioral parameters, particularly those related to human development, has not been established by these authors. 908. Sjoden, P. 0., and Soderberg, U. (1972) Sex-dependent effects of prenatal, 2,4,5-trichlorophenoxy-acetic acid on rats' open field behavior. Physio1. Behav. 9:357-360. Behavioral development was studied in rats exposed to 2,4,5-T in utero. Pregnant Wistar rats (25) were administered 100 mg/kg of 2,4,5-T (less than 1 ppm TCDD contaminant) orally in maize oil on day 7, 8, or 9 of gestation. Control (pregnant) rats were administered oil only. All offspring were observed for anatomical abnormalities grossly at birth and by autopsy at the end of the experiment. At about 90 days of age offspring were selected from the experimental and from the control groups and subjected to an open field behavior test. For this test, each rat was placed in the center of a grid and observed for 2 minutes. Behavior was recorded which included ambulation (number of squares traversed), rearing (frequency rat rose on hind legs), defecation, latency (time to leave starting square), and grooming. The test was repeated on 2 separate days and data were compared for all treated males and control males, and for treated and control females. No anatomical abnormalities were observed in any group and no behavioral differences were observed between the 2 female groups. Litter size and perinatal mortality were adversely affected by treatment. The experimental males showed significantly increased behavior for all parameters except defecation. The authors concluded that the experimental males were more explorative than male controls. The authors concluded that a behavioral effect could be elicited in male rat development at a dose of 2,4,5-T that did not elicit anatomical effects. The significance of the effect presented here is difficult to extrapolate to other situations without more information regarding the relationship of explorative behavior to other aspects of behavioral development. The effect described does not appear to be an adverse effect or the result of a change in the rate of behavioral development, although the authors did not address these issues. 328 �909. Smith, A, E. (1978) Relative persistence of di- and tri-chlorophenoxy alkanoic acid herbicide in Saskatchewan soils. Weed Res. 18(5):275-279. [Background material.] 910. Smith, A. E. (1976) The hydrolysis of herbicidal phenoxyalkanoic esters to phenoxyalkanoic acids in Saskatchewan soils. Weed Res. 16:19-22. [Background material.] 911. Smith, F. A., Schwetz, B. A., and Nitschke, K. D. (1976) Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in CF-1 mice. Toxicol. Appl. Pharmacol. 38:517-523. The teratologic and embryotoxic effects of TCDD were evaluated in CF-1 mice. Doses of 0.001 to 3.0 ug/kg/day of TCDD in corn oil-acetone (98.2) were administered by oral gavage to pregnant mice on days 6 to 15 of gestation. On day 18 live, dead, and resorbed fetuses were counted and the maternal liver was weighed. Fetuses were examined for gross malformations and for visceral malformations after fixation with Bouin's fluid or skeletal malformations after staining with alizarin red. The maternal liver weight to body weight ratio was significantly increased only in the highest dose group, compared with vehicle controls. Resorptions per implantations increased significantly only in the 1.0 ug/kg/day group, while no significant changes were observed in the percentage of litters with resorptions, maternal or fetal weights, fetal length, or number of implantation sites.. Increased incidences of cleft palate in the highest two groups and of dilated renal pelvis in the highest group were observed. The non-teratogenic dose of TCDD was 0.1 ug/kg/day. 912. Smith, L. L., and Rose, M. S. (1977) A comparison of the effects of paraquat and diquat on the water content of rat lung and the incorporation of thymidine into lung DNA. Toxicology 8 (2):223-230. The effects of diquat administration to rats on lung edema, pulmonary cell proliferation associated with fibrosis, and mortality are described. A group of 50 Alderley Park rats were administered 105 u mole/kg diquat dichloride intraperitoneally and daily incidence of mortality was recorded. Controls were administered, saline [ H]-thymidine was administered intraperitoneally, and DNA synthesis was determined by the amount of radioactivity incorporated into the DNA fraction of lung tissue in 1 hr. in vivo. Pulmonary edema was determined by the loss in weight of the lung tissue when it was dried to a constant weight. About 70% of the rats died within 14 days of diquat treatment and 20% died within 2 days. On days 1, 2, 4, and 8 after diquat treatment, H-thymidine incorporation was measured and found to be reduced to about 1/2 of control levels. Small decreases in 329 �water content of the lung were detected on the first 2 days, only. effects of paraquat were also studied. The authors concluded that diquat did not produce acute damage to the lung (with edema) or fibrosis which were produced by paraquat. 913. The Smith, R, J. (1978) Dioxins have been present since the advent of fire, says DOW. Science 202:1166-1167. [Editorial.] 914. Smith, P., and Heath, D. 4(3):411-445. (1976) Paraquat. CRC Grit. Rev. Toxicol. [Review article.] 915. Sadykov, R. E., Rabochev, V. K., and Strokov, Y. N. (1972) The effect of butyl 2,4-D treatment of pastures on the reproductive functions of sheep. Zhivotnovodstvo 34(l):73-74. The effect of 2,4-rD butyl ester on reproduction was studied in sheep. A pasture was sprayed by helicopter at the rate of 3 kg per hectare with 2,4-D butyl ester. Groups of 25 mature ewes with 2-3 month old lambs were grazed on the field 72 hr., 144 hr. and 288 hr. After spraying, and a control group grazed on unsprayed pastureland nearby. Animals grazed for 1 month. Clinical hematology and body weights were assessed. The only effects observed in exposed animals were changes in the lambs in the first 4 days of exposure and included an increase in body temperature of 0.5° and a 50% increase in leukocyte counts. All ewes were mated artificially and then spontaneously and reproductive parameters were assessed. No statistical differences were observed in the rate of conception, numbers of newborns or stillborns and birth weights between treated and control groups and no malformations occurred. The authors reported that other farm experiments had indicated that sheep exposed to pastures sprayed with 2,4-D butyl ester showed decreased fertility as high incidences of sterility, abortions, and stillborns, fetal malformations, decreased male mating behavior, and abnormal sperm resulted. No other details or references were provided for these results. The authors concluded that animals should not graze on sprayed pastureland for 20 days for adult and for 45 days for newborns before weaning. 916. Somers, J. D., Moran, E. T., and Reinhart, B. S. (1978a) Influence of hen dietary calcium and phosphorous on the integrity of the egg shell as it would influence hatching success and the consequences of preincubation 2,4,5-T spraying with and without a high TCDD level. Bull. Environ. Contain. Toxicol. 19(6) :648-654. The teratogenicity of 2,4,5-T alone and with 2.0 ppm TCDD sprayed on normal chicken eggs and eggs with deficient shells was studied. 330 �Chickens were fed diets deficient in calcium (with 50% of the normal dietary calcium) or deficient in phosphorous (with 74% of the normal phosphorus) or deficient in both minerals. Females were maintained on the experimental diet for 40 weeks and during the last 3 weeks they were artificially inseminated. Resultant eggs were sprayed at the rate of 746 1/h with 2,4,5-T (with less than 0.1 ppm TCDD) or 2,4,5-T with 2 ppm TCDD. The age(s) of the eggs when they were sprayed was not reported. .Hatchability and frequency of malformations were recorded. Egg shells, non-viable embryos, and chicks were analyzed for 2,4,5-T contents by gas-liquid chromatography . Eggs from hens fed low calcium diets had fewer eggs and the egg shells reduced porosity and reduced strength. The low phosphorus diet did not cause these effects. No reductions in hatchability or increases in malformations were observed after the deficient eggs were sprayed with 2,4,5-T with or without TCDD. No increase in 2,4,5-T levels were observed in shells, eggs, embryos, or chicks from the low-calcium diet group compared to levels from the control diet group treated with 2,4,5-T. Mean 2,4,5-T levels were 66 ppm in shells before incubation, 0.35 ppm inside these eggs, and 0.24 ppm in hatched chicks. The authors concluded that 2,4,5-T was unable to penetrate the egg shells from the experimental groups. The crude methods of treating eggs and measuring effects combined with the lack of reported pertinent details limits the usefulness of this report . 917. Somers, success 2,4,5-T after a J. D. , Moran, E. T. , and Reinhart, B. S. (1978b) Reproductive of hens and cockerels originating from eggs sprayed with 2,4-D, and picloram followed by early performance of their progeny comparable in ovo exposure. Bull. Environ. Contam. Toxicol. The reproductive effects of spraying 2,4-D, 2,4,5-T or picloram on White Leghorn chicken eggs was evaluated. Eggs (fo generation) were sprayed with one of the three herbicides on day 0, 4, or 18 of gestation. Commercial preparations of herbicides were used and were sprayed at the rate of 746 1/ha. The dioxin content of 2,4,5-T was not reported. Vehicle controls were treated with the inert ingredients of the formulations and a genetic control and a control sprayed with water were also included in the experiments. After fo generation eggs hatched, the chicks were allowed to develop and egg-laying was monitored for the fo hens mated with males of the same group. Egg weight and shell porosity (measured as weight loss after 10 days of storage) and strength (measured as the extent of deformation produced by a 500 g weight) were also determined. Sperm counts were determined by comparing the optical density of sperm samples from males with the optical density of standard boar sperm. At 55 weeks of age the gross appearance and weight of the testes were determined. Eggs (fl generation) were collected from the fo hens artificially inseminated with sperm from males of the same treatment group. Half of the fl eggs were treated on day 0 with the same treatment their parental generation received and the other half remained untreated. The viability, incidence of malformations, and weight gain of the fl -generation through 4 weeks of age were monitored. No adverse effects were 331 �produced by any of the herbicides on any of the parameters presented in this study. The design of the experiments were obscured in the poorly written report and the crude techniques used to dose the eggs and evaluate reproductive parameters severely limit the value of this publication. A previous report by these authors documented that only a minor portion of 2,4,5-T sprayed under the conditions of the present study enters the egg. 918. Somers, J. D., Moran, E. T., and Reinhart, B. S. (1978c) Hatching success and early performance of chicks from eggs sprayed with 2,4-D, 2,4,5-T and picloram at various stages of embryonic development. Bull. Environ. Contam. Toxicol. 20(3):289-293. The effects of 2,4-D, 2,4,5-T, and picloram sprayed on chicken eggs on the hatchability, teratogenicity, chick growth, and mortality was studied. Commercial preparations of each herbicide were sprayed at a rate of 746 1/ha (10 times the recommended rate) on day 0, 4, or 18 of incubation. Eggs stored for up to 24 days prior to incubation were also sprayed. The percentage of hatched eggs, of gross malformations in chicks, of chick mortality, and chick weight gain at 4 weeks of age were monitored. None of the herbicides produced any adverse effects on any parameters studied, regardless of the embryonic age when spraying occurred. A subsequent report by this group of authors (Somers et al, 1978a) demonstrated that 2,4,5-T does not penetrate the egg under the conditions used in the current experiment, which provides one explanation for the negative results presented here. 919. Source assessment: Pesticide manufacturing air emissions-overview and prioritization. (1978) U.S. Environmental Protection Agency. Environmental Protection Technology Series No. EPA-600 (2-78-004d). 135 pp. [Not available.] 920. Sparschu, G. L., Dunn, F. L., Lisowe, R. W., and Rowe, V. K. (1971) Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid oh fetal development in the rat. Fd. Cosmet. Toxicol. 9:527-530. The ef-fects of high doses of 2,4,5-T on fetal rat survival and development were studied. Groups of 25 pregnant rats were administered either 50 mg/kg 2,4,5-T (0.5 ppm TCDD contaminant) in Methocel by oral intubation on days 6-15 or 100 mg/kg on days 6-10 of gestation. On day 20 fetuses were removed and observed for skeletal and visceral malformations. Corpora lutea, implantation sites, and resorptions were counted. Maternal toxicity, including 80% mortality was observed in groups treated with 100 mg/kg 2,4,5-T. One of the four pregnant rats from this group had viable fetuses (13) on day 20. A higher incidence of poor or delayed ossification was observed in these fetuses, but was considered a reversible manifestation of no teratologic significance because 3-week-old rats from other experiments from this lab failed to 332 �show skeletal abnormalities even though delayed cranial ossification was seen in 20-day fetuses from the same group. No statisticallysignificant decrease in any parameters, except delayed cranial ossification, was observed for the 50 mg/kg treatment group. The authors concluded that 2,4,5-T was not teratogenic at doses below those producing maternal toxicity. 921. Sparschu, G. L., Dunn, F. L., and Rowe, V. K. (1971) Study of the teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Fd. Cosmet. Toxicol. 9:405-412. The teratogenicity of TCDD was evaluated in the rat. TCDD in corn oil-acetone (9:1) at doses from 0.03 to 8.0 ug/kg was administered by oral gavage on day 6 to day 15 of gestation to pregnant Sprague-Dawley rats. Viable and dead fetuses, early and late resorptions, corpora lutea, and implantation sites were counted on day 20 of gestation. Fetuses were weighed, examined for gross malformations, hemorrhages in the gastrointestinal tract, and for either skeletal malformations after alizarin red staining or visceral malformations after fixing in Bouin's fluid and histological changes after staining with hematoxylin and eosin. Maternal vaginal hemorrhages were often observed in rats given doses of 2-8 ug/kg/day of TCDD and the maternal weights and numbers of resorptions per litter of the groups that received 0.5-8 ug/kg/day were adversely altered compared with vehicle controls. Tn the highest dose group all implants had resorbed in early pregnancy. The numbers of implantation sites and corpora lutea were not altered by any dose of TCDD. Fetal weights of the 0.125 and 0.5 ug/kg/day groups were significantly lower than controls. The frequencies of intestinal hemorrhage and subcutaneous edema showed dose-related frequencies. No renal malformations were observed. 922. Sparschu, G. L., Dunn, F. L., and Rowe, V. K. (1970) Teratogenic study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Toxicol. Appl. Pharmacol. 17:317. [Abstract, only.] 923. Spencer, E. Y. (1973) Guide to Chemicals Used in Crop Protection Research Institute, U. of Western Ontario, London, Ontario. p, 414. [Not available.] 924. Sponsors of Science Inc. (1974) Sponsors of Science Inc. on the safety of 2,4,5-T and dioxin. Clin. Toxicol. 7(4):413-421. [Editorial.] 333 �925. SRI International. (1981) A case-control study of the relationship between exposure to 2,4-D and spontaneous abortion in humans. National Forest Products Association, Washington, DC: 116 p. t The incidence of spontaneous abortions among women whose husbands were occupationally exposed to 2,4-D was investigated. A study population, which was composed of farmers, forest workers and herbicide applicators in Oregon and Washington, were issued questionnaires by mail which requested information regarding the occurrence of miscarriages and exposure to 2,4-D. Telephone interviews were then conducted with all respondents that indicated having had at least 1 miscarriage and with a select group of those that reported live births. The telephone interviews addressed the time and extent of 2,4-D exposure of both parents, relative to the time of conception, and other confounding factors, including smoking habits, drug consumption and illnesses. 8,287 of the 14,747 questionnaires were returned. 3,787 eligible cases were identified, of which 1,098 were selected for telephone interviews, 604 interviews were completed; 134 cases of miscarriages and 311 controls (cases of live births were validated and studied. No positive association was obtained between 2,4-D exposure of the father and subsequent spontaneous abortions in the wife, for the total group or for groups of only farmers or of forest and commercial workers. A correlation of these 2 variables was observed in a group of 21 young forest and commercial workers, with 54 corresponding controls, but not for the corresponding group of farmers of the same age group. The authors concluded that there was no evident relationship between 2,4-D use and spontaneous abortions, the result obtained in young forest and commercial workers warrants further study, but does not justify imposing restrictions on 2,4-D use until the study is completed. 926. St. John, L. E., Wagner, D. G., and Lisk, D. J. (1964) Fate of atrazine, kuron, silvex, and 2,4,5-T in the dairy cow. J. Dairy Science 47:1267-1270. The rate of excretion of 2,4,5-T was determined in the cow. One Holstein cow was fed a diet that contained 5 ppm 2,4,5-T for 4 days. Milk and urine samples were collected for 6 days after the start of the experimental diet and these samples were analyzed for 2,4,5-T by gas chromatography. The level of sensitivity for 2,4,5-T was 0.5 ppm. The daily amounts of 2,4,5-T excreted in urine were 93, 108, 112, 104, 9, and 4 mgs for the 6 days of collection, respectively. The total urinary excretion of 2,4,5-T accounted for 430.7 mg, which approximated the total dosage of 454 mg administered. Urinary 2,4,5-T was in the form of soluble salts. No results were reported for levels of 2,4,5-T detected in milk. Atrazine, kuron, and silvex excretion were also studied. The authors concluded that all of the administered 2,4,5-T was excreted intact in urine. 334 �927. Stalling, D. L., and Hackins, J. N. (1978) Metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) in bluegills and water. J. Agr. Fd. Chem. 26(2):447-452. The metabolism of 2,4-D by fish in a plastic pool of water outdoors was evaluated over a 12-week period following a single application. Thirty bluegill fish (Lepomis machrochirus) were placed in a pool with 2 mg/L [ C]-2,4-D dimethylamine salt. Another pool without fish served as a control. Both pools were partially (30%) shaded by dark screens and life forms were allowed to develop in the water for 2 weeks. Water samples were removed weekly and fish removed after 1, 5, 8 and 12 weeks and separated into the fillet and the head-viscera. All analyzed for radioactivity. CO^ in the! water samples was trapped and counted. Tissues were extracted to separate the amino acid plus glycogen fraction, neutral lipids, free fatty acids, and acid soluble fraction. Each fraction was analyzed for radioactivity. After 12 weeks, 49% and 16% of the radioactivity remained in the experimental and control pools, respectively. CO- made up none and 89% of the radioactivity in the experimental and control pools, respectively. The experimental pool contained a heavy algae bloom by the end of the test period. About one-third of the radioactivity in the fish was in the fillet and radioactivity increased in the fish over the period. None of the radioactivity was presented as 2,4-D. After 84 days, the distribution of radioactivity in fillet was 59%, 22%, 10%, and 3% for the amino acids, neutral lipid, acid-soluble, and free fatty acid fractions, respectively, and for the head-viscera were 45%, 18%, 10% and 28%, respectively. The authors concluded that the 2,4-D aromatic portion had been completely degraded by microorganisms and other factors within the pools and were then incorporated into biochemicals synthesized by the fish. Rapid degradation of 2,4-D was implied by the observation that no labeled 2,4-D metabolites were detected in any sample. 928. Stehl, R. H., and Lamparski, L. L. (1977) Combustion of several 2,4,5-trichlorophenoxy compounds: Formation of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Science 197(4307):1008-1009. [Background material.1 929. Stellman, S., and Stellman, J. (1980) Health problems among 535 Vietnam veterans potentially exposed to toxic herbicides. Society for Epidemiological Research: Abstracts. 444. [Abstract, only.] 930. Sterling, T. D. (1975) Toxic and teratogenic effect of 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Summary of Hearings of the Assembly Committee on Natural Resources of the State of Wisconsin, March 19, 1975, 24 p. [Background material.] 335 �931. Sterling, T. D. (1971) Difficulty of evaluating the toxicity and teratogenicity of 2,4,5-T from existing animal experiments. Science 174:1358-1359. 174:1358-1359". [Editorial.] 932. Stevens, J. T., DiPasquale, L. G., and Farmer, J. D. (1976) The acute inhalation toxicology of cacodylic acid. Toxicol. Appl. Pharmacol. 37:0412. [Abstract, only.] 933. Stevens, J. T., DiPasquale, L. C., and Farmer, J. D. (1979) The acute inhalation toxicology of the technical grade organoarsenical herbicides, cacodylic acid and disodium methanearsonic acid; a route comparison. Bull. Environ. Contam. Toxicol. 21(3):304-311. The toxicity of cacodylic acid, administered by various routes of exposure, is described. Swiss-Webster mice and Sherman rats were exposed to a commercial preparation of cacodylic acid (Phytar 138; 65.6% cacodylic acid), and to Fisher- or Ansul-purified cacodylic acid (95.5% and 99.5% purity, respectively). Groups of 10 animals of each sex were exposed to an atmosphere of up to 10 mg/liter of cacodylic acid (Phytar 138), produced in an inhalation chamber by a dust generator for 2 hours and then were observed for clinical signs for the next 14 days. At death, or at 14 days for the survivors, animals were necropsied. Of groups of 10 male rats, two died within 14 days of exposure to 4.1 mg/liter cacodylic acid, none died after exposure to 6.9 mg/liter, and one died after exposure to 10.8 mg/1. The L^Q for female rats was calculated at 3.9 mg/liter. Mice were exposed to one concentration, 6.4 mg/liter and one death occurred in 14 days. Respiratory distress, rhinorrhea, and porphyrin-like encrustation occurred during exposure and diarrhea, erythematous lesions, and decreased weight gain occurred after exposure. Gross pathology observed in fatal cases included dark spots and redness of the lungs, impacted caecum, and blood and mucous in the intestine. Respiratory irritation was measured as a decrease in respiratory rate in mice (three per group) exposed to cacodylic acid aerosol for 5 minutes. Results were expressed as the concentration of cacodylic acid that produced a 50% decrease in respiratory rate ( D . . . Control mice were R,,) exposed to diatomatous dust. The RDcn for cacodylic acid was 3.15 mg/liter and equal amounts of cacodylic acid from either the commercial or a purified preparation (99.5% purity) were equally effective. Exposure to 3.5 mg/liter of diatomaceous earth produced a minimal response on respiratory rate. Acute doses of 400-625 mg/kg of Fisher-purified cacodylic acid were administered to groups of 30-80 animals intraperitoneally or intravenously and intraperitoneal LD values were calculated as 720, 520, 520, and 600 mg/kg for male and female rats and male and female rats, respectivelv, and 470 mg/kg for female rats dosed intravenously. In addition to pathological changes observed after inhalation exposure, loss of the righting reflex, 336 �rigidity, and decrease in body temperature occurred; the thymus was reduced in size and appeared red and the adrenals appeared dark. All deaths occurred within 4 days of the injection. The authors concluded that the cacodylic acid aerosol produced some pulmonary irritancy that complicated interpretation of pulmonary toxicity data. 934. Stevens, J. T., Hall, L. L., Farmer, J. D., DiPasquale,,L. C., Chernoff, N., and Durham, W. F. (1977) Disposition of C and/or As-cacodylic acid in rats after intravenous, intratracheal, or peroral administration. Environ. Health Perspect. 19:151-157. The pharmacokinetics of cacodylic acid absorption, distribution, and elimination are described in the rat following administration by 3 routes. All studies were performed on male Sherman rats except for the experiment to identify sex differences which also included female Sherman rats and the,study of placental transfer with pregnant CD rats. A dose of 78 tng/kg [ C]-cacodylic acid was administered by tracheal cannula and the lungs and trachea were removed after <j>r^ min. and assayed for remaining activity. A dose of 60 mg/kg f C]-cacodylic acid was administered perorally and after 4 hours the gastrointestinal tract was removed and assayed for remaining activity. The half-time for absorption was calculated to be 2.2 min. from the pulmonary route and 248 min. from the oral route. Tissue concentrations of cacodylic acid were calculated from the levels of radioactivity detected in various tissues of groups of 3-4 rats (rats weighed 280-380 g each). Tissue levels were determined 0.1 to 168 hrs. after 200 mg/kg [ C]-cacodylic 9gid was administered intravenoulsy,75 min. to 60 days after 33 ug of [ C]-cacodylic acid plus 3.5 ug of f As 1-cacodylic acid were administered intravenously was, 104 days after,1 or 5 oral doses of 33 ug of [ C]-cacodylic acid plus 6.9,ug of [ AS]-cacodylic acid were administered or 1 dose of 33 ug of f C]-cacodylic acid plus 13.8 ug of [ AS]-cacodylic acid was administered intratracheally. One hour the after both the high and low intravenous doses about 11-15%, 1.2% and 0.8% of each dose was recovered in whole blood, liver, and kidney respectively and less than 0.5% remained in the lung, brain and spleen. The only differences in distribution between the 2 doses occurred at 15 minutes, wit a higher level in the liver after the high dose than low dose and a higher level in the kidney after the,low dose than high dose. No differences in tissues distribution of [ Cl compared to [ As] occurred after intravenous administration. At 168 hr. after the high doses, 10% of the dose remained the blood and less than 0.5% remained.in any other tissue. After 105 days, the concentration of [ C] in the spleen and kidney were highest after intravenous dosing than by other routes and were highest in the liver and blood after oral dosing than by other routes while all other tissue levels were the same, regardless of route of administration. Comparing tissue distribution after 1 and 5 doses, the spleen, liver, and brain retained more of the administered doses after the multiple dosage regimen than after the single dosage regimen. Tissue distribution of 24-34 mg/kg of [ C]-cacodylic acid administered intravenously was the same for male and female rats. Levels of [ C] were analyzed in both 337 �fetal and maternal tissues 24 hrs. after a pregnant rat was administered 33 ug [ ]-cacodylic acid plus [ Asl-cacodylic acid on day 21 of gestation. Levels were comparable for maternal tissues and the corresponding tissues in the fetus. Blood samples collected from rats from each treatment group described for the experiments on tissue distribution were separated into plasma and red blood cells for separate analysis of radioactivity and the results were analyzed pharmacokinetically. The data for plasma levels of [ C] after the high intravenous dose was administered were triphasic, with clearance half-times of 0.014, 0.22 and 3.42 hr. for the 3 phases. Peak plasma levels were reached 5 min., 10 min., and 1 hr. after the intravenous, intratracheal, and oral doses, respectively. Clearance from,whole blood was 92, 76, and 90 days, respectively. Excretion of f Cl from the low intravenous dose and from doses given by the other routes were determined by analysis of radioactivity in urine, feces, expired air, and bile collected from cannulated bile ducts and the amount in the whole body after 24 hr. Recovery of administered doses were 88-93% and absorption was 66% for the oral dose and 92% for the intratracheal dose. After oral dosing, over 31% of the dose was recovered in feces and less by other routes. Biliary secretion was demonstrated. From 60-71% of the doses given by the other routes.were excreted in urine and an insignificant amount was excreted as C0_ by the lungs after dosage by any route. The authors concluded that cacodylic acid was absorbed slower after oral administration than after other routes were used and had a high affinity for erythrocytes. The authors also concluded that cacodylic acid was not converted to inorganic arsenic, that biliary secretion accounted for fecal excretion after intravenous administration of cacodylic acid, and that cacodylic acid readily crosses the placenta. 935. Strange, J. R., Allred, P. M., and Kerr, W. E. (1976) Teratogenic and toxicological effects of 2,4,5-trichlorophenoxyacetic acid in developing chick embryos. Bull. Environ. Oontam. Toxicol. 15(6):682-688. The embryolethality and teratogenicity of 2,4,5-T was studied in fertilized chicken eggs. The results, published elsewhere (Strange, J. and Kerr, W.E.; 1976), indicated that 2,4,5-T administered in DMSO altered.hatchability with LDc« values of 68 mg/kg when injected on day 0 and 62 mg/kg when injected on day 5. No malformations were observed in embryos exposed to 50 mg/kg 2,4,5-T, for 2 days. In this paper (Strange, J.R., Allred, P.M., and Kerr, W.E., 1976) an additional experimental group given 25-150 mg/kg 2,4,5-T in acetone and control groups given acetone alone were injected on day 0. The LD for 2,4,5-T in acetone was 133 mg/kg while the maximum amount of acetone used as the 2,4,5-T vehicle produced no effect on mortality. The authors concluded that DMSO and 2,4,5-T acted synergistically, producing a lower LD^ than 2,4,5-T in acetone. (The solubility of 2,4,5-T in each vehicle was not indicated.) 338 �936. Strange, J. R., and Kerr, W. E. (1976) Teratogenic and toxicological examination of 2,4,5-trichlorphenoxyacetic acid in developing chiek embryos. Toxicology 6:35-40. The teratogenic and embryolethal effects of 2,4,5-T were studied in chick embryos. Fertilized eggs were injected with 12-125 mg/kg 2,4,5-T into the air space on day 0 or day 5 of incubation and the number of hatched eggs was recorded. 2,4,5-T contained less than 0.1 ppm dioxin contaminant and was administered in dimethyl sulfoxide (DMSO). Control included eggs that were untreated, eggs that were drilled only, and eggs that were injected with distilled water or DMSO. Teratologic effects were studied in 12 embryos administered 50 mg/kg 2,4,5-T and 12 control eggs (drilled only), which were incubated for 48 hours. Embryos were fixed in Bouin's fluid, stained, and examined for malformations. The LD for eggs injected on day 0 was 62 mg/kg and on day 5 was 68 mg/kg. DMSO did not produce toxicity at levels below 100 mg/kg. The LD-Q for DMSO was 210 mg/kg. No abnormalities were observed in the eggs examined for teratologic effects. However, at the developmental stage examined, the kidney was not developed sufficiently to reveal the types of malformations reported in rodents. 2,4,5-T at 50 mg/kg prevented 37% of the eggs in the embryo lethality study from hatching, but all of the eggs examined for malformations were viable 2 days after exposure, suggesting that death occurred late in development. The authors estimated that 41 mg/sq. ft. of 2,4,5-T would be distributed on a field from application of 2,4,5-T at recommended agricultural rates, resulting in exposure of 4.1 mg per egg externally. 937. Strik, J. J. T. W. A. (1979) Porphyrins in urine as an indication of exposure to chlorinated hydrocarbons. Ann. N. Y. Acad. Sci. 320:308-310. The results of urinalysis of TCDD-exposed people for porphyrins is reported. Twenty months after the explosion in Seveso, Italy which released a cloud of TCDD, urine was collected from people in the most polluted area. Total porphyrin values from this group were not significantly different from control groups from other countries. Thin layer chromatography of the porphyrins revealed an abnormal pattern indicating chronic hepatic porphyria type A. No other details of the group examined or the results were given. Other samples were analyzed for porphyrins that were collected from groups exposed to chlorinated hydrocarbons. The levels of urinary porphyrin excretion were not elevated in these groups but the patterns of porphyrins excreted in general was indicative of chlorinated hydrocarbons. 939. Stroo, W. E., McCormack, K. M., and Hook, J. B. (1979) Renal functional effects of 2,4,5-trichlorophenoxyacetic acid and silvex after acute and prolonged exposure. J. Toxicol. Environ. Health 5(5):845-854. Renal clearance of organic ions in vivo and uptake of organic ions in vitro were compared after acute and chronic doses of 2,4,5-T and Silvex 339 �were administered to rats. Sprague-Dawley rats were administered 99% pure herbicides with less than 0.05 ppm dioxin contaminant subcutaneously in 95% ethanol or orally in the diet. Renal cortical slices in vitro were incubated in a medium with C-labeled organic ions and then uptake was determined by counting radioactivity in the solubilized tissue and in the medium. Clearance in vivo was.determined in rats infused with saline containing \ H]-inulin and \ C] para-aminohippurate (PAH). Renal parameters were studied during several clearance periods in which herbicide-treated rats were given a saline load or high (1.14 mg/min) or low (0.114 mg/min) PAH infusion rates. The in vitro uptake of PAH or N-methyl nisotinamide chloride (NMN) was altered in renal slices from rats given 800 ppm Silvex orally but not in rats given 200 ppm silvex or 2,4,5-T. PAH and 2,4,5-T uptake was decreased to the same extent in slices of kidneys removed 24 hours after the last of 14 doses or 1 dose of 100 mg/kg 2,4,5-T. Decreases in uptake of PAH or tetraethylammonium were observed 4 hours after one or 14 doses of 20 mg/kg 2,4,5-T but not after 24 hours. These decreases also occurred 4 hr. after a single dose of 50 mg/kg of Silvex was given. The results from in vivo studies showed the same trends; a large (100 mg/kg) single dose or series of 14 doses of 2,4,5-T caused decreased PAH and TEA clearance and increased filtration fraction (i.e. PAH clearance/ inulin clearance) 24 hours after administration. The effects of a low dose (20 mg/kg) of 2,4,5-T or (50 mg/kg) Silvex observed after 4 hours did not persist for 24 hours, even after 14 consecutive doses of 2,4,5-T had been administered. Alterations in electrolyte excretion occurred with changes in PAH clearance. The authors concluded that 2,4,5-T probably did not accumulate in vivo from chronic exposure and suggested that decreased anion transport reflected the presence of 2,4,5-T in the kidney but was not a manifestation of toxicity. To strengthen the conclusions pertinent to 2,4,5-T accumulation, the authors would have to demonstrate that the single dose of herbicide produced a submaximal effect that could potentially be increased by multiple doses (or a higher single dose) and that the lack of increased effect by repeated doses is not the result of compensatory mechanisms. 940. Stumm, W., and Morgan, J. J. John Wiley and Sons], p. 69. (1970) Aquatic chemistry. [New York: [Background material.] 941. Styles, J. A. (1974) Studies on the effects of paraquat and diquat on cells in culture. Viability of macrophages and fibroblasts incubated with paraquat and diquat. Br. J. Exp. Pathol. 55(1):71-77. The viability of rat alveolar macrophages and cloning efficiency of mouse fibroblasts were studied in cells cultured in the presence of diquat. Stationary and suspension cultures of rat alveolar macrophages, rat peritoneal macrophages and mouse L929 fibroblast cells were cultured in medium that contained 10 M to 10 M diquat dibromide for up to 24 hr. Viability was evaluated by exclusion of trypan blue of suspended cells and by eosin exclusion for stationary cultures. 340 �After 24 hr. of exposure of suspended cells to 10 M diquat, 60% of each type of macrophage survived and 0-15% survived 24 hr. exposure to 10 M diquat. Exposure of stationary cells to 10 M diquat for 30 min. resulted in a decrease in macrophage viability of 40% during the subsequent 2-3 days. Higher concentrations of diquat and,long periods of exposure produced larger effects on viability. At JO M", for 24 hr. diquat killed all fibroblasts and between 10 and 10 M, produced a dose-related decrease in fibroblast cloning efficiency. The cytotoxic effects of paraquat were also studied. The authors concluded that diquat toxicity to macrophages in vitro was equivalent to paraquat toxicity and to fibroblast cloning was substantially higher than paraquat toxicity and suggested that the reverse order of toxicity observed in vivo reflected different distributions of the two compounds to the lung. 942. Styles, J. A. (1973) Cytotoxic effects of various pesticides in vivo and in vitro. Mutat.Res. 21:50-51. [Abstract, only.] 943. Sudak, F. N., Claff, C. L., and Cantor, M. (1966) Body temperature regulation in rats treated with 2,4-dichlorophenoxyacetic acid. Arch. Int. Pharmcodyn. 160(2):253-264. The ability of 2,4-D treated rats to maintain a normal body temperature when exposed to the warm or cold was studied. Male Wistar rats were administered 2,4-D intraperitoneally or intracisternally and control rats were administered saline. After 45 minutes, skin and colonic temperatures were monitored, carbon dioxide (CO-) in expired air was measured, and electrocardiograms and electromyograms were obtained from animals exposed to the cold, heat, or ambient (thermoneutral) temperatures. Some rats were acclimatized to the cold by exposure to 6°C for 4 weeks. 2,4-D produced a dose-related decrease in body temperature in rats placed in the cold (2°C) after treatment. Rats injected with 300 mg/kg 2,4-D were unable to maintain their body temperature in the cold and showed a steady decrease in metabolism (measured as CO- production) during 90 minutes in the cold, no increase in blood circulation to the skin (measured as the difference between skin and colonic temperatures), and no increase in shivering (measured as increased activity in the electromyogram). Statistically significant increase in C0_ production over controls was observed for treated rats at ambient temperature, but only a small increase in colonic temperature occurred. At 35 °C, colonic temperature and C0_ production increased in 2,4-D treated rats causing death to all animals after 30-105 minutes of heat exposure. None of the controls died within 120 minutes. Cold-acclimatized rats treated with 2,4-D had significantly lower body temperatures and a smaller increase in COproduction than acclimatized saline controls. Rats given 3 mg/kg intracisternally under chloralose anesthesia showed the same extent of impairment of temperature regulation in the cold as rats given 300 mg/kg 2,4-D (without anesthetic). At colonic temperatures below 20°C, 341 �no heart activity was detected. After rewarming, all animals survived. The authors suggested that 2,4-D impaired heat regulation by acting on central mechanisms that control heat production and loss. 944. Sugar, J., Toth, K., Csuka, 0., Gati, E., and Somfai-Relle, S. (1979) Role of pesticides in hepato-carcinogenesis. J. Toxicol. Environ. Health 5(2/3):183-191. This report presents some of the same data as the reports by Toth et al., 1978, and Toth et al., 1979. Data for Swiss H/RIOP mice fed combinations of trichlorophenoxyethanol (TCPE) and TCDD were used; the data from mice fed TCDD alone were not reported here. The authors noted that significantly increased number of liver tumors in male mice was apparent in the two groups fed the highest dose of TCPE (67 or 70 mg TCPE/kg) and that the TCDD doses (0.112 ug/kg or 0.007 ug/kg) were apparently unrelated to the production of liver tumors. Another group given 7.0 mg TCPE/kg with 0.07 ug TCDD/kg did not show an increased liver tumor incidence. The authors also noted that this particular strain of mouse had an unusually high rate of spontaneous liver tumors (average incidence in controls of 26.33 percent). There was no predominance of any particular type of chemically induced liver tumor, not did cirrhosis ever precede the formation of tumors. Because the incidence of liver tumors was significantly greater in male than in female mice, the authors considered a possible sex-linked effect on aryl hydrocarbon hydroxylase (AHH) induction by TCPE. Because of the contamination of TCPE by TCDD, TCDD's AHH inducing activity was also looked at using the fluorometric procedure described by Nebert and Belboin (1968) to determine AHH activity, it was found that in male mice activity increased over the course of the experiment, while in females it leveled off after the third day and then declined. Induction increased in direct proportion to TCDD content. The authors note that TCDD, which is a strong inducer of AHH, was also shown ineffective in producing tumors, while quite the reverse is the case for TCPE. 945. Sugiura, K. , Matsumoto, N., Washino, T., Mihara, Y., and Goto, M. (1979) Ecological chemistry XVII. Accumulation of organochlorine compounds in fishes. Distribution of 2,4,5-T, alpha-HCH, beta HCH, gamma HCH and 2,4,6,2'4'6'-hexachlorobiphenyl in tissues. Chemosphere (6) .-365-368. The authors studied the accumulation and elimination of C-2,4,5-T and several other organochlorine compounds in carp using whole body autoradiography. In the accumulation,experiment, a carp was placed in a 40 liter tank containing 0.056 ppm C-2,4,5-T (purity unspecified) for 6 days after which the fish was processed for autoradiography. In the elimination experiments, the fish were removed from the treated water after six days and placed in 2,4,5-T free water for 2 days after which the fish were processed. Numbers of fish in each group were not reported. Accumulation of 2,4,5-T occurred only in the gall bladder and was almost entirely eliminated within 2 days. 342 �946. Summary of Dioxin Planning Conference. , 25-26. 15 pp. (1974) Washington, DC. July [Background material.] 947. Summary of the Statement of Mr. Cleland before the Subcommittee on Medical Facilities and Benefits of the Veterans Affairs Committee, House of Representatives, February 25. (1980). [Background material.] 948. Sundell, L., Rehn, M., Axelson, 0. concerning herbicides. (1973) An epidemiological study This study reports findings similar to those presented in Axelson et al.; Herbicide exposure and tumor mortality: an updated epidemiological investigation on Swedish railroad workers and Axelson et al., Herbicide exposure and tumor incidence: an epidemiological investigation on Swedish railroad workers, 1974. 949. Suschetet, M., and Causeret, J. (1973) Pathophysiologic and nutritional effects of amino-4-trichloro--3,5,6-picolinic acid (picloram) in rats. CR Acad. Sci. (Paris) 276(11):1775-1777. [Foreign language.] 950. Suskind, R. R. (1980) TCDD contamination in the United States case study. Presented at the National Academy of Sciences International Workshop of Areawide Chemical Contamination, March 17. 13 p. [Review article.] 951. Suskind, R. of 2,4,5-T. Prepared by Laboratory, R. (1979) A study of workers involved in the manufacture Protocol for Nitro, West Virginia Study, June 1979. the Department of Environmental Health, Kettering Cincinnati, Ohio. 29 pp. [Background material.] 952. Suskind, R. R. (1978) Chloracne and associated health problems in the manufacture of 2,4,5-T. Report to the Joint Conference, National Institute of "Environmental Health Sciences and International Agency for Research on Cancer, WHO, Lyon, France, January 11. 7 pp. The health of workers at a plant in Nitro, West Virginia, is described. The plant manufactures 2,4,5-T and in 1949 an accident occurred in which a relief valve of a reaction vessel opened, releasing vapors into 343 �the factory. Five years later, 117 persons had chloracne, which was traced to this incident. A total of 228 persons, including workers' family members and medical personnel, were affected 5 years after the manufacture of 2,4,5-T had been started. Following the explosion, the typical symptoms described included headache, dizziness, and vomiting within hours, a facial rash within 1 week, which developed into acne during the next week, and a tendency to feel tired. One month after the explosion, aching muscles were reported, leading to inability to walk; insomnia, and extreme irritability. Three months after the explosion, hyperpigmentat ion of the skin appeared. In three out of the four hospitalized cases, the liver was palpable. Nerve biopsies showed destruction of myelin sheaths and nerve fibers, although muscle biopsies showed no abnormal features. Total serum lipid values tended to be high, and prothrombin concentration was one-half of the normal value. Therapy was unsuccessful for acne or for pain, although tranquilizers were successful for treating nervousness and irritability. Hepatic changes were temporary, and resolved in a few months. At the time of the incident in this factory, TCDD was not identified as the causative agent, precluding its hasty removal from the work environment. Following the experimental application of 2.5-5.0 percent trichlorophenate to human subjects, acne developed and persisted for 3-4 months. 953. Sweeney, G. D., Jones, K. G., Cole, F. M., Basford, D., and Krestynski, F. (1979) Iron deficiency prevents liver toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Science 204(4390):332-335. The toxic effects of TCDD in mice maintained on an iron deficient diet were ascertained. C57B1 mice were fed an iron deficient diet and 1.6 to 2.0 ml of blood was removed over a 4 week period, prior to TCDD treatment. Weekly intraperitoneal doses of 25 ug/kg TCDD in corn oil were administered for 11 weeks to mice continued on the iron deficient diet and to mice fed a normal diet. Urinary porphyrin levels were monitored during the TCDD treatment period. After the 11 week treatment period, mice were weighed and liver weights, iron levels, and enzyme levels were determined. Liver tissue was also examined histopathologically. Prior to TCDD treatment, hemoglobin levels dropped from 10.4 to 5.5 g/dl but recovered to 10.2 g/dl in mice exposed to TCDD and to 13.4 g/dl in mice fed the iron deficient diet but not exposed to TCDD. No mice died after the start of the TCDD exposure, but the mice exposed to TCDD on both diets weighed about 10% less than controls fed the corresponding diet. Non-TCDD-treated mice on iron deficient diets weighed about 20% less than controls. Liver hypertropy occurred in both TCDD-treated groups, but only the TCDD-exposed mice on the control diet developed elevated hepatic and urinary porphyrins (primarily coproporphyrin), hepatic histopathology, a decrease in uroporphyrinogen decarboxylase, to 20% of controls, and a deterioration of the condition of the fur. Mixed function oxidase activity was induced by TCDD in both groups, but to a slightly smaller extent in the iron deficient group. The authors concluded that tissue iron in a form different from that of circulating iron is essential for TCDD to elicit toxicity. 344 �954. Szocs, J., Dr., Molnar, V., Balogh, E., Ajtay, M., and Fulop, I. (1970) Experimental data on the toxic effects of 2,4-D (diclordon) herbicide. Revista Medicala 16;91-93. The acute and subacute tpxicites of 2,4-D were studied in the rat. Male rats (12-50 rats per group) were administered a single dose of 625 mg/kg 2,4-D sodium salt by stomach tube and were killed 2-48 hr. later; other groups of rats were administered doses of 125 mg/kg (total number of doses not indicated) and were killed 21 days later (did not specify whether this referred to 21 days after the first or last dose of the subacute regimen). Blood and liver samples were analyzed for various enzyme activities by histochemical analyses or unspecified methods and serum proteins were analyzed by paper chromatography. Serum and liver aldolase levels were decreased by 35-40% in all groups and all time points below the control value. Glutamic oxaloacetic transaminase levels were elevated maximally at 6 hours in the liver and 12 hours in the serum after acute exposure and slightly increased after subacute exposure. Fluctuations in both directions were observed for glutamic pyruvic transaminase, catalase, cholinesterase, and succinic dehydrogenase, activities, and in various protein fractions. Alkaline phosphatase activity in the cytoplasm increased and acid phosphatase activity decreased, as evidenced from histochemical staining. Fatty infiltration of the liver and dystrophia were also observed. The authors concluded that 2,4-D produced subacute hepatitis as well as neurotoxicity (reported by others) and recommended that safeguards be instituted for occupational handling. 345 �955. Tarrant, R. F., and Allard, J. (1972) Arsenic levels in urine of forest workers applying silvicides. Arch. Environ. Health 24(4):277-280. The levels of arsenic excreted by forest workers involved in applying cacodylic acid to trees is presented. Six men who applied cacodylic acid by 2 different methods (with an injection hatchet, 3 men; by squirt bottle, 3 men) provided urine samples at the start of each work week and at the end of each 5 day work week, over 9 weeks. Arsenic in the urine samples was analyzed by a colorimetric method. Clean clothing and eye protection were provided daily to the workers. A control group of 3 workers in the same area that were not exposed to chemicals provided urine samples on the same schedule as the exposed workers for arsenic analyses. The mean levels of arsenic at the start and end of the work week was 0.04 ppm and 0.07 ppm, respectively for controls and 0.08 and 0.41 ppm for workers using the squirt method of application for 5 weeks in which all subjects provided samples and 0.2.1 and 0.63 ppm respectively for the group that used the injectionhatchet, for the 2 weeks in which all subjects provided samples. The means for both exposed groups was 0.56 ppm arsenic. All levels for exposed groups were significantly higher than control levels at the end of each week, but the levels for the 2 exposure groups were not significantly different from each other. Arsenic levels were also presented for workers exposed to monosodium methanearsonate. The authors concluded that forest workers were occasionally exposed to . excessive quantities of arsenic, in which total arsenic exceeded 0.3 ppm in urine. 956. Taylor, C. (1974) Chemical toxicity and mental disorder, flatter to the editor] Am. J. Psych. 731(5):609. [Editorial.] 957. Taylor, J. S. (1979) Environmental chloracne: Ann. N. Y. Acad. Sci. 320:295-307. Update and overview. [Review article.] 958. Taylor, J.S. 13:585-591. (1974) Chloracne—a continuing problem. Cut is [Review article.] 959. Taylorson, R., and Kleisath, J. (1961) Cacodylic acid investigations. BL Technical Memorandum 9-24. Crops Division, Ft. Detrick, MD. DTIC No. AD254825L. 23 pp. [Not available.] 346 �960. Teitelbaun, P. J., and Poland, A. P. (1978) Studies of the hepatic uptake of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse. Fed. Prpc. Fed. Am. Soc. Exp. Biol. 37(3):692. [Abstract, only.] 961. Telegina, K. S., and Bikbulatova, L. I. (1971) Affection of the follicular apparatus of the skin in workers occupied in production of butyl ether of 2,4,5-trichlorophenoxyacetic acid. Vestn. Dertnatol. Venerol. 44(12):35-39. [Foreign language.] 962. Tenchini, M. L., Crimaudo, C., Simoni, G., De Carli, L., Giorgi, R., and Nuzzo, F. (1979) Approaches to the evaluation of genetic damage after a major hazard in chemical industry: preliminary cytogenetic findings in TCDD exposed subjects after the Seveso accident. Part of a special project to investigate TCDD-exposed pregnancies. Task Force from Universita di Milano (Italy). Supported by the Assessorato alia Sanita, Regione Lombardia (Italy).. The authors report on the cytogenetic analysis of peripheral blood and aborted tissues obtained from 25 pregnant women who were exposed to TGDD during the Seveso accident. Peripheral blood cultures and explants from embryos, placentas, umbilical cords were established. Chromosome preparations were made from 24-72 hour peripheral blood cultures and from 9-40 day explant cultures. The number of metaphases of peripheral blood cells examined for each individual varied from 90-150. No control were reported. The numbers of chromosome aberrations observed were in the normal range according to the authors. In fetal tissues, larger numbers of chromosomal aberrations were observed. The chromosomal damage was higher in embryonic tissue than in placental or umbilical explants. No conclusion can be made on these results because no controls of unexposed fetal tissues were included in the experiment. It is possible that chromosomal effects observed in these tissues were a result of culturing conditions. 963. Tenchini, M. L., Giorgi, R., Crimaudo, C., Simoni, G., Nuzzo, F., and de Carli, L. (1977) Approaches to examination of genetic damage after a major hazard in chemical industry: preliminary cytogenetic findings on TCDD-exposed subjects after Seveso accident. Presented at the Expert Conference on Genetic Damage Caused by Environmental Factors, Oslo, Norway, May 11-13, 1977. [Not available.] 347 �964. Thiess, A. M., and Frentzel-Beyme, R. (1977) Mortality study of persons exposed to dioxin following an accident which occurred in the BASF on 13 November 1953. Presented at the Fifth International Conference of Medichem - Occupational Health in the Chemical Industry, 9 pp. The author performed a 20-year followup investigation of 75 BASF workers exposed to trichlorophenol and dioxin (the structures of these compounds were not given) during an industrial accident in 1953. The historical prospective design utilized both internal and external cohort groups for comparison of mortality and neoplastic incidence. Three populations were selected for use as external comparison groups. 1.) Total population of Ludwigshafen population 180,000 where approximately 25% of persons were workers at BASF, 2.) Total governmental district Rhinehessia Palatinate 3,700,000, 3.) Total Federal Republic of Germany (FRG) 60 million. The internal comparison group was derived from BASF matched by age and date of entry into the factory. An analysis of the 3 external and internal matched comparison groups showed that the dioxin exposed group had a lower mortality rate than Ludwigshafen, but a higher rate than the internal matched comparison population, the administrative district of Rhinehessia Palatinate and the Federal Republic of Germany as a whole. The dioxin exposed group demonstrated 6 neoplasms at a rate twice that of the total German Republic and a third greater than the internal controls. Four of the neoplasms were of the stomach which was greater than found in all comparison groups and significantly greater than 3 comparison groups. The statistical method utilized was the Poisson distribution. The author reported that the number of malignant neoplasms is increased above expectation in the age group 65-75. 965. Thigpen, J. E. , McConnell, E. E., Moore, J. A., and Faith, R. E. (1977) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on host resistance to infectious agents. Environ. Health Perspec. 20:245. Abstract, only.] 966. Thigpen, J. E., Faith, R. E., McConnell, E. E., and Moore, J. A. (1975) Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infection and Immunity 12(6):1319-1324. The effect of subacute administration of TCDD on resistance of mice to infectious agents is described. Male C57BL 16Jfh mice were administered TCDD (99+% purity) in acetone-corn oil (1:6) by gastric intubation. Doses of 0.5-20 ug/kg were administered once weekly for 4 weeks. Two weeks after the last dose of TCDD was given, mice were injected intraperitoneally with Salmonella bern or Herpesvirus suis (pseudorabies virus; PRV). During the following 14 days, general appearance of the animals, weight gains, mortality rates and time from infection to death were determined and necropsies were performed on some mice. The highest dosage group gained significantly less weight 348 �than the non-TCDD-treated group. Mortality rates were 25% for vehicle controls, about 65% for the 1, 5 and 10 ug/kg dosage groups, and 95% for the 2.0 ug/kg dosage group. The time interval to death was shortened from 8.4 days for non-TCDD-treated mice to 3.0 days for the highest dosage group. No alterations in response to the PRV infection were observed in TCDD-treated mice, compared to non-TCDD-treated mice. Thymic atrophy and a hepatic necrosis were observed in mice that received the highest dose of TCDD. Lesions attributed to the bacterial infections were observed in animals infected with S. bern. The authors concluded that doses of TCDD which did not elicit clinical or pathological changes were able to affect host defense. 967. Thomas, J. A. (1974) Actions of pesticides and other drugs on the male reproductive system. U.S. Environmental Protection Agency Report, National Technical Information Service Publication, ISS No. PB-237-381. 30 pp. 3 The effect of 2,4,5-T on the biotransformation of "H-testosterone by the mouse prostate gland was studied. Three groups of rats were administered 6.25, 12.5, or 25.0 mg/kg 2,4,5-T by gastric intubation daily for 10 days. On the next day, H-testosterone was administered intraperitoneally and the anterior prostate glands were excised 5 minutes later, weighed, and analyzed by thin layer chromatography for various radioactive metabolites of testosterone. For other experiments hepatic microsomes of the pretreated mice were incubated for 60 min. with H-testosterone and then radioactivity associated with 6 beta gamma-, 7 gamma-, and /6 gamma-hydroxytestosterone were determined. The low, intermediate, and high doses of 2,4,5-T caused decreases of 14%, 19%, and 35% respectively, in the total amounts of radioactivity associated with the prostate. The proportions of the total radioactivity associated with each of the four metabolites isolated were not altered. The weights of the seminal vesicles and prostate glands were not altered by the treatment, and prostate fructose levels were not altered. Radioactivity (per mg liver tissue) associated with hydroxytestosterone derivatives was not altered by 2,4,5-T pretreatment. The authors concluded that 2,4,5-T did not alter blood levels of androgens (since organ weights were not altered) but decreased testosterone assimilation by the prostate possibly by a direct inhibition of hormone binding to target tissue. Other organochloride herbicides showed effects resembling those of 2,4,5-T while other classes of herbicides tested did not alter androgen metabolism. 968. Thomas, J. A., and Lloyd, J. W. (1973) Organochlorine pesticides and sex accessory organs of reproduction. Pestic. Environ: Continuing controversy, Pap. Inter. Am. Conf. Toxicol. Occup. Med. 8th pp. 43-51. [Not available.] 349 �969. Thomas, P., and Amor, 0. F. (1968) A case of diquat poisoning in cattle. Vet. Rec. 83(26):674-676. The toxic effects of diquat were described in cattle. A drum that originally contained diquat had been discarded on a farm near pastureland. Four years later, 3 cows that grazed this pasture died and the toxic symptoms and tissue analyses of one heifer for diquat indicated that the deaths were probably from acute oral ingest ion of diquat. Tissue levels of the heifer were from 9 to 21 ppm for the liver, kidney, and abomasum and 2 ppm of brain. The total ingested dose was estimated from these data to be 50-100 ml. The viscera of a second cow that died contained no detectable diquat and death was assumed to have occurred after diquat had been excreted. Symptoms observed before death in the heifer included dehydration, sunken eyes, rapid respiration, and hind-leg incoordination. The animal became comatose and died the night after symptoms were recognized. The cow also displayed symptoms of hind-leg incoordination and sunken eyes on the day prior to death. Microscopic examination of tissues from a second heifer that died from the same farm revealed edema, congestion, and inflammation of the mucous of the gastrointestinal tract and cranial and cardiac hemorrhages. Only minor lesions were observed in the cow at autopsy. The authors did not state clearly whether the clinical symptoms, histopathology, and diquat tissue levels were all from the same heifer, as 2 heifers and one cow died, but diquat was confirmed in the tissues of one heifer, only and was the presumed cause of death of the other two animals. 970. Thomas, P. T., and Hinsdill, R. D. (1979) The effect of perinatal exposure to tetrachlorodibenzo-p-dioxin on the immune response of young mice. Drug and Chem. Toxicpl. 2(1 & 2):77-98. The reproductive effects of TCDD administration prior to mating and during gestation is described, and immune competence of offspring is evaluated. Female Swiss Webster mice were fed diets of 1-20 ppb TCDD for 4 weeks and then were bred with male Swiss Webster mice and were maintained on the experimental diet throughout gestation and for the first 3 weeks of lactation; uncontaminated feed was administered on the fourth week. Litters were reduced to 7-8 offspring 1 week after birth and mice were weaned at 4 weeks and tested for immunologic function starting 1 week after weaning. Fetal and newborn mortality, maternal appearance and newborn weights were recorded. Serum of offspring immunized twice with sheep red blood cells (SRBC) was assayed for hemolysin titers and the number of anti-SRBC plaque-forming scleen cells was determined in vitro. 2,4-Dinitro, 1-fluorobenzene (DNFB) was applied to 1 ear of DNFB-sensitized mice and the thickness of the ear 6-72 hours later was compared to the thickness of the other ear which received only vehicle. Spleen cells were cultured with concanavalin A (Con A) or E. coli lipopolysaccharide (LPS) and.the proliferative response was measured as the incorporation of [ Hl-thymidine into DNA. Some offspring were inoculated with Listeria monocytogenes or Salmonella typhimurium LPS and mortality over the subsequent 14 days was recorded. White blood counts, organ weights and histopathological 350 �changes were determined. Only the highest dosage group of adult mice showed signs of toxicity. These signs appeared by 6 weeks of treatment and included facial alopecia and edema but not reduced body weights. No effects on conception rate or litter size occurred in treated groups, while survival during the first week after birth was decreased in the 2 highest groups (fed 10 and 20 ppb TCDD diets). Offspring showed the same signs of toxicity as adult mice. These signs appeared in the 3 highest dosage groups (5-20 ppb); decreased weights were also observed in some offspring by 4 weeks of age. A. dose-related decrease in plaque-forming cells was observed in TCDD-treated offspring, but no decrease in lymphocyte counts or antibody levels occurred. TCDD treatment did not elicit changes in DNFB sensitivity, blastogenic response to mitogens, survival of Listeria inoculation, spleen or liver weights, or differential white cell counts. A marked increase in sensitivity to Salmonella endotoxin, thymic atrophy, and hepatic necrosis were produced by 1-5 ppb TCDD feeding regimens. The authors concluded that doses of TCDD that failed to elicit toxic signs on adult mice impaired immunologic functions of a specific subpopulation of T lymphocytes in offspring. The authors also indicated that the doses used in these experiments were relevant to chronic human exposure to environmental TCDD and their results suggest that these doses induce toxicity. Details of mating frequencies, rate of successful matings and other reproductive and teratogenic parameters that are not reported would have been useful to relate these doses and effects reported here with those of other studies. 971. Thomas, W. L. (1974) The effects of herbicides in South Vietnam. Part B. Working papers: Economic Stress and Settlement Changes. National Academy of Sciences-National Research Council. AD-779 021. 61 pp. [Background material.] 972. Thomasson, W. A. (1979) Deadly legacy: veteran. The Bulletin pp. 15-19. Dioxin and the Vietnam [Editorial.] 973. Thompson, D. J., Emerson, J. L., and Sparschu, G. L. (1971) Study of the effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on rat and rabbit fetal development. Teratology 4:243. [Abstract, only.] 351 �974. Thompson, D. J., Emerson, J. L., Strebing, R. J. , Gerbig, C. G., and Robinson, V. B. (1972) Teratology and postnatal studies on 4-amino-3,5,6-trichloropicolinic acid (picloram) in the rat. Food Cosroet. Toxicol. 10(6): 797-803. The effect of picloram on the fetal and neonatal development of the rat were studied. Groups of 35 pregnant rats were administered 500, 750 or 1000 mg picloram /kg by oral gavage from days 6-15 of gestation. A control group of 35 rats received the corn oil vehicle only. Up to 3/4 of each group of rats were killed on day 20 of gestation and corpora lutea, resorptions, live fetuses, fetal weights and gross malformations were determined. Fetuses were fixed in Bouin's fixative or stained with alizarin red and observed for visceral or skeletal malformations, respectively. The remaining litters were allowed to deliver and fetal growth weights and viability were recorded to 21 days of age. The skeletal development of two male and two female weanling rats from each group was examined. Maternal toxicity and mortality occurred from the two highest dosages, of picloram. No reductions in litter size or pup weights or increases in the numbers of resorptions or corpora lutea occurred in any treatment group. Malformations with statistically significant higher incidences in treatment groups compared to controls were Unilateral hydroureter, unossified fifth sternebrae, and accessory ribs. The gestation period, duration of labor, viability, lactation indices, body weights, and skeletal development was normal for the two lowest dose groups. The viability and lactation indices from birth to day 5 was decreased in the highest dose group, which included a litter of 12 pups with 10 deaths, reported to be from maternal neglect. No other parameters were abnormal for the high dosage group. The authors concluded that picloram was not teratogenic or detrimental to postnatal development under their experimental conditions. 975. Thunberg, T., Ahlborg, U. G., and Johnsson, H. (1979) vitamin A (retinol) status in the rat after a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch. Toxicol. 42(4):265-274. Retinol levels in the serum and livers of TCDD-treated rats were measured. Male Sprague-Dawley rats were administered 10 ug/kg TCDD in corn oil by oral gavage. Rats were fed diets containing 3.6 mg of retinol per kg feed and food consumption and body weights were recorded twice per week. From 2 days to 8 weeks after TCDD treatment, rats were killed and the livers and serum were removed. Liver homogenates and sera were extracted with ether, dissolved in methanol, and analyzed for retinol by liquid chromatography. The body weights of TCDD-exposed rats were 61 g less than controls after 8 weeks and food consumption was also decreased, corresponding to 1.0 mg of retinol less consumed in 8 weeks by treated rats. Serum levels of retinols varied widely and showed no consistent trends while hepatic retinol concentrations steadily increased in controls over 8 weeks and remained constant in treated rats. Retinol storage in livers of treated rats, expressed as a percentage of storage in control rats, decreased steadily to 30% of controls after 8 weeks. . The authors concluded that this difference represented an increase in retinol storage by control rats that was 352 �blocked after TCDD treatment which the authors suggested was caused by an enzyme-induced increase in retinol turnover. No systemic toxicological significance or indications of hepatic toxicity seem to be associated with the altered vitamin A. concentrations and storage levels in liver reported in this study. 976. Tinsley, I..J. Chemical concept s in pollutant behavior. Wiley-Interscience, 1979),265 p. 977. Todd, R. L. 52:663-664. (New York: (1962) A case of 2-4D intoxication. J. Iowa Med. Soc. A case involving dermal exposure of a 52-year-old man to 2,4-D is described. Two exposures, one on the arm and one on the leg were followed by episodes of nausea and vomiting during the following 5-10 days. The second episode also produced a low-grade fever. A general feeling of weakness noted after the second exposure developed into paralysis of the leg muscles, weakness of the arm and hand muscles, and losses of deep pain sensations, vibratory sensations, and deep tendon reflexes. Severe pain in the posterior portion of the thigh was treated with analgesics. After 6 months of physical therapy the patient was able to walk aided by crutches, and after 2 years was able to walk unaided. A transcent bone-marrow depressed was also observed accompanied by leukopenia and granulocytopenia of the peripheral blood. 978. Tognoni, G. (1977) Health survey at Seveso. NATO Ecotoxicology Workshop, Univ. of Surrey, Guilford, Surrey, U.K. [Not available.] 979. Toth, K., Somfai-Relle, S., Sugar, J., and Bence, J. (1979) Carcinogenicity testing of herbicide 2,4,5-trichlorophenoxyethanol containing dioxin and of pure dioxin in Swiss mice. Nature 278(5704):538-549. This report describes the results of a feeding study, the preliminary results of which were reported in Toth et al., 1978. Twelve groups of 10-week-old Swiss/H/Riop mice weighing 24-30 g were g'iven 2,4,5trichlorophenoxyethanol (TCPE) and TCDD, TCDD alone, or only the vehicle. The groups receiving a combination of TCPE and TCDD consisted of 100 males and 100 females, while those receiving TCDD or the vehicle alone consisted of 45 males. The study was conducted in three phases, consisting of groups 1-3, 4-8, and 9-12; the first two phases considered animals receiving various combinations of TCPE and TCDD; controls received carboxymethylcellulose as the vehicle. The third phase dealt with animals receiving TCDD only; controls received sunflower oil as the vehicle. TCPE containing TCDD was suspended in a salt solution of 0.5 percent carboxymethylcellulose, and TCDD given alone was dissolved in sunflower oil. The animals were given various 353 �doses by gastric tube once a week for a year and observed for their lifetimes. The TCPE administered to group 1 was 99.7 percent pure. The LDc0 of TCPE containing .1 ppro. TCDD is 1,320 mg/kg when an acute, peroral dose is given. The maximum tolerated dose was determined to be 70 mg/kg; this was the largest dose that produced no noticeable tissue lesions in the mice during 6 months of treatment. Moribund animals and those died spontaneously were autopsied and their organs were histologically examined. Sections stained with haematoxylin and eosin were examined by light microscopy. Electron microscopy and polarized light were used to identify amyloid deposits stained with Congo red. Statistical analysis was made of the pathological findings. The cumulative data indicate that the only significant difference between treated groups and controls was in the incidence of liver tumors in males. Groups 1, 2, and 10 showed the highest incidence of liver tumors (48, 58, and 48 percent, respectively)—about twice that of their control groups (3 and 12). Group 1 received TCPE at 67 mg/kg and TCDD at .112 ug/kg, in the vehicle given at 50 mg/kg; group 2 received 70.0 mg TCPE per kg and .007 ug TCDD per kg, in the vehicle given at 50 mg/kg; and group 10 received TCDD at .7 ug/kg in the vehicle given at 10 mg/kg. Smaller doses of TCPE and TCDD caused less frequent tumors; groups 5 and 6 showed insignificant differences from control groups 7 and 8. TCDD when given alone was found to have a significant liver tumor-enhancing effect only at the second highest dose (group 10 - .7 ug/kg: 48 percent incidence compared to 18% in controls). Group 9, which received the largest dose of TCDD (7.0 ug/kg) showed a considerable decrease in the average life span, and severe, chronic, ulcerous skin lesions followed by generalized lethal amyloidosis. Skin lesions and amyloidosis were observed in all groups fed TCDD alone. The authors concluded that both TCPE and TCDD enhance liver tumors in male mice, and that this effect is dose-dependent. Severe skin lesions resulting form TCDD may eventually lead to amyloidosis. 980. Toth, K., Sugar, J., Somfai-Relle, S., and Bence, J. (1978) Carcinogenic bioassay of the herbicide 2,4,5-trichlorophenoxyethanol (TCPE) with different 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) content in Swiss mice. Progress in Biochemical Pharmacology 14:82-93. The authors studied the hepatic effects in mice of 2,4,5-trichlorophenoxyethanol (TCPE) and TCDD. Five groups of 200 Swiss H/RIOP mice, half of them male and the rest female were treated with a .variety of combinations of 2,4,5-trichlorophenoxyethanol (TCPE) and TCDD. Three additional groups receiving only TCDD consisted of 50 males each. The mice were 10 weeks old and weighed 25-30 g at the beginning of the experiment, and they received the compound through gastric intubation once a week for 1 year. They were observed for 1 year following cessation of treatment. Moribund mice, those that died spontaneously, and a healthy mouse from each treatment group were autopsied, and their organs were examined histologically. Tissue samples were fixed in formol and set in paraffin, stained with hematoxylin and eosin, and examined by light microscopy. Amyloid deposits were stained with Congo red, and examination under the polarizing microscope showed greenish birefringence. Liver specimens were fixed in a 2.5 percent solution of 354 �glutaraldehyde and then in 1 percent osmium tetroxide. They were then dehydrated in alcohol and embedded in durcupan, and stained with uranyl acetate and lead citrate. These specimens were examined under electron microscope. TCPE containing TCDD was suspended in a salt solution of .05 percent carboxymethyl cellulose, and TCDD alone was dissolved in sunflower oil. The LD_ 0 of TCPE containing 0.1 ppm TCDD was found to be 1,320 mg/kg when an acute peroral dose was given to 6 mice. The maximum tolerable dose was determined to be 70 mg/kg. Three groups of 100 male and 100 female mice served as controls; 1 received only the carboxymethyl cellulose vehicle, and 2 were untreated. A fourth control group of 50 male mice received the sunflower oil vehicle. The doses given to the treated animals,were as follows: Groups 1 and 2 were fed 70 mg TCPE/kg and 7 x 10 or 7 x 10 mg TCDD/kg, _? respectively; Group 4 mice received 7.0 mg TCPE/kg with 7 x 10 mg TCDD/kg; Group 5 received 0.7 mg TCPE/kg with 7 x 10 mg TCDD/kg; Group"6 received 7.0 mg TCPE/kg with 7 x 10 mg_TCDD/kg; and Groups 9, 10, and 11 received only TCDD in doses of 7 x 10~ mg/kg, 7 x 10~ mg/kg or 7 x 10 mg/kg, respectively. The male mice in groups 1 and 2 showed twice the incidence of liver tumors (43% and 54%) as did the controls (15-28%) after the 2d year of observation. Decrease in the dose of TCPE resulted in a lesser incidence of liver tumors (15-21%) that was comparable to the controls. The dose of; TCDD given alone did not appear to be related to the development of liver tumors in any of the mice. There were noticeable similarities between the tumors of control and treated animals on both a macro- and microscopic«level. Tn one group which was administered only TCDD, at a dose of 10 mg/kg, following autopsy of 19 mice, there were 8 cases of dermatitis associated with arayloidosis involving at least one organ. Three of these animals had post-necrotic liver cirrhosis, and in some cases focal necrosis was apparent. Lower doses of TCDD did not have these effects. The observed effects of TCDD alone were not examined in depth, as this portion of the test was still in progress. 981. Townsend, J. C., Bodner, K. M., VanPeene, P. F. D., Olson, K. D., and Cook, R. R. (1981) Survey of reproductive events of wives of employees exposed to chlorinated dioxins. Unpublished draft report of Dow Chemical Co., Inc. 39 p. 982. Toxic liquid wastes now biodegradable. April 1981, 2 p. (J981) Research News, (USDA) [Editorial.] 983. Toxicology Information Response Center. (1979) Health effects and environmental fate of 2,3,7,8-tetrachlorodibenzodioxin (TCDD) - A Bibliography, 1976-1979. 40 pp. [Bibliography.] 355 �984. Truhaut, R., Pham-Hau-Chanh, Van Haverbeck, G., Azurn-Gelade, M. C., Saint Ruf, G., and Lareng, L. (1974) Acute toxicity of tetrachlorodibenzo-p-dioxin in rats; structural, ultrastructural, and emzymological study of the liver. C. R. Hebd. Acad. Sci. Ser. D. 279:1565-1569. [Not available.] 985. TRW Inc. (1978) At sea incineration of Herbicide Orange on board the M/T Vulcanus. (1973) Prepared for the US-EPA, Industrial Environmenta1 Research Laboratory, Office of Energy, Minerals, and Industry, Research Triangle Park, NC. NTIS Publication No. PB281690. [Background material.] 986. Tsapko, V. P. (1966) The herbicide 2,4-D as a health hazard in agriculture. Gig. Sanit. 31:449-450. Clinical symptoms that developed in farmers exposed to 2,4-D and the toxicity of 2,4-D in mice were reported. Farmers (number not given) who started to work in a field sprayed with 1 kg/hectare of 2,4-D 1 hour previously complained of general weakness, headache, vomiting, chest pain, and loss of consciousness after 40 minutes. Three days after another field was sprayed with 2,4-D from a tractor, farmers that worked in the field for up to 1 hour complained of headache, burning of the mouth, vertigo, vomiting, general weakness, and fever. A single intragastric dose of 370 rag/kg of 2,4-D produced 100% mortality in mice, while 182.5 rag/kg was not lethal to any mice (numbers of mice tested were not reported). The animals died 1-2 days after they received 2,4-D and exhibited paresis of the limbs, clonic-tonic spasms, shallow respiration, and began comatose prior to death. The authors concluded that the commercial preparation they tested was more toxic than other 2,4-D preparations reported elsewhere because it contained toxic impurities and recommended standardizing the compositions of these preparations as well as requiring that protective clothing be worn by workers in fields 1-2 days after 2,4-D spraying. 987. Tschirley, F. H. (1969) Defoliation in Vietnam. Science 163:779-786. [Review article.] 988. Tuchmann-Duplessis, H. (1978) Pollution de 1' environment et descendance. A propos de 1' accident de Seveso. Med. et Hyg. 36:1758-1766. [Review article.] 356 �989. Tucker, D. P. (1978) Bromacil and diuron residue levels in Florida citrus soils. Pest. Monit. J. 12(2):47-50. The authors present data on bromacil and diuron residues in citrus soils following herbicide application for 7-8 years. Two paired blocks of citrus, 10 acres each, were selected as sampling sites. In one block weeds were controlled by tillage while in the block herbicides were used. Herbicide application generally occurred once per year. Soil types differed in each sampling location. The soil in the Polk County grove was Astatula fine sand; the soil at the Hardee County grove was Mayakka fine sand. Each site averaged 114-127 cm rainfall per year. The Hardee County site had permanent supplemental irrigation (30-50 cm/yr), while the Polk county grove recieved only supplemental irrigation. Herbicides were sprayed as wettable powder formulations of bromacil (3.61 kg/ha) and diuron (1.8 kg/ha). Soil samples from 0-15 cm and 15-30 cm depths were collected in rows between trees, at the drip line or under the tree canopy. Each sample was a composite of 10 subsamples. Samples were collected three times in Polk County and in Hardee County, and were analyzed by microcoulometric gas chromatography (bromacil) or colorimetrically after chromatographic cleanup (diuron). Limits of detection of these analytic methods were not reported. Residue levels expressed as percentages of total herbicide applied over 7-8 years ranged from 0.3-3.9% for bromacil and from 3.7-13.1% for diuron. As percentages of only the last application, the residue levels range from 2.5-31% for bromacil and from 33.6-84.6% for diuron. According to the authors, this is an indication that a large part of the herbicide application remains after 1 year. Bromacil was more evenly divided among the soil layers sampled than diuron, probably a reflection of the greater solubility of bromacil in water. Overall residues were higher in Mayakka fine sand than in Astatula fine sand. The authors conclude that the data indicate that levels of these herbicides that would be toxic to citrus fruit would not accumulate in the soils tested. 990. Tulp, M. Th. M., and Hutzinger, 0. polychlorinated dibenzo-p-dioxins. (1978) Rat metabolism of Chemosphere 9:761-768. Metabolites of polychlorinated dibenzo-p-dioxins were identified. Male Wistar rats were administered a single oral dose of 250 mg/kg of 1 of 8 dibenzo-p-dioxins. Urine and feces were collected for the subsequent 7 days and were analyzed for metabolites. Metabolites were isolated by thin layer chromatography and identified by gas chromatography-mass spectroscopy. The dibenao-p-dioxins administered included 1 nonchlorinated, 2 mono-chlorinated, 2 di-chlorinated, 1 tri-chlorinated, I tetra-chlorinated and 1 octa-chlorinated compound; 2,3,7,8-TCDD was not tested. The monochlorinated compounds and non-chlorinated compound were excreted as sulfur-containing metabolites. All compounds, except the octa-chloro compound, were metabolized to mono- and dihydroxy derivatives. Primary hydroxylation occurred only at the 2,3,7 or 8 position. None of the compounds were metabolized by cleavage of a carbon-oxygen bond. The octa-chloro ompound was not metabolized. The authors concluded that dibenzo-p-dioxin metabolism resulted exclusively 357 �by formation of 2,3-epoxides and that in the 2,%7,8-TCDD the relevant carbons are chlorinated, blocking this metabolic pathway and preventing its metabolism. 991. Tung, T. T. (1973) Primary cancer of the liver in Viet Nam. Chirurgie 99(7):427-436. The authors reported that between 1955 to 1961, 159 patients with primary liver cancer out of a total of 5,492 cancer patients were admitted to hospitals in Hanoi, North Vietnam. Between 1962 and 1968, the prevalence of liver cancer cases increased to 791 patients out of a total of 7,911 cancer patients. The increased prevalence of liver cancer in North Vietnam was attributed to the spraying of herbicides in South Vietnam which began in 1961. The appearance of liver cancer in the north was believed to be the result either of population mixing between the north and south regions, or by transport of the herbicide to the north by wind or contaminated mammals, birds, or insects. The report also described methods of diagnosis and treatment of liver cancer. A viral origin of the liver cancer was considered unlikely following analysis of the patients' blood for the presence of antigen which was found in 2.05% of the patients. The authors provided no rationale as to why this particular test eliminated the possibility of a viral etiology. No other possible etiology was considered (i.e., changes in diet or environment) and no proof was provided to support the possible relationship between the prevalence of liver cancer in North Vietnam and herbicide spraying in South Vietnam. 992. Tung, T. T. (1973) Primary carcinoma of the liver in Viet Nam. Pesticide Abstracts 7(6):4-1355. [Abstract, only.] 993. Tung, T. T., Anh, T. K., Tuyen, B. Q., Tra, D. X., and Huyen, N. X. (1971) Clinical effects of massive and continuous utilization of defoliants on civilians. Vietnamese Studies 29:53-81. Health effects of South Vietnamese refugees to North Vietnam are presented. A group of 179 refugees out of a total of 903 refugees in Hanoi were selected for this study. (The basis for the selection was not described.) The refugees lived in sprayed areas for 2 months to 5 years. The refugees described the effects of spraying that were elicited immediately after exposure as well as symptoms that persisted for months or years. Chromosomal aberrations were investigated in peripheral blood smears. The content of the spray or the frequency of exposures for individuals reporting symptoms was not addressed. Immediately after spraying, refugees experienced rhinorrhea, continuous sneezing, vomiting, headache and asthenia. These symptoms do not last past 4 days. Other patients experienced swelling of the eyelids, and a sensation of burning on the skin. Prolonged symptoms included asthenia, visual difficulties in reading, and chromosomal aberrations. 358 �For each patient, 300 cells were analyzed and a total of 1600 cells of spray victims (apparently from 5-6 patients) were analyzed. These cells had a higher anomaly rate (5.9 per 100 cells) than the control group rate of 1.1 per 100 cells). The control population was not described. Three cases of Trisomy 21 were described; in only 1 case the time of exposure was considered - the mother left South Vietnam 7 months before the premature birth of the Trisomy 21 child. Other information needed to determine whether the mother or father was exposed to any spray at relevant times was not provided. One case of malformations was described, also without accounting for when exposure occurred relative to conception. The authors concluded that defoliant spraying in Vietnam produced ocular lesions and genetic defects. The correlation between herbicide exposure and adverse health effects in this report is not convincing. The authors do not document exposure to herbicides specifically nor have they demonstrated a causal effect between spraying and chromosomal aberrations or illnesses in the exposed group (which does not appear to be selected in an unbiased manner). 994. Tung, T. T., Lang, T. D., and Van, D. D. (1980) The problem of mutagen effects on the 2nd generation after exposure to herbicides. The reproductive effects of North Vietnamese populations were studied. Former South Vietnamese soldiers who subsequently migrated to North Vietnam and married women from North Vietnam villages that were not sprayed with chemicals were interviewed regarding the numbers and types of birth defects among their children. Information for some locations were obtained from hospital obstetric records which did not include information on the military service of the father. Half of the 30 birth defects among couples from Yen-Bai were in children whose father was a veteran from South Vietnam and whose mother was from this northern region (the proportion of parents fitting this description for all of the births during 1975-78 in this province was not disclosed). Of these 15 children, five children had limb defects, 6 had anencephaly (usually with other deformities) and the remaining children had cleft palate (1), other cranial abnormalities (1) or abdomenal abnormalities ( ) Birth defects in the 15 children, whose fathers were not southern 2. veterans included 4 cases of hydrocephaly, 8 cases of Batrachian abdomen, 3 cases of hare lip, 1 each of cleft palate, club foot, and anal imperforation. In Quy Mang district, all 9 birth defects of the 233 births between 1976-1978 were in children whose fathers were southern veterans. Thirty families of the 4500 inhabitants (number of families not specified) had fathers that were southern veterans. The birth defects occurred in 6 families and included 3 anencephalies (all in the same family), 1 cleft palate, 1 hydrocephaly, 3 hare lips and 5 cases of limb abnormalities. No other information, including descriptions of families, herbicide exposure potential, sources of information or clinical conditions were presented for these people. The incidence of birth defects, abortions and premature deliveries and sterility were all reported to be higher among families of veterans from the south than the north. The authors concluded that the southern veteran was more likely to have.offspring with birth defects than other 359 �Vietnamese fathers. The types of records examined (and their reliability), the clinical definitions of malformations, the family histories, ages of parents, any evidence that herbicide exposure was different among fathers, other concomitant exposures and many other factors, important in evaluating the etiology of these birth defects were not reported. 360 �995. Ulanova, I. P. (1975) Toxicometry and prophylactic toxicology. In Methods Used in the USSR for Establishing Biologically Safe Levels of Toxic Substances. Paper presented at a World Health Organization meeting held in Moscow, December 12-19, 1972; 45-55. [Review article.] 996. U.S. Air Force. (1979) Aircraft Sampling Westover AFB MA. USAF OEHL Technical Report: OEHL 79-59. USAF, OEHL, AFSG, Brooks AFB, Texas, 5 p. [Background material.] 997. , U.S. Air Force (1979) Herbicide Orange site treatment and environmental monitoring. USAF OEHL Report: OEHL TR-79-169. USAF, OEHL, Brooks AFB, Texas. 37 p. [Background material.] 998. U.S. Air Force Armament Laboratory. (1968) Biological effectiveness of stull bifluid and orange. Technical Report No. AFATL-TR-68-122. [Background material,] 999. U.S. Air Force Armament Laboratory. (1969) Comparison Test of Defoliants. Vol. II. Appendix III. Biological effectiveness of stull bifluid and Orange No. ADTC-TR-69-30. [Background material.] 1000. U.S. Air Force Armament and Development Test Center. of Defoliants Vol. I. Authors. No. ADTC-TR-69-30. (1969) Comparison [Background material.] 1001. U.S. Air Force. (1966) An ecological study on the effects of certain concentrations of cacodylic acid on selected fauna and flora. No. APGC-TR-66-54. 23 p. [Background material.] 1002. U.S. Air Force. (1975) Studies of the ecological impact of repetitive aerial applications of herbicides on the ecosystem. AD-A032 773. 127 P[Background material.] 361 �1003. U.S. Department of Health, Education, and Welfare. (1978) Bioasaay of picloram for possible _carcinog_e_ni_city. National Cancer Institute Careinogenesis Report Series No. 23. 91 pp. In this report, the carcinogenicity of technical grade picloram (90% pure) was evaluated in mice and rats. Osborne-Mendel rats (50 animals of each sex per dosage group) or B6C3F1 mice (80 animals of each sex per dosage group) were fed picloram for 80 weeks. Only 10 animals of each sex were used as matched controls. The low-dose rats were fed 10,000 ppm picloram for 39 weeks, then 5,000 ppm for 41 weeks for a time weighted average of 7,437 ppm. The high-dose group of rats received 20,000 ppra picloram for 39 weeks and 10,000 ppm for 41 weeks. Animals were observed for another 33 weeks before termination of the study. In mice, the low-dose group was fed 5,000 ppm for 1 week and 2,500 ppm for 79 weeks, while the high-dose group was fed 10,000 ppm for 1 week and 5,000 ppm for 70 weeks. Time weighted averages were 2,531 and 5,062 ppm for the low- and high-dose groups, respectively. Animals were observed for 10 weeks after treatment was terminated. Picloram was mixed with the standard lab diet with acetone and corn oil each at 2% of the final feed weight. Treated feed was kept at 17°C for no longer than 1 week. Samples of formulated feed were analyzed for picloram concentration at intervals through the experimental period. The mean analytical concentrations for the tested samples was within 2.5% of the calculated concentration; the coefficient of variation did not exceed 6.7%. Pood and water were supplied to the animals ad libitum. During the course of the study all animals were observed for signs of toxicity, tumor formation, and body weight gain. No blood chemistry of liver enzyme analyses were performed. Pathological evaluation of animals who were killed or died during the study included gross and microscopic examination of major organs and tissues, and all lesions. Signs of toxicity in rats began to be observed during the second 6 months of treatment and included diarrhea, hematuria, and rough coats. During the second year of the study, additional signs of toxicity occurred: pale mucous membranes, dermatitis, alopecia, tachypnea, discolored urine, diarrhea, and vaginal bleeding. However, no significant differences in survival were observed between control and treated groups. A relatively high incidence of follicular hyperplasia, c-cell hyperplasia, and c-cell adenoma of the thyroid was observed in both sexes. However, statistical tests did not show evidence for association of these conditions with picloram treatment. There was some evidence of liver effects associated with picloram treatment. Females were more effected than males. Increased incidences of hepatic neoplastic nodules, a benign lesion, were observed in treated animals of both sexes compared with untreated controls, which was statistically significant (p=0.016) in females but not males. In addition, both males and females possibly developed a treatment related lesion, foci of cellular alteration, that is frequently associated with induction of hepatic carcinoma and neoplastic nodules in rates. No statistics on this lesion were presented. In mice, signs of toxicity associated with picloram treatment were not observed until the second year of the study. These included slight hyperactivity, rough hair coat, and abdominal distention. No differences in survival occurred. No tumors associated with 362 �picloram treatment were found in either male or female mice. From this bioassay, the report concluded that picloram was not carcinogenic in male Osbourne-Mendel rats or B6C3F1 mice. In female rats, however,' incidence of benign tumors was associated with picloram treatment. 1004. U.S. Department of Health and Human Services. [Fourth progress report of Interagency Work Group made public.] News Release, August 1, 1980. 5 pp. [Background material.] 1005. U.S. Environmental Protection Agency. (1979) Preliminary report of assessment of a field investigation of six-year spontaneous abortion rates in three Oregon areas in relation to forest 2,4,5-T spray practices. Prepared by the Epidemiologic Studies Branch Benefits and Field Studies Division OPP, OTS, EPA. The report describes a retrospective study of 2,4,5-T exposure and the incidence of spontaneous abortion. Three cohort groups were selected from a highly sprayed control forest region (Study) a bordering nonforested agricultural region (Urban), and a control area with little reported use of 2,4,5-T. To estimate exposure rates information or when, where, and how much 2,4,5-T was sprayed was obtained. Incidence data were abstracted by hospital personnel and staff epidemiologists at hospitals in each.of the three areas. Physician interview data supplemented abstracted data. A spontaneous abortion index with a 5-month moving average was used to describe incidence. Statistical methods used in the covelational analaysis included: analysis of variance, frequency tables by chi-square, power spectrum analysis, crosscorrelation analysis, both parametric and non-parametric statistics and linear multiple regression fitted to a sine wave model. Data show the following: significantly higher spontaneous abortion rate for the study group than either the control or urban group; a statistically significant seasonal cycle in the abortion index with approximate 4-month peaking period observed in the study group in June; a significant cross-correlation between the study group spontaneous abortion index rates and exposure by month with an lag of 2 or 3 months. 1006. U.S. Environmental Protection Agency, Office of Pesticides Programs. (April 21, 1978) Rebuttable presumption against registration and cont'd registration of pesticide products containing 2,4,5-T. Federal Register 43:78. pp. 6500-6501. [Background material.] 363 �1007. U.S. Environmental Protection Agency. (1978) Pesticide programs Rebuttable presumption against registration and continued registration of pesticide products containing 2,4,5-T. Federal Register 43(78):17116-17147. [Review article.] 1008. U.S. General Accounting Office, The Use of Herbicides and Other Chemicals in Vietnam. Letter and attachments to R. H. Metcalfe, U. S. House of Representatives, on August 16, 1978. 14 pp. [Background material.] 364 �1009. Van Logten, M. J., Gupta, B. N., McConnell, E. E., and Moore, J. A. (1980) Role of the endocrine system in the action of 2,3,7,8-TCDD on the. thymus. Toxicology 15(2) : 135-144. The toxic effects of TCDD were assessed in adrenalectomized rats and hypophysectomized rats. Female Fricher rats were administered one dose of 10-20 ug/kg TCDD in cprn oil by oral gavage. Some rats underwent adrenalectomy or hypophysectomy at least one week prior to TCDD treatment and some of the hypophysectomized rats were administered 0.25 mg growth hormone subcutaneously daily for 11 days, beginning 1 day prior to TCDD treatment. Three or 10 days after TCDD was given to experimental rats or corn oil was given to controls, rats were killed, hematological analyses were performed in blood samples, and tissues were removed, weighed, and examined histologically. After 3 and 10 days, body weights and thymus weights relative to body weights for both adrenalectomized rats and normal rats given 10 ug/kg TCDD were significantly below corresponding rats that did not receive TCDD. After the higher dose was administered, these effects were more pronounced and were accompanied by decreased ulcerine weight and increased liver weight, both relative to body weight, thymic involution, and hepatic necrosis. Hypophysectomy even accompanied by growth hormone administration failed to prevent weight loss or thymic involution induced by TCDD treatment. Upon histologic evaluation, the thymus of hypophysectomized rats given TCDD was more affected than the thymus of normal rats treated with TCDD and rats given growth hormone and TCDD. No increases in peripheral blood lymphocytes or polymorphonuclear leukocytes occurred in TCDD-treated rats. The authors concluded that the adrenal-thymus axis was not involved in the TCDD effect because the spleen and adrenals were not affected. Thymic atrophy from TCDD treatment was also not mediated by the adrenals or the pituitary gland. 1010. Van Miller, J. P., and Allen, J. R. (1977) Chronic toxicity of 2,3,7.8-tetrachlorodibenzo-p-dioxin in rats. Fed. Proc. Fed. Am. Soc. Exp. Biol. 36:396. [Abstract, only.] 1011. Van Miller, J. P., Lalich, J. J., and Allen, J. R. (1977) Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8tetrachlorodibenzo-p-dioxin. Chemosphere (9):537-544. The authors investigated the carcinogenicity of TCDD in rats. Male Sprague-Dawley rats (10 per dose level) weighing about 60 g each were fed a diet containing 0, 1, 5, 50, or 500 ppt or 1, 5, 50, 500, or 100 ppb TCDD. Diets were formulated by adding TCDD in suspended acetone and dissolved in corn oil to standard lab chow. No analysis of the food was made to determine if the formulations were correct or if formulations degraded under storage conditions. The animals were fed the TCDD containing diet for 78 weeks, at 65 weeks laparotomies were performed on surviving animals to determine if tumors were present. 365 �Animals were observed up until 95 weeks. Complete necropsies were performed on all animals and microscopic examination of major tissues was performed. Animals receiving 50, 500, and 1000 ppd died during the first 4 weeks of the experiment. Gross pathological examination revealed atrophy of the thymus and spleen, dilation of common bile ducts, and gastrointestinal tract hemorrhage. Microscopic examination of tissues detected severe liver necrosis and cellular proliferation of the common bile ducts. Decreased spermatogenesis was also observed in many of the animals. All of the animals in the 1 and 5 ppb groups died by the 90th week of the experimental, while 4-8 animals died per group at the 0-500 ppt dose levels. No death-related dose effect was observed. The overall incidence of neoplasms in the 5ppb was 38%. No tumors were found in the 1 ppt on the control groups. The number of neoplasms or the number of animals with neoplasms detected did not increase with increasing TCDD dose. Liver tumors occurred only in animals receiving 1 or 5 ppb TCDD. While tumor incidence of treated groups was higher than control groups, the experiment uses too few animals to prove conclusively that TCDD is carcinogenic. The authors concluded that the data suggest that TCDD may be carcinogenic. 1012. Van Miller, J. P., Marlar, R. J. , and Allen, J. R. (1976) Tissue distribution and excretion of tritiated tetrachlorodibenzo-p-dioxin in non-human primates and rats. Fd. Costnet. Toxicol. 14:31-34. The distribution of TCDD in tissues of adult and infant Rhesus monkeys and in rats is presented. Single intraperitoneal doses of 400 ug/kg [ H]-TCDD in corn oil were administered to 3 adult female monkeys, 4 male infant monkeys (2-4 months of age) and 5 adult male Rprague-Dawley rats. Urine and feces were collected for 7 days. Body fluids and tissues were analyzed for radioactivity and tissue samples were prepared for light microscopy and liver samples for light microscopy. Seven days after exposure, the adult monkeys, infant monkeys and rats lost 11%, 21%, and 11% respectively of the initial body weights. Thymic atrophy and fatty infiltration of the liver were observed in the rats, hypertrophied hepatocytes were observed in adult monkeys, and proliferation of the smooth endoplasmic reticulum was observed in all livers. The percentages of the dose of radioactivity excreted in urine were 1.1, 2.0, and 0.5%, in adult monkeys, infant monkeys and rats, respectively, and in feces were 4, 1, and 5% respectively. Total recovery of radioactivity was 59-72% for the 3 groups. Differences in tissue distribution of TCDD were observed for the 2 species. Over 40% of the administered dose was retained in rat liver at 7 days and 10% was retained in the liver of monkeys. The fat, skin and muscle of monkeys had relatively higher levels of radioactivity than these tissues from rats. The authors concluded that the higher rat hepatic levels were in accordance with smooth endoplasmic reticular proliferation and hepatotoxicity in the rat and the higher skin levels for monkeys correlated with dermal lesions seen in monkeys but not rats. 366 �1013. Verhulst, H. L., and Grotty, J. J. (1968) Deaths from chlorinated phenoxyacetic acids (2,4-D, 2,4,5-T; MCPA). National Clearinghouse for Poison Control Centers, Bulletin. Atlanta, Communicable Disease Center, pp. 1-4. A brief review of reports of poisoning by 2,4,5-T and 2,4-D to the National Clearinghouse for Poison Control Centers is presented. The bulletin mentioned that several cases of skin rash and of peripheral neuritis were reported after exposure to these herbicides. After two sisters played in a heavily sprayed area for several hours, shortly after the spraying was done, inflammation of the mouth, lips, eyelids, and mucous membranes and a skin rash developed; poison oak was also a suspected cause of these illnesses. A Michigan farmer repeatedly unplugged an herbicide (specific compound was not mentioned) sprayer and developed peripheral neuropathy 4 days later. Other symptoms associated with unspecified chlorinated phenoxyacid derivatives were listed. A fatal suicide case of a 48-year-old man was described. The victim had ingested "a cupful" of weed killer which contained 20% 2,4-D and 40% 2,4,5-T. He vomited within 1 hour and became dazed. After 1 day, the patient was febrile, had low blood pressure, was hyperventilating, and had elevated blood urea nitrogen level. Drug therapy failed to reverse the clinical systems and an erythemia and anuria developed. The patient died 46 hours after the ingestion, of cardiac arrest. Symptoms were compared to a case of MCPA poisoning. The authors concluded that some controversy has arisen regarding the toxicity of 2,4-D and 2,4,5-T. 1014. Verrett, J. (April 7 and 15, 1970) Effects of 2,4,5-T on man and the environment. Hearings of the Committee on Commerce (U.S. Senate). (Washington, DC: U.S. Gov't. Printing Office), p. 190. [Not available.] 1015. The Veterans Administration. (1981) Advisory Committee on HealthRelated Effects of Herbicides - Transcript of Proceedings. Feb 4, 1981. 158 pp. [Background material.] 1016. The Veterans Administration. 1(1). 4 p. (1980s) Agent Orange Bulletin, December [Background material.] 1017. The Veterans Administration. (1980b) Advisory Committee on HealthRelated Effects of Herbicides - Transcript of Proceedings (Third Meeting, Dec. 12, 1979), 123 pp. [Background material.] 367 �1018. The Veterans Administration. (1980c) Advisory Committee on Health-Related E f f e c t s of Herbicides - Transcript of Proceedings (Fourth Meeting, April 23, 1980), 123 pp. [Background material.] 1019. The Veterans Administration. (1980d) Advisory Committee on HealthRelated Effects of Herbicides - Transcript of Proceedings (Fifth Meeting, Aug. 6, 1980), 136 pp. [Background material.] 1020. The Veterans Administration. (1980e) Advisory Committee on HealthRelated Effects of Herbicides - Transcript of Proceedings (Sixth Meeting, Nov. 6, 1980), 144 pp. [Background material.] 1021. The Veterans Administration. (1980f) "Agent Orange" A Selected Bibliography. 2nd ed. 16 pp. [Bibliography.] 1022. The Veterans Administration. (1980h) Proceedings from the 2d Continuing Education Conference on Herbicide Orange. Washington, DC, May 28-30. [Background material.] 1023. The Veterans Administration. (1979) [Agent Orange! News Release, June 1, 1979. 4 pp. [Background material.] 1024. The Office of the White House Press Secretary. News Release, Dec. 11, 1979. 1 p. (1979f) fAgent Orange! [Background material.] 1025. The Veterans Administration. (1979b) [Decision to study exposed Vietnam veterans] News Release, May 29, 1979. 2 pp. [Background material.] 368 �1026. The Veterans Administration. (1979c) [Health related effects of herbicides.] News Release, June 8, 1979. 3 pp. [Background material.] 1027. The Veterans Administration. (1979d) [Health related effects of herbicides.] News Release July 22, 1979. 3 pp. [Background material.] 1028. The Veterans Administration. (1979e) [Testing of Agent Orange carried in body fat.] News Release Feb. 7, 1979. 2 pp. [Background material.] 1029. The Veterans Administration. (1979g) Transcript of Proceedings - In the Matter of: Advisory Committee on Health-Related Effects of Herbicides. June 11, 1979. 143 pp. [Background material.] 1030. The Veterans Administration. (1979h) Transcript of Proceedings - In the Matter of: Advisory Committee on Health-Related Effects of Herbicides (Second Meeting, Sept. 24, 1979), 69 pp. [Background material.] 1031. Veterans Administration Office of the Administrator of Veterans' Affairs. "Agent Orange and Veterans' Health." [Statement of Administrator of Veterans' Affairs] Washington, DC. 3 pp. [Testimony.] 1032. Vinopal, J. H., and Casida, J. E, (1973) Metabolic stability of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mammalian liver microsomal systems and in living mice. Arch. Environ. Contam. Toxicol. 1(2):122-132. Biotransformation of TCDD by hepatic microsomes from rabbit, rat, and mice in vitro and by mice in vivo are described. Liver microsomes were prepared from male New Zealand rabbit, male Sprague-Dawley rat and male Swiss-Webster mouse tissue and were incubated in the presence of [ H]-TCDD. In the absence or presence of added NADPH, all of the radioactivity was recovered by extraction into organic solvents (i.e., as the unmetabolized compound). No metabolites ws>re identified after the incubation period by thin-layer or gas chromatographics. Male mice (2-6 per group) were administered 130 ug/kg [ H]-TCDD in olive oil, 369 �intraperitoneally. Urine and feces collected for 3 days and liver and kidneys removed 1-20 days after exposure were analyzed for radioactive metabolites by thin-layer and gas chromatographics. No metabolites were detected in any samples. Trace amounts of radioactivity were detected in the urine and kidney and 13% of the dose was recovered in the feces. About 15% of the dose was recovered in the liver after 1 and 4 days, 27 and 22% after 8 and 11 days, respectively, and 10% at 15 and 20 days. The microsomal fraction had the highest concentration of radioactivity, of the various subcellular fractions of hepatic tissue. The authors concluded that TCDD was preferentially localized in the endoplasmic reticulum was resistant to biotransformation, and was excreted primarily in feces, potentially from biliary excretion. 1033. Vogel, E., and Chandler, J. L. R. (1974) Mutagenicity testing of cyclamate and some pesticides in Drosophila melanogastin. Experientia 30:621-624. The authors tested 2,4-D and 2,4,5-T Na salt in a sex linked recessive lethal test in Drosophila melanogaster. Adult 2-day-old Berlin K males were fed 2,4-D (4.5 or 9.0 mM) or 2,4,5-T (3.6 or 7.2 mM) for 3 days. Following treatment the males were mated to 2 females for 3 days (brood 1). Males were mated with new females for an additional 3-day brood (brood 2) and a 4-day brood (brood 3). Results of the study were analyzed by the X test with criteria for a positive result being p less than 0.01. Neither 2,4-D nor 2,4,5-T induced increased numbers of recessive lethals compared to controls. However, a decline in fertility in broods 2 and 3 was observed in flies treated with 7.2 mM 2,4,5-T. 1034. Vos, J. G. (1978) 2,3,7,8-Tetrachlorodibenzo-para-dioxin: Effects and mechanisms. In Chlorinated Phenoxy Acids and Their Dioxins, C, Ramel, ed. (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978) p. 165-176. [Review article.] 1035. Vos, J. G. (1977) Immune suppression as related to toxicology. CRG Critical Reviews in Toxicology 5(1):67-101, [Review article.] 1036. Vos, J. G., Kreeftenberg, J. G., Engel, H. W. B., Minderhoud, A., and Van Noorle Jansen, L. M. (1978) Studies on 2,3,7,8-tetrachlorodibenzo-p-dioxin induced immune suppression and decreased resistance to infection: Endotoxin hypersensitivity, serum zinc concentrations and effect of thymosin treatment. Toxicology 9:75-86. The effects of TCDD on thymic atrophy in thymosin-treated mice, on endotoxin hypersensitivity and on macrophage function are described. Swiss mice were administered 1.5-100 ug/kg (98.6% purity) in 370 �acetone-arachis oil (1:9) orally. After 2-5 days E_. coli endotoxin was administered and serum zinc levels and mortality were assessed 48 hours after endotoxin administration. Listgria monocytogenes migration to the spleen was assessed in male mice inoculated 4 days after 4 weekly doses of 50 ug/kg TCDD had been administered. Peritoneal macrophages were harvested from male mice after 4 weekly doses of 50 ug/kg TCDD was administered; glucose oxidation rates of macrophages were estimated by a nitro-blue tetrazolium dye technique. Mice were treated with 10 ug/kg TCDD at 1, 4, 8, 11, 15 and 18 days of age. Thymosin was administered daily for 3 weeks. Thymus cells were isolated-and their response to mitogens was determined in vitro by assessing pH]thymidine incorporation. Thymogen treatment did not increase mitogenic responsiveness of thymic cells or alter thymic weight or serum zinc concentrations in TCDD-treated mice. Susceptibility to endotoxin was enhanced by TCDD treatment, while macrophage oxidation and Listerla phagocytosis and transport to the spleen remained unaltered. The authors concluded that immunosuppression by TCDD resulted from an unknown effect on T-lymphocytes, since no changes in macrophage phagocytosis viability or metabolism occurred which could account for observed endotoxin hypersensitivity. 1037. Vos, J. G., and Moore, J. A. (1974) Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8 tetrachlorodibenzo-p-dioxin. Int. Arch. Allerg. Appl. Immunol. 47:777-794. The effects of perinatal exposure of rats and mice to TCDD on cellular immunity is described. Female Fisher 344 rats and C57B1/6 Sch mice were administered 1-5 ug/kg TCDD in acetone-corn oil (1:6) by oral intubation. Maternal treatments were administered to rats on days 11 and 18 of gestation and on postnatal days 4, 11 and 18, or only postnatally, on days 0, 7 and 14. Maternal treatments to mice were administered on days 14 and 17 of gestation and postnatal days 1, 8 and 15. Male 1-month-old mice were administered 4 weekly doses of 1, 5 or 25 ug/kg TCDD and male 4-month-old mice were administered 6 weekly doses of 1, 5 or 25 ug/kg TCDD. Spleen and thymus lymphocytes were removed from treated animals and cultured in the presence of phytohemaglutinin (PHA) or conconavalin A (Con A) mitogen and [ H]-thymidine incorporation into DNA was measured to assess the proliferative response of cultures to mitogens. Other lymphocytes were exposed to TCDD in vitro only. Graph versus host assays were performed by injecting C57B1/6 donor mouse spleen cells into the right hind foot pad of (C57B1/6 x DBA-2) hybrid recipient mice. The ratio of the weights of the right to left popliteal lymph nodes was used to determine GVH activity. For rat GVH studies, Fisher-344 donor cells were administered to (F-344 x BN) hybrid recipients. Tail skin graphs from DBA-2 mice or (F-344 x BN) hybrid rats were grafted to C57B1/6 mice and F-344 rats, respectively; autografts were also performed. Allograft rejection times were measured. In some experiments hematological parameters and histopathology were evaluated. Rats in the 5 ug/kg TCDD treatment group pre- and postnatally or postnatally only had reduced spleen, thymus and body weights and high fetal mortality; decreased organ weights, but no mortality occurred in the 1 ug/kg 371 �dosage group. Postnatal TCDD treatment resulted in reduced responsiveness of spleen and thymus cells to PHA and decreased GVH activity in rats and mice. Peripheral lymphocyte counts and levels of serum proteins were not significantly altered by postnatal treatment. Skin graft rejection times in mice that were treated pre- and postnatally with 2 and 5 ug/kg doses of TCDD were prolonged. Lymphocytes from one-raonth-old mice treated with 25 ug/kg TCDD showed decreased responsiveness to PHA and decreased thymus weights, while the 4-month-old mice treated with this dose had decreased thymus weights, only. No decrease in GVH activity or in other organ weights occurred in 1- or 4-month-old mice treated with any dose of TCDD. In vitro treatment of lymphocyte with up to 0.02 ug TCDD per ml. medium had no effect on proliferative response to mitogens. Thymus atrophy was confirmed histopathologically in treated mice and rats, although liver damage was not observed. Decreased eosinophilia of acidophilic cells of the adenohypophysis was observed in treated rats. The authors concluded that TCDD-induced suppression of cell-mediated immune responsiveness was an age-related phenomenon which, in the neonate, resembled the effects of thymectomy. The authors postulated that cellular-immunosuppression may be the cause of death in mouse and rat neonates and in adult guinea pigs, all of which fail to develop major liver pathology after TCDD treatment. 1038. Vos, J. G., Moore, J. A., and Zinkl, J. G. (1974) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin fTCDD) in C57B1/6 mice. Toxicol. Appl. Pharmacol. 29:229-241. The acute and subchronic toxicities of TCDD were evaluated in the mouse. Male C57B1/6 mice were administered TCDD (99+% purity) in acetone-corn oil (1:6) by gastric intubation. Single doses of 100, 150 or 200 ug/kg TCDD or vehicle only were administered to 58 mice in an acute study and weekly doses of 0.2-2.5 ug/kg TCDD were administered for 2-6 weeks to 100 mice in a subacute study. Body weights and some organ weights were recorded and necropsies, histologic, and hematologic examinations were performed. Liver sections were stained with periodic acid-Schiff reagent for lipids and iron and was examined by fluorescence microscopy for porphyrins. The oral LD,.- was 114 ug/kg. The mean survival time was about 3 weeks for mice treated with 150 or 200 ug/kg TCDD. Body weight losses occurred, but were followed by terminal increases which were attributed to fluid accumulation. The mean body weight of survivors was 11% below controls 2 months after treatment. Ocular lesions, atrophy of the spleen and thymus, hemorrhagic and distended intestines, hepatic necrosis, lipid vacuoles, and evidence of excessive amounts of hepatic porphyrins were reported after acute exposure. After subacute exposure to 5 or 25 ug/kg doses of TCDD, significant losses in body weight, increases in liver to body weight ratios, and decreases in thymus to body weight ratios occurred. Increases in neutrophils, hemoglobin, mean corpuscular hemoglobin, and total serum protein concentrations occurred in the highest dosage group. Tissue changes after subacute exposure resembled those that occurred after acute exposure. Lipid accumulation in the liver showed a dose-related pattern that occurred from even the lowest dose of 372 �0.2 ug/kg TCDD. The authors suggested that the toxic dose of TCDD to C57B1/6 mice was age-dependent based on their unpublished observations. 1039. Vos, J. G., Moore, J. A., and Zinkl, J. G. (197.3) Effect of 2,3,7,8tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals. Environ. Health Perspec. 5:149-162. The effects of TCDD on cell-mediated and humoral immunity were studied in the guinea pig, rat, and mouse. Female Hartley guinea pigs (10 per group) were administered 8 weekly doses of 0.008-1.0 ug/kg TCDD in acetone-corn oil, orally. Humoral immunity was determined by measuring serum tetanus-antitoxin concentrations by radial iramunodiffusion following 2 subcutaneous injections of tetanus toxoid to guinea pigs. Delayed hypersensitivity to tuberculin was measured as the thickness of the skin at the site of tuberculin injections to sensitized guinea pigs. Pooled serum samples were also analyzed for cortisol and corticosterone levels. Delayed hypersensitivity was also tested in female CD rats (10 per group) administered 6 weekly doses of 0.2-5 ug/kg TCDD, orally. Donor spleen cells were prepared from male C57B1/6 mice administered 4 weekly doses of 0.2-25 ug/kg TCDD, orally. Donor cells were injected into the right hind foot pads of C57B1/6 x DBA-2 hybrid mice. The ratio of weights of the right to left the popliteal lymph nodes of the recipient after 7 days was used as a measure of cell-mediated immunity. Thymic atrophy was evident in guinea pigs administered 0.2 ug/kg TCDD, while the higher dose produced 100% mortality and this group was not studied further. Corticosteroid levels were not altered by TCDD and serum tetanus antitoxin levels were decreased in the 0.2 ug/kg treatment group, only. Skin reactions to tuberculin showed a TCDD dose-related decrease in response in guinea pigs but no effect was seen in the rat. The highest dose of TCDD administered to rats produced thymic atrophy, but no deaths occurred. Severe thymic atrophy was observed in mice treated with 5 and 25 ug/kg and at 5 ug/kg a highly significant decrease in graft versus host activity was observed; spleens of the 25 ug/kg treatment group were too small to perform the experiment. The authors concluded that sublethal doses of TCDD suppressed cell-mediated immunity in the guinea pigs and mice which may have been the cause of death in these species; TCDD also caused a slight suppression in humoral immunity in guinea pigs. 1040. Vos, J. G., et al. (1978) TCDD accident at a chemical factory in the Netherlands. Working Papers. IARC, Lyon - Joint NIEHS/IARC working group report. [Not available.] 373 �1041. Wade, N. 204:817. (1979) Viets and vets fear herbicide health effects. Science [Editorial.] 1042. Wagner, S. L., and Weswig, P. (1974) Arsenic in blood and urine of forest workers as indices of exposure to cacodylic acid. Arch. Environ. Health 28(2):77-79. The levels of exposure of 5 forest workers to cacodylic acid and the corresponding urinary excretion of arsenic are reported. The numbers of hours of exposure and amounts of cacodylic acid used by 5 white men were recorded for an 11-week period. Prescribed clothing and plastic masks were worn by the workers most of the time (the level of compliance was not estimated). Blood samples and 24-hour urine samples were collected at the end of every 5-day work week and were analyzed for cacodylic acid. Blood and urine were analyzed from a control group of 5 forest workers not exposed to cacodylic acid. Physical examinations and blood chemistries, hematological and urine analyses performed just prior to and following the 11 week exposure period revealed no unusual findings. No workers lost time from work for health reasons and the only clinical symptoms reported were nausea, diarrhea and general aches for 2 days by 1 worker. Average duration of exposure was 23.7 hours per week to 817 grams (dry) cacodylic acid per man per week. Both blood and urine levels rose during the first weeks of exposure. Urine levels dropped on the ninth week, when exposure ended. Blood levels dropped after week 4 and the only change that correlated with this drop was a severe heat wave which was suggested to have resulted in higher arsenic excretion in the sweat. No correlation coefficients were calculated for individual cacodylic acid exposure levels and urine or blood arsenic levels. The correlation between urine and blood arsenic levels was low (coefficient = 0.14). The maximum level reached for exposed workers was 0.27 ppm of arsenic in blood and for controls was 0.08 ppm. The maximum urine levels for exposed workers was 519 ppm and for controls was 201 ppm. The authors concluded that cacodylic acid did not cause intoxication when proper handling methods were used. The authors suggested that a strong garlic odor in sprayed areas which was reported by all workers indicated that forest workers may be exposed to the toxic metabolite, arsine gas after spraying cacodylic acid. 1043. Walker, A. E,, and Martin, J. V. (1979) Lipid profiles in dioxinexposed workers. Lancet; 446-447. [Abstract, only.] 1044. Walker, A., and Smith, A. E. (1979) Persistence of 2,4,5-T in a heavy clay soil. Pestic. Sci. 10:151-157. The authors report on the persistence of 2,4,5-T in a heavy clay soil when temperature and soil moisture are varied. Regina heavy clay soil, �0-5 cm layer,, was collected and stored for 3 months. In the laboratory experiments, C-2,4,5-T (2ug/g soil) was used; in field experiments unlabelled 2,4,5-T (5 mg/20x20 cm plot) was used. In the laboratory, degradation of 2,4,5-T (2 ug/g soil) was measured at 8-34% moisture at 10-35 C for up to 70 days. Duplicate samples were analyzed for 2,4,5-T at 7-14 day intervals. Regardless of temperature, at 8% soil moisture little to no 2,4,5-T was degraded. The half life of 2,4,5-T in this soil sample varied from 4 days at 35C and 34% moisture to 60 days at at IOC and 20% moisture. Under field conditions the soil has 40% moisture. In field experiments in the spring, 90% degradation of 2,4,5-T occurred in 50 days when the temperature averaged 16C and rainfall was high (amounts not specified). Under dry summer conditions, degradation was slower even though temperatures were higher. In the fall, when rainfall was low and temperatures averaged IOC, degradation was also slow. These data are consistent with trends observed in laboratory models. The authors concluded that when field and laboratory experiments were compared, the laboratory models generally overestimated the degradation patterns of 2,4,5-T. From these experiments, it appears that degradation of 2,4,5-T is dependent on temperature and moisture content of the soil. The authors did not discuss the importance of biological, chemical, and physical losses which may explain these patterns. 1045. Walker, E. M., Gadsden, R. H., Atkins, L. M., and Gale, G. R. (1972) Some effects of 2,4-D and 2,4,5-T on Ehrlich ascites tumor cells in vivo and in vitro, Industr.Med. 41(l):22-27. The effects of 2,4-D and 2,4,5-T on Ehrlich ascites tumor growth and synthesis of DNA, RNA, and protein by tumor cells were evaluated. Ehrlich ascites tumor cells were maintained in BALB/c mice and daily intraperitoneal doses in dimethyl sulfoxide of 45-75 mg/kg 2,4-D (99% purity) or 62-85 mg/kg 2,4,5-T (99% purity) were administered for 5-6 days. Controls received vehicle only. Average change in body weight, mortality, total packed tumor cell volume, and survival time were recorded. The effects of 2,4-D and 2,4,5-T on de noyo RNA purine synthesis were evaluated in tumor cells in vivo, [ C]-Formate was administered intraperitoneally 24 or 48 hours after a single dose of 25-100 mg/kg of 2,4-D and 2,4,5-T in DMSO was administered intraperitoneally. Three hours after formate was administered, the cells were removed and radioactivity-associated with adenine and guanine were determined. Incorporation of H-uridine, H-thymidine and C-leucine into RNA, DNA, and protein, respectively, was determined in vitro after a 20 minute pulse period by radioactive analysis of TCA-precipitable cell contents. 2,4-D and 2,4,5-T (10 M each) were introduced into the cultures 0-2 hours before the precursors. Both 2,4-D and 2,4,5-T produced ion inhibition of tumor growth in vivo, with 30-73% inhibition of total packed cell volume for all doses of both compounds tested. Survival time was increased by 26% for 2,4-D and 41% for 2,4,5-T. Effects of both compounds on RNA synthesis in vivo were variable and slight increases in RNA, DNA, and protein synthesis were observed for treated cultures. The authors concluded that an appreciable inhibitory effect on Ehrlich ascites tumor growth occurred from 2,4-D and 2,4,5-T 375 �treatments but the changes observed in the remaining biochemical parameters were inadequate to explain the growth inhibition. 1046. Wallis, W. E., Van Poznak, A., and Plum, F. (1970) Generalized muscular stiffness, fasciculations, and myokymia of peripheral nerve origin. Arch. Neurpl. 22:430-439. A clinical description is made of a 21-year old male who suffered from a syndrome of generalized muscular stiffness, fasciculations and myokymia. He developed these symptoms after having sprayed 2,4-D. For one year, the patient had been employed as a farmhand to spray sugar cane fields with 2,4-D herbicide. During spraying, he utilized no protective mask or clothing and handled herbicide freely. Paresthesis of the hands and feet were the first symptoms to develop, followed by painful musclar stiffness in all four limbs. The pain disappared after a week, but the muscular stiffness gradually progressed over the next two years, causing impairment of gait and loss of manual dexterity. Upon clinical examination, the patient exhibited abnormalities in skeletal muscle function and motility, deep tendon reflexes and posture. Passive flexion of the limbs met with a steady uniform resistance that gradually increases as the flexion continued. The facial, massiter, trunk and extremity muscles all displayed myokymio when at rest. The patient was treated with 1,500 mg of diphenylhydantion (DPH) given intravenously. The clinical symptoms improved dramatically and continued treatment with 0.3 gm of DPH daily alleviated practically all of the muscular stiffness, myokymice and postural deformities. To determine the physiologic origin of this nueromuscular disorder, electromyography, peripheral nerve block, nerve conduction velocities, treatment with curare, and muscle and nerve biopsies were performed on the patient prior to treatment with DPH. Based on results of these tests, the authors concluded that the cause was not in the central nervous system since the muscular stiffness was unchanged by sleep, spinal anesthesia, and deep barbituate anesthesia. The authors also concluded that the myokymice did no originate in the post-synaptic neuromuscular junctions since curare eliminated the muscle tetany and all electromyographic activity. The fact that DPH, which acts specifically on motor nerve terminals, alleviated the syndrome led the authors to conclude a peripheral motor nerve origin. Depressed motor nerve conduction velocities and degenrative changes in the biopsy of the sural (sensory) nerve supported the authors conclusions. They stated that the actual cause of the patient's affliction was unknown, but damage to peripheral nerves from exposure to chemicals such as 2,4-D may play an etiologic role. 1047. Walsh, J. (1977) Seveso: The questions persist where dioxin created a wasteland. Science 197:1064-1067. [Review article.] 376 �1048. Ward, C. T., and Matsumura, F. (1978) Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a model aquatic environment. Arch. Environ. Contain. Toxicol. 7:349-357. The authors describe the persistence and metabolic degradation of \TCDD in lake sediment and water under laboratory conditions. Four sediment and lake water samples were used in the study, three from Lake Mendota and one from Lake Wingra in Wisconsin. Samples were prepared by filtering portions of sediment and water to obtain damp sediment and clear lake water. Five grams of sediment and about 18 ml of water were placed in glass tubes to provide an anaerobic environment. TCDD was added to the tubes at 0.71, 1.0, or 1.83 ppm of C-TCDD (label position unspecified) for one hour to 589 days. The authors also reported that 2,4,5-T (100 ppm) was added to some samples, however, no results were presented. Analysis of C-TCDD and metabolites present in sediment or lake water was by thin-layer chromatography. Most of the radioactivity, 94-96%, was recovered from the sediment. TCDD disappeared faster in sediment and lake water together than in lake water alone. The half-life of TCDD in Lake Mendota sediment was calculated to be about 600 days. If TCDD was added to lake water without sediment, approximately 71% of the radioactivity was recovered after 589 days. Nearly all of the TCDD recovered was the parent compound. Percent recovery of radioactivity was related to water loss, suggesting that water-mediated evaporation of TCDD takes place. Of the small amount of radioactivity present in lake water from the sediment plus lake water samples, 33-98% of the radioactivity recovered was metabolites of TCDD. However, of the total original TCDD added, metabolites accounted for only 1-4%. When samples were exposed to low levels of illumination, no significant photolysis of TCDD occurred. The author explained this by the fact that most of the C-TCDD was associated with the sediment and was not exposed to light. The authors concluded that under the laboratory conditions of this study, TCDD is a persistent chemical in lake water sediment and is resistant to microbial attack. 1049. Wassora, J. S., Huff, J. E., and Loprieno, N. (1977/1978) A review of the genetic toxicology of chlorinated dibenzo-p-dioxins. Mutat. Res. 47:141-160. [Review article.] 1050. Watson, A. P., Van Hook, R. I., and Reichle, D. E. (1976) Toxicity of organic and inorganic arsenicals to an insect herbivore. Environ. Sci. Tech. 10(4):356-359. The authors report on the toxicity of two cacodylic acid formulations to meadow katydids. Fourth instar nymphs of Conocephalus fasciatus were collected in the field and placed in experimental cages (10 per cage) with fresh fescue clippings and water containing the herbicide. Cacodylic acid (65% dimethyl arsenic acid, 35% elemental arsenic) was introduced into drinking water at 1.5, 15, 150, 1,500, and 15,000 ppm. 377 �Phytar 560 (4% free cacodylic acid, 23% sodium cacodylate, 73.5% inert ingredients, 12.7% elemental arsenic) was tested at 0.15, 15, 150, and 1,500 ppm. Chronic oral toxicities of both compounds were calculated at 7 and 14 days. LD,-., values obtained were 34.4 ppm (7 days) and 7.5 ppm (14 days) for cacodylic acid, and 10 ppm (7 days) and 3.5 ppm (14 days) for Phytar 560. At 14 days, there was no difference between the two compounds in the elemental arsenic dose required to produce the LD value. The authors concluded that toxicity of these compounds was due to digestive assimilation of elemental arsenic. The authors also concluded that at recommended applications rates, Phytar 560 would be toxic to herbivorous insects, such as the katydid. 1051. Way, J. M. (1969) Toxicity and hazards to man, domestic animals, and wildlife from some commonly used dioxin herbicides. Residue Rev. 26:37-62. [Review article. ] 1052. Weakley, B. S. (1977) How dangerous is sodium cacodylate? Microscopy 109, Pt. 2:249-251. Journal of [Editorial, ] 1053. Weber, J. B. (1972) Interaction of organic pesticides with particulate matter in aquatic and soil systems. In: Fate of Organic Pesticide in the Aquatic Environment. Advan. Chem. Ser. No. Ill; Am. Chem. Soc., Washington, DC: pp.- 55-120. [Background material.] 1054. Weber, J. B., Monaco, T. J., and Worsham, A. D. (1973) What happens to herbicides in the environment? Weeds Today 4:16-22. [Background material.] 1055. Weimer, J. T., Ballard, T. A., Owens, E. J., and McNamara, B. P. (1970) Toxicological studies on herbicide "White" in animals. US NTIS Technical Report No. AD 712 317. 28 pp. The inhalation, dermal, and oral toxicities of single and repeated exposure of rabbits, and rats to herbicide White are described. A commercial preparation of herbicide White and 2 samples from a military source (which were found to be identical chemically and toxicologically to the commercial sample) were used and contained 11.7% triisopropanolamine salt of 4-amino-3,5,6-trichloropicolinic acid, 44.9% triisopropanolamine salt of 2,4-D and water; other ingredients were not revealed by the manufacturer. Compounds were applied under temperate 378 �conditions of moderate temperature and humidity, and tropical conditions of high temperature and humidity. Undiluted White was applied as a single dose or 5 successive daily doses to clipped, undamaged rabbit skin and to sateen cloth tapped to clipped rabbit skin for 48 hr. White was applied to one eye of rabbits and in some cases procedures to prevent infection were implemented. The sites of application were examined twice daily for 30 days. Rats and rabbits were administered undiluted White by oral gavage and ^13 values were calculated from mortality rates at 48 hr. and 5 days. Rats and rabbits were exposed to 1230-1419 rag. White aerosol per cubic meter air from 120 to 270 min. and mortality rates were calculated. Erythema resulted from single exposure of bare skin to 0.03-0.50 ml of White, while necrosis appeared after 5 doses and cleared x^ithin 6 days of the last dose. Doses of 0.02 ml, applied under tropical conditions, produced necrosis by the third day that had not cleared in 10 days. Many rabbits succumbed to the tropical conditions (total number was not reported and this experiment was terminated). Applications to clothed skin under temperate conditions resulted in necrosis only after the highest dose was administered 5 times and occurred in 1 of the 6 rabbits. Single doses of 0.01 ml or higher to the eye, under temperate or tropical conditions, produced eye irritation that was diminished (tropical blepharitis) or disappeared in 10 days. At 0.05 ml or above, bilaterial corneal opacity occurred, which cleared in 21-28 days. The oral LDc0 was 1.67 ml/kg for rabbits and 4.17 ml/kg for rats; all deaths occurred within 24 hours and inactivity was the only observed sign of toxicity. The LCe,, was 150,982 mg-min/cubic meter in rabbits. Deaths occurred up to 14 days after exposure. Signs of toxicity included inactivity during the exposure and blepharitis 4-5 days after exposure. Rats were also inactive during the inhalation exposure, but no deaths or other toxic signs resulted. The authors concluded that White is not likely to produce toxicity by oral, dermal, or inhalation exposures, except for temporary eye irritation and corneal opacity after direct contact to the eye and temporary local skin damage from repeated exposures. 1056. Weirich, J. (1969) Intoxication with diquat (reglone). Dtsch. Gesundheitsw. 24(42):1986-1988. A case report of diquat poisoning is described. A 43 year old man sprayed 4 liters of diquat in 600 liters water per hectare for 6 hours (at the rate of 2 hectares per hour) under windy conditions. On the following day, he experienced fever, dizziness, head and eye pain, and diarrhea, and polyuria. Although the headaches remained 5 days later, he again sprayed diquat (20 liters over 3 hours) under the same conditions. The other symptoms returned and 4 days after the second exposure, these symptoms were alleviated, except for headaches and a feeling of general weakness. These conditions persisted for an additional week, at which time the patient was admitted to the hospital. Disturbances in liver and kidney function and in EKG stimulation were noted and were all reversible over the next 23 days (without treatment). Respiratory effects were never observed. The authors concluded that the windy weather conditions resulted in higher 379 �than usual exposure to diquat, which had rarely been reported to produce signs of toxicity in workers. 1057. Weiss, S. U. , and Beckert, W. H. (1975) Herbicide effects on cultured animal cells. Abstracts 15th Annual Meeting Am. Soc. Cell Biol. p. 451a. [Not available.] 1058. Weissberg, J. B., and Zinkl, J. G. (1973) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin upon hetnostasis and hematologic function in the rat. Environ. Health Perspect. 5:119-123. Hematologic parameters were evaluated in rats 10 and 14 days after daily administration of TCDD began. Female CD rats were orally administered 10 ug/kg TCDD in acetone-corn oil daily for 2 weeks. After 10 and 14 days, blood was drawn by cardiac puncture under methoxyflurane anesthetic from four treated and four vehicle-control rats. Smears of bone marrow preparations were stained for megakaryocyte identification. Bleeding times were estimated by lacerating an ear and observing the time required for bleeding to stop with periodic blotting. Clot extraction, platelet factor III activity, platelet aggregation, prothrombin times, factor X assay, and fibrinogen degradation products were among the hematologic parameters that were determined. The parameters that were significantly elevated in TCDD-treated groups were cell volumes, erythrocyte counts, reticulocyte count (at 14 days only, due to a low control value), neutrophil counts, and times for prothrombin consumption assay with and without inosithin. Significantly lowered values were obtained for TCDD-treated groups than controls for mean corpuscular volume and mean corpuscular hemoglobin at day 10 only, platelet counts and clot retraction. Other parameters were comparable in control and treated animals. The authors suggested that the observed hemoconcentration was consistent with dehydration; alterations in leukocytosis and red cell indices were nonspecific, indicative of widespread toxicity; depression of blood platelets was suggested to result from an antibody response, as normal marrow megakaryocyte levels and absence of fibrinogen degradation products eliminated other possibilities. The authors were unable to explain the prolonged prothrombin consumption time in the absence of altered prothrombin times. Depression of hematopoiesis in monkeys fed toxic fat (reported by others) was concluded to indicate species variability or causative agent other than TCDD. No other systemic effects in the treated rats were mentioned in relation to the hematologic findings and no discussion of the appropriateness of the selected time points was provided. 1059. West, I. (1979) Toxicity of the herbicide 2,4,5-T. editor] Western J. Med. 131(4):335-336. [Editorial.] 380 [letter to the �1060. Westing, A. H. (1979) The safety of 2,4,5-T. Science 206(4423):1135-1136. [letter to the editor] [Editorial.] 1061. Westing, A. H. Ecological considerations regarding massive environmental contamination with 2,3,7,8-tetrachlorodibenzo-p-dioxin. In Chlorinated Phenoxy Acids and Their Dioxins, C. Pamel, ed. (Ecol Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978) p. 285-294. [Review article.] 1062. Westing, A. H. (1972a) Herbicidal damage to Cambodia. In Harvest of Death, J. B. Neilands, ed. (Free Press, 1972) pp. 177-201. [Review article.] 1063. Westing, A. H. (1972b) Herbicides in war: doubt. Biolog. Conserv. 4(5):322-327. Current status and future [Review article.] 1064. Westing, A. H. (1971) Ecological effects of military defoliation on the forests of South Vietnam. Bioscience 21(17)-.893-898. [Review article.] 1065. Wheeler, J. A. (1968) Herbicides in the perspectives of 20 months and 20 years. Science 161:255-256. [Background material.] 1066. Whitehead, C. C. and Pettigrew, R. J. (1972a) The effect of 2 ,4-dichlorophenoxyacetic acid on laying hens. Br. Poult. Sci 13:191-195. The effect of 2,4-D on reproduction was studied in hens. Hens (10 per group) were administered 6.2 or 18.7 mg (acid equivalents) of 2,4-D butoxyethyl ester in ethanol orally in a gelatin capsule daily from 28 to 34 weeks of age. Controls (10) received capsules only. Egg production, egg and yolk weights and shell thickness were recorded from 22 weeks of age. From 34 to 48 weeks of age, hens were artificially inseminated and hatchability, gross malformations, and 21 day survival of chicks were observed. Non of the hens died, no adverse effects were observed for any parameter examined and no malformations occurred. The authors concluded that the doses used for these experiments, equivalent 381 �to 50 and 100 mg/kg of 2,4-D, were below the doses that cause reproductive or teratogenic effects in chickens, but are doses likelv to be encountered in normal agricultural practice. 1067. Whitehead, C. C., and Pettigrew, R. J. (1972b) The subacute toxicity of 2,4-dichlorophenoxyactetic acid to chicks. Toxicol. Appl. Pharmacol. 21:348-354. The subacute oral toxicity of 2,4-D and of 2,4,5-T are described for chicks. Broiler chickens (4 weeks of age) in groups of 2-10 were administered single peroral doses of 250-900 rag/kg of 2,4-D butoxyethyl ester or 2,4,5-T butoxyethyl ester in ethanol (agricultural grade of both herbicides were used). Mortality, body weights and food intake were monitored during the subsequent week. Mortality was high (40-50?) for only the groups given the highest dosage of each herbicide. Transient decreases in food consumption were observed for all groups. Groups of 10 chicks (1 day of age) were fed diets with 10-7,500 mg per kg feed of either phenoxy ester for 3 weeks. In addition to the parameters observed after acute exposures, necropsies were performed. Chicks fed at least 2,000 mg/kg 2,4-D or 1,000 mg/kg 2,4,5-T had reduced food consumption and body weights. Levels of 5,000 mg/kg or higher of 2,4,5-T caused 90-100% mortality. Levels of 5,000 mg/kg 2,4-D did not cause any deaths but histological changes including swollen kidneys and mottled spleens occurred. Histological changes after 2,4,5-T treatment were not mentioned. Groups of four chicks (2 weeks of age) were administered diets with 1,000 or 5,000 mg/kg of feed of either herbicide for 1 week. Three control groups were used, including two groups that were pair-fed with the groups that received the higher dosage of each herbicide. Plasma calcium and magnesium levels were measured at the end of the exposure period. Groups of chicks fed 5,000 mg/kg of either herbicide for 1 week were fed control diets for 3 subsequent weeks and body weights were recorded. No alterations in calcium or magnesium levels were observed between exposed groups and their corresponding control groups. All chicks fed high levels of herbicide for 1 week recovered and resumed normal growth when they were administered control diets. When offered a choice, chicks consumed control diets rather than diets contaminated with 5,000 mg/kg of either herbicide. The authors concluded that chicks were able to tolerate high dosages of 2,4-D or 2,4,5-T administered subacutely and toxic symptoms that occurred were reversible. 1068. Williams, R. F., Inman, Q. S., and Ulberg, L. C. (1979) Development of isolated mammalian embryo techniques for toxic substance screening. U.S. Environmental Protection Agency, Research Triangle Park, NC.: Report No. EPA-600/1-79-007. 72 p. [Background material.] 382 �1069. Willis, G. H., Rogers, R. L., and Southwick, E. M. (1975) Losses of diuron, linuron, fenac, and trifluralin in surface drainage water. J. Environ. Qual. 4(3):399-402. The authors studied the loss of diuron in surface drainage water from agriculture plots. Diuron was applied by ground spraying 0.84 kg/ha on two experimental cotton plant plots. The plots were 7.3 by 61 m with a slope of 0.2% and were composed of Mhoon silty clay loam soil. Three annual applications were made. Surface runoff was analyzed for diuron by gas chromatography (detection limit 10 ppb) for 3 months after application. Diuron was present in surface runoff from the plots but at concentrations less than 10 ppb. Two to 3 months after application, no diuron was detected in surface runoff. The authors conclude that the data suggest that diuron properly applied poses little threat to aquatic areas adjacent to sprayed areas. 1070. Wilson, J. G. (1977) Teratogenic effects of environmental chemicals. Fed. Proc. 36(5):1698-1703. [Not available.] 1071. Wilson, J. G. (1972a) Abnormalities of intrauterine development on non-human primates. WHO Research and Training Centre on Human Reproduction, Stockholm. The use of non-human primates in research on human reproduction. WHO Symposium, ed. E. Diezfolvsy and C. C. Standley. pp 261-292. Data from an experiment on the teratologic effects of 2,4,5-T on Rhesus monkeys is presented, along with a review of teratology observed in non-human primate studies. Seventeen pregnant Rhesus monkeys (4-5 per group) were orally administered doses of between 5 and 40 mg/kg 2,4,5-T three times per week between days 20 and 48 of gestation. On day 100, fetuses were removed by hysterotomy and were weighed and observed for gross, internal, and skeletal malformations, methods were not described. One abortion occurred, on day 61 in the highest treatment group. Compared to fetuses from the control group of 7 monkeys, fetuses from groups treated with 10 mg/kgations were not reported. No conclusions were stated by the authors related to the teratogenicity of 2,4,5-T. 1072. Wilson, J. G. (1972b) Report on treatment of pregnant Rhesus monkeys with 2,4-D and CMPA. Report to Swedish Poisons and Pesticides Board, [Not available.] 1073. Wilson, J. G. (1972c) Teratological potential of 2,4,5-T. Proc. South Weed Sci. Soc. 25:26-30. [Not available.] 383 �1074. Witschi, H. (1977) Environmental agents altering lung biochemistry. Fed. Proc 36(5):1631-1634. [Review article. ] 1075. Witschi, H., Kacew, S., Hirai, K. I., and Cote, M. G. (1977) In vivo oxidation of reduced nicotinamide-adenine dinucleotide phosphate by paraquat and diquat in rat lung. Chem. Biol. Interactions 19(2):143-160. Male Sprague-Dawley rats were administered 40 mg/kg f C]-diquat and tissue concentrations of diquat were determined from 0.1 to 24 hrs. later. All tissue levels fell rapidly with no selective pulmonary uptake observed. The ratio of NADPH/NADP in control lung was determined by an enzymatic assay procedure to be 3.4-4.8 and this ratio dropped substantially in the presence of diquat, indicating that oxidation occurred. Incorporation of f H.l-glycine by lung in vivo was determined as a measure of de novo synthesis of adenine-containing compounds, including pyridine nucleotides, and was not elevated 4 hours after diquat was administered. (The specific activity of free glycine was not altered by diquat administration). In the presence of 100% oxygen, the ratio of pulmonary NADPH/NADP was decreased for low doses of diquat (compared to the ratio for diquat treatment to rats in air) but no effect was observed when high doses of diquat were used. Light microscopy of pulmonary tissue from diquat-treated rats revealed no lesions, while electron microscopy revealed damage to type I alveolar cells. The effects of paraquat were also described. The authors concluded that a cause-and-effeet relationship between NADPH oxidation (and the resulting biochemical consequences) and diquat mediated lung damage was not clearly established. 1076. Wojtalik, T. A., Hall, T. F., and Hill, L. 0. (1971) Monitoring ecological conditions associated with wide-scale applications of DMA 2,4-D to aquatic environments. Pestic. Monit. J. 4(4):184-203. In this study, the concentrations of 2,4-D and biological response to its application at the Nickajack and Guntersville Reservoirs were measured. In June-April 1969, the reservoirs, located on the Tennessee River were sprayed with about 170,000 gallons of dimethylamine salt of 2,4-D containing 4 Ib of 2,4-D acid equivalent per gallon to control Eurasian watermilfoil. The herbicide was applied by helicopters at rates of 20 to 40 Ib acid equivalent per acre on about 18,000 acres. Four areas on the Guntersville Reservoir that were treated and one area that was not treated were sampled before and after treatment. Water samples, plankton, chlorophyll, carbon-14, Eurasian watermilfoil and other plant samples were collected. Temperature, dissolved oxygen, alkalinity and other physical parameters were sampled to determine the efficacy of the herbicide. Organisms were collected in the reservoir and adjacent riverbed to determine the toxicity and accumulation of 2,4-D. Two weeks after spraying, the 2,4-D concentrations ranged from less than 0.001 mg/1 to 0.72 mg/1. No living watermilfoil were found 384 �in the area 1 year after treatment but there were no negative effects on most of the other aquatic plants. No fish kills were observed and there were no indications of acute toxicity. Macro-invertebrate samples did not indicate toxicity but did show evidence of population changes. The authors attribute this to the collapse of the milfoil. Mussel samples taken below the reservoirs shortly after application showed levels of less than 0.050 mg/kg to 0.97 mg/kg net weight with accumulations decreasing downstream. The authors indicate there is no known source of the high concentration of 2,4-D in a pretreatment mussel sample. Samples taken from raw and treated water at water treatment plants indicated that the facilities did not remove significant amounts of 2,4-D. Plankters adsorbed or absorbed 24% of 2,4-D 1 hour after application and almost 100% 24 hours after application. The authors conclude that reduced amounts of liquid 2,4-D are effective in controlling watermilfoil if flow and habitat characteristics are considered. Underwater injection of 2,4-D near the rootcrowns could achieve effective control. 1077. Wolfe, W. H. (1980b) Human health effects following exposure to the phenoxy herbicides and TCDD. Presented at the Educational Conference on Herbicide Orange, U.S. Veterans Administration, Washington, DC, May 28-30. [Review article.] 1078. Wong, A. S., and Crosby, D. G. (1978) Decontamination of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) by photochemical action. Monograph of the Giovanni Lorenzini Foundation. 1:185-189. [Review article.] 1079. Wood, A. E. (1972) Interrelations of humans, dogs, and rodents. Science 176:437. [Editorial.] 1080. Wood, T. E., Edgar, H., and Salcedo, J. (1976) Recovery from inhalation of diquat aerosol. Chest 70(6):774-775. The clinical symptoms of human exposure to diquat aerosol were described. The subject, a 45-year-old man, had been spraying diquat when the nozzle clogged and then suddenly discharged a cloud of aerosol into his face. Myalgia, headaches, a cough that produced thick red sputum, fever, and neck stiffness developed. The patient was admitted to a hospital 4 days after the accident and was then observed to be in a confused state and showed consolidations of the lung. An erythematous rash developed on one arm. After prednisone therapy was initiated, all clinical symptoms disappeared. Oxygen, which was administered before exposure to diquat was recognized, was considered to have been potentially detrimental. 385 �1081. Woods, J. S. (1973) Studies on the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on mammalian hepatic -aminolevalinic acid synthetase. Environ. Health Perspec. 5:221-225. The porphyrogenic activity of TCDD was studied in rats by measuring hepatic delta-aminolevulinic acid (ALA) synthetase activity after exposure in vivo and in vitro. Male rats, pregnant rats 3 days prior to expected delivery, and newborn rats (3 and 11 days of age) were administered a dose of 5-25 ug/kg TCDD in corn oil-acetone (6:1) 1-28 days prior to being killed. One group of control rats received vehicle only and the second group received 400 mg/kg allylisoproplacetamide (AIA) subcutaneously. Hepatic ALA synthetase activities of adult, newborn and fetal liver homogenates and of mitochondrial and extramitochondrial fractions of liver cells were determined. ALA synthetase activity was also assayed after in vitro exposure of liver homogenates, isolated,mitochondria, and ALA synthetase from prophyric rat liver to 10 -10 M TCDD. No change in ALA synthetase activity occurred in liver homogenates prepared from adult male rats 1-28 days after TCDD exposure. Twenty-four hours after ALA exposure, ALA synthetase activity was increased seven-fold and returned to normal 2 days later. TCDD exposure did not result in altered ALA synthetase activity in liver of rats of any age, in any subcellular fraction or after in vitro exposure. The authors concluded that the induction of ALA synthetase activity observed in embryonic chick liver (reported elsewhere) and lack of induction in rats in the current study represents a maior difference in species susceptibility. 1082. Woolson, E. A. (1977a) Fate of arsenicals in different environmental substrates. Environ. Health Perspec. 19:73-81. [Review article.] 1083. Woolson, E. A. (1977b) Generation of alkylarsines from soil. Weed Sci. 25:412-416. The author measured alkyarsines generated from soils treated with cacodylic acid. Flasks containing 100 g of silt loam soil and 6 g of ground soybean meal.were treated with 13.33 moles cacodylic acid, which included 1.1 (u)Ci C-cacodylic acid (specific activity 1.2 (u)Ci/mg). Both aerobic and anaerobic soils were studied in duplicate. Gases coming from soil samples were analyzed for 160 days. Volatile organo-arsenicals were formed most rapidly under aerobic conditions. Gases trapped from aerobic soils contained 18% of the activity of the original treatment, while gases from anaerobic soils contained only 7.8% of the activity. Volatile gases were identified as dimethyl arsine and trimethylarsine. In addition, organo-arsenicals remaining in the soil were also measured. In both treatments, most of the extractable arsenic was present as a trimethyl arsenical. The authors concluded that volatilization of organo—arsenicals in soil is an., important part of the arsenic cycle in soils. The formation of CO2 was not measured in this study. 386 �1084. Woolson, E. A. Organoarsenical herbicides. In Herbicides: Chemistry, degradation and mode of action. Vol 2'eds., P. C. Kearney and D. D. Kaufman (New York: Marcel Dekker Inc., 1976) pp. 741-776. [Review article.] 1085. Woolson, E. A., Ensor, P. D., Reichel, W. L., and Young, A. L. (1973) Dioxin residues in lakeland sand and bald eagle samples. Advan. Chem. Ser. 120:112-118. The authors report on TCDD residues in soil profile samples collected at Eglin APB in an area where massive herbicide spraying occurred and in bald eagle samples collected from 15 States. Sample analysis was conducted by gas chromatography (detection limit, 1 ppb). Twelve soil cores 36 in long were collected in 1970: 2 cores from an area receiving 2,4-D and 2,4,5-T at 947 Ib/acre for each herbicide (1962-1964); 4 cores from an area receiving 584 Ib/acre of each herbicide (1964-1966); and 6 cores from an area receiving 160 Ib/acre 2,4,5-T and 183 Ib/acre 2,4-D (1968-1969). Small amounts of 2,4-D ( . - 15.4 ppb) and 2,4,5-T (8.0-8.4 ppb) were detected in the cores 08 samples. However, no TCDD was detected in any of the samples. No TCDD was detected in any of the bald eagle samples (detection limit 0.05 ppm). 1086. Woolson, E. A., and Isensee, A. R. (1981) Soil Residue accumulation from three applied arsenic sources. J. Weed Sci. 29:17-21. The authors report on the accumulation of arsenic in the soil often repeated applications of cacodylic acid. Herbicide was applied to Matapeake silt loam soil at 11.2, 22.4, and 112 kg/ha, which correspond to Ix, 2x, and lOx the recommended application rate. To devise a "worst possible case" situation, cacodylic acid was sprayed on bare ground, which was then rototilled, and soybeans and radishes were planted to indicate phytotoxicity. Under normal application conditions, herbicide is sprayed on vegetation for weed control and planting does not occur until the following year. Twelve core soil samples (0-15 cm and 15-30 cm) were collected from each plot and composited at the end of the growing season. Arsenic (As) residues in the samples were determined by colorimetric methods. Each year total As losses from the soil were about 15%, probably as a result of volatilization of alkylarsines, according to the authors. Soil As levels increased with increasing application rates. Accumulation of arsenic in the soil was gradual. The authors calculated that at the maximum recommended rate of cacodylic acid application, equilibrium would be reached after about 22 years of application. Soybeans and radishes were adversely affected by the highest application rate of cacodylic acid but not by the two lower rates. The authors concluded that use of recommended application rates of arsenical herbicides should not adversely affect plants because of soil accumulation. 387 �1087. Woolson, E. A., and Kearney, P. C. (1981) Brief overview of cacodylic acid in soils and man. Public Statement by TJ. S. Dept. of Agriculture, 3 p. [Background material.] 1088. Wgolson, E. A., and Kearney, P. C. (1973) Persistence and reactions of C-cacodylic acid in soils. Environ. Sci. Techno1. 7:47-50. The authors studied the persistence and degradation of cacodylic acid in three soil types, Lakeland loamy sand, Hagerstown silty clay loam, and Christiana clay loam. The authors also examined the distribution of cacodylic acid into water soluble, iron, aluminum, and calcium fractions in the three soils. C-cacodylic acid (0.2uCi) was added to each air-dried soil sample at 1, 10, and 100 ppra. One set of soils was brought to 75% of field moisture capacity to measure aerobic degradation. Both sets of soil samples were incubated at 25C for 32 weeks. The rate of disappearance of C-cacodylic acid was dependent on the soil type. After 32 weeks, 23% of the original C remained in Christiana soil, 53% in Hagerstown soil, and 62% in lakeland soil. The rate of application, however, did not have any appreciable effect on cacodylic acid disappearance. Under aerobic conditions, approximately 35% of the original cacodylic acid was converted to a volatile organoarsenical and 41% converted to CO- and AsO, in 24 weeks. Under anaerobic conditions, 61% of the cacodylic acid was converted to an organo-arsenical. According to the authors, the degradation was probably due to microbial activity. 1089. Workers' exposure to 2,4-D studied. April 1981, 3 p. [Editorial.] 1090. (1981) Research News, (USDA), , Wright, T. C. (1979) Agent Orange Dioxin: TCDD. L C Science Tracer Bullet No. TB 79-10. 9 pp. [Bibliography.] 388 �1091. Yamauchi, H., and Yakamura, Y, (1979) Urinary inorganic arsenic and tnethylarsenic excretion following arsenate-rich seaweed ingestion. Jap. J. Ind. Health 21:47-54. [Abstract, only.] 1092. Yang, K. H., and Peterson, R. E. (1976) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on plasma disappearance and biliary excretion of Ouabain in rats. Pharmacologist 18(2):246. 1093. Yockim, R, S., Isensee, A. R., and Jones, G. E, (1978) Distribution and toxicity of TCDD and 2,4,5-T in an aquatic model ecosystem. Chemosphere (3):215-220. The authors evaluated the toxicity of TCDD and the environmental fate of TCDD and 2,4,5-T in an aquatic model ecosystem. Silt loam soil (pH 5.3, 4009) was treated with.either ring-labeled C-TCDD (460 mCi/g) at 0.1 ppm or carboxy-labeled C-2,4,5-T (280 mCi/g) at 0.1, 1.0 or 10.0 ppm. The compounds were added to the soil in benzene, after which the soil was air-dried. Treated soil in triplicate was added to ecosystem tanks which were then flooded with 16 liters of water. Untreated soil was used as a negative control. The possible effects of the benzene solvent were not discussed. Each tank received 100 water fleas, 15 snails, Ig algae, and 15 mosquito fish, which were isolated from the other species to prevent predation. Water and tissue samples were removed for scintillation counting on days 1, 3, 7, 15, and 32). Radioactivity in the water from TCDD-treated tanks reached equilibrium by day 1 (2-4 ppt), while 2,4,5-T levels did not reach equilibrium during the experiment. Bioaccumulation ratios for organisms in TCDD treated tanks were 2-6x10 while ratios of less than 50 were observed in 2,4,5-T treated tanks. The first group of fish added to the TCDD tanks died by day 15 having tissue levels of 7.2 ppb TCDD. A second group of 15 fish were then added to the TCDD tanks. These fish all died by day 32 with TCDD tissue levels of 4.4 ppb. Nasal hemorrhaging and listless swimming were observed in the dying fish. Three pregnant females added to the TCDD treated tanks with the second group lost external signs of pregnancy and hemorrhaged shortly before their deaths on day 15. TCDD at the concentration tested had no apparent effect on any other organism tested. No toxicity induced by 2,4,5-T was observed. In addition, in a single trial experiment, the authors added 105 ppb TCDD to aquatic soils and measured TCDD persistence. Levels of TCDD decreased by 18% at the end of the 180-day experimental period. The TCDD concentration used in these experiments was 10 - 10 times higher than,what would occur in a real world situation. The behavior of 10 - 10 lower levels of TCDD cannot be assessed by this experimental design. 389 �1094. Yoder, J., Watson, M., and Benson, W. W. (1973) Lymphocyte chromosome analysis of agricultural workers during extensive occupational exposure to pesticides. Mutat. Res. 21:335-340. The authors performed chromosomal analysis on lymphocytes from persons occupationally exposed to herbicides and insecticides. The study was part of an ongoing survey by the Idaho Community Study on Pesticides. Peripheral blood samples were drawn from male Caucasians only. Persons with recent histories of viral infections, chemotherapy or x-ray treatment or histories of malignancies were rejected. Controls were matched for age and physical characteristics (undefined) as closely as possible. The high exposure group was defined as spraying with pesticides 4 hours per day on 3 or more days per week during the peak spraying season of July 15 through August 30th. The control group was composed of 16 persons with no known pesticide exposure, mean age 39.8. The exposed group was composed of 26 persons employed by weed control agencies with a mean age 36.5 and a mean herbicide exposure of 6.3 years. These workers were exposed primarily to 2,4-D, amitrole and atrazine. Peripheral blood samples were obtained from each person during the peak spraying season and during the midwinter "off season." Lymphocyte cultures were prepared from each blood sample and cultured for 48 hours after which chromosome spreads were made. Scoring for chromatid gaps and breaks was performed on coded samples to avoid bias. Twenty-five metaphase spreads from each person were examined for chromosomal aberrations. Chromosomal aberrations in the unexposed group did not differ markedly when off season and spraying season samples were compared. During the peak spraying season the exposed group had nearly 4 times as many gaps per person and 25 times as many breaks per person compared to the mid winter sampling. However, the winter sampling of the herbicide exposed group had approximately half the number of gaps and one fourth the number of breaks compared to the unexposed group. The authors speculated that enhanced chromosome repair may be taking place at this time as compensatory protection. No statistical analysis of the results were presented. Ranges of chromatid gaps as well as chromatid breaks overlapped in all groups tested. No conclusions can be reached on the effects of 2,4-D alone because exposure was to unknown quantities of several herbicides. 1095. Yoshida, T., and Castro, T. F. (1975) Degradation of 2,4-D, 2,4,5-T and picloram in two Philippine soils. Soil Sci. Plant Nutr. 21(4):397-404. The authors report on the degradation of 2,4-D, 2,4,5-T, and picloram in two Philippine rice field soils. Samples of Maahas clay soil (pH 6.6, 2.0% organic matter) and Louisiana clay soil (pH 4.7, 3.2% organic matter) were collected from rice paddies, air dried, passed through a 2 mm sieve and placed in test tubes at 20 g per tube. Herbicides were added to soil in acetone: 20 ppm 2,4-D, 10 ppm 2,4,5-T or 1 ppm picloram. The solvent was evaporated before incubation began. One half the samples were submerged to a depth of 3 cm to mimic flooding conditions, the other samples (Upland samples) were maintained at 80% field moisture capacity. Amounts of herbicides were measured at 0, 2, 390 �4, and 6 weeks for 2,4-D; 0, 2, 4, 8, and 12 weeks for 2,4,5-T; and 0, 3, and 6 months for picloram. Soil samples were analyzed for herbicide residues by gas chromatography. In upland Maahas soil, 2,4-D was degraded nearly completely in 6 weeks. Submerged samples did not degrade 2,4,-D as rapidly. However, by 4 weeks over 50% of the herbicide had disappeared. In Louisiana soil, 2,4-D degraded more slowly. More than 60% 2,4-D had degraded after 6 weeks. 2,4,5-T also degraded rapidly in upland Maahas soil, and had disappeared by 12 weeks. In submerged samples, slight amounts of 2,4,5-T residues were being detected at 12 weeks. Degradation of 2,4,5-T in upland Louisiana soil was complete by 8 weeks. In submerged samples, degradation was about 60% in 12 weeks. Picloram was the most persistent of the herbicides studied. The amount of picloram recovered in upland or submerged Maahas soil was less than 50% in 3 months. However, on Louisiana soil, degradation did not begin for 3 months. By 6 months, less than half of the original picloram remained in the soil. In order to determine if microbial activity was responsible for herbicide degradation in the soils tested, the authors sterilized soil samples by autoclaving or cobalt irradiation. 2,4,5-T was added to autoclaved and unautoclaved samples of all 4 soil types at 9-10 ppm. By 12 weeks, the nonautoclaved'samples had completely or nearly completely degraded the herbicides. Little to no degradation was observed in nonautoclaved samples. Similar results were observed with irradiated and nonirradiated samples using both 2,4-D and 2,4,5-T. The authors concluded that the major agent for degradation of these pesticides in soil is microbial activity. 1096. Young, A. L. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA, Docket No. 415 et. seq., June 1980. [Testimony.] 1097. Young, A. L. (1980) Use of herbicides in South Vietnam, 1961-1971. Presented at the 2d Continuing Education Conference on Herbicide Orange, Washington, DC, May 28-30. [Background material.] 1098. Young, A. L. (1974) Ecological studies on a herbicide-equipment test area (TA-C-52A), Eglin AF Base Reservation, Fla. US NTIS Publication ISS No. 780517/9GAT 141 pp. [Background material.] 1099. Young, (1978) orange Health A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. The toxicology, environmental fate, and human risk of herbicide and its associated dioxin. USAF Occupational and Environmental Laboratory Report No. USAF OEHL - 78 -92. 262 pp. [Review article.] 391 �1100. Young, A. L., Thalken, C. E., Arnold, E. L., Cupello, J. M., and Cockerham, L. G. (1976) Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the environment: Summary and decontamination recommendations. Headquarters Air Force Logistics Command Report No. AFA-TR-76-18. 41 pp. [Background material.] 1101. Young, A. L., and Wolverton, B. C. (1970) Military herbicides and insecticides. Technical Notes AFATL-TN-70-1. Air Force Armament Laboratory, Eglin AFB, Florida. 59 pp. [Not available.] 1102. Young, J. F., and Haley, T. J. (1977) Pharmacokinetic study of a patient intoxicated with 2,4-dichlorophenoxyacetic acid and 2-methoxy-3,6-dichlorobenzoic acid. Clin. Toxicol. 11(5):489-500. A pharmacokinetic evaluation was described based on^blood and urine data from a woman who ingested 100 ml (estimated) of a mixture of 2,4-D (20.1 gm) and 2-methoxy-3,6-dichlorobenzoic acid (1.9 gm; Dicamba). The clinically stabilized patient underwent lavage and had been treated with 3 drugs the nature of whose interactions with the herbicides were unknown. The concentrations of the 2 chemicals in blood and urine collected during the acute phase of intoxication were reported in a separate publication. These data were analyzed kinetically on an analog computer interfaced to a digital computer. The model that generated data that provided the best fit for the experimental data for 2,4-D was a one-compartment model. Clearance from the single compartment representing the blood content was described by three rate constants, 2 related to urinary elimination and 1 related to fecal elimination (although fecal levels of 2,4-D were not measured). Initially Dicamba was cleared by a urinary excretory route that became available to 2,4-D only after the relative concentrations of the 2 compounds favored 2,4-D, This pathway was hypothesized to be glycine conjugation. The plasma half life of 2,4-D was 59 hours initially and 17 hours when the route originally saturated by Dicamba was available to 2,4-D. The volume of 2,4-D distribution was estimated to be 10.2 liters. 1103. Young, J. F., and Haley, T. J. (no date) Simultaneous human pharmacokinetics of 2,4-dichlorophenoxyacetic acid and 2-methoxy-3,6-dichlorobenzoic acid. Fed. Proc. 38:679. [Abstract, only.] 392 �1104. Zack, J. A., and Suskind, R. R. (1980) The mortality experience of workers exposed to tetrachlorodibenzodioxin in a trichlorophenol process accident. J. Occup. Med. 1-4. The mortality experience, of 121 plant workers exposed to TCDD in a 1949 TCP process accident was studied. Data were obtained from plant safety, medical, and workmens compensation records. The development of chloracne was the criteria used to select the study population. Followup of the population verified 89 living and 32 dead. Death certificates were obtained on deceased cohorts and with the underlying cause of death coded using the 8th revision of the ICDA. Analyses were performed by the modified life table method using the Monson program. A standard mortality ratio (SMR) of .69 for all deaths was found with 32 observed deaths with 46.41 expected. This was the only statistically significant difference found. 1105. * Zandvoort, R., Van Den Born, G. W., Braber, J. M., and Smelt, J. H. ,(1980) Leaching of the herbicide bromacil after application on railroads in the Netherlands. Water Air Soil Pollut. 13:363-372. The authors measured the movement of bromacil through railroad-bed soils one year after application. A commercial preparation of bromacil (Hyvar X) was sprayed at 2.2 and 4.4 kg/ha yearly from 1972 until 1976 or at 2.2 kg/ha in alternate years. Soil core samples were collected down to a depth of 80 cm and analyzed for bromacil by gas chrooatography. The highest concentrations of bromacil were found in the top 40 cm of the soil column. After 4 yearly sprayings of 2.2 kg/ha, residues of bromacil in the 0-80 cm soil columns were 3.1, 1.7, 2.5, and 1.4 for 1974 through 1977. Similar results were observed at the other close levels. It appears that bromacil does not accumulate in the soil to any great extent. In addition, the authors also studied the leaching of bromacil through soil columns under laboratory conditions. Two soil columns were used: one made of untreated air-dried soil from the railroad bed and the other made of untreated air-dried soil of the 20-90 cm layer of soil from the railroad bed. Bromacil (1.24 mg Hyvar X) mixed in sand was then added to the top of each column which was then wetted to field capacity. Elutions of 10 cm of water were applied for 2 days.. Both the eluate and 5 cm sections of each of the columns was analyzed for bromacil. A large quantity of bromacil was found in the leachate. In the top layer soil column 34% of the recovered bromacil was found in the leachate and in the deep layer soil column 74%. Most of the bromacil remaining in the soil column was found in the application layer or in the 15-30 cm depths. From these experiments the authors concluded that bromacil is highly mobile in soil and can possibly leach below 100 cm or even into groundwater in one year. They did not verify these conclusions experimentally. 393 �1106. Zelikov, A. K., and Danilov, L. N. (1974) Occupational dermatoses (acne) in workers engaged in production of 2,4,5-trichlorophenol. Sov. Med. 7:145-146. A brief case report of chloracne occupational exposure to 2,4,5trichlorophenol is given. The patient presented with clinical symptoms of chloracne 3 months after working in the production of 2,4,5trichlorophenol. No internal pathology was detected, and blood urine analyses gave normal results. Based on the histological skin findings and the history of contact with 2,4,5-trichlorophenol, the authors diagnosed chloracne. The worker was removed from exposure to the chlorinated compound and received treatment. Sanitary and engineering changes were in the plant to prevent other workers from being exposed. No new cases were observed in the subsequent 1-1.5 years. 1107. Zepp, R. G., Wolfe, N. L., Gordon, J. A., and Baughman, G. L. (1975) Dynamics of 2,4-D esters in surface waters. Hydrolysis, photolysis and vaporization. Environ. Sci. and Technol. 9(13):1144-1150. [Background material.] 1108. Zetterberg, G. (1978) Genetic effects of phenoxy acids on microorganisms. In Chlorinated Phenoxy Acids and Their Bioxins, C. Rattiel, ed. (Ecol. Bull, No. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 193-204. The authors studied the tnutagenicity of 2,4-D and 2,4,5-T in a haploid strain of yeast, Saccharonyces cerevisiae RAD18, which is auxotrophic and requires histidine for growth. Upon exposure to a mutagen RAD18 reverts from histidine dependence to histidine independence. To test the sensitivity of the yeast cells to 2,4-D, several growth stages of yeast cells, pH levels and concentrations of 2,4-D were used. 2,4-D caused increased mutations in log phase cells, but not in stationary cells. Increased mutation frequencies were observed only at concentration of 0.2 mg/ml or higher and pH less than 4.5. In addition, no controls or numerical data reported, and no statistical analysis performed. Criteria for a positive result was not presented by the authors. Under these conditions survival of the yeast cells was extremely low, 1% or less. In order to prove that the revertants observed in these experiments were "true" revertants and not 2,4-D resistant spontaneous revertants, the authors performed reconstruction experiments. Ten revertant colonies and 10 his- colonies were inoculated in growth medium and then treated with 2,4-D. Both types of of cells had similar sensitivities to 2,4-D (0.1-0.25 mg/ml) proving that the increased mutation frequency was due to a mutagenic effect. The mutagenicity of 2,4,5-T was also dependent on pH. At pH values greater than 4,5, no mutagenic effects were observed. However, at pH 4.3 and cell survival less than 1%, mutation frequencies increased. 394 �1109. Zetterberg, G., Busk, L., Elovson, R., Starec-Nordenhammar, I., and Ryttman, H. (1977) The influence of pH on the effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) on Raccharomyces cerevisiae and Salmonella typhimurium. Mutat. Res. 42:3-18. The authors studied the genetic effects induced by 2,4-D on Saccharomyces cerevisiae and Salmonella Typhimurium in vivo and in vitro. Two strains of S. cerevisiae were employed: D4, a diploid strain heteroallelic at the ade 2 and trp 5 loci requiring adenine and tryptophan for growth and D5, another diploid strain requiring adenine for growth. Strain D4 detects mitotic gene conversion while D5 detects mitotic crossing over. In addition, S. typhimurium strains TA1530, TA1535, TA1531, and TA1538, which detect point mutations, were included in the test battery. TA1530 and TA1535 revert from histidine requiring to prototrophy by base pair substitutions, while TA1531 and TA1538 revert by frameshift mutations. In order to assess the genetic effects of 2,4-D in vivo, a host mediated assay in mice was performed using male CBA mice (309 each) treated orally with 2,4-D. Ethyl methane sulfonate was used as a positive control in all the in vitro experiments. In S. cerevisiae D4, 2,4-D (0.6 mg/ml) at pH 4.5 significantly increased the frequency of mitotic gene conversion for the ade 2 locus (p less than 0.05), but not at the trp 5 locus. A pH greater than 4.6 no increase in mitotic gene conversion was observed. From this the authors speculated that yeast cells can only take up the undissociated form of 2,4-D which occurs at pH 4.5 or lower. In S. cerevisiae strain D5, at pH 4.3, 0.3 mg 2,4-D/ml increased the frequency of mitotic recombination. However, in S. typhimurum 2,4-D (0.5 mg/ml) did not induce an increase in the number of revertants at either pH 4.3 or pH 6.8. No exogenous metabolic activation was included in the experiment. In the host mediated assay, 6 mg 2,4-D was administered in a single oral dose to each of 4-8 CBA mice that had been iniected intraperitoneally with S. cerevisiae D4, TA1531 or TA1530. The dose of 2,4-D was close to the maximum tolerated dose according to the authors. After 3 hours exposure, the indicator organisms were recovered from the peritoneal cavity and plated on minimal medium. No increase in mitotic gene convertants of histidine independent revertants were observed. This, however, cannot be taken as a conclusive result. The compound may not have reached the indicator cells or the near neutral pH conditions in the peritoneal cavity prevented 2,4-D from being taken up by the cells. Furthermore, no positive control was included in the experimental design to ensure that the test systems were working properly. 1110. Zielinski, W. L., Jr., and Pishbein, L. (1967) Gas chromatographic measurements of disppearance rates of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice. J. Agr. Fd. Chem. 15:841-844. The rates of elimination of 2,4-D, 2,4-D butyl ester, 2,4-D isooctyl ester and 2,4,5-T were studied in the mouse. Female C57RL/6 mice were administered 100 mg/kg of 2,4-D acid or ester or 2,4,5-T in dimethylsulfoxide and were killed (6 mice per group) from 0 to 24 hours later. The amount of herbicide in the whole body was determined by 395 �analyzing an aliquot of a whole-body hotnogenate by gas chromatography. Some mice were pretreated with 5 daily doses (100 mg/kg each) of herbicide prior to the test dose on the sixth day. Herbicide levels were expressed as a percentage of the last administered dose (100 mg/kg in each case). The percentage of the dose recovered at time 0 was 111% for the 2,4,5-T pretreated group and ranged from 77.1% to 81.8% for all other groups. The rates of disappearance were highest for the 2,4-D esters and lowest for 2,4,5-T. The rates for 2,4-D esters were faster after pretreatment and for 2,4,5-T were slower after treatment. The half times were not calculated from the same interval for each compound. The values calculated for 2,4-D butyl ester were 1.1 hours (0.9 after pretreatment) 0-2 hours after treatment; for 2,4-D isooctyl ester were 3.5 hours (3.2 after pretreatment) 0-4 hours after treatment; for 2,4-D was 4.1 hours between 0-16 hours; for 2,4,5-T was 14.1 hours (31.8 after pretreatment) for 0-24 hours. No 2,4-dichlorophenol metabolite was detected after administration of any of the 2,4-D compounds. The authors concluded that 2,4-D was excreted at a slower rate than the 2,4-D esters but much more rapidly than 2,4,5-T and pretreatment (with the same herbicide) enhanced 2,4-D butyl ester clearance only. 1111. Zimmerman, F. K. (1971) Induction of mitotic gene conversion by mutagens. Mutat. Res. 11:327-337. [Review article.] 1112. Zingeser, M. R. (1979) Anomalous development of the soft palate in rhesus macaques (Macaca mulatta) prenatally exposed to 3,4,7,8tetrachlorodibenzo-p-dioxin, (sic). Teratology 19(2):54A-55A. [Abstract, only.] 1113. Zinke, P. J. (1974) The effects of herbicides in South Vietnam. Part B. Working papers: Effect of herbicides in soils of South Vietnam. National Academy of Sciences-National Research Council. AD-779 024. 39 pp. [Not available.] 1114. Zinkl, J. G., Vos, J. G., Moore, J. A., and Gupta, B. N. (1973) Hematologic and clinical chemistry effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Perspec. 5:111-118. The effect of TCDD on serum enzyme levels, biochemistries, and hemotology was evaluated in the rat, mouse, and guinea pig. Daily oral doses of 0.1 -10 ug/kg of TCDD were administered to female CD rats for 30 days. Blood was collected between 3 and 31 days from the start of the feeding regimen. A single oral dose of 1-50 ug/kg TCDD was 396 �administered to CD-I mice and blood was drawn 1-5 weeks later. Oral weekly doses of 0.008-1.0 ug/kg TCDD were administered to female Hartley guinea pigs for 8 weeks. At 8 weeks or when the animals in the highest dosage group became moribund, blood was collected. Half of each treatment group was also administered tetanus toxoid and the other half, mycobacterium tuberculosis. Blood was analyzed for hematologic parameters, blood chemistries, and serum enzymes. Serum bilirubin, cholesterol, glutamic pyruvate transaminase, and glutamic-oxaloacetic transaminase activities were elevated in rats that received the highest dosage, after 7-31 days and lactic dehydrogenase was elevated after 24 days. Alkaline phosphatase levels were normal in all groups of rats. Blood glucose concentrations were depressed between days 10-24. Serum protein levels fluctuated. The only significant dose-related responses were decreased blood glucose levels on days 10-24 and cholesterol elevations on days 17 and 24. Hemoconcentration was observed in the high dosage group on days 17 and 24 and thrombocytopenia occurred in all treatment groups. In guinea pigs and in mice, dose-related decreases in leukocyte and lymphocyte counts were observed 1 week after treatment. Mouse blood chemistries were not analyzed. The authors suggested that hemoconcentration (which occurred just prior to death) resulted from shock and dehydration; other blood parameters reflected liver damage. The authors concluded that the main toxic effect of TCDD in the rat was hepatocellular necrosis and was accompanied by an important platelet effect. In mice and guinea pigs, TCDD was immunosuppressive, as evidenced by lymphopenia in these animals after treatment. 1115. Zitko, V. (1972) Absence of chlorinated dibenzodioxins and dibenzofurans from aquatic animals. Bull. Environ. Contain. Toxicol. 7(2/3):105-110. The authors reported on the absence of TCDD residues in tissues from aquatic animals. Muscle and liver of white shark, eggs of double crested cormorants and herring gulls, muscle of eel and chain pickerel and commercial samples of herring oil and groundfish-herring fishmeal were analyzed for TCDD by gas chromatography (detection limit 0.04 ug/g). The authors did not specify whether any of the animals had been exposed to herbicides. 1116. Zitko, V., Hutzinger, 0., and Choi, P. M. K. (1972) Contamination of the Bay of Fundy-Gulf of Maine area with polychlorinated biphenyls, polychlorinated terphenyls, chlorinated dibenzodioxins, and dibenzofurans. Environ. Health Perspec. 1:47-50. The authors analyzed an unspecified number of fish and bird samples from the Bay of Fundy for the presence of TCDD. Muscle and liver of white shark, eggs of cormorants and gulls, commercial herring oil, and groundfish herring fishmeal were analyzed by gas chromotography (detection limit 0.04 ug/g wet weight). No TCDD was found in any of the samples. 397 �INDEX Abstracts - 2, 34, 41, 59, 62, 63, 67, 68, 72, 89, 106, 117, 118, 128, 130, 139, 154, 194, 24, 208, 233, 240, 265, 281, 290, 314, 320, 338, 346, 347, 355, 372-274, 389, 404, 420-422, 439, 444, 497, 505, 506, 530, 532, 541, 557, 562, 566, 583, 658, 663, 665, 679, 686, 706, 716, 733, 735, 780, 804, 807, 819, 829, 859, 864, 901, 922, 929, 932, 942, 960, 965, 973, 992, 1009, 1043, 1091, 1092, 1103, 1112. Acute toxicity - 3, 49-52, 54, 73, 82, 84, 96, 101, 115, 116, 126, 134, 138, 144, 280, 418, 610, 743, 842, 970, 146, 305, 447, 617, 746, 874, 975, 147, 319, 455, 627, 751, 878, 977, 153, 164, 168, 173, 201, 203, 205, 222, 243-246, 271, 272, 324, 327, 331, 344, 345, 370, 381, 382, 395, 401, 406, 462, 500-503, 520, 554, 555, 561, 564, 565, 574, 603, 609, 631, 637-641, 650, 653, 654, 660, 661, 681, 690, 696, 727, 755, 762, 765, 767, 770, 779, 797, 800, 809, 836, 839, 879, 889, 906, 912, 917, 933, 939, 941, 943, 954, 969, 986, 1008, 1012, 1037, 1046, 1055, 1075, 1080. Aquatic toxicity - 88, 94, 151, 289, 328, 469, 471, 662, 677, 726, 810, 822, 832, 851, 898, 945, Background material - 16, 26, 30, 53, 74, 186, 187, 206, 229, 230, 260, 335, 341, 351, 427, 440, 448, 449, 463, 464, 472, 479-482, 507, 625, 626, 700, 702, 712, 719, 799, 824, 891, 940, 946, 971, 998-1003, 1007, 1014-1029,' 1065, 1068, 1087. Bibliographies - 5, 85, 109-112, 248-253, 267, 293, 468, 490, 865, 983, 1090. Chronic toxicity - 409, 451, 549, 551. Editorials - 9, 65, 102, 132, 176, 182, 239, 258, 259, 292, 294, 306, 323, 438, 453, 465, 587, 593, 602, 616, 731, 760, 769, 796, 813, 828, 847, 982, 1041, 1052, 1059, 1060, 1079, 183, 217, 220, 231, 232, 236, 238, 349, 350, 352, 396, 397, 424-426, 436, 634, 644, 670, 676, 687, 694, 695, 853-855, 886, 913, 924, 931, 956, 972, 1089. Environmental fate - 13, 24, 25, 45, 47, 107, 210-214, 247, 255, 285, 297, 312, 336, 353, 363, 387, 391, 487-489, 509, 512, 591, 624, 629, 648, 655, 664, 675, 680, 689, 705, 725, 736, 739, 861, 871, 872, 884, 890, 897, 904, 910, 989, 1048, 1050, 1053, 1054, 1076, 1083, 1085, 1086, 1093, 1098, 1100, 1107, 1115, 1116. Industrial accidents and military exposure - 38, 58, 99, 160, 225, 273, 274, 362, 366, 367, 383, 405, 416, 491, 492, 494-496, 538-540, 573, 652, 745, 763, 764, 782, 795, 817, 818, 827, 834, 870, 937, 952, 964, 981, 993, 1104, 1106. �Metabolism - 10, 21, 39, 44, 60, 61, 64, 75, 80, 81, 119, 120, 125, 140, 145, 157, 158, 165, 169-171, 177, 192, 227, 287, 288, 298-300, 313, 316, 318, 330, 337, 358, 361, 376, 379, 380, 390, 392, 393, 399, 400, 410, 446, 450, 458-461, 470, 476, 477, 504, 510, 526, 543, 547, 552, 553, 563, 567-570, 577, 578, 581, 588, 589, 592, 604, 606, 607, 613-615, 630, 646, 647, 668, 697, 714, 732, 741, 742, 747, 749, 750, 759, 774, 775, 777, 783, 784, 787-793, 801, 808, 835, 837, 840, 857, 858, 860, 888, 905, 926, 934, 990, 1011, 1031, 1081, 1102, 1110. Monitoring in humans and animals - 55, 202, 263, 268, 546, 556, 579, 580, 590, 678, 707, 843, 885, 927, 955, 1042. Monitoring in the environment, food, and drinking water - 4, 11, 12, 56, 98, 108, 113, 148, 149,, 166, 167, 174, 317, 364, 394, 518, 525, 597, 599, 622, 692, 704, 717, 737, 740, 803, 873, 877, 909, 1044, 1069, 1088, 1105. Mutagenicity - 7, 8, 28, 71, 86, 87, 92, 142, 155, 215, 234, 235, 339, 375, 475, 493, 628, 632, 633, 703, 773, 812, 831, 863, 880, 882, 883, 893, 896, 962, 1032, 1094, 1108, 1109. Oncogenicity - 32, 35, 37, 78, 90, 121, 175, 190, 256, 257, 296, 411, 413-415, 456, 478, 550, 571, 572, 699, 753, 944, 948, 979, 980, 991, 1010. Reproductive toxicity - 14, 22, 29, 42, 66, 91, 100, 141, 143, 178, 180, 195, 196, 198-200, 224, 241, 264, 266, 275, 291, 301, 308-310, 356, 385, 402, 408, 441-443, 457, 5.29, 531, 558, 560, 584, 594, 605, 608, 621, 669, 685, 701, 721-723, 728, 761, 821, 833, 852, 856, 868, 875, 892, 907, 908, 911, 915, 916, 918, 920, 921, 925, 930, 935, 936, 967, 974, 994, 1004, 1036, 1066, 1071. Review articles - 1, 15, 17, 20, 23, 31, 33, 36, 40, 48, 69, 79, 83, 93, 103, 104, 114, 124, 127, 133, 137, 150, 161, 184, 189, 191, 193, 209, 218, 219, 221, 226, 237, 254, 261, 262, 276, 283, 284, 286, 295, 302, 307, 321, 322, 329, 332-334, 340, 342, 348, 354, 357, 359, 371, 378, 423, 428-432, 454, 466, 467, 483, 484, 486, 498, 499, 513, 515, 519, 521, 523, 524, 527, 528, 534-537, 586, 598, 600, 611, 612, 619, 620, 623, 635, 636, 642, 649, 651, 657, 666, 671-673, 682, 684, 688, 711, 715, 718, 720, 734, 752, 756, 766, 768, 772, 785, 786, 794, 798, 805, 806, 815, 816, 820, 830, 838, 866, 869, 876, 881, 894, 900, 903, 914, 950, 957, 958, 976, 987, 988, 995, 1006, 1033, 1034, 1047, 1049, 1051, 1061-1064, 1074, 1077, 1078, 1082, 1084, 1099, 1111. Subacute toxicity - 18, 19, 95, 97, 105, 162, 188, 228, 242, 269, 270, 278, 326, 369, 398, 403, 445, 514, 542, 5448, 618, 667, 748, 757, 758, 778, 826, 850, 887, 953, 966, 1035, 1039, 1045, 1058, 1067, 1114. Testimonies and statements - 27, 76, 77, 172, 181, 216, 279, 343, 407, 656, 683, 691, 693, 709, 710, 713, 730, 841, 847, 1030, 1096. Use and release of herbicides - 6, 57, 131, 152, 159, 179, 223, 325, 386, 417, 419, 473, 474, 545, 585, 674, 738, 744, 771, 781, 928, 985, 996, 997, 1097. *U.S. BOVEBMMEHT PRIK2ING OFFICE: 1981-0-522-610/22 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5778 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Description An account of the resource <strong>Corporate Author: </strong>JRB Associates, McLean, Virginia Date A point or period of time associated with an event in the lifecycle of the resource 1981-10-01 Title A name given to the resource Review of Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins, Volume II: Annotated Bibliography ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/87113f62cbc7e6cad68b3aa86c067db7.pdf d6b485aa833374411443f3769b9298b7 PDF Text Text Item D Number 0*777 D MotSGannBd Author Corporate Author JRB Associates, McLean, Virginia RflUOrt/ArtlClB TltlB Review °f Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins, Volume I: Analysis of Literature Journal/Book TltlB 1981 Month/Day Color Numhar of Images Oct °ker D ° Descripton Notes Thursday, March 28, 2002 Page 5777 of 5780 �Veterans Administration Review of Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins Volume I Analysis of Literature Department of Medicine and Surgery �VA Contract Number: V101(93)P-823 JRB Project Number: 2-816-03-744-00 Review of Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins Volume I This report was prepared under a cost reimbursement-type contract awarded on a competitive bid basis on December 15, 1980. The contract will run for nine and one-half months and is funded at $111,743. Prepared for Contracting Officer's Technical Representative: Barclay M. Shepard, M.D. Special Assistant to the Chief Medical Director for Environmental Medicine (102) Department of Medicine and Surgery Veterans Administration 810 \fermont Avenue, N.W. Washington, D.C. 20420 Submitted by: JRB Associates 8400 Westpark Drive McLean, Virginia 22102 �FOREWORD Public Law 96-151 enacted December 20, 1979, mandated the Veterans Administration to conduct "...a comprehensive review and scientific analysis" of the worldwide literature on Agent Orange and other phenoxy herbicides. The need to conduct such a review was in response to an increasing awareness among veterans the Congress and the public of the potential long-term health consequences of exposure to these herbicides and the contaminant dioxin. This report was prepared wholly and exclusively by JRB Associates, Inc., and represents an independent assessment of the current state of science relating to the herbicides used in conjunction with the Vietnam conflict. The publication of this document does not signify that the contents necessarily reflect the views and policies of the Veterans Administration. �PREFACE The controversy surrounding the tactical use of herbicides in Southeast Asia during the Vietnam conflict has now extended into its third decade. Few environmental or occupational health issues have received the sustained national attention that has been focused on "Agent Orange". The controversy centered first on the actual employment and subsequent ecological effects of herbicides in South Vietnam, then on the question of the safe disposal of surplus herbicide following the conflict, and lastly, on whether herbicides were responsible for health problems reported among Vietnam veterans. As each facet of the controversy came to the attention of the public, more government agencies were tasked to deal with the associated issues. Hence, today a great many branches and agencies of the Federal government are involved in seeking resolutions of the scientific, medical, legal and social problems surrounding "Agent Orange." In addition, many state governments have enacted legislation relating to this highly controversial issue. The basis for resolving the Agent Orange controversy must in large measure stem from the results of scientific inquiry. Appropriately, the preparation of extensive reviews and summaries of the scientific literature have appeared in the past, e.g., Midwest Research Institute Report (1967), National Academy of Science Report (1974) and United States Air Force Technical Report (1978). The present report therefore has benefited from the information developed in previous reports as well as from recently published scientific data. The volume of available scientific literature pertaining to the herbicides used in Southeast Asia has increased almost geometrically since the 1967 report. Especially important has been the increase in the scientific data on the toxic contaminant 2,3,7,8-tetrachlorodibenzo-£-dioxin (TCDD). The present report is published in two volumes. Volume I presents a detailed scientific assessment of the literature, and Volume II contains an annotated bibliography of the relevant literature. Although much is known about the toxicity of the herbicides used in Vietnam, a number of gaps in the scientific knowledge are still present. It is hoped that this two-volume report may serve to focus the continuing public dialogue and future scientific inquiry on those aspects of the problem in which substantial doubt or gaps in information remain. This report should also assist researchers in identifying opportunities for the systematic development of new knowledge based on what is now known and accepted as fact. VETERANS ADMINISTRATION October 1981 11 �CONTENTS Page Foreword ii Figures vii Tables viii 1 Introduction 1-1 Background of this Report 1-1 Methods 1-3 Limitations 1-4 Conclusions and Gaps in Current Knowledge 1-6 Recommendations 2 1-13 The Military Use and Application of Herbicides in Vietnam 2-1 Summary of Tactical Use 2-1 Description of Herbicides Used in Vietnam 2-2 The HERBS Tape 2-11 Geographical and Temporal Distribution of Herbicides in South Vietnam 2-11 Quantitative and Qualitative Use Distribution of Herbicides in South Vietnam 2-18 Release of TCDD in South Vietnam during Spraying of Herbicides . . 2-20 Release of Esters of 2,4,5-T 2-22 Release of Esters of 2,4-D and the Triisopropanolamine Salt of 2,4-D 2-23 Release of the Triisopropanolamine Salt of Picloram 2-23 Release of Cacodylic Acid and its Sodium Salt 111 2-24 �CONTENTS (Continued) Page 3 3-1 Environmental Fate and Monitoring of 2,4-D, 2,4,5-T, Picloram, and Associated Esters and Salts 3-1 Environmental Fate and Monitoring of TCDD 3-8 Environmental Fate of Cacodylic Acid 3-27 Conclusions 3-28 Metabolism, Enzyme Induction, and Mechanism of Action 4-1 2,4-D 4-1 2,4,5-T 4-4 TCDD 4 Environmental Fate and Monitoring 4-7 Diquat . 4-16 Diuron and Monuron Bromacil 4-18 Cacodylic Acid 4-18 Summary and Conclusions 4-19 Human Exposure to TCDD from Industrial and Military Uses 5-1 Industrial Explosions 5-1 Occupational Exposure that did not Involve Explosions 5-15 Human Exposure to TCDD from Industrial Waste Disposal, and Laboratory Exposure 5-21 Human Exposure to TCDD from Military Use of Herbicides 6 4-17 Picloram 5 4-17 5-26 Acute Toxicity » 6-1 Mortality 6-1 Dermal Lesions 6-17 Pulmonary Lesions 6-18 IV �CONTENTS (Continued) Hepatotoxicity .......................... 6-20 Neurotoxicity ........................... 6-23 Nutrient Absorption and Utilization Hematological Effects ................ ............... Structure and Function of Lymphatic Tissues Renal Effects ...... Mortality Hepatotoxicity Hematological Effects ............ Renal Effects 7-11 7-13 7-17 ........... 7-20 ............ 7-22 ........................... Cardiovascular Effects .................. Summary and Conclusions .......... Reproductive Toxicity . 7-23 . . . . 7-23 ........... ....................... ............. ... .......... ..... ............................... ............. .............................. ............................. 7-23 8-1 .............................. Combinations of 2,4-D, 2,4,5-T, and TCDD Picloram 7-1 ................ Structure and Function of Lymphatic Tissues Diquat 7-1 ................... Nutrient Absorption and Utilization TCDD 6-37 .................. . 6-33 6-37 ....................... ...... 6-32 . . 6-36 ............................. Dermal Lesions .... 2,4,5-T ..... .................... Subacute and Chronic Toxicities 2,4-D . ...................... Summary and Conclusions. . 8 ....... ......................... Cardiovascular Effects 7 , 6-27 8-1 8-3 8-7 8-12 8-18 8-18 �CONTENTS (Continued) Page Dalapon Bromacil 8-19 Diuron 8-19 Cacodylic Acid . 8-19 Summary and Conclusions 8-19 Mutagenicity 9-1 Human Cytogenetic Studies 9-1 Cytogenetic and Host-mediated Studies in Mammals 9-6 Mutagenicity Studies in Drosophila 9-9 Mutagenicity in In Vitro Systems 9 8-18 9-12 Conclusions Carcinogenicity 10-1 Epidemiologic Studies 10-1 Animal Studies 10-8 Summary of Carcinogenic Potential of Herbicides 10 9-20 10-19 APPENDIX: Ongoing Epidemiologic Research VI A-l �FIGURES Number Page 2-1 Chemical Structures of Parent Herbicides 2-3 2-2 2-3 2-4 2-5 Chemical Structures of Herbicide Esters and Salts, Used in Vietnam 2-4 Chemical Structure of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) 2-5 Chemical Structures of Herbicides Used in Small Quantities in Vietnam 2-5 South Vietnam 2-7 2-6 Defoliation Missions 2-12 2-7 Crop Spraying Missions 2-13 3-1 Semi-logarithmic Plot of Soil Concentrations (Parts per Trillion) of TCDD in Herbicide Orange Biodegradation Studies at Eglin AFB, Florida, and Hill AFB, Utah VII 3-22 �TABLES Number Page 2-1 Application of Herbicides in Vietnam by Year. 2-16 2-2 Annual Number of Acres Sprayed in Vietnam 2-17 2-3 Estimated Acreage Sprayed One or More Times, 1965-1971 2-19 3-1 Physical Properties of Herbicides 3-2 4-1 Enzymes Responsive to TCDD Induction 4-12 5-la References on Industrial Explosions that Involved TCDD 5-2 5-lb References Which Review Industrial Exposures to TCDD 5-2 5-2a References on Industrial Exposures to TCDD 5-3 5-2b Other Industrial Exposures 5-4 5-3 References on Other Human Exposures to TCDD . . . . . 5-5 5-4 Health Effects from Industrial Accidents Involving TCDD 5-11 5-5 Health Effects of Occupational Exposure to TCDD 5-18 5-6 Health Effects of Human Exposure to TCDD 5-22 6-1 LD5Q Values (in mg/kg) for 2,4-D 6-2 6-2 LD5Q Values (in mg/kg) for 2,4,5-T 6-3 6-3 References on the Acute Toxicity of 2,4-D 6-4 6-4 References on the Acute Toxicity of 2,4,5-T 6-8 6-5 LD5Q Values (in mg/kg) for TCDD 6-10 6-6 References on the Acute Toxicity of TCDD 6-11 6-7 Acute Human Expousres to 2,4-D 6-25 7-1 References on the Subacute and Chronic Toxicities of 2,4-D. . . . 7-2 7-2 References on the Subacute and Chronic Toxicities of 2,4,5-T. . . 7-5 7-3 References on the Subacute and Chronic Toxicities of TCDD . . . . 7-7 8-1 References on the Reproductive Effects of 2,4-D in Mammals. . . . 8-2 Vlll �TABLES (Continued) Number 8-2 Page References on the Reproductive Effects of 2,4,5-T (less than 1 ppm TCDD) . . , 8-5 References on the Reproductive Effects of TCDD Exposure to Females 8-11 References on the Reproductive Effects of 2,4,5-T (with Unknown Levels of TCDD) 8-13 9-1 Summary of Short-term Test Results 9-1 9-2 Cytogenetic Effects of Herbicides on Human Cells 9-2 9-3 Summary of Cytogenetic and Mutagenic Effects of Herbicides on Mammals 9-7 Summary of Mutagenic Effects of Herbicides on Drosophilia Melanogaster 9-10 9-5 Assays of Herbicides on In Vitro Mutagenicity Assays 9-13 9-6 Results of In Vitro Assays for Detecting Mutagenicity of Herbicides 9-14 8-3 8-4 9-4 IX �CHAPTER 1 INTRODUCTION The potential long-term health consequences of exposure to herbicides used during the Vietnam Conflict has been an issue of increasing public concern for the past decade. Among the environmental and occupational health and safety issues raised during the 1960s and 1970s, few have received the sustained and growing national attention that has been focused on "Agent Orange." The concern over U.S. military personnel exposures to herbicides in Vietnam has attained national significance for a variety of reasons. The problem was unexpected: the herbicides selected for use as defoliants in Vietnam had been widely used within the U.S. for many years, were considered only moderately toxic, and were believed to be safe and effective when handled with care. The presence of a highly toxic trace contaminant in one of the compounds was not recognized until the late 1960s, when the defoliation program was well established in Vietnam. The number of persons potentially exposed is almost unprecedented: an estimated 2.4-2.8 million U.S. military personnel served in Vietnam. The number and range of health effects attributed to exposure are great: from chloracne, to headaches, excessive tiredness, sleeplessness, and increased susceptibility to colds and flus, to birth defects in offspring, and cancer. The problem does not admit to a ready solution: in almost all cases, the magnitude and duration of individual exposures to herbicides in Vietnam cannot be determined, usually cannot even be estimated with accuracy, and may be confounded by the wide-spread use of the same herbicides within the U.S. The search for answers to this dilemma has itself been complicated by the enormous public concern. The scientific literature varies widely in detail, quality, and findings. Researchers' suggestions and conclusions have been seized and debated, sometimes without sufficient attention to the data underlying those statements, the methods of collection used to obtain the data, and the approaches to analysis used to interpret them. All of these elements of scientific inquiry have limitations. "Good" research is necessarily careful research, and careful research is most often conservative of statement. Only recently have researchers addressing the problem of herbicide exposures in Vietnam had sufficient time since the recognition of the problem to begin to benefit from the opportunities for knowledgedevelopment provided by lines of inquiry systematically pursued. 1.1 BACKGROUND OF THIS REPORT Fifteen different herbicides were shipped to and used in Vietnam between January 1962 and September 1971. Eight of these formulations contained the phenoxy herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) 1-1 �or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or both. herbicides used in Vietnam included: The fifteen • PURPLE: a formulation of 2,4-D and 2,4,5-T used between January 9, 1962, and late 1964 • PINK: containing 2,4,5-T and used between 1962 and 1964 GREEN: containing 2,4,5-T and used between 1962 and 1964 • ORANGE: a formulation of 2,4-D and 2,4,5-T used between January 1965 and April 1970 • WHITE: a formulation of picloram and 2,4-D • BLUE: containing cacodylic acid • ORANGE II: a formulation of 2,4-D and 2,4,5-T used in 1968 and 1969 • DINOXOL: a formulation of 2,4-D and 2,4,5-T; small quantities were tested in Vietnam between 1962 and 1964 TRINOXOL: containing 2,4,5-T; small quantities were tested in Vietnam between 1962 and 1964 • DIQUAT: small .quantities tested in Vietnam between 1962 and 1964 • BROMACIL: small quantities tested in Vietnam between 1962 and 1964 • TANDEX: small quantities tested in Vietnam between 1962 and 1964 • MONURON: small quantities tested in Vietnam between 1962 and 1964 • DIURON: small quantities tested in Vietnam between 1962 and 1964 • DALAPON: small quantities tested in Vietnam between 1962 and 1964. Over 80 percent of the herbicides sprayed in Vietnam was "Agent Orange." After 1965, Herbicide Orange was used almost exclusively. Those phenoxy herbicides containing 2,4,5-T also contained trace contaminants produced in the manufacturing process, including: • 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) • 2,7-dichlorodibenzodioxin • Penta-, hexa-, hepta-, and octachlorodibenzodioxins. Phenoxy herbicides generally contain other dioxin contaminants in proportions of between 2-to-l and 10-to-l of the quantities of TCDD contained. The levels 1-2 �of contamination of these dioxins in the phenoxy herbicides used in Vietnam is unknown. The U.S. Air Force (1979) estimates the maximum TCDD concentration in 2,4,5-T produced during the period was 15-47 parts per million (ppm); the Air Force estimates the mean concentration was 1.91 ppm. It is the presence of the dioxin contaminant, TCDD, in 2,4,5-T that has been the focus of much of the debate about the health consequences of exposure to herbicides used in Vietnam. Although other dioxin contaminants were probably present in the phenoxy herbicides used, no other dioxin has been shown to be as toxic as 2,3,7,8-tetrachlorodibenzo-para-dipxin. In January of 1980, in response to the national concern over exposures to herbicides in Vietnam, the U.S. Congress included provisions in Public Law 96-151 mandating that the Veterans Administration conduct a critical review of the worldwide literature on the phenoxy herbicides. In December of 1980, again in response to the national concern, the Veterans Administration expanded that mandate and initiated a critical review of the worldwide literature on all the herbicides used in Vietnam, including the phenoxy herbicides and their dioxin contaminant. This volume and the accompanying annotated bibliography constitute the final report of that critical review. For those who want to go beyond this report, several very good general reviews and references have been published, including: NRC, 1974; IARC, 1977; Joint IARC/NIEHS Working Group, 1978; National Research Council of Canada, 1978; Young et al., 1978; -Bovey and Young, 1980; and Esposito et al., 1980. 1.2 METHODS The methodology used in compiling this critical review of the literature included five principal activities: • Literature identification • Literature retrieval and control • Document indexing and abstracting • Literature analysis • Report preparation. Initially, three assumptions guided the development of the effort: • The Congress was primarily interested in the health consequences of exposure to the herbicides used in Vietnam (i.e., the substances). • The Congress was primarily concerned with health effects among otherwise healthy adult men (i.e., the population). 1-3 �• The Congress was seeking information and scientific judgment on the state of knowledge concerning the likelihood and severity of health effects resulting from exposure to the herbicides used in Vietnam (i.e., the outcomes). These assumptions led to the development of a set of relevancy criteria which guided the culling of the literature. The activity used both automated and manual search techniques, including tree-searches of cited references. It was facilitated by the willing cooperation of a number of involved Federal agencies, including the Department of Agriculture, the Environmental Protection Agency, the Food and Drug Administration, the Center for Disease Control, and the Department of Defense. Using an expanding and reiterative search strategy and the relevancy criteria developed, over 1,400 documents were acquired and examined. About 1,200 were judged to fall within the relevant parameters, were thoroughly reviewed, and are included in the accompanying bibliography. Over 900 of these documents were annotated in detail by staff scientists; relevant abstracts, summaries, review articles, and editorials were included in the bibliography, but were not annotated. This body of culled literature was used by staff scientists in the compilation of this report. Particular attention was payed to the quality of the data presented and the methods used in its collection and analysis. Attention was also directed toward conflicting findings and gaps in available information. The draft re.port, exclusive of final conclusions, was reviewed by a number of senior scientists and expert consultants. This report was developed exclusively by the contractor, JRB Associates, without consultation, advice, or direction from the Veterans Administration. The conclusions, opinions, judgments, and recommendations presented are those of the contractor, and may not reflect the opinions, positions, or policies of the Veterans Administration. The goal of this report is to present a balanced and critical review of the current state of published scientific knowledge relevant to the problem of exposures to herbicides, and particularly phenoxy herbicides, in Vietnam. This report cannot solve such a complex problem. It may, however, serve to focus and direct future discussion to those aspects of the problem in which substantial doubt or lack of information remains, and thereby contribute to the systematic pursuit of promising opportunities for knowledge-development. 1.3 LIMITATIONS As this report devotes considerable attention to the limitations inherent in the work of others, it is important that one attempt to recognize the report's own inherent limitations. First, no search of the literature in so volatile a subject area as this can in fact be complete. New research continues to be published, the 1-4 �automated data bases used to search the literature are often as much as a year behind the journals they abstract, and any automated search is necessarily limited by the intelligence that originally abstracted and keyed the journals' contents. The contents of the accompanying bibliography have been brought as much up to date as possible by manual searches of recent issues of key journals and the advice and input of expert readers. It is inevitable that some published and many unpublished reports are not included here. It is hoped that few of these contain critically relevant or important, supported findings not duplicated elsewhere. Second, all research efforts must yield to some limitations of time and resources. In this case, both the time (nine-and-a-half months) and the resource limitations were real concerns. There are documents that, although identified and considered relevant, could not be obtained within the time available. These are relatively few. There are documents that might have proven relevant if examined, but that were judged beyond the criteria of relevance when identified and so were not acquired. There are documents that, almost certainly relevant, simply were uncovered too late to be included in the schedule. Several reports recently declassified by the U.S. Air Force fall into this category. Ultimately, it is again hoped that few of these omissions contain new, important, supported findings not duplicated elsewhere. Third, realities of time, resources, and practicality served to limit the scope of the literature search in very specific terms, as well: • Substances similar, in structure-activity relationships to the herbicides used in Vietnam were not considered. These substances include Silvex, paraquat, and dioxins other than 2,3,7,8-TCDD. • The fate and effects of the metabolites of the Vietnam herbicides were not considered. For example, the fate and toxicological effects of cacodylic acid (Herbicide Blue) are described, but not the fate and toxicological effects of inorganic arsenic administered in the absence of cacodylic acid. A final element critical to the veterans and public concern over exposures to herbicides in Vietnam was judged beyond the scope of the present study: the estimation of exposure potential inherent in the Vietnam experience. Approximately 2.6 million U.S. military personnel served in South Vietnam between January 1, 1965, and March 31, 1973, the period of heaviest herbicide use (GAO, 1979a). The personnel that appear to have had the greatest risk of exposure were the people who took part in the actual spraying of the herbicides. This would include the flight mechanics of RANCH HAND aircraft who generally operated the spraying units; persons operating spraying units on helicopters, trucks, and boats; and persons using back-pack sprayers. Although most of the herbicide spraying was done aerially, and up to 87 percent by RANCH HAND fixed-wing aircraft, up to 4 percent may have been applied in ground operations (Craig, 1975). Although it is apparent that exposures did 1-5 �occur among spray operators and RANCH HAND aircrews (Young et al., 1978; GAO, 1979b), little can be said about the quantity and quality of such exposures. Many variables alter the rate of absorption of 2,4,5-T by workers. Some of these factors, including type of occupation, rate of spraying, type of protective clothing, and rates of absorption by both dermal and inhalation routes, were considered in developing a model for estimating potential dosages of 2,4,5-T absorbed by workers (RPAR Assessment Team, 1979). The Assessment Team used several different values for each parameter, based on assumptions regarding the conditions of exposure. They then performed exposure assessments for occupational situations. However, Leng (1978) has challenged some of the assumptions used by the RPAR Assessment Team to calculate their exposure assessment, including the extent of skin exposure and dermal absorption rates. Nisbet (1980) has also presented estimates of human exposures in the general population. Since the assumptions used for these exposure assessments apply to occupational use of herbicides, but not military use, the results are not necessarily related to assessments of potential exposure in Vietnam. The General Accounting Office (1979b) further attempted to estimate troop deployment in spray areas, and aborted missions and dumped herbicide cargos have also been reported. Once again, it is apparent that ground troop exposures occurred in Vietnam, but it is beyond the scope of this report to attempt to assess the magnitude and duration of such exposures; this work must be carried out by others. 1.4 CONCLUSIONS AND GAPS IN CURRENT KNOWLEDGE This section presents summary statements of the conclusions supported by the available literature and gaps in current knowledge identified during the literature review. These summary statements are arranged by topic areas addressed in subsequent chapters of this report: • Metabolism • • • • • • 1.4.1 Human exposure to TCDD Acute toxicity Subacute and chronic toxicities Reproductive toxicity Mutagenicity Carcinogenicity. Metabolism (Biodynamics and Biotransformation) Conelus ions • Pharmacokinetics of 2,4-D and 2,4,5-T in humans have been described. �• Both 2,4-D and 2,4,5-T are cleared rapidly from the blood after they are absorbed, with half-lives for plasma clearance in humans of 12-23 hours. • Both compounds are excreted by the kidney primarily as the unmetabolized compounds. • Renal clearance rates for phenoxy acids in animals decrease at high doses that cause nephrotoxicity and saturate the renal transport system. • The clearance rate of 2,4-D in humans decreases when the urinary pH is low. • Neither 2,4-D nor 2,4,5-T has been shown to accumulate in animal fat. • Both compounds reach fetal tissues after they are administered to pregnant animals. • TCDD is cleared slowly, with half-lives for body clearance of 2-3 weeks in animals. • TCDD undergoes biotransformation and the metabolites are rapidly excreted in bile. • TCDD is retained in the liver of the rat, a species that shows an hepatotoxic response to TCDD, to a far greater extent than in the livers of two other species which do not show liver lesions after TCDD administration. • Diquat is absorbed by the lung, but is not retained in the lung and is rapidly cleared by animals. • Free radical formation does not appear to be diquat's mechanism of toxicity under conditions of normal oxygen tension. • Diuron and bromacil undergo biotransformation prior to excretion; diuron is excreted by the kidney. TCDD Enzyme Induction and Receptor Binding • TCDD is a potent inducer of various microsomal enzymes; the induced enzymes show elevated levels over a long period of time, • In certain strains of mice, TCDD binds to a cytosol receptor, the gene product of the Ah locus. 1-7 �Gaps in Information Information on the following topics is incomplete or missing in the literature reviewed for this analysis: • Patterns of biotransformation, distribution, and excretion of TCDD in humans • The chemical structures of TCDD metabolites in bile • Differences in distribution and biotransformation of TCDD for a wide range of species • Differences in TCDD-receptor binding capacity and extent of enzyme induction in a wide range of species • Pathways for the biotransformation of cacodylic acid and the relative importance of each pathway • Biodynamics, including pathways and rates of elimination in humans or animals for: bromacil, picloram, dalapon, monuron, tandex. 1.4.2 Incidents of Human Exposure to TCDD Conclusions • Chloracne is the most consistently reported health effect of TCDD exposure in humans; in severe cases, chloracne has lasted for 28 years; milder cases have gone undetected or have disappeared in less than a year. • Neurasthenia, a series of subjective complaints including irritability, fatigue, and insomnia, has been reported after many industrial accidents and exposures; in 1 instance these complaints occurred in the absence of chloracne; a 2-year latency period between TCDD exposure and the onset of neurasthenia has been reported. • Other neurological disorders (as peripheral neuritis) and hepatic disorders (as hepatomegaly) have been reported after several of the incidents. • The earlier accidents and exposures were associated with a wider variety of symptoms and more severe symptoms than the later incidents. • Porphyria cutanea tarda and gastrointestinal problems have not been commonly reported and seem to be associated with long-term exposure. • An increased risk among exposed people has not been established in mortality studies; increases in any particular cause of death has not been observed for more than 1 study group, so far. 1-8 �No data have been systematically collected for a clearly defined study group from Vietnam; health effects are usually claimed by individuals, without documentation by health professionals; exposure to herbicides in Vietnam (and potentially TCDD) is presumed in these studies and exposure levels are unknown; these data have not been compared to any control groups, in general; and symptoms reported often have been nonspecific and may be associated with other factors present in combat situations. Gaps in Information The following information is missing from most accounts of human exposure to TCDD. • The number of exposed people who were not affected • Health status of exposed workers that did not develop chloracne • Incidences of conditions other than chloracne and comparison of these data with data from control groups • Standardization of methods of evaluating symptoms of neurasthenia for purposes of comparison among different studies • Conditions that could be detected by the examination methods used, but which did not occur (especially for conditions reported in other incidents) • Sufficient mortality data for analysis (due to the short period of time that has lapsed since some of the incidents occurred and the relatively small number of workers exposed) • Exposure levels • Human health effects from use of defoliants in Vietnam have not been systematically documented. 1.4.3 Acute Toxicity Conclusions • For both 2,4-D and 2,4,5-T, the single oral dose lethal to 50 percent of exposed animals (the oral LDc/j) is between 350-800 mg, based on published data, almost all of which was published 20-30 years ago. • The cause of death from lethal doses of 2,4-D or 2,4,5-T to animals is unknown; both compounds produce several non-specific effects, such as mild weight loss. • 2,4-D produces neurotoxicity in humans and animals. 1-9 �• The LDcQ values for TCDD are extremely low (between 1-300 ug/kg) and vary widely among different species. • A long latency period, of about 3 weeks, occurs between TCDD administration to test animals and death, and the cause of death is usually not known. • The in vivo characteristics of TCDD intoxication suggest toxicity on a cellular level, although TCDD toxicity has not been demonstrated in cultured cells. • Thymic atrophy (without a corresponding loss in immune function) and severe weight loss have been observed in many species after TCDD exposure. • Weight loss does not result from decreased food consumption, disturbances in absorption of nutrients from the gastrointestinal tract, or a stress reaction mediated by endocrine glands. • TCDD produces hepatotoxicity only in some species. • The oral LD,-0s for monuron and diuron in animals are about 1,000 mg/kg; both produce neurotoxicity; death usually occurs 1 day after exposure, from respiratory or cardiac failure. • The oral LD,ns for picloram and dalapon in animals are between 2,000-8,000 mg/kg;. death occurs within hours of a lethal dose of dalapon. • The oral LD-Q for diquat in animals is between 30 and 200 mg/kg; doses in this range produce severe gastrointestinal lesions and death within 2 weeks; doses 4-5 times higher produce neurotoxicity and death within several hours. • Values ranging from 200 to 3,000 mg/kg have been reported for the oral LDcn for cacodylic acid in rodents. Gaps in Information The following information has not been reported or is not adequate in published literature: • Effects of acute exposure to 2,4,5-T in humans • LDsn values for 2,4,5-T samples with less than 0.1 ppm TCDD • Verification of the LD5Q values for 2,4-D that were published 20-30 years ago 1-10 �• LD-Q values for cacodylic acid, published in a refereed journal, with descriptions of details on sample purity, methods used, and patterns of toxicity that could be compared to those of inorganic arsenic poisoning • The causes of death and target organs for picloram and dalapon • Information on the acute toxicity of tandex. 1.4.4 Subacute and Chronic Toxicities Conclusions • 2,4-D and 2,4,5-T are not cumulative toxicants. • Subacute toxicity of both compounds resemble their acute toxicities, except that subacute doses of 2,4-D do not produce myotonia, but cause bleeding of the gums in dogs. • TCDD is a limited cumulative toxicant; cumulative effects of doses administered within a month of each other have been observed, but not for doses administered beyond about one month. • The subacute effects of TCDD that are not observed after acute doses are porphyria and depletion of blood cells; iron deficiency protects TCDD-treated animals from the porphyrinogenic effects. • Chronic doses of diquat cause cataracts in two species tested (dog and rat). Gaps in Information • 1.4.5 The subacute effects of cacodylic acid, monuron, diuron, bromacil, and tandex have not been described thoroughly or at all. Reproductive Toxicity Conclusions • No human reproductive effects have been verified to date from male or female exposure to 2,4-D, 2,4,5-T, or TCDD. • In the two experiments that involved exposure of males only to phenoxy acids prior to conception, no evidence of reproductive effects was observed; (combinatinons of 2,4-D, 2,4,5-T, and TCDD were administered in one study and of 2,4,5-T with an unknown level of TCDD contamination in another study). • After 2,4-D is administered to pregnant animals, decreased fetal growth rates have occurred. 1-11 �• After 2,4,5-T (with less than 0.1 ppm TCDD) is administered to pregnant animals, decreased fetal growth rates have occurred and at higher doses in mice, cleft palate is produced; these effects are observed in the absence of maternal toxicity, this teratogenic effect of 2,4,5-T has not been observed in the rat, hamster, monkey, or rabbit. • After TCDD is administered to pregnant mice, cleft palate and renal abnormalities is fetuses have occurred. • Synergistic effects may occur in mice when the level of TCDD added to 2,4,5-T exceeds 5 ppm; this effect pertains to the incidence of cleft palate. • Diquat, dalapon, and diuron produce adverse effects on development only when they are administered at .doses that cause maternal toxicity. • Bromacil and picloram have not produced effects on development at any doses tested. Gaps in Information The following types of studies have not been conducted and published to date: • The effects of human male exposure during a limited time prior to conception on reproductive outcome of the resultant pregnancy, for documented exposure to 2,4-D, 2,4,5-T, and/or TCDD • The effect of exposure of males of mammalian animal species to any single herbicide or dioxin, alone, on reproductive performance. 1.4.6 Mutagenicity Conclusions • 2,4-D and 2,4,5-T produce weak mutagenic effects. • TCDD has shown mutagenic effects in bacteria and yeast systems, which have not been confirmed yet in mammalian in vivo tests. • Cacodylic acid, bromacil, and monuron have not produced mutagenic effects in in vitro tests. » Diquat and diuron have produced mutagenic effects in vitro, which have not been confirmed yet in vivo. 1-12 �Gaps in Information The following gaps in information remain: • The in vivo mammalian mutagenic effects of TCDD, diquat, and diuron • The mutagenic potential of dalapon, picloram, and tandex in any system. 1.4.7 Care inogenic ity Conclusions • Evidence from human studies suggest that exposure to phenoxy acids, with concomitant exposure to many other pesticides and to TCDD, may lead to an increased risk of soft-tissue sarcoma; the etiologic role specifically of phenoxy acids has not been elucidated. • Mortality studies of groups of human workers exposed to TCDD has not revealed an increased carcinogenic risk in these people, although the numbers of deaths in these groups have been exceedingly small to date. • Animal studies have not produced any evidence that 2,4-D, 2,4,5-T, cacodylic acid or picloram are carcinogenic. • TCDD appears to act secondarily or indirectly in enhancing the careinogenicity of other components (usually unidentified) in animal studies. • Carcinogenic effects of monuron have been observed in animals; further studies of the careinogenicity of this compound are being conducted. Gaps in Informat ion Information on the carcinogenic potential of diquat, diuron, dalapon, bromacil, picloram, and tandex and on only 2,4-D, 2,4,5-T, or TCDD, without concomitant exposure to trichlorphenol or other herbicides in humans is missing. 1.5 RECOMMENDATIONS This section presents recommendations for further study drawn from the review of the literature addressed in this report. • Dalapon and bromacil are compounds that were used in small amounts in Vietnam and have not been shown to pose a significant risk; no further studies are recommended on these compounds. �• Picloram also has a low order of toxicity. The carcinogenic potential of monuron is currently under investigation. Monuron was not used extensively in Vietnam and, other than the carcinogenic potential, has a low order of toxicity. No additional studies are recommended for these compounds. • Diquat has a moderate toxicity and has been well studied. The only study recommended on this compound is in vivo mammalian mutagenicity testing, in light of positive effects observed in in vitro tests. This compound does not produce effects that would be likely to place humans at high risk after exposure. • The information on cacodylic acid is conflicting and not adequately documented. Its toxicity and metabolism in relationship to the extent of biotransformation to inorganic arsenic after absorption and the toxicological impact of this metabolism should be investigated. • No information on the toxicology of tandex was found. Low usage of this compound in Vietnam, however, does not make it a likely target of concern. • The effects of 2,4-D, 2,4,5-T, and TCDD administered in combination have generally not been compared to the individual effects to determine whether the combination produces additive, potentiating, or synergistic effects; an exception is the effect of cleft palate in mice by 2,4,5-T, which was potentiated by doses of TCDD. The effects of combined doses should be investigated. The major concern of veterans in Vietnam that has not been adequately addressed in published literature to date is the potential for human exposure to TCDD to produce the same health effects with the same potency as those observed in animal studies. The wide variation of responses to TCDD among different species and a lack of understanding of the mechanisms of its toxicity and metabolism have led to this situation. The remaining recommendations address this issue. • Procedures for evaluating both exposure levels and health effects from occupational exposures and accidents should be established by an international agency. These procedures should be available before another incident occurs, so the most useful types of information can be collected on a timely basis and the same type of data could be obtained from different accidents for purposes of comparison. Any protocol should consider the items listed above as Gaps in Information in previous accounts; information on cholesterol levels and other parameters discussed in other recommendatons should be studied. �The relative importance of the rates and pathways of biotransformation and tissue distribution in various species should be addressed. Studies should be initiated to: - Identify the biliary metabolites of TCDD - Compare in various species the pathways of TCDD metabolism (based on the types of metabolites formed) and the rates of metabolism with TCDD toxicity in that species, as was done by Gasiewicz and Neal (1979) for the hamster - Determine the relative importance of the proportion of TCDD distributed to specific tissues with the toxicity in that tissue, (if disproportionate distribution to specific human tissues occurs, this should become apparent as TCDD levels in autopsy samples become available). The potential for the inductive effects of TCDD to alter lipid metabolism and cause depletion of fat stores has not been adequately considered. TCDD produces a long-term elevation of serum cholesterol (in animals and humans), a long-lasting induction of certain enzymes, and a long latency period after exposure and before death occurs, during which time animals become emaciated. The possibility that enzymes that degrade lipid stores are induced and no longer respond to regulatory mechanisms should be investigated. The biochemical events that precede chlorance have not been adequately considered and may in time lead to the development of useful therapy. Humans have been proposed to be less sensitive than animals to the toxic effects of TCDD (Crow, 1980). Recent experiments by Poland and Glover (1980) demonstrated that the presence of (1) cytosol receptors for TCDD, (2) sensitivity to enzyme induction by TCDD, .and (3) sensitivity to the toxic effects of TCDD, including cleft palate and thymic atrophy, all segregated together in certain strains of mice and were all absent in others. If this approach were extended to different animals species and these parameters were shown to correlate in different species, a basis for extrapolating the inductive potential and receptor-binding capacity (which potentially could be measured in vitro in human tissue) to the likelihood of toxic effects in humans may be able to be established. By understanding the mechanisms of TCDD toxicity, the degree of correlation of receptor binding, enzyme induction and toxicity, and the role of metabolism in altering toxicity in animals, extrapolations of these parameters to man may become feasible. 1-15 �CHAPTER 1. REFERENCES Associate Committee on Scientific Criteria for Environmental Quality. (1978) Phenexy Herbicides—Their effects on environmental quality. (Ottawa, Canada: National Research Council of Canada) 440 p. Bovey, R. W., and Young, A. L. (1980) The Science of 2,4,5-T and Associated Herbicides. (New York: John Wiley and Sons.) Comptroller General of the United States. (1979) U.S. ground troops in South Vietnam were in areas sprayed with Herbicide Orange. U.S., Washington, DC., No. FPCD-80-23. 12 pp. Comptroller General of the United States. (1979) Health effects of exposure to Herbicide Orange in South Vietnam should be resolved. U.S., Washington, DC., No. CED-79-22. 38 pp. Craig, D. A. (1975) Use of herbicides in Southeast Asia. Historical report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 pp. Crow, K. D. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA Docket No. 415 et al., Nov. 14. Esposito, M. P., Tiernan, T. 0., and Dryden, F. E. (1980) Dioxins. U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory, Office of Research and Development. US-EPA, Cincinnati, Ohio. 351 pp. Gasiewicz, T. A., and Neal, R. A. (1979) 2,3,7,8-Tetrachlorodibenzo-p-dioxin tissue distribution, excretion, and effect? on clinical chemical parameters in guinea pigs. Toxicol. Appl. Pharmacol. 51:329-339. International Agency for Research on Cancer. (1977) IARC monographs on the evaluation of the carcinogenic risk of chemicals to man: Some fumigants, the herbicides 2,4-D and 2,4,5-T, chlorinated dibenzodioxins and miscellaneous industrial chemicals. 15:41-299. Joint NIEHS/IARC Working Group Report. (1978) Long-Term Hazards of Polychlorinated Dibenzodioxins and Polychlorinated Dibenzofurans. World Health Organization - International Agency for Research on Cancer, Lyon. No. 78/001. 57 pp. 1-16 �Leng, M. L. (1978) 2,4,5-T RPAR - Review of EPA's rationale and their calculations for exposure to 2,4,5-T and TCDD. [Unpublished paper] DOW. 7 p. National Research Council. (1974) The Effects of Herbicides in South Vietnam: Part A. Summary and Conclusions. National Academy of Sciences, Washington, D.C. AD-774-749. Nisbet, I. C. T (1980) Direct testimony before the U. S. Environmental Protection Agency, FIFRA Docket Nos. 415, et al. Poland, A., and Glover, E. (1980) 2,3,7,8-tetrachlorodibenzo-p-dioxin: Segregation of toxicity with the Ah locus. Molec. Pharmacol. 17:86-94. U.S. Air Force (1979) Herbicide Orange site treatment and environmental monitoring. USAF OHEL Report: OHEL TR-79-169. USAF, OEHL, Brooks AFB, Texas. 37 p. Young, A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. 1-17 �CHAPTER 2. THE MILITARY USE AND APPLICATION OF HERBICIDES IN VIETNAM This chapter will deal with the history of tactical herbicide use in Vietnam. The herbicides used and their various components are described, along with the distribution of the compound for defoliant or crop destruction purposes. Types of herbicidal missions, methods of application, and potential human exposures are described as well. 2.1 SUMMARY OF TACTICAL USE In 1961, the military conducted tests in Vietnam on the feasibility of using commercially available herbicides for defoliation and crop destruction. Further tests were conducted in Vietnam from 1962 to 1968, in Thailand in 1964 and 1965, and at Eglin AFB in Florida from 1962 to 1970, on the effectiveness of various herbicide mixtures and for methods of herbicidal application (Young et al., 1978). The three maior military obiectives of herbicide use in Vietnam were (Young et al., 1978; NRG, 1974):' • Defoliation for offensive purposes - removal of trees and vegetation from enemy controlled or heavily infiltrated areas, caches, supply routes, and communication lines to increase visibility prior to reconnaissance, air strikes, or ground operations • Crop destruction - removal of food sources from enemy controlled or heavily infiltrated areas in order to hinder the establishment of permanent bases and large scale military offensives by the enemy • Defoliation for defensive purposes - removal of trees and vegetation along major water transportation routes, highways, and communication lines, and around the perimeters of military bases, supply depots, and landing zones, to prevent cover for ambush. Herbicides were sprayed in Vietnam for defoliation and crop destruction from 1962 to 1971 in a military operation named RANCH HAND. The total area sprayed between 1962 and 1965 was small, accounting for less than 7 percent of the total acreage sprayed during the Vietnam conflict. Rapid yearly increases in the annual number of acres sprayed occurred from 1962 to 1967. The annual number of acres sprayed reached a maximum in 1967, leveled off slightly in 1968 and 1969, and declined rapidly in 1970 prior to the termination of spraying in 1971. During this time more than 20 million gallons of herbicides were sprayed over 6 million acres, some of which were sprayed more than once. More than 3.5 million acres of South Vietnam—approximately 8.5 percent of the country—were sprayed one or more times. Herbicide was applied at about 1.0 to 1.5 gallons per acre from January 1962 to July 1964. After July 1964 until �the termination of spraying in 1971, herbicide agents were generally applied at approximately 3 gallons per acre (NRC, 1974). The major herbicides sprayed in Vietnam were assigned code names corresponding to the color of identification bands painted on the storage drums. During the initial stages of light herbicide use in Vietnam, from 1962 through 1964, the most commonly used herbicides were Purple and Pink. Orange, White, and Blue, which were introduced into the Vietnam conflict after 1964, rapidly replaced the use of Purple and Pink, and became the most widely used herbicides in Vietnam (Young et al., 1978). Heavily sprayed areas included inland forests near the demarcation zone; inland forests at the junction of the borders of Cambodia, Laos, and South Vietnam; inland forests north and northwest of Saigon; mangrove forests on the southernmost peninsula of Vietnam; and mangrove forests along major shipping channels southeast of Saigon. Crop destruction missions were concentrated in northern and eastern central areas of South Vietnam (NRC, 1974). Most of the herbicides used in Vietnam were sprayed by fixed-wing aircraft, although a substantial number of missions were also carried out by helicopter, particularly after mid-1970. Only small amounts of herbicides were sprayed by ground sources such as river boats, trucks, and personnel wearing back-pack sprayers (NRC, 1974). The aerial spraying of herbicides rapidly declined in 1970 after reports were released concerning the possible teratogenicity of 2,4,5-trichlorophenoxyacetic acid 2,4,5-T, a component of Orange. Some of the reported teratogenicity of 2,4,5-T was later attributed to 2,3,7,8-tetrachlorodibenzopara-dioxin (TCDD), a common contaminant of 2,4,5-T and its derivatives. The last spraying of herbicide by airplane in Vietnam occurred in January of 1971. After that, sprayings were done primarily for defensive defoliation purposes and were carried out by helicopter or on the ground; these operations also were terminated by the end of 1971. Military stockpiles of Orange were incinerated on shipboard in the Pacific Ocean near Johnston Island in 1977 (Young et al., 1978). Further details on the herbicides used in Vietnam and the quantitative, temporal, and geographical distribution of the herbicides are presented in subsequent sections of this chapter. 2.2 DESCRIPTION OF HERBICIDES USED IN VIETNAM The herbicides most widely used in Vietnam, in terms of gallons sprayed and acres covered, were Orange, White, and Blue. Other color-coded herbicides used in Vietnam included Purple, Pink, Orange II, and Green (Young et al., 1978). The major non-solvent chemical components of the color-coded herbicides were mixtures of esters or salts of herbicides widely used in agriculture and forestry in the U.S., such as 2,4-D, 2,4,5-T, picloram, and cacodylic acid. The structures of these herbicides are given in figure 2-1. The structures of the most important esters and salts of 2,4-D, 2,4,5-T, picloram, and cacodylic acid used in Vietnam are given in figure 2-2. The structure of TCDD, a toxic contaminant of 2,4,5-T and its derivatives is given in figure 2-3 (Young et al., 1978). 2-2 �/—\ / Cl - / 0 \ 0-CHII V 2 C- 2,4-Dichlorophenoxyacetic Acid (2,4-D) ( Cl 0 V °~CH? C~OH O 2,4,5-Trichlorophenoxyacetic Acid (2,4,5-T) •N o V C~OH CH - AS - OH II 0 FIGURE 2-1. 4-Amino-3,5,6-Trichloropicolinic Acid (Picloram) Hydroxydimethylarsine Oxide (Cacodylic Acid) CHEMICAL STRUCTURES OF PARENT HERBICIDES 2-3 �0 II 0-CH2 C-0-CH2 n-Butyl Ester of 2,4-D /Orange Component \ ( Orange II Component \Purple Component / 0-CH2 C-0-CH2 n-Butyl Ester of 2,4,5-T /Orange Component^ ( Purple Component) \Pink Component / 0-CH- C-O-NH (C, H- OH) 2 J o J Triisopropanolamine Salt of2,4-D (White Component) C-O-NH (C, H, OH), J o -> Triisopropanolamine Salt of Picloram (White Component) 0-C-O-CH Cl Isobutyl Ester of 2,4,5-T /Purple Component^ \Pink Component / Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl CH,, Cl Figure 2-2: Chemical Structures of Herbicide Esters and Salts, Used in Vietnam 2-4 �2,3,7,8-Tetrachlorodibenzo-para-Dioxin (TCDD) Figure 2-3: Chemical Structure of 2,3,7,8-Tetrachlorodibenzopara-Dioxin (TCDD) 2-5 �The physical properties ( e . g . , solubility, volatility) of the esters and salts made them generally better suited than the parent herbicide acids for storage, application, or forest canopy penetration. However, the effectiveness of the esters and salts indicated that they either maintained most of the herbicidal activity of the parent herbicide or were rapidly transformed to the parent herbicide in the environment after application. The physical and chemical properties of 2,4-D, 2,4,5-T, picloram, cacodylic acid, corresponding esters and salts, and TCDD that are related to environmental fate or biological assimilation are discussed in chapter 3. In addition to the color-coded herbicides, small amounts of the following herbicides were tested and possibly used to a limited extent in Vietnam (Young et a l . , 1978): The chemical structures of these compounds are given in figure 2-4. • Dinoxol, mixture of the butoxyethanol esters of 2,4-D and 2,4,5-T • Trinoxol, 40% butoxyethanol ester of 2,4,5-T • • Diquat, [6,7-dihydrodipyridol]pyrazidinium Bromacil, 5-bromo-3-sec-butyl-methyluracil • Monuron, 3-[p-chlorophenyl]-l,l-dimethylurea • Diuron, 3-[3,4-dichlorophenyl]-l,l-dimethylurea • • Tandex, [3,3-dimethylureidol-phenyltertbutyl carbamate Dalapon, 2,2-dichloropropionic acid. The physical descriptions and approximate chemical compositions of the major color coded herbicides used in Vietnam are presented below along with measured levels of TCDD in Orange and Purple. Orange is described by Young et al. (1978) as a reddish-brown or tan liquid which was essentially insoluble in water, but soluble in diesel fuel and various organic solvents. It had a specific gravity of approximately 1.28 at 25°C and was moderately volatile compared to White or Blue. The vapor pressure at 25°C of the n-butyl eater of 2,4-D, which made up close to 50 percent of Orange, is reported to be 8.4 x 10 mm (Hameker and Rerlinger (1969; in NRC, 1974) and 3.9 x 10- mm at 25°C (Zepp et al., 1975); the vapor pressure of the n-butyl ester of 2,4,5-T, which made up close to the other 50 percent of Orange, is estimated to be similar (Hameker and Rerlinger, 1969; in NRC, 1974). �o o Diquat CH,. Broraacil N-CH-CH, Br Cl /CH3 O /-NH-C-N' Monuron Diuron Cl Cl I ELC-C-COOH Dalapon 3 Figure 2-4. Chemical Structures of Herbicides Used in Small Quantities in Vietnam 2-7 �The specified chemical formulation of Orange was 50:50 mixture by weight of the n-butyl esters of 2,4-D and 2,4,5-T (NRC, 1974). The military specifications were as follows (Young et al., 1978): • n-butyl ester of 2,4-D 49.49% • n-butyl ester of 2,4,5-T 48.75% • 2,4,5-T 1.00% • 2,4-D 0.13% • inert compounds 0.62% One gallon of Orange contained approximately 4.2 Ibs. of active (as free acid plus acid part of the esters) 2,4-D and 4.4 Ibs. of active 2,4,5-T (Young et a l . , 1978). However, the chemical composition of Orange varied somewhat for d i f f e r e n t lots from different manufacturers, due to the presence of unreacted precursors, chemical byproducts, and contaminants. Analysis of Orange samples from various lots stored at Gulfport, Mississippi and Johnston Island showed the following typical variations and mean composition (Fee et al., 1975). Orange Sample Components Variation n-butyl ester of 2,4-D 4 2 . 6 % - 46.2% n-butyl ester of 2,4,5-T 39.3% - 44.9% miscellaneous butyl esters of 2,4-D and 2,4,5-T 4.0%- miscellaneous octyl esters of 2,4-D and 2,4,5-T 0.3% - 5.8% 2.0% 2,4-D 0.2% - 0.8% 0.5% 2,4,5-T 0.1% - 0.8% 0.6% other components (e.g., butanol, toluene, trace TCDD) 9.1% 7.0% 3.6% It should be noted that the analyses were performed on samples taken from lots which had often been stored for several years and which may have undergone more changes in chemical composition than most of the Orange sprayed in Vietnam. Chemical formulations which contain 2,4,5-T or its derivatives are generally contaminated xvith the dioxin TCDD, which is substantially toxic in mammals. The concentration of TCDD in 42 samples of Orange taken from Gulfport in 1972 varied from 0.05-13.0 ppm with a mean concentration of 1.77 ppm. The concentration of TCDD in 238 samples of Orange taken from storage in 2-8 �Gulfport, Mississippi in 1975 varied from 0.02-15.0 ppm with a mean concentration of 2.11 ppm. The concentrations of TCDD in samples taken from Orange that had been shipped to Johnston Island from Vietnam in 1972 were similar. The concentration of TCDD in 200 samples of Orange taken from Johnston Island in 1972 varied from 0.05-47.0 ppm with a mean concentration of 1.91 ppm. The concentration of TCDD in ten samples of Orange taken from Johnston Island in 1974 varied from 0.07-5.3 ppm with a mean concentration of 1.68 ppm. However, only four of the 200 samples contained TCDD concentrations greater than 15.0 ppm. Some of the Orange sampled at Johnston Island may have been Orange II (Young et al., 1978). White White is described by Young et al. (1978) as a dark brown liquid with high viscosity, which was essentially insoluble in diesel fuel and various organic solvents, but was soluble in water. White had a specific gravity of approximately 1.12 at 258C and is described as non-volatile compared to Orange (Young et al., 1978). The specified chemical formulation of White was an approximate 50:40:10 mixture by weight of the solvent triisopropanolamine, triisopropanolamine salt of 2,4-D, and the triisopropanolamine salt of picloram. The approximate military specifications were (Young et al., 1978): • Triisopropanolamine, other inert components 50.2% • Triisopropanolamine salt of 2,4-D 39.6% • Triisopropanolamine salt of picloram 10.2% The triisopropanolamine salts of 2,4-D and picloram have much higher aqueous solubilities but much lower vapor pressures than the parent compounds 2,4-D and picloram (NRC, 1974). One gallon of White contained approximately 2.0 Ibs. of active 2,4-D and 0.54 Ibs. of active picloram (active meaning as free acid plus acid portion of the salts) (Young et al., 1978). Blue Blue is described by Young et al. (1978) as having been a clear yellowish-tan liquid that was soluble in water, but insoluble in diesel fuel and various organic solvents. Blue had a specific gravity of approximately 1.32 at 25°C and was involatile compared to Orange (Young et al., 1978). 2-9 �The active components of Blue are primarily the sodium salt of cacodylic acid and cacodylic acid. The military specifications for Blue were (Young et al., 1978): • Water 59.5% • Sodium salt of cacodylic acid 26.4% • Cacodylic acid 4.7% • Sodium chloride 5.5% • Surfactant 3.4% • Antifoam agent 0.5% One gallon of Blue contained approximately 3.1 Ibs. of active (acid plus acid portion of salt) cacodylic acid (Young et al., 1978). Orange II, Purple, Pink, Green The specified chemical formulations of Orange II, Purple, Pink, and Green are given below. The chemical formulations of Orange II and Purple are similar to Orange (Young et al., 1978). • Orange II - 50:50 by weight isooctyl ester of 2,4,5-T and n-butyl ester of 2,4-D • Purple - • Pink - 60:40 by weight n-butyl ester of 2,4,5-T and isobutyl ester of 2,4,5-T • Green - mostly n-butyl ester of 2,4,5-T. 50:30:20 by weight n-butyl ester of 2,4-D, n-butyl ester .of 2,4,5-T, and isobutyl ester of 2,4,5-T Orange II, Purple, Pink, and Green all contain esters of 2,4,5-T. Therefore, all four herbicides were probably contaminated with TCDD. Of the four herbicides, only one sample of Purple has definitely been analyzed for TCDD. The one sample of Purple analyzed had a TCDD concentration of 45 ppm, which is generally much higher than for Orange samples. Young et al. (1978) postulate that both Purple and Pink generally may have contained higher levels of TCDD than Orange. They base this postulate primarily on the comparison of TCDD levels in the soils of test grid sites receiving predominantly Orange spray to those receiving predominantly Purple or Pink spray. 2-10 �2.3 THE HERBS TAPE The most detailed information found in the literature on the distribution of herbicides was the National Research Council (NRC), National Academy of Sciences Report on the Effects of Herbicides in South Vietnam (1974). However, the information in the NAS report is based primarily on data stored on the HERBS computer tape, supplied by the Department of Defense, which is incomplete. The HERBS tape contains information on most of the herbicidal missions flown by fixed-wing aircraft from August 1965 to the last such flight in January 1971, and on crop missions flown by helicopter between June 1968 and March 1971. It does not include information on any herbicidal missions performed prior to August 1965, non-crop related herbicidal missions flown by helicopter, crop missions flown by helicopter prior to June 1968, nor any non-aerial herbicidal missions (e.g., river boat, truck, back sprayers). It also does not contain information on helicopter missions flown by the South Vietnamese military. Despite its deficiencies, the HERBS tape contains data on the spraying of approximately 17.6 million gallons of Orange, White, and Blue, 88.0 percent of the estimated total of 20 million gallons of herbicide spraying in Vietnam. The information on the tape accounted for the spraying of approximately 5.9 million acres, 92.2 percent of an estimated total of 6.4 million acres sprayed in Vietnam from 1962 through 1971. Based on other data supplied to NRC, it was estimated that approximately 1.25 million gallons of herbicides were sprayed on approximately 400,000 acres prior to August 1965, and that approximatley 600,000 gallons of herbicides were sprayed on crops by helicopter between August of 1965 and June of 1968. In addition, approximately 13,000 acres of crops were sprayed by helicopter with approximately 36,000 gallons of White and Blue between March and October of 1971 (NRC, 1974). The data supplied in the remaining sections of this chapter are taken primarily from the NRC (1974) report. The numbers of gallons sprayed and acres covered were estimated by NRC (1974) from the numbers of missions and airplanes on the missions, as recorded on the HERBS tape. Estimates of gallons sprayed are based on tank capacities of aircraft taking part in the missions. Estimates of areas sprayed in defoliation and crop destruction methods were based on an assumed swath width of 80m and the length of spray line recorded on the HERBS tape. Estimates of areas sprayed during missions over perimeters, waterways, caches, etc., were based on the estimated number of gallons sprayed and an assumed application of three gallons per acre. 2.4 GEOGRAPHICAL AND TEMPORAL DISTRIBUTION OF HERBICIDES IN SOUTH VIETNAM South Vietnam was divided into four major military regions (MR I through MR IV) north to south. A map of South Vietnam, presented in figure 2-5, shows the boundaries of the four military regions and the provinces. Figures 2-6 and 2-7 show the outline of Vietnam and darkened regions which represent approximate areas of defoliation and crop destruction missions, respectively. These maps were developed from maps supplied in the NAS report, which were based on the HERBS tape. 2-11 �nc Ntkhen Phinomv* S*NOA N*hKoi I OCMARCtnON UNI ; \ ' t : DO'BJ Ha fcx f ry • *Muon|N6ng v MR I ^ THUA THIEN ,_, H»u<uw 5,5a Nan, T H A pUANGJsTtN ^ ^nW.Wp f ..~', r \ KONTUM^ MR BINH —^—N uNhon Stung < Ttngy Xirf^U 0«»U I .A M SB O ,¥ N^x > DARLAC Thorn ..^'., sb^r*, DUC ^ompong Cham / *J tin Ring ?B1NH ''THUAN BINH [T.k.o I I TUY i S«7R»"1 >han Thiat -'MUt KE OA /pUuoc ^ ^lm : : C _eu uo HWI *1T^ \M DuonjOonit!;' — KIEN MO V t m« ouoc V 'Vl*'T"m' MR III 5'' ~ : •V SOUTH VIETNAM * _,t-B-> inttmttion*! boundary "• "•-- Province bountftry Qu>n Lonj,; • MR IV ConSon^ _ -,w CON SOX boundary ® Nition»lc»pit«l ^•— •• "•• Railroad 'most Vntntmi ftctioni inopirabit) ~ Ro«d — — — — Trii) e 0 . 0 Figure 2-5. South Vietnam 2-12 ( 25 2^ 50 50 75 100 Mil«s 75 100 Kiiomctt'. �Herbicide Defoliation Missions—Fixed Wing 1965-1970. Data from HERB 01 file SOUTH VIETNAM 28 SO STATUTi MILJS Figure 2-6. Defoliation Missions 2-13 r_,7!t �Herbicide Crop Destruction MissionsFixed Wing and Helicopter 1965-1971. Data from HERB 01 file. SOUTH VIETNAM Figure 2-7. Crop Destruction Missions 2-14 �Examination of figures 2-5, 2-6, and 2-7 indicates that the greatest number of defoliation missions flown between August 1965 and January 1971 were directed against: • Inland forests in MR I, especially along the demarcation line • Inland forests in MR II along the Laotian and Cambodian borders • Inland forests in MR III northeast, north, and northwest of Saigon • Mangrove forests in MR IV on the Ca Mau Peninsula • Mangrove forests in the Rung Sat region of the province southeast of Saigon, which encompassed major shipping channels. Crop destruction missions between August 1965 and February 1971 were primarily directed against fields in MR I and the eastern half of MR II. Crop destruction missions were generally prohibited in the Mekong Delta region, which makes up most of MR IV. During the initial periods of Operation RANCH HAND, from January 1962 through 1964, Purple and Pink were the most widely used herbicides. According to Young et al. (1978), based on a DOD memorandum, approximately 145,000 gallons of Purple and 123,000 gallons of Pink were procured and disseminated in South Vietnam from January 1962 through December 1964. After 1964, the most widely used herbicides were Orange, White, and Blue. The approximate annual and total volumes of Orange, White, and Blue sprayed in Vietnam from August 1965 through February 1971 are listed in table 2-1. Table 2-1 is adapted from table S-I in the NRC report and is based only on the HERBS tape. Part of the 11.3 million gallons of Orange sprayed was probably Orange II. Approximately 950,000 gallons of Orange II were shipped to Vietnam during 1968 and 1969; some of this was not used in Vietnam and was shipped from Vietnam to Johnston Island in 1972. Table 2-2 lists the estimated numbers of acres sprayed annually in Vietnam from January 1962 through December 1972. The values for 1962 through 1965 and for 1971 are taken from Westing (1976). The values for 1966 through 1971 are taken from table III-B-2 in the NAS report and are based on the HERBS tape. Acres which are sprayed more than once are counted as additional acres sprayed. It can be seen from Table 2-2 that the estimated acreage covered annually from January 1962 through December 1964, when Purple and Pink were the herbicides primarily used, is small compared to the acreage covered after 1965, when Orange/Orange II, White, and Blue were the herbicides primarily used. The annual number of acres sprayed approximately quadrupled from 1965 to 1966, and doubled from 1966 to 1967, when it reached a maximum. The maximum in 1967 was followed by a small decline in 1968, a leveling-off in 1969, and a rapid decline in 1970 prior to termination of spraying in 1971. The number of acres sprayed in 1970 was less than 20 percent of the acres sprayed in 1969. 2-15 �TABLE 2-1: APPLICATION OF HERBICIDES IN THE VIETNAM WAR BY YEAR Millions of Gallons 1962July 1965 Aug-Dec 1965 1966 1967 1968 1969 1970 1971 Total Orange NAa .37 1.64 3.17 2.23 3.25 .57 .00 11.22 White NAa 0 .53 1.33 2.13 1.02 .22 .01 5.24 Blue NAa 0 .02 .38 .28 .26 .18 .00 1.12 .37 2.19 4.88 4.63 4.53 .97 .01 18.95 Year Total 1.27 S Not Available. (Table S-I in NRG 1974) 2-16 �TABLE 2-2: ANNUAL NUMBER OF ACRES SPRAYED IN VIETNAM21 Year Acres 1962 5,724b 1963 24,920b 1964 93,869° 1965 221,552° 1966 608,106° 1967 1,570,114° 1968 1,365,479° 1969 1,365,754° 1970 294,925 V V 1971 a Acres sprayed more than once are counted as additional acres sprayed. b Westing (1976) c NRG (1974) 2-17 �2.5 QUANTITATIVE AND QUALITATIVE USE DISTRIBUTION OF HERBICIDES IN SOUTH VIETNAM As mentioned in section 2.4, the maior targets of herbicide spraying in South Vietnam were inland forests, crops (cultivated land), and mangrove forests. Table 2-3 lists the distribution of herbicide spraying among inland forests, cultivated land, and mangrove forests. Table 2-3 is taken completely from table S-III in the NRC report, and is based on the HERBS tape. From August 1965 to February 1971, more than 11.3 million gallons of Orange/Orange II, 5.3 million gallons of White, and 1.1 million gallons of Blue were sprayed in South Vietnam. The volume use distribution that can be estimated for Orange, White, and Blue during the period August 1965 through February 1971 is presented below and is based on table III-B-1 in the NRC report, as derived from the HERBS tape. • Orange/Orange II - 89.5% defoliation, 8.6% crop destruction - 0.9% other (cache searches, communication lines, etc.) - 0 . 9 % perimeter defense, 0.3% waterway defense. • White - 95.3% defoliation, 1.1% crop destruction - 1.9% perimeter defense, 0.3% waterway defense - 1.4% other • Blue - 39.3% defoliation, 53.1% crop destruction - 4.2% perimeter defense, 0.4% water defense - 3.0% other • Average . - 87.9% defoliation,, 9.2 % crop destruction . - 1.4% perimeter defense, 0.3% waterway defense - 1.2% other. The HERBS tape does not include an estimated 600,000 gallons of herbicide agents sprayed on crops by helicopters from August 1965 to June 1968. When these are included, the percentages for the average are 85.4 percent defoliation, 12.2 percent crop destruction, 1.4 percent perimeter defense, 0.3 percent waterway defense, and 0.7 percent other. The HERBS tape also does not include an estimated 1.25 million gallons of herbicides sprayed prior to August of 1965, including 145,000 gallons of Purple and 123,000 gallons of Pink. Both Orange/Orange II and White were used on woody species. White was preferred over Orange in areas where spray drift was a concern, as it is not volatile compared to Orange. However, Orange was often favored over White in 2-18 �TABLE 2-3: Vegetation Type Inland forest ESTIMATED ACREAGE SPRAYED ONE OR MORE TIMES, 1965-1971C Total in SVN in 1953 Millions of Acres Percent Number of Times Sprayed Aug. 1965-Mar. 1971 Millions of Acres 1 2 3 4 + Total Sprayed One or More Times Millions Percent of Acres 25.91 62.4 1.72 0.62 0.22 .11 2.67 10.3 7.80 18.8 0.20 0.04 0.01 0.00 0.26 3.2 .72 1.7 0.14 0.07 0.03 0.02 0.26 36.1 Other 7.07 17.1 0.31 0.07 0.02 0.00 0.39 5.5 Total 41.50 100.0 2.37 0.80 0.28 0.13 3.58 8.6 Cultivated land Mangrove forest Does not include coverage of missions before August 1965 (1.27 million gallons) and missions after that date for which location information is incomplete (1.1 million gallons), representing about 12.5% of the total gallonage accounted for. Compare tables III C-l and III C-2, and related text. Inland forests include those areas classed as dense forest, secondary forest, swidden zones, bamboo forests, open dipherocarp, Lagerstroemia and Leguminosae forests. "Other" include pine forests, savanna and degraded forests, grasslands and steppes in higher elevations, dunes and brushland, grass and sedge swamps and areas of no vegetation (urban areas, roads, water courses, etc.). Classification and area figures follow Bernard Rollet (1962). See tables II-E and III B-3 and the accompanying text. (Table S-III in NRC, 1974) 2-19 �areas where spray drift was of little concern because it is a faster defoliant than White. Orange was also used on a wide variety of broadleaf crops. The persistence of picloram in soil made the use of White for crop destruction generally disadvantageous. A large percentage of Blue was used for crop destruction, particularly on cereal or green crops. 2.6 RELEASE OF TCDD IN SOUTH VIETNAM DURING SPRAYING OF HERBICIDES TCDD is a byproduct of the alkaline hydrolysis (under high temperature and pressure) of 1,2,4,5-tetrachlorobenzene to form 2,4,5-trichlorophenol, which is the industrial precursor of 2,4,5-T (Young et al., 1978; Esposito, 1980). TCDD is, therefore, a common contaminant of 2,4,5-T and its various esters. Conditions such as those used to synthesize 2,4,5-trichlorophenol favor the formation of highly chlorinated dioxins such as TCDD (Young et al., 1978; Esposito, 1980). However, 2,4-dichlorophenol (which is the industrial precursor of 2,4-D) and picloram are not synthesized under alkaline conditions at high temperature and pressure (Young et al., 1978). Therefore, contamination of 2,4-D or picloram by TCDD is unlikely. TCDD was not detected to a detection limit of .0005-.02 ppm in samples of 2,4-D or picloram (Young et al., 1978), but other dibenzo-p-dioxins have been detected in both 2,4-D and 2,4,5-T (Esposito, 1980). Although a mechanism has been proposed for the possible photochemical generation of TCDD from 2,4,5-T, Crosby et al. (1973) could not detect TCDD in 2,4,5-T that had been irradiated with sunlight or simulated sunlight. The failure to detect TCDD may be due to a more rapid rate of TCDD photodecomposition than rate of formation (Crosby et al., 1973). The major source of release of TCDD in South Vietnam appears to have been as a contaminant of herbicides such as Orange/Orange II, Purple, and Pink, all of which contained 2,4,5-T and various esters of 2,4,5-T. The spraying of herbicides in Vietnam was occasionally done as a drying step prior to deforestation by fire (Collins, 1967). Therefore, reports that TCDD can be produced from the pyrolysis of 2,4,5-T or its butyl ester are of interest. Buu-Hoi et al. (1971) reported that TCDD was formed during the pyrolysis of 2,4,5-T, its butyl ester, and vegetation previously treated with 2,4,5-T or its butyl ester. Saint-Ruf (1972) reported that TCDD was formed from the pyrolysis of the herbicide Rilvex which contains 2,4,5-T. However, Langer et al. (1973) could not detect TCDD in the sodium salts of either 2,4,5-T or Silvex that had been heated to 300°C and 350°C respectively. Stehl and Lamparski (1977) detected TCDD in the burned residues of grass that had been previously treated with approximately 12 Ibs per acre of 2,4,5-T. Ahling et al. (1977) detected TCDD levels in the burned residues of wood chips covered with 2,4,5-T prior to burning at 500°C. Close to 500 samples of Orange/Orange II have been analyzed for TCDD. The concentration of TCDD in the samples varied from 0.02 to 15.0 ppm (except for four samples greater than 15.0 ppm). The mean concentration of TCDD in the Orange/Orange II was approximately 2.0 ppm. One gallon of Orange/Orange II (specific gravity of Orange 1.28 at 25°C, density of Orange II similar) 2-20 �weighed approximately 10.7 Ibs. Assuming that the Orange/Orange II sampled was representative of the Orange/Orange II sprayed in South Vietnam, one gallon of Orange/Orange II sprayed in^Vietnam could be expected to have generally contained between 2.1 x 10 Ibs and 1.6 x 10 Ibs of TCDD or an the average of 2.1 x 10 Ibs of TCDD. Therefore, during one spraying of Orange/Orange II at three gallons/acre, forests or crops_7would theoretically be_gxpected to have received generally between 6.3 x 10 Ibs/acre and 4.8 x 10 Ibs/acre or an average 6.3 x 10 Ibs/acre TCDD. However, the targeted forests and crops would probably generally receive less TCDD than calculated above due to drifting of spray. Furthermore, the amount of TCDD reaching ground level in heavily forested areas would be far less than the amount striking the upper canopy. Both Purple and Pink contained esters of 2,4,5-T and were apparently contaminated with TCDD. Although only one sample of Purple (45.0 ppm TCDD) and none of Pink had been analyzed for TCDD, Young et al. (1978) presented circumstantial evidence that both Purple and Pink generally contained much higher levels of TCDD than Orange/Orange II. The comparison of TCDD residues in soil from adjacent test grids receiving Orange, Purple, or Pink, supports that postulate, however, evidence they present to support their estimate of TCDD levels in Purple and Pink greater than 30.0 ppm and 60.0 ppm respectively is somewhat weaker and is, therefore, discussed below. Young et al. (1978) based their estimate on the average TCDD concentration in Purple on the assumption that the four samples of Orange/Orange II with levels of TCDD greater than 15.0 ppm were actually Purple labeled as Orange by mistake. They averaged the TCDD levels in those four samples with the TCDD level in the one sample of verified Purple that was analyzed. Their assumption that the four samples of Orange/Orange II with TCDD levels greater than 15.0 ppm (all taken from Johnston Island) were actually Purple was based on several factors. These included: the high TCDD level in the one Purple sample analyzed; reports that as many as 20 drums of Purple were redrummed into cans labeled as Orange just prior to the shipping of Orange/Orange II from Vietnam to Johnston Island; reports that substantial quantities of the isobutyl ester of 2,4,5-T, a component of Purple but not of Orange/Orange II, had been detected in a few of the samples from Johnston Island; and the fact that all four samples of Orange/Orange II with levels of TCDD greater than 15.0 ppm were taken from Johnston Island. The estimate of the average TCDD concentration in Pink was based on the relative percentages of the acid equivalent of 2,4,5-T in Purple and Pink and the assumption that both Purple and Pink were formulated from batches of 2,4,5-T with similar TCDD levels. Based on the TCDD levels in 2,4,5-T manufactured from 1958 to 1963, the authors also estimated that the average concentration of TCDD in Purple formulated prior to 1964 may have been as low as 5.0 ppm. Since the spraying of Purple in Vietnam was essentially terminated after 1964, most of the Purple sprayed would have been formulated prior to 1964. If the levels of TCDD in Purple and Pink were generally as high as those estimated by Young et al. (1978), the amount of TCDD released per acre during the spraying of Purple or Pink would have been far greater than during the 2-21 �spraying of Orange/Orange II. This is despite the fact that the application rates for most of the Purple and Pink sprayed (1-1.5 gallons per acre prior to July 1964) were lower than for Orange/Orange II (three gallons per acre). However, as pointed out by Young et al. (1978), most of the Purple and Pink were sprayed prior to 1965 before the massive build-up of U.S. military personnel. Furthermore, only an estimated 90,000 acres were sprayed with Purple or Pink compared to over three million acres sprayed with Orange/ Orange II (Young et al., 1978). Overall, the spraying of more than 11.^ million gallons of Orange/Orange II from August 1965 through February 1971 is estimated to have released close to 240 pounds of TCDD, assuming that the average concentration of TCDD in Orange/Orange II was 2 ppm (2.1 x 10 pounds per gallon). 2.7 RELEASE OF ESTERS OF 2,4,5-T The major herbicides sprayed in South Vietnam that contained esters of 2,4,5-T were Orange/Orange II, Purple, and Pink. One gallon of Orange contained approximately 4.6 pounds of the acid equivalent of 2,4,5-T. The specified formulation for Orange was 50 percent by weight of the n-butyl ester of 2,4,5-T. Assuming one gallon of Orange weighed 10.7 pounds, one gallon of the specified formulation of Orange would contain approximately 5.4 pounds of .the n-butyl ester of 2,4,5-T. Therefore, Orange applied at three gallons per acre is estimated to have released a maximum of 13.8 pounds per acre of the acid equivalent of 2,4,5-T and 16.2 pounds per acre of the n-butyl ester of 2,4,5-T per spraying. The actual amount of 2,4,5-T released per acre was probably less than estimated because, actual samples of Orange contained less than 50 percent by weight of the n-butyl ester of 2,4,5-T. Furthermore, spray drift and volatilization generally reduced the amount deposited per acre. Canopy cover in heavily forested areas would, as in the case of other chemicals, greatly reduce the amount of ester reaching ground level. As stated previously, some of the approximately 11.3 million gallons of Orange sprayed in Vietnam was probably Orange II, since 950,000 gallons of Orange II were shipped to Vietnam during 1968 and early 1969 (Young et al., 1978). The specified formulation for Orange II was 50 percent by weight of the isooctyl ester of 2,4,5-T, instead of 50 percent of the n-butyl ester of 2,4,5-T, as in Orange. Assuming that one gallon of Orange II, like Orange, weighed approximately 10.7 pounds, one gallon of the specified formulation of Orange II would contain approximately 5.4 pounds of the isooctyl ester of 2,4,5-T. Therefore, Orange II applied at three gallons per acre is estimated to have released a maximum of 16.2 pounds per acre of the isooctyl ester of 2,4,5-T per spraying. The densities of Purple and Pink could not be found in the literature. Assuming the densities of Purple and Pink were similar to Orange, one gallon of Purple or Pink would have weighed approximately 10.7 pounds. The specified formulation of Purple was 30 percent by weight the n-butyl ester of 2,4,5-T and 20 percent by weight the isobutyl ester of 2,4,5-T, so one gallon of the specified formulation of Purple would contain approximately 3.2 pounds of the n-butyl ester of 2,4,5-T and 2.1 pounds of the isobutyl ester of 2,4,5-T. 2-22 �Therefore, Purple applied at 1.5 gallons per acre is estimated to have released a maximum of 4.R pounds per acre of the n-butyl ester of 2,4,5-T and 3.2 pounds per acre of the isobutyl ester of 2,4,5-T. The specified formulation of Pink was 60 percent by weight the n-butyl ester of 2,4,5-T and 40 percent by weight the isobutyl ester of 2,4,5-T, so one gallon of the specified formulation of Pink would contain approximately 6.4 pounds of the n-butyl ester of 2,4,5-T and 4.3 pounds of the isobutyl ester of 2,4,5-T. Therefore, Pink applied at 1.5 gallons per acre is estimated to have released a maximum of 9.6 pounds per acre of the n-butyl ester of 2,4,5-T, and 6.5 pounds per acre of the isobutyl ester of 2,4,5-T. 2.8 RELEASE OF ESTERS OF 2,4-D AND THE TRIISOPROPANOLAMINE SALT OF 2,4-D The major herbicides sprayed in South Vietnam that contained esters or salts of 2,4-D were Orange/Orange II, White, and Purple. One gallon of Orange/Orange II weighed approximately 10.7 pounds and contained approximately four pounds of the acid equivalent of 2,4-D. The specified formulation of both Orange and Orange II was 50 percent by weight the n-butyl ester of 2,4-D, so one gallon of the specified formulation of Orange/Orange II would contain approximately 5.4 pounds of the n-butyl ester of 2,4-D. Therefore, Orange/ Orange II applied at three gallons per acre is estimated to have released a maximum of 12 pounds per acre of the acid equivalent of 2,4-D and 16.2 pounds per acre of the n-butyl ester of 2,4-D. One gallon of White (density of 1.12 at 25°C) weighed approximately 9.3 pounds, and contained approximately 2 pounds of the acid equivalent of 2,4-D. The specified formulation of White was 40 percent by weight of the triisopropanolamine salt of 2,4-D, so one gallon of the specified formulation of White would contain approximately 3.7 pounds of the salt. Therefore, White applied at three gallons per acre is estimated to have released a maximum of 11.1 pounds per acre of the triisopropanolamine salt of 2,4-D per spraying. The specified formulation of Purple was 50 percent by weight the n-butyl ester of 2,4-D. Assuming that one gallon of Purple weighed approximately the same as one gallon of Orange (10.7 pounds), one gallon of the specified formulation of Purple would contain 5.4 pounds of the n-butyl ester of 2,4-D. Therefore, Purple applied at 1.5 gallons per acre is estimated to have released a maximum of 8.1 pounds per acre of the n-butyl ester of 2,4-D per spraying. 2.9 RELEASE OF THE TRIISOPROPANOLAMINE SALT OF PICLORAM One gallon of White weighed approximately 9.3 pounds and contained approximately 1.54 pounds of the acid equivalent of picloram. The specified formulation of White was approximately 10 percent by weight the triisopropanolamine salt of picloram, so one gallon of the specified formulation of White would contain approximately 0.93 pounds of the triisopropanolamine salt of picloram. Therefore, White applied at three gallons per acre is estimated to have released a maximum of 2.8 pounds per acre of the triisopropanolamine salt of picloram. 2-23 �2.10 RELEASE OF CACODYLIC ACID AND ITS SODIUM SALT One gallon of Blue (specific gravity 1.32 at 25°G) weighed approximately 11 pounds and contained approximately 3.1 pounds of the acid equivalent of cacodylic acid. The approximate formulation of Blue was 5 percent by weight cacodylic acid and 26.5 percent by weight the sodium salt of cacodylic acid, so one gallon of Blue contained approximately 0.5 pounds of cacodylic acid and 2.9 pounds of the sodium salt of cacodylic acid. Therefore, Blue applied at three gallons per acre is estimated to have released a maximum of 9.3 pounds per acre of the acid equivalent of cacodylic acid, 1.5 pounds per acre of cacodylic acid, and 8.7 pounds per acre of the sodium salt of cacodylic acid. 2-24 �CHAPTER 2. REFERENCES Ahling, B., Lindskog, A., Jansson, B., et al. (1977) Formation of polychlorinated dibenzo-p-dioxins and dibenzofurans during composition of a 2,4,5-T formulation. Chemosphere 33:461-468. Buu-Hoi, N. P., Saint-Ruf, G., Bigot, P., et al. (1971) Preparation, properties and identification of dioxin (2,3,7,8-tetrachlorodibenzopara-dioxin) in the pyrolysate of defoliants containing 2,4,5-T and its esters and in contaminated vegetation. C.R. Acad. Sci., Paris Ser. D. 273:708-7111. (French) Collins, C. V. (1967) Herbicide operations in Southeast Asia, July 1961-June 1967. DTEC 67-0020. Pacific Air Force, APO San Francisco DTIC No. AD 779796. 76 pp. Crosby, D. G., Moilanen, K. W., and Wong, A. S. (1973) Environmental generation and degradation of dibenzodioxins and dibenzofurans. Environ. Health Perspec. 5:259-265. Esposito, M. P., Tiernan, T. 0., and Dryden, F. E. (1980) Dioxins. U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory, Office of Research and Development. US-EPA, Cincinnati, Ohio. 351 pp. Fee, D. C., Hughes, B. M., Taylor, M. L., Tierman, T. 0., and Hill, C. E. (1975) Analytical methodology for herbicides orange. Volume II: Determination of origin of USAF stocks. Aerospace Research Laboratories. Technical report ARL TR 75-0110. 30 p. Langer, H. G., Brady, T. P., and Briggs, P. R. (1973) Formation of dibenzodioxins and other condensation products from chlorinated phenols and derivatives. Environ. Health Perspect. 5:3-7. National Research Council. (1974) The Effects of Herbicides in South Vietnam: Part A. Summary and Conclusions. National Academy of Sciences, Washington, D.C. AD-774-749. Saint-Ruf, G. (1972) Formation of dioxin in the pyrolysia of sodium a-(2,3,7,8-trichlorophenoxy)-propionate. Naturwissen Schaften 59(12):648. 2-25 �Stehl, R, H., and Laraparski, L, L. (1977) Combustion of several 2,4,5-trichlorophenoxy compounds: Formation of 2,3,7,8tetrachlorodibenzo-p-dioxin. Science 197(4307):1008-1009. Westing, A. H. (1972a) Herbicidal damage to Cambodia. In Harvest of Death, J. B. Neilands, ed. (Free Press, 1972) pp. 177-201. Young, A. L. (1974) Ecological studies on a herbicide-equipment test area (TA-C-52A), Eglin AF Base Reservation, Fla. US NTIS Publication ISS No. 780517/9GA. 141 pp. Young, A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. Zepp, R. G., Wolfe, N. L., Gordon, J. A., and Baughman, G. L. (1975) Dynamics of 2,4-D esters in surface waters. Hydrolysis, photolysis and vaporization. Environ. Sci. and Technol. 9(13):1144-1150. 2-26 �CHAPTER 3 ENVIRONMENTAL FATE AND MONITORING In order to assess environmental effects of herbicide use, this chapter looks at the mechanisms by which herbicides are distributed in the air, soil, and water. These include wind drift after initial spraying, and contamination of waters through leaching of treated soils, soil or sediment/water partitioning, or water erosion of contaminated soil particulates. Transformation of the chemicals in the environment by such processes as photolysis, hydrolysis, and volatilization are studied. Finally, the ultimate impact of the distribution of herbicides in the environment is investigated: the biological accumulation, concentration, and magnification of the chemicals in terrestrial and aquatic plants and animals. 3.1 3.1.1 ENVIRONMENTAL FATE AND MONITORING OF 2,4-D, 2,4,5-T, PICLORAM, AND ASSOCIATED ESTERS AND SALTS Physical and Chemical Properties The physical properties of 2,4-D, 2,4,5-T, picloram, and associated esters and amine salts are listed in table 3-1. The chemical structures of 2,4-D, 2,4,5-T, and picloram are depicted in figure 2-1. The chemical structures of the esters and salts are depicted in figure 2-2. The n-butyl esters of 2,4-D and 2,4,5-T, which are the primary components of Orange, undergo nonbiological and biological hydrolyses to 2,4-D and 2,4,5-T, respectively (Zepp et al., 1975; Smith, 1972; Grover, 1973 in Crosby, 1976; Smith, 1976). Picloram, 2,4-D, and 2,4,5-T are susceptible to photodecomposition by radiation at wavelengths above 290 nm which is the approximate ground-level cutoff of solar radiation (Mosier and Guenzi, 1973; Crosby and Tutas, 1966; Baur and Bovey, 1974; Zepp et al., 1975; Baur et al., 1973; Crosby and Wong, 1973). They are also susceptible to various biological transformations in plants and soil, with transformation rates generally in the order 2,4-D > 2,4,5-T > picloram (Young et al., 1978; NRC, 1974). 3.1.2 Hydrolysis Zepp et al. (1975) determined the hydrolysis rates of the methyl and n-butoxyethyl esters of 2,4-D in water as a function of pH. In general, 3-1 �TABLE 3-1. PHYSICAL PROPERTIES OF HERBICIDES Compound Vapor Pressure Aaueous Solubilitv 2, 4-D Much lower than Ester 620 mg/1 n-Butyl Ester 3.9 x 10-1 mm(2) at 25° C™ 8.4 x 10 mm at 25° C 1 mg/1 at 25°C Triisopropanolamine Salt 2,4,5-T Lower than Ester n-Butyl Ester Picloram Trlisopropanolamine Salt Lower than Ester >620 mg/1 250 mg/1 Comparable to n-Butyl Ester of 2,4-D<D 6.2 x 10"7 mm(1) at 35° C 430 mg(1)/l at 25° C Lower than Picloram 430 mg(1)/l at 25° C 1. NRC (1974) 2. Zepp et al. (1975) 3-2 <250 mg/1 �the overall rate of hydrolysis of the esters was given by Zepp et al. (1975) as: d[c] db - - (k. [H+] + kM + k_ [OH"]) [c] A N (3-D B Where k., kN, kg = rate constants for acid catalyzed, neutral and base catalyzed hydrolysis [C] = ester concentration [H ], [OH ] = hydrogen, hydroxide ion concentration. The measured rates indicated that k [OH ] was much greater than k^ or k.[H+] over the normal pH range for natural waters of 6-9. Therefore, in natural waters, the hydrolysis rate for the esters can probably be given approximately by: • -k_ [OH"] [C] (3-2) D Assuming that the pH of a well-buffered natural water remains constant, [OH ] remains constant and the hydrolysis rate becomes pseudo-first order with a half-life independent of concentration and given by t1/2 = In 2/kB[OH~] (3-3) Using the measured value of kg for the methyl ester of 2,4-D and assuming that structural reactivity relationships for the esters of 2,4-D were similar to those observed for acetic acid esters, Zepp et al. (1975) estimated that the value of kR for the base-catalyzedhydrolysis of the n-butyl ester of 2,4-D was approximately 3.7 molL-1 sec . Substituting the estimated value of k^ into equation 3-3 along with hydroxy ion concentrations at pH 6 and 9, Zepp ec al. estimated that the hydrolysis half-life of the n-butyl ester of 2,4-D in natural water would range from 220 days at pH 6 to 5.2 hours at pH 9. The estimated hydrolysis half-lives at pH 7 and 8 are respectively 22 days and 52 hours. Smith (1972) and Grover (1973) in Crosby (1976) experimentally determined the hydrolysis half-life of the n-butyl ester of 2,4-D to be 100 hours in neutral water and much less in aqueous soil suspensions. Smith (1976) reported that the n-butyl ester of 2,4-D undergoes almost complete hydrolysis to 2,4-D in less than 24 hours in moist soil. Similar results were reported for the hydrolysis of the n-butyl and isooctyl esters of 2,4,5-T to 2,4,5-T. Aly and Faust (1969) reported that esters of 2,4-D were completely hydrolyzed to 2,4-D within 9 days in lake water. Therefore, rates of biological hydrolysis appear to be greater than rates of chemical hydrolysis. 3-3 �Based on the above discussion, the esters of 2,4-D and 2,4,5-T released to the soil or biologically active water in Vietnam probably underwent rapid hydrolyses to 2,4-D and 2,4,5-T. 3.1.3 Photodecomposition Phenoxy herbicides such as 2,4-D and 2,4,5-T can undergo several different photo-reactions in water, including photo-oxidation of the phenoxy side chain to form chlorophenols, photo-nucleophilic displacement of Cl by OH to form chlorophenols, and photo-reductive dechlorination involving the replacement of Cl with H to form phenoxyacetic acid (Crosby 1976; Akermark, 1978). Zepp et al. (1975) estimated the average photolytic half-life of 2,4-D in shallow clear water as follows. The rate of photolysis was assumed to be given by (3-4) k. = average rate of sunlight absorption & s quantum yield. The average rate of sunlight absorption was calculated by integrating sunlight intensity data over a 12-hour period of sunlight for September on a clear day at latitude 34°N. If the quantum yield is assumed to be independent of wavelength, or is an average value, the rate of photolysis becomes psuedo-first order with a half-life given by (Zepp et al. , 1975): In 2/kfl <S (3-5) Substituting the value of k. and a reported quantum yield for 2,4-D in water into equation 2-5, Zepp et al. estimated that the photolytic half-life of 2,4-D in clear, shallow water in September at latitude 34°N, exposed to 12 hours of unobstructed sunlight, would be 20 days. Estimates of half-lives under cloud cover or in deeper water would be much greater. Crosby and Wong (1973) determined the photodecomposition rate of 2,4,5-T in water exposed to summer sunlight at Davis, California. Aqueous solutions of 2,4,5-T were irradiated with sunlight for approximately 12 hours per day for 4 days. After 48 hours of irradiation, approximately 17 percent of tthe 2,4,5-T had undergone photodecomposition. Studies on the photodecomposition rates of 2,4-D and 2,4,5-T exposed to artificial light at wavelengths above 290 nm showed that the photodecomposition rate of 2,4-D was generally well over twice that of 2,4,5-T under the experimental conditions used. Although there have been reports that 2,4-D and 2,4,5-T are relatively stable to photolysis under dry conditions, Baur et al. (1974) and Baur et al. (1973) have shown that substantial losses of dried films of 2,4-D, 2,4,5-T, �and picloram on glass occur over a period of several days when the films are irradiated with low intensity light (700 to 1,100 uW/cm ) at 356 nm. For example, after 7 days of irradiation, 41-79 percent of the picloram, 57-97 percent of the 2,4,5-T, and 30-70 percent of the 2,4-D were lost from petri dishes. The percentage lost for each compound depended upon the amount initially applied, which varied from approximately 10 ug to 100 ug. Other experiments conducted in the dark indicated that volatilization could not be responsible for the large losses observed during irradiation. Experiments by Hosier and Guenzi (1973) indicate that picloram in aqueous solution exposed to radiation at wavelengths above 300 nm will undergo rapid photodecomposition. Irradiation of a 2.1 x 10 M aqueous solution of the sodium salt of picloram resulted in a 99 percent loss of picloram in less than 72 hours. The intensity of the radiation was not given. 3.1.4 Biological Transformations 2,4-D undergoes a wide variety of relatively rapid biological transformations in plants and in soil, including cleavage of the ether bond, hydroxylation, and ring cleavage (Allebone, 1975). Picloram and 2,4,5-T also undergo biological transformation in plants and soils, but transformation rates are generally lower than for 2,4-D (Young et al., 1978; NRG, 1974). The persistence of picloram, 2,4-D, and 2,4,5-T in soil and plants is discussed in section 3.1.7 3.1.5 Volatility The n-butyl esters of 2,4-D and 2,4,5-T, which were the primary components of Orange, possess much higher vapor pressures than 2,4-D, 2,4,5-T, picloram, or associated amine salts. Therefore, they are generally much more volatile. Grover et al. (1972) reported that 25 to 30 percent of the n-butyl ester of 2,4-D applied to the ground for testing was collected as vapor drift up to 246 feet downwind from the application point, within 30 minutes after application. However, only negligible amounts of the dimethylamine salt of 2,4-D were detected as vapor drift. Zepp et al. (1975) used the following equation from Mackay and Leinonen (1975) to estimate the evaporative half-life of the n-butyl ester of 2,4-D as a function of depth: t 1/2 - d In 2/kL where d • depth in meters IL * liquid mass transfer coefficient (in m/hr). (3-6) �The liquid mass transfer coefficient is related to Henry's constant and the molecular weight. Henry's constant was estimated from the ratio of the vapor pressure to the aqueous solubility. They estimated the evaporative half-life of the n-butyl ester of 2,4-D from water in days to be approximately l.ld, where d is the depth of the water in meters. For example, the estimated evaporative half-life from water that is 1-10 m deep was 1.1 to 11 days for the n-butyl ester of 2,4-D. Evaporative half-lives of the n-butyl ester of 2,4,5-T from water should be comparable. However, the n-butyl esters of 2,4-D and 2,4,5-T also undergo hydrolysis in water to 2,4-D and 2,4,5-T. Baur et al. (1973) observed negligible volatilization of 2,4,5-T or picloram from aqueous solution of the sodium salts incubated at 30°C for 7 days. Baur et al. (1974) observed similar results from the sodium salt of 2,4-D in aqueous solution. Therefore, the evaporative removal of the 2,4-D and 2,4,5-T moieties from water may depend upon the relative rates of evaporation and hydrolysis of the n-butyl esters. 3.1.6 Adsorption to Soils, Leaching, and Water Transport Studies have indicated that the acidic herbicides picloram, 2,4-D, and 2,4,5-T are weakly adsorbed to soil (O'Connor and Anderson, 1974; Weber, 1972). The pK values for 2,4-D and 2,4,5-T are, respectively, 2.80 and 2.84. Since the pH of most soils is greater than 4.5, and that of most natural waters is greater than 6 (Tinsley, 1979; Sturam and Morgan, 1970), the acid herbicides will exist primarily in anionic forms in the environment. Therefore, the negatively charged anionic herbicide molecules are probably not readily adsorbed to soil because of the overall negative charge on the soil surface (Weber, 1972). Studies by Weber (1972) on the adsorption of 14 herbicides to soil indicated that the acidic herbicides 2,4-D and picloram were not adsorbed to soil as much as basic or cationic herbicides. O'Connor and Anderson (1974) measured the adsorption of 2,4,5-T from water to four soils taken from the western United States. Adsorption to the soils fit Freundlich isotherms of the form X = KG 1/n (3-7) Where X = amount of 2,4,5-T adsorbed in units of ug/g soil C = concentration of 2,4,5-T in units of ug/ml K = binding constant 1/n = empirical constant. Although values of 1/n were not reported, they appeared from the graph to be fairly close to 1. Therefore, the observed binding constant K approximated sediment/water equilibrium partition coefficients. The value of K varied from 0.31 to 3.0 and was generally greatest for soils with the highest organiccontent. Chemicals having sediment/water partition coefficients under 10 are 3-6 �classified as being weakly adsorbed (Mill, 1980). The values for the adsorption of 2,4,5-T to soils was obviously much less than 10 , so the adsorption of 2,4,5-T to the soils tested was extremely weak. Herbicides such as piclorara, 2,4-D, and 2,4,5-T, which possess moderate aqueous solubilities (430 rag/1, 620 mg/1, 251 mg/1, respectively) but weakly adsorb to soil, should have some potential for leaching to surface runoff or groundwater (Tinsely, 1979; Bovey and Young, 1980). Although several field studies have indicated that 2,4-D and 2,4,5-T are susceptible to moderate leaching, this does not appear to be a major factor in the distribution of 2,4-D and 2,4,5-T in the environment since the highest concentrations of the compounds generally remain in the upper layers of the soil (Bovey and Young, 1980, in Associate Committee, 1978). For example, in tests conducted by the U.S. Air Force, 4,000 pounds per acre of Herbicide Orange were applied at a depth of 15 cm to soil plots in Utah. Although 2,4-D and 2,4,5-T residues were detected to a depth of 90 cm after 282 days, more than 90 percent of the residues remained in the top 30 cm of soil (Young et al., 1974, in Associate Committee, 1978). Studies by Barnett et al. (1967; in Associate Committee, 1978) on the movement of 2,4-D residues in soil under simulated rainfall showed that most of the 2,4-D remained at a depth of 0-8 cm, although some was present at a depth of 8-15 cm. Only negligible amounts were detected below 15 cm. The vertical displacement and leaching potential of picloram in some soils appears to be somewhat greater than for 2,4,5-T. Scifres et al. (1977) reported that no residues of 2,4,5-T could be detected in soil at a depth greater than 15 cm in a Southwest United States watershed that receives annual sprayings of 1:1 picloram and 2,4,5-T at approximately 1 pound per acre. However, low levels of picloram were detected to a depth of 60 cm. Although most of the 2,4,5-T detected was in the upper 2.5 cm of soil, substantial levels of picloram were detected to a depth of 15 cm. Lutz et al. (1973) reported similar results for a North Carolina watershed receiving between 2 and 4 pounds per acre of 2,4,5-T and picloram. One hundred days after spraying, most of the 2,4,5-T residues remained in the top 8 cm of soil during the study, although some 2,4,5-T was detected to a depth of 45 cm. Although close to 70 percent of the picloram residues also remained in the top 8 cm of soil, a substantially higher proportion of picloram was detected at lower depths than for 2,4,5-T. Based on studies performed on the movement of picloram in Texas and Puerto Rico soils, Bovey et al. (1969) concluded that substantial quantities of picloram can be leached from soils, particularly under conditions of heavy rainfall. The leaching of picloram and 2,4,5-T may lead to trace contamination of groundwater, as evidenced by the detection of low levels (less than 1 ppb) of picloram and 2,4,5-T in groundwater underlying an area of Texas that had received picloram and 2,4,5-T spray every 6 months for 2 1/2 years, at 2 pounds per acre. There have been a number of studies on the removal of 2,4-D, 2,4,5-T, and picloram from soils by surface runoff water, either through leaching or by �erosion of soil particulates to which the herbicides are adsorbed (Barnett et al., 1967; Trichell et al., 1968; Edward and Glass, 1971; Sheets and Lutz, 1969; all in Bovey and Young, 1980; Lutz et al., 1973; Lawson, 1976). Although substantial quantities of the herbicides are often detected in runoff occurring soon after herbicide applications, concentrations rapidly decline in succeeding runoffs. Total losses of herbicides due to runoff over substantial periods of time do not generally account for more than 5 percent of the applied herbicide. For example, although concentrations of picloram, 2,4-D, and 2,4,5-T in initial runoff from recently sprayed experimental plots in North Carolina were often relatively high (0.3-4.2 ppm), concentrations in succeeding runoffs rapidly declined (Sheets and Lutz, 1969, in Bovey and Young, 1980). Total herbicide loss after several runoffs typically accounted for less than 1 percent of the applied herbicides. Nevertheless, substantial quantities of herbicides can be transported by surface runoff from watersheds receiving heavy herbicide spraying and heavy rainfall. 3.1.7 Environmental Persistence and Monitoring in Soil and Water Several studies have shown that the relative persistence in soils of picloram, 2,4-D, and 2,4,5-T is generally picloram > 2,4,5-T > 2,4-D (Yoshida and Castro, 1975; Lutz et al., 1973; Altom and Stritzke, 1973). For example, Lutz et al. (1973) studied the persistence of picloram and 2,4,5-T applied at approximately 2 pounds per acre to North Carolina soils. Fifteen days after spraying, an average of approximately 40 percent of the picloram remained, compared to 10 percent of the 2,4,5-T. After 100 days only negligible amounts of 2,4,5-T remained, whereas approximately 10 percent of the original picloram remained. Persistence was related to initial concentration. Altom and Stritzke (1973) reported that the average half-lives of picloram, 2,4,5-T, and 2,4-D in three Oklahoma soils were, respectively, >100 days, 20 days, and 4 days. Byast and Hance (1975) studied the degradation of C-labeled 2,4,5-T in four South Vietnamese soils under laboratory conditions. Two soils were taken from an agricultural area in Bien Hoa Province. The other two soils were mangrove swamp soil from either the Rung Sat region or the Can Maw pennisula. After 49 days of incubation of the 2,4.5-T with the various.soils, between 64.5 percent and 69.5 percent of the C applied as 1 ppm C-2,4,5-T had evolved to C0~. .After 168 days' incubation, between 76 percent and 79 percent of the C applied as 15 ppm C-2,4,5-T had evolved to C02> The initial concentration of 15 ppm 2,4,5-T is comparable to the estimated concentration the acid equivalent of 2,4,5-T would have if it were applied as the n-butyl ester of 2,4,5-T in Orange at 3 gallons per acre to a depth of 3 inches and assumed to be uniformly mixed (NRC, 1974). Blackman et al. (1974) analyzed 11 soil samples taken in 1971 from a site in the Rung Sat region of Vietnam which received heavy herbicide spraying. The site had received at least 86 pounds per acre 2,4-D, 79 pounds per acre 2,4,5-T, 3 pounds per acre picloram, and 9 pounds per acre cacodylic acid. Four of the 11 samples contained 2,4-D concentrations equivalent to an application rate between 0.007 and 0.04 pounds per acre. All 11 samples contained 2,4,5-T concentrations equivalent to an application rate between 0.005 and 3-IT �0.079 pounds per acre and picloram concentrations equivalent to an application rate between 0.002 and 0.01 pounds per acre. Picloram was not detected in filtered water samples taken from the lower part of the main shipping channel to Saigon in 1972 but was detected in suspended sediments removed from the water. Schultz and Harman (1971) determined the persistence of 2,4-D in the water and mud of nine ponds located in Florida, Georgia, and Missouri. The ponds were sprayed with the dimethylamine salt of 2,4-D at the rate of 2, 4, or 8 pounds per acre acid equivalent of 2,4-D. Residues of 2,4-D declined in Florida and Georgia ponds, from maximums of 0.35 and 0.69 mg/1, respectively, observed 3 days after spraying, to less than 0.005 mg/1 within 14 and 28 days, respectively, after spraying. Residues in Missouri ponds declined from a maximum of 0.63 mg/1 to less than 0.005 mg/1 within 56 days after spraying. 2,4-D residues in the mud of the Georgia and Florida ponds never exceeded 0.05 mg/kg and declined to less than 0.005 mg/kg within 56 days after spraying. A maximum value of 0.170 mg/kg 2,4-D residue was found in the mud of Missouri ponds but no 2,4-D residues could be detected in the mud of the Missouri ponds past 28 days after spraying. (Norris, 1966) studied the persistence of 2,4-D and 2,4,5-T in forest floor litter under laboratory conditions. Approximately 85 percent of 2,4-D applied to red alder forest floor litter was decarboxylated within 300 hours. Less than 25 percent of the applied 2,4,5-T was decarboxylated within 300 hours, but 53 percent was degraded after 690 hours. Norris et al. (1977) studied the persistence of 2,4,5-T in a forest in the Northwest United States that had been treated with 2 pounds per acre of the isooctyl ester of 2,4,5-T. Residues of 2,4,5-T in vine maple, blackberry vines, grass, and Douglas Fir branches declined from minimum concentrations of from 11 ppm to 115 ppm (obtained almost immediately after spraying) to less than 0.5 ppm within 1 year after spraying. Maximum residues of 2,4,5-T on the forest floor (obtained approximately 1 month after spraying) declined 50 percent within 6 weeks and 90 percent within 6 months. 3.2 ENVIRONMENTAL FATE AND MONITORING OF TCDD 3.2.1 Physical and Chemical Properties TCDD is a colorless crystalline solid at 25°C, with a melting point at 305"C and a molecular weight of 322 (Crummett and Stehl, 1973, in Esposito, 1980). The solubility of TCDD in water is approximately 0.2 ppb (0.2 ug/l); this is presumably at 20-25°C, but no temperature is given (Crummett and Stehl, 1973, in Esposito, 1980). The solubility of TCDD in Herbicide Orange is approximately 580 ppm, again presumably at 20-25°C (NRC, 1974). The vapor pressure and octanol/water partition coefficient could not be found in the literature. However, the low volatility of dried films of TCDD on glass and soil (Crosby et al., 1971) indicate that the vapor pressure is extremely low. The chemical structure of TCDD is depicted in figure 2-3. TCDD is stable to acid and base treatment and its thermal decomposition temperature is above 750*C (NRC, 1974). Significant rates of nonbiological hydrolysis or oxidation 3-9 �under normal environmental conditions are unlikely due to its chemical stability (EPA, 1979). The major routes of environmental transformation for TCDD appear to be photolysis and biotransformat ion. Rapid rates of TCDD photolysis by reductive dechlorination are observed in the presence of such adequate hydrogen donors as apparently are contained in Herbicide Orange (see section 3.2.2). Rates of biotransformat ion are slow by comparison but apparently can lead to substantial transformation over a period of several months to a year (see section 3.2.3). 3.2.2 Photolysis TCDD has a solar radiation absorption maximum of 307 nm (Crosby et al., 1971), which is well above the approximate 290 nm limit of solar radiation which actually reaches the earth's surface (Tinsley, 1979). The compound exhibits a wide range of photolysis rates which are dependent upon the surrounding medium. Crosby and Wong (1977) performed important photolysis experiments relative to the environmental fate of TCDD in Vietnam. They determined the photolysis rate of 15 ppm TCDD in Herbicide Orange irradiated by summer sunlight in California. The Herbicide was spread in thin layers (5 mg/cm ) 2 over borosilicate glass, as drops over excised rubber plant leaves (1^7 mg/cm and 6.7 mg/cm ), and on the surface of Sacramento loam soil (10 mg/cm ). Samples were taken after various periods of irradiation of up to 6 hours, and benzene extracts were analyzed by gas chromatography for TCDD levels. After 6 hours of irradiation by sunlight, the following percentages of the TCDD originally in the Herbicide Orange remained: • In layers over glass: 40 percent 2 • As drops on rubber plant leaves at 1.3 mg/cm : 25 percent 2 • As drops on rubber plant leaves at 6.7 mg/cm : negligible • On Sacramento loam soil: 85 percent. The authors postulate that the slower photolysis rate of TCDD in Orange spread on soil compared to that spread on glass or rubber plant leaves is due to shielding of soil-absorbed Herbicide Orange from the sunlight. The time required to remove 50 percent of the Orange on rubber plant leaves in both cases was less than 4 hours. The authors postulate that the apparent difference in photolysis rates for TCDD in Orange spread at different levels on the rubber plant leaves is primarily due to daily variations in sunlight intensity. Controls incubated in the dark were used to check on possible loss of TCDD by volatilization or through absorption to leaves or soil. The recovery of TCDD from controls stored in the dark for 6 hours was generally well over 98 percent, which indicated that neither volatilization nor poor extraction efficiency contributed significantly to the observed losses of TCDD in Orange irradiated by sunlight. J-iU �Crosby et al. (1971) determined the apparent photolysis rates of TCDD at 5 mg/1 methanol, irradiated by sunlight and by UV light that approximately simulated sunlight. After various periods of irradiation, they analyzed samples by gas chromatography for TCDD levels. After 9 hours of irradiation by the UV lamp, less than 30 percent of the original TCDD in solution remained, and after 24 hours of irradiation only negligible amounts of TCDD remained. The major product of the TCDD photolysis was 2,3,7-trichlorodibenzo p-dioxin, although small amounts of a dichlorodibenzo-p-dioxin were also detected. Similar but faster TCDD photolysis rates were observed during irradiation by sunlight. After 4 hours of irradiation by sunlight, less than 40 percent of the original TCDD in solution remained, and after 7 hours only negligible amounts of TCDD remained. The results for experiments carried out in open flasks in sealed tubes were similar, which indicates that the contribution of volatilization to the removal of TCDD was negligible. Stehl et al. (1973, in Esposito, 1980) reported that TCDD in isooctane and octanol irradiated by artificial sunlight had a half-life of less than 40 minutes. In contrast to the rapid photolysis rates observed for TCDD in Orange or various organic solvents, Crosby et al. (1971) observed negligible photolysis rates for TCDD in aqueous suspensions or on soil after solvent evaporation. They applied 2.4 ppm TCDD in methanol on 2.5 cm layers of soil. After the methanol was evaporated, both dry and water-moistened soil layers were irradiated with artificial sunlight for 96 hours. In another experiment, aqueous suspensions of TCDD were irradiated. The decomposition of TCDD was reported to be negligible in both the soil and the water. TCDD spread on glass .irradiated for 14 days was also stable to photolysis. Similar results were reported by Ward and Matsumura (1978). Twenty samples of lake water and sediment were incubated with C-labeled TCDD for 39-40 days. Twelve samples were irradiated and eight samples were incubated in the dark. After the incubation period, the sediment and water were separated and extracted with various organic solvents. Thin layer chromatography was used to determine the percentage of recovered radioactivity that was in the form of C-TCDD. The average recovery of TCDD from the samples incubated in the dark was 93.3 percent +_ 7 percent. For samples irradiated with light, it was 89.5 percent +^17.7 percent (sic). The difference in the recovery percentages does not appear to be statistically significant, so photolysis over the 39-40 day incubation period appears to have been negligible. However, the authors note that over 90 percent of the TCDD was recovered in sediments; thus, the sediment could have shielded much of the TCDD from the light. Also, although the authors do not specify the type of light used, they do indicate that the light intensity was low (50 to 80 foot-candles). The products of TCDD photolysis typically appear to be less chlorinated and generally less toxic dioxins than TCDD, and continued irradiation can lead to decomposition of the dibenzo-p-dioxin structure. Crosby et al. (1971) indicated that continued irradiation of a methanol solution by sunlight for 26 hours after the complete photoreductive dechlorination of TCDD led to the decomposition of the dibenzo-p-dioxin structure as evidenced by the loss of solar radiation absorption above 290 nm. 3-11 �The available literature indicates that TCDD undergoes rapid photolysis by direct sunlight when it is associated with Orange or various organic solvents, which can act as hydrogen donors during reductive dechlorination. This fact has prompted several groups to use organics on TCDD-contaminated soils to increase the TCDD photolysis rate (Esposito, 1980). In contrast, TCDD in water or left as a film on glass or soil after the evaporation of hydrogen-donating solvents appears to undergo negligible rates of photolysis. Therefore, TCDD released in Vietnam probably underwent rapid photolysis as long as it was associated with Orange or other herbicidal agents and was exposed to direct sunlight. However, based on the slow photolysis rate of TCDD not associated with organic solvents, the photolysis rate for any TCDD remaining after the evaporation of the more volatile n-butyl ester components of Orange probably would be much slower, and possibly negligible. Another important aspect of predicting the rate of TCDD photolysis in Vietnam is the effect of shade. Orange, as well as other herbicide agents containing TCDD, did not cause complete defoliation until 1 to 2 months after application (Young et al., 1978). Therefore, TCDD in herbicidal agents penetrating the upper canopy of dense forests or vegetation may have remained shaded for several weeks. Unfortunately, little is known about the effect of shade on the rate of TCDD photolysis. Nash and Beale (1978) reported that TCDD is emulsifiable and that granular formulations of Silvex underwent photolysis in both shade and direct sunlight. However, they present only an average time required after volatilization to remove 50 percent of the TCDD for all conditions combined (7.7 days in emulsion formulation, 13.5 days in granular formulation). 3.2.3 Microbiological Degradation The percentage of microorganisms capable of biodegrading TCDD appears to be low. Matsumura and Benezet (1973) tested the ability of approximately 100 microbiological strains to degrade TCDD. All of the strains previously had been shown to degrade pesticides normally considered persistent. However, only five of the strains tested were shown to have the ability to biodegrade TCDD. Although no direct measurements of biotransformat ion rates for TCDD could be found in the literature, the rates appear to be slow based on reported persistence of TCDD in soil, sediments, and water (Kearney et al., 1972; Ward and Matsumura, 1978). Since the persistence of TCDD depends not only upon the rate of biotransformation, but also on photolysis rates and rates of transport, persistence studies are discussed separately in section 3.2.7. The major products of the biotransformation of TCDD are apparently unknown. However, studies by Kearney et al. (1972) indicate that complete oxidation to C0? is negligible. They applied 1.78, 3.56, and 17.8 ppm C-labeled TCDD to two Maryland soils. After a year of incubation, 52-89 percent of the original C was recovered by combustion, the recovery percentage varying with soil type and initial TCDD application. According to the authors, the experimental design was such that loss of C could occur only through volatilization of TCDD or its metabolites or the evolution of 3-12 �CO- after complete oxidation of the C-labeled TCDD. However, only negligible amounts of CCL were detected in the trapping solutions. 3.2.4 Leaching The characteristics of a compound which primarily determine its leaching potential are its aqueous solubility and its adsorption to soil (Tinsely, 1979). TCDD has a low aqueous solubility (approximately 0.2 ppb), but its adsorption to soil is moderately strong (section 3.2.5). Therefore, the potential for substantial leaching of TCDD appears to be low. One method of experimentally estimating the leaching potential of a chemical is to determine its Rf value in thin layer chromatography using soil as the adsorbent layer, water as the solvent, and the chemical as the solute (Tinsley, 1979). Helling (1970, in Helling et al., 1973) studied the leaching potential of TCDD in five soils of various organic content with thin layer chromatography. He reported that the leaching potential for TCDD in all five soils was low. Studies by the Air Force on the vertical distribution of TCDD in soils that received large quantities of herbicide agents during tests tend to confirm that significant leaching of TCDD in soils does not generally occur. For example, samples from a soil plot in Utah, 282 days after the application of approximately 4,000 pounds per acre of Orange (3.7 ppm TCDD average) to a depth of 6 inches, were analyzed for TCDD at various depths. The analysis showed the following average concentrations of TCDD: 0-6 inches in depth, 15,000 ppt; 6-12 inches, 3,000 ppt; 12-18 inches, 90 ppt; and 18-24 inches, 120 ppt. Therefore, over 82 percent of the TCDD detected remained at the 6-inch depth to which it had been originally applied. Furthermore, the detection of some, and perhaps most, of the TCDD present at lower depths was due to contamination during sampling. Analysis of the same plot 4 years later showed a reduction in average TCDD concentrations at all depth intervals, but most of the TCDD detected was still in the upper 0-6 inch level (Young et al., 1978): 0-6 inches, 6,600 ppt (56 percent reduction); 6-12 inches, 200 ppt (93 percent reduction); 12-18 inches, 14 ppt (88 percent reduction). Analysis of soil samples taken from the Utah plot indicated that substantial leaching had not occurred over a 4-year period; but the average annual rainfall at the Utah plot was only 10 inches/year (Young et al., 1978). However, analysis of soil samples taken from plots receiving TCDD at Eglin AFB in Florida, where the average annual rainfall is 60 inches/year, were similar (Young et al., 1978). Samples taken 414 days after the application of Orange at 4,000 pounds per acre to a depth of 6 inches (TCDD concentration not specified) showed the following average concentration of TCDD (Young et al., 1976): 0-6 inches, 250 ppm; 6-12 inches, 50 ppm; 12-36 inches, <25 ppm (which was detection limit). Again, most of the TCDD was in the original 6-inch surface layer to which it had been applied. Finally, Young (1975) determined the concentration of TCDD as a function of depth in soil samples taken in 1974 from a testing grid at Eglin AFB that had received 1,900 pounds per acre of Purple in various sprayings 10-12 years earlier. The analysis showed the following average TCDD concentrations as a function of depth: 0-1 inch, 150 ppt; 1-2 inches, 160 ppt; 2-4 inches, 3-13 �700 ppt; 4-6 inches, 44 ppt; 6-36 inches, <10 ppt (which was the detection Limit). Therefore, even after 10 to 12 years, at an average of 60 inches of rainfall per year, most of the TCDD detected was in the upper 6 inches of soil. Leaching experiments performed by Matsumura and Benezet (1973) on soil columns, and by Nash and Beall (1978) on a model ecosystem, also indicate limited vertical dislocation of TCDD in soil to which water is continually applied. However, the experiments were not as long-term as the Air Force studies and significant quantities of TCDD were detected in the leachate. For example, levels of TCDD in the leachate from the ecosystem studied by Nash and Beall (1978) may have been as high as .06 ppb, which is approximately 25 percent of the aqueous solubility of TCDD. Levels of the TCDD in eluates from the soil column studied by Matsumura and Benezet (1973) were as high as 0.8 ppb, which is four times the aqueous solubility of TCDD and indicates that the leachate may have contained suspended soil matter to which TCDD was adsorbed. Therefore, the amount of TCDD that actually orginated from leaching was probably far less than the total TCDD in the leachate. Although TCDD appears to have a very limited leaching potential, there is evidence that it may have leached from a landfill in New York containing 3,300 tons of trichlorophenol, from a landfill in Arkansas containing wastes from 2,4,5-T production, and from a dump site of the Hooker Chemical Company in Michigan (Esposito, 1980). However, the TCDD has been found primarily in the water and sediment of surrounding surface waters, indicating that TCDD . transport may have occurred by soil erosion from contaminated surface layers instead of by leaching. The results of the Air Force studies indicate that substantial contamination of groundwater in Vietnam by leaching of TCDD from soil was unlikely, even for periods of several years after spraying. Although the rainfall in Vietnam, particularly in the rainy season, may have been generally heavier than in Florida, the applications of TCDD in the Air Force experiments were far greater even than in areas of Vietnam receiving multiple spraying. Although there is evidence of some leaching of TCDD in the work of Matsumura and Benezet (1973), Nash and Beall (1978), and in reports of TCDD in the water and sediments of water bodies adjacent to dumping sites, most of the TCDD involved, may have been transported by erosion of soil particulates to which TCDD was adsorbed. 3.2.5 Sediment or Soil/Water Partitioning 14 Isensee and Jones (1975) studied the partitioning of C-labeled TCDD between soil, water, and aquatic species in a model aquatic ecosystem after equilibrium had been obtained. In various experiments, they added between .01 and 149 ug of TCDD to between 20 and 420 g of Lakeland sandy loam soil, Matapeake silt loam soil, or a mixture of the two soils at the bottom of an aquarium. They then added 4 liters of water and various aquatic species. After 30 days, they removed samples of soil and water from the aquarium. They removed suspended soil from water samples.by centrifuge, and water from soil by drying, and analyzed the samples for C. Thin layer chromatography of 3-14 �14 extracts from.some of the samples indicated that most of the C was still in the form of C-labeled TCDD. Therefore, estimates of the TCDD concentration in soil and water samples are based on the measured C activity. Data for the equilibrium adsorption of a chemical from water to soil can frequently be fitted to an empirical Freundlich equation of the form (EPA, 1979): C = K C 1/n s sw w (3-8) where C = equilibrium concentration of chemical in soil in mass/mass units C = equilibrium concentration of chemical in water in the same mass/mass units = dimensionless sediment/water equilibrium partition coefficient K 1/n m empirical exponent. Isensee and Jones (1975) did not attempt to fit their data to a Freundlich equation, so 1/n was not determined. However, in many cases 1/n = 1 (EPA, 1979). JRB calculated soil/water equilibrium partition coefficients from the data presented by Isensee and Jones (1975) by assuming 1/n « 1 in equation 3-8 and rearranging the equation to give K • C /C sw s w The calculated values of K 2.1 x 10*. (3-9) 4 for six sets of data varied from 1.1 x 10 to Karickhoff et al. (1979) derived the following relationship from a linear least squares analysis of the binding of several organics with low aqueous solubilities to soils or sediments with varying organic content: log K - -0.54 log S + 0.44 (3-10) where K oc ' Ksw/oc K = soil or sediment/water equilibrium partition coefficient oc • organic fraction of the soil or sediment s » aqueous solubility in mole fraction. TCDD has an aqueous solubility of approximately 0.2 ppb (0.2 ug/1) and_a molecular weight of 322, so the molar solubility is approximately 6.2 x 10 TCDD moles/1. Therefore, the mole fraction,solubility of TCE in water is approximately 6.2 x 1 0 / 5 5 . 6 , or 1.1 x 10 . Substituting that value for the 3-15 �aqueous mole fraction solubility in equation 2-10 and solving for K gives K = 2.3 x 10 . The organic fractions of the Lakeland sandy loam soil and the Matapeake silt loam soil used by Isensee and Jones (1975) were respectively 0.009 and 0.015. Therefore, the theoretical values for K for Lakeland and Matapeake soil are 2.1 x 10 and 3.5 x 10 , respectively, which are within a factor of 2 of the ones calculated from the data presented by Isensee and Jones (1975). The model aquatic ecosystem of Isensee and Jones (1975) was allowed to approach at least an approximate equilibrium. However, most natural aquatic systems are far from equilibrium (Stumm and Morgan, 1970), so care must be taken in predicting typical soil or sediment/water partitioning from soil or sediment/water equilibrium partition coefficients. Nevertheless, some general guidelines have been suggested based on an empirical comparison of actual soil or sediment/water partioning to soil or sediment/water equilibrium partition coefficients (Mill, 1980). If a compound has a soil or sediment/water partition coefficient greater than 10 for a given soil or sediment it will generally exhibit high soil or sediment/water partitioning in natural systems where the given soil or sediment predominates. If the equilibrium partition coefficient is less than 10 for a given soil or sediment, the compound will exhibit low soil or sediment/water partitioning. The K values calculated for TCDD above.were for soils with moderately low organicwcontents and ranged from 1.1 x 10 to 3.5 x 10 . Soils with higher organic contents would be predicted to exhibit proportionately higher soil or sediment/water equilibrium partition coefficients for the adsorption of TCDD. Therefore, based on the guidelines presented by Mill (1980), TCDD would be predicted to exhibit moderately high to high soil or sediment/water partitioning in most natural systems, depending upon the organic content of the predominant soil or sediment. The apparently moderately high soil or sediment/water partitioning of TCDD probably inhibits the leaching of TCDD from soil to groundwater or surface water, as discussed in section 3.2.4. However, it may occasionally lead to transport of larger quantities of TCDD to surface water than would be predicted from its low aqueous solubility and leaching potential, by waterborne transport of suspended soil or sediment particulates to which TCDD is adsorbed. Matsumura and Benezet (1973), Ward and Matsumura (1978), and Isensee and Jones (1975) have detected TCDD levels several times greater than its aqueous solubility in unfiltered or filtered but uncentrifuged water samples from model ecosystems. Therefore, most of the TCDD in the water samples may have been adsorbed to suspended particulate matter. The model ecosystems were probably somewhat more static than most natural water systems, so the concentration of suspended particulate matter in those model systems was probably far less than would be observed in natural aquatic systems such as rivers, or in flood or rain water flowing over areas contaminated with TCDD. Therefore, it is conceivable that some TCDD could be transported from contaminated areas to rivers, streams, lakes, or ponds by water erosion of soil particulates to which TCDD is adsorbed. Furthermore, once the TCDD-contaminated particulates reached a river or stream, they could be transported farther downstream, or deposited as sediments which could be resuspended and transported downstream during periods of heavy flow. 3-To �As mentioned previously, the TCDD detected in the water and sediments of surface waters surrounding landfills and dump sites could have been transported by water erosion of TCDD-contaminated soil. Young et al. (1978) indicate that 10-35 ppt levels of TCDD in the silt of a pond and stream adjacent to a test area in Eglin AFB, Florida that had received heavy herbicidal spraying was probably primarily due to soil erosion; the TCDD was detected only at points where eroded soil entered the water. Bartleson, Harrison, and Morgan (1975; in Esposito, 1980) measured TCDD levels in the soil of the test area at Eglin AFB and reported that the highest levels of TCDD in the soil were generally found in low-lying areas and the lowest levels in areas of loose soil. Again, this supports the postulate that TCDD transport had occurred by water erosion of TCDD-contaminated soil (Esposito, 1980). Shadoff et al. (1977) measured an average 5 ppt TCDD in the mud and 0.2 ppt TCDD in the water (each based on two samples) of a pond in Arkansas that received rainfall and irrigation runoff from rice fields treated with 1.25 pounds per acre of 2,4,5-T at various times for 18 years prior to the study. However, the pond water was repeatedly reused for irrigation of recently sprayed rice fields, so the low levels of TCDD detected could be explained by leaching alone, although some of the TCDD may have been transported by soil erosion. They also measured an average 3 ppt TCDD in the mud and 0.1 ppt TCDD in the water (each based on two samples) of an impoundment in Texas that received drainage from large areas receiving between 0.5 and 4 pounds per acre of 2,4,5-T for mesquite and brush control. Again, however, although some TCDD transport by water erosion of soil could have occurred, the low levels of TCDD measured from a large drainage area could probably be accounted for by leaching alone. 3.2.6 Volatilization and Transport in Air Volatilization does not appear to be a significant short-term removal pathway for TCDD in the environment. Although the vapor pressure of TCDD could not be found in the literature, several short-term experiments have indicated that it is non-volatile. Crosby et al. (1971) applied 2.4 ppm TCDD in methanol to soil and, after evaporating the methanol, irradiated the soil for 96 hours with a UV lamp. After the irradiation, almost all of the TCDD was recovered from the soil. The authors concluded that TCDD losses due to both photolysis and volatilization were negligible. Crosby and Wong (1977) applied 15 ppm TCDD in Orange to soil and exposed rubber plant leaves. Some of the soil and leaf samples were irradiated with sunlight for 6 houra and some were kept in the dark as controls. Although there were substantial losses of TCDD by photolysis from samples exposed to sunlight, TCDD losses from the dark controls were negligible. Therefore, the authors concluded that losses due to volatilization were negligible. The time period between application of the Orange and extraction for the control samples was not given, but it was at least 6 hours. Although there have been suggestions in the literature that volatilization may contribute significantly to the removal of TCDD over longer time periods, the evidence is inconclusive since it does not take into consideration other possible routes of removal. For example, Isensee and Jones (1971) 3-17 �applied C-labeled TCDD in an aqueous surfactant to the leaves of a soybean plant and to oats. The leaves and oats were analyzed for C content 2S 7, 14, and 21 days after application. After 2 days, the soybean,leaves contained 92 percent and the oats contained 83 percent of the applied C. No further, loss of C was observed in the soybean leaves, but oats continued to lose C slowly, and contained 63 percent of the applied C after 21 days. Although the authors attribute C losses to the volatilization of TCDD, other routes of removal were possible, including photolysis, as suggested by Crosby and Wong (1977), volatilization of TCDD metabolites, and translocation in the plants. Ward and Matsumura (1978) added C-labeled TCDD to samples of lake sediment and incubated the sediments with lake water in 20-ml glass culture tubes with loosened screw caps. Different samples were incubated for different lengths of time, up to 589 days. After incubation, sediments were separated from water (usually by filtration), the sediment and water were extracted with organic solvents, and the radioactivity of the solvents was measured to determine the loss of radioactivity from the sediment/water samples. Water loss from the samples was also measured. The authors detected a direct relationship between the total loss of radioactivity and the loss of water from various samples. The authors suggested that the observed relationship indicated that the loss of radioactivity may have been related to water-mediated evaporation of TCDD. However, the loss of radioactivity may have been due primarily to volatilization of metabolites of C-TCDD, which are more^ volatile than TCDD, or to the evolution of CO- during metabolism of CTCDD. The graph the authors present depicting Toss of radioactivity versus water loss is similar to the graphs they present depicting radioactivity loss versus incubation time. Therefore, the observed relationship between radioactivity loss and water loss could be due to the dependence of both water loss and production of volatile metabolites from TCDD on length of incubation. Transport of TCDD in the air is not dependent upon volatilization alone. Other possible mechanisms of air transport include wind erosion of TCDDcontaminated soil, spray drift of herbicides such as Orange that contain TCDD, and the introduction of TCDD-contaminated particulates into the air by burning. Some of the horizontal dispersion of TCDD (discussed in the previous section) that was attributed to leaching or water erosion of soil could have been due to wind erosion of TCDD-contaminated soil (Esposito, 1980). Tests by Harrigan (1970) indicated that as much as 13 percent of the Orange sprayed at targets under operational parameters similar to those used in Vietnam missed test areas because of both spray drift and volatilization. As mentioned previously, some of the herbicide missions flown in Vietnam were for the purpose of drying areas prior to massive fire bombing. Reports of problems in the fly ash of municipal incinerators (Esposito, 1980) and the relatively high thermal decomposition temperature of TCDD indicate that introduction of some TCDD-contaminated particulates into the atmosphere during the burning of forests and crops was possible. 3.2.7 Environmental Persistence The persistence of TCDD at a given location in a single phase of the environment will depend upon its tranformation rate within the phase, its rate of transport within the phase, and its rate of transport to other phases. 3-18 �Although it is sometimes possible to estimate qualitatively the relative importance of various removal processes, it is rarely possible to determine quantitatively the relative contribution of each process to the overall rate of removal. Therefore, determinations of the persistence of TCDD in the environment are discussed separately from discussions of specific removal processes. Kearney et al, (1972) determined the persistence of TCDD in Lakeland, Maryland loamy sand soil (organic matter 0.9 percent) and in Hagerstown, Maryland silty clay loam soil (organic matter 2.5 percent) under laboratory conditions. Initial concentrations of 1, 10, and 100 ppm TCDD were established in both soils by application of benzene solutions of TCDD to 100 g samples of soil. Soils were sampled on the day of application and 20, 40, 80, 160, and 350 days afterwards. The samples were extracted with hexane-acetone and analyzed for TCDD by gas chromatography. The percentage recovery of applied TCDD decreased with time. After 350 days, the percentages of TCDD recovered from initial concentrations of 1, 10, and 100 ppm in Lakeland soil were, respectively, 54, 57, and 56 percent. Therefore, the persistence of TCDD in the Lakeland soil appeared to be independent of initial concentrations of TCDD, which would be consistent with first order and pseudofirst order kinetics. After 350 days, the percentages of TCDD recovered from initial concentrations of 1, 10, and 100 ppm in Hagerstown soil were, respectively, 54, 63, and 71 percent. Therefore, in Hagerstown soil, there did appear to be some dependence of persistence on the intial concentrations of TCDD. Also, the persistence of TCDD applied at 10 and 100 ppm in Hagerstown soil appeared to be slightly longer than in Lakeland soil, even though the authors postulated that the microbiological population in Hagerstown would be greater than in Lakeland soil because of the higher percentage of organic matter in Hagerstown soil. However, the apparent dependency of TCDD persistence on initial TCDD concentrations or on soil type may instead be due to differences in efficiency of extraction. For example, soils with higher organic content, such as Hagerstown soil, would be expected to bind TCDD more stongly that soils with lower organic content, such as Lakeland soil (Karickhoff et al., 1979). Therefore, the efficiency of extraction may have been lower for Hagerstown soil than for Lakeland soil. Although the authors give an average extraction efficiency of 85 percent, they do not give extraction efficiencies for each initial TCDD concentration in each soil. Since the average extraction efficiency was 85 percent, the persistence of TCDD in the soil tested was considerably greater than indicated by the recovery percentages. Therefore, the time required to remove 50 percent of the applied TCDD in the soils tested was much greater than 1 year. Furthermore, if the removal processes approximate first order or pseudo-first order kinetics, the time required to remove 50 percent of the applied TCDD will be independent of applied concentration and, therefore, always greater than 1 year. Ward and Matsumura (1978) determined the persistence of TCDD in Lake Menelotaj,Wisconsin water and sediment under laboratory conditions. They applied C-labeled TCDD to glass tubes containing approximately 5 grams of wet sediment and 18 ml of water. After incubation, the sediment and water were separated by filtration and extracted with organic solvents. The C content of the extracts was determined by liquid scintillation counting. The C content of the sediments was also determined by counting the CO evolved and trapped during combustion. Some of the sediment and water organic extracts were evaporated and the remaining residues were dissolved by ether or 3-19 �acetone and analyzed by thin layer chromatography (TLC) to determine the percentage of the recovered C that was still C-TCDD. After 588 days of incubation, between 44.5 and 52.2 percent of the applied C was recovered from the sediment by combustion and between 42.1 and 49.3 percent by extraction. Analysis by TLC indicated that between 95.6 and 99 percent of the C recovered from sediments was still C-TCDD. The results of the analyses of the water over the sediment after 588 days of incubation were not given. However, after 167 days of incubation with another type.pf Lake Menelota sediment, less than 2 percent of the orginally applied C was in the aqueous phase. Therefore, it is probable that most of the C remaining after 588 days incubation was in.the sediment phase. Since slightly less than 50 percent of the applied C was,recovered from the sediment after 588 days and since most of the recovered C was C-TCDD it appears that the time required under laboratory conditions to remove 50 percent of the applied TCDD in the Lake Menelota sediment tested was approximately 600 days. Ward and Matsumura (1978) also determined the persistence of TCDD in Lake Menelota water without sediment. After approximately 590 days of incubation, between 67.1 and 75.8 percent of the applied C was recovered, of which over 98.4 percent was shown by TLC to be TCDD. Therefore, the persistence of TCDD in the tested lake water was longer than in the tested lake sediment under laboratory conditions. In 1972, the U.S. Air Force began field tests on soil plots in Utah and in Florida to determine the persistence of TCDD in soil (Young et al., 1978; Young et al., 1976; Commoner and Scott, 1976). The Utah soil was clay loam with 1.4 percent organic content and a pH of approximately 7.8 (Young et al., 1976). The Florida soil was sandy loam with 0.5 percent organic content and a pH of approximately 5.6. Approximately 4,000 pounds per acre of Orange that contained TCDD were applied to the plots in both Utah and Florida. However, Commoner and Scott (1976) point out that the actual application level was higher, since the Orange was deposited uniformly, to a depth of six inches, in narrow swaths within the plots. The average concentration of TCDD in the Orange used in Utah was 3.7 ppm, but the concentration of TCDD in the Orange used in Florida was not reported (Commoner and Scott, 1976). As mentioned previously in section 3.2.4, most of the recovered TCDD remained in the top 6 inches of soil even after several years. The concentration of TCDD in composite soil samples taken from the plots in Utah and Florida at various times after Orange application are given below (Young et al., 1976): Days After Applications Florida Utah ___ 5 282 414 513 637 707 780 1,000 1,150 375 ppt 15 ppb 250— ppt 75 ppt — — 7.3 ppb 46— ppt — — — 3-20 5.6— ppb 3.2 ppb 2.5 ppb �Under conditions of low pollutant concentration, the kinetics of biodegradation will sometimes approximate a second order reaction of the form (EPA, 1979): ^ = - k, [C] [X] dt ^ (3 ~ll) Where v = second order rate constant C = pollutant concentration X » cell population. Under conditions of low pollutant concentration and high cell population, the cell population can sometimes be assumed to remain relatively constant (after the log period) to give a pseudo-first order equation of the form: "b " \ tCl ~ dt ( 3 Where k, =» pseudo-first order rate constant = k, [X] Integration of equation 2-12 gives In C = -\t+ In C (3-13) Where C « Initial pollutant concentration. Therefore, a plot of In C versus t for a process following pseudo-first order kinetics should be linear with a slope equal to ( , ). k Figure 3-1 is a plot of ln/2.3 versus t for both the Utah (concentrations in ppb) and Florida (concentrations in ppt) experiments with TCDD persistence in soil. The plot is derived from the kinetic data given above and is taken from the semi-logarithmic plots given in figure III-2 of Young et al. (1978). The lines drawn are linear least squares fitted to the data points. The data points from the Utah experiments all fit closely to the line, which indicates that the degradation rate may have followed closely psuedo-first order kinetics over the time period covered (282 days after Orange application to 1,150 days after Orange application). The slope of the line_gives k, ,„ ,^ for the Utah experiments, which is equal to -2.1 x 10 days . Contrary to the low scatter of the Utah points, the four data points from the Florida 3-21 �20,000 Hill AFB, Utah Eglin AFB, Florida V., 10,000 a o (0 p. 1,000 § g •H 4J n) Vl 4-1 g O O O 100 o en 10 200 400 600 800 1,000 1,200 Time (Days After Incorporation) Figure 3-1. SEMI-LOGARITHMIC PLOT OF SOIL CONCENTRATIONS (PARTS PER TRILLION) OF TCDD IN HERBICIDE ORANGE BIODEGRADATION STUDIES AT EGLIN AFB, FLORIDA, AND HILL AFB, UTAH (Figure III-2, Young et al., 1978) 3-22 �experiment are widely scattered. Therefore, another linearly fitted line was drawn through the last three data points which reduced the scatter_somewhat. The slope of the line based on three points is equal to -5.31 x 10 days , which we assume to be equal to k, /_,, . , \. The scatter of the data points from the Florida experiment may have been due to the initial presence of a lag time during which cell acclimation could take place and which would not follow pseudo-first order kinetics until acclimation had occurred. Note that the first Florida data point is well below the line linearly fit to the last three data points. There is no apparent lag time from the Utah data, but in contrast to the first Florida data point at 5 days after Orange application, the first Utah data point is at 282 days after Orange application. Therefore, the lag time may have occurred prior to 282 days. However, Young et al. (1978) estimate that the initial concentration of TCDD in the Utah soil was 148 ppb based on calculations but not on actual measurement. If their calculations are accurate, the initial rate of TCDD degradation prior to 282 days would have been much greater instead of much slower than the apparently constant pseudo-first order rate after 282 days. During the periods that the rate processes follow pseudo-first order kinetics, the half-lives can be calculated from the following equation derived from equation 2-13 by substituting 1/2 Co for C and rearranging: C l/2 In 2/k 1 (3-14) Where t]/2 • half-life during time periods in which pseudo-first order kinetics are followed. Substituting the values of k, ,T .v and k, , , . . -. into equation 3-6 gives: t. ,, ,Ilt.aVx " 330 days and t ,„ /-,__.J,\ - 130 days. The t , /„,.„,,% value is the same as the one estimateiTbjrYounget al. (1978), but tn4Z vucat1' /_, . , s .. ,»,*. ^Florida; estimate is shorter because they based their estimate on a . ' linear fit to all four data points. The half-lives calculated for time intervals over which first order kinetics appear to be approximately followed are, of course, not valid for time intervals in which first order kinetics are not followed. For example, the time required to remove 50 percent of the applied TCDD from the Florida plot is obviously greater than the calculated half-life, assuming first order kinetics over the last three data points. However, if the estimated initial concentration of 148 ppb TCDD (Young et al., 1978) in the Utah plot is accurate, the time required to remove 50 percent of the applied TCDD was much less than the calculated half-life, assuming first order kinetics for the data points presented. Based on the Air Force studies, it appears that TCDD may be removed from some soils by pseudo-first order biological processes after an initial period of acclimation. The half-life for pseudo-first order kinetics is independent 3-23 �of the TCDD concentration. Therefore, even though TCDD concentrations in the Air Force studies were probably far higher than the concentrations which would have been found in Vietnamese soils, the estimated pseudo-first order halflives for the Air Force study may serve as a useful estimate of pseudo-first order half-lives in Vietnamese soils with the following limitations: 1) the pseudo-first order half-lives should be dependent on the quantity and type of cell populations in the soil and temperature, which may be quite different in Vietnamese soil; and 2) initial non-first order degradation rates will depend upon initial TCDD concentrations in the soil. 3.2.8 Bioaccumulation, Bioconcentration, and Biomagnification by Aquatic Organisms Several groups have determined the potential bioaccumulation (uptake), bioconcentration (ratio of tissue to water concentrations), and biomagnification (increase in tissue concentrations at succeedingly higher trophic levels) of TCDD by aquatic organisms in model ecosystems. Matsumura and Benezet (1973) determined wet weight bioconcentration factors (ratio of TCDD concentrations in wet tissue to TCDD concentrations in water) for daphnia and ostracod, which are freshwater crustaceans, and for brine shrimp, mosquito larvae, and silverside fish. The bioconcentration factors for daphnia and ostracod were determined as follows: a thin film of C-labeled TCDD was applied to the inner surface of a glass container and algae, which is a food source for daphnia and ostracod, was grown for 24 hours in the container.. The algae was then transferred along with the culture medium to an aquarium containing the crustaceans for 4 to 7 days. The C content of,the water was determined,by extracting with chloroform and counting the C in the extract. The C content in tissues was determined either by homogenizing and then extracting the TCDD from the tissues, or by tissue construction followed by measuring the evolved CO in a trapping solution. In all cases, the, C content of the tissues and water were assumed to be still in the form of C-TCDD but verification of the assumption was either not done or not discussed. The measured bioconcentration factors for daphnia and ostracod were, respectively, 2,198 and 107 g, based on reported water concentrations of 0.4 ppb and 2.6 ppb, respectively. However, the reported water concentrations were higher than the reported aqueous solubility of TCDD (0.2 ppb), so some of the measured water concentration was probably TCDD adsorbed to suspended particulate matter and not necessarily available for uptake. Bioconcentration factors based on the aqueous solubility of TCDD (0.2 ppb) would be 4,395 and 1,395 g for daphnia and ostracod, respectively. TCDD levels in the algae were not reported, so estimates of biomagnification potential cannot be made. Matsumura and Benezet (1973) determined bioconcentration factors for brine shrimp, mosquito larvae, and silverside fish as follows. A thin film of C-labeled TCDD was deposited on 1 g of sand and then added to an aquarium containing the,test organisms. After 4 to 7 days, the tissue and water were analyzed for C content as described previously. The C content was again assumed to be entirely C-labeled TCDD. The measured bioconcentration factor for the brine shrimp based on a TCDD concentration in water of 0.1 ppb was 1.57 x 10 . The bioconcentration factors for the mosquito larvae and the 3-24 �3 silverside fish were, respectively, 2.85 x 10 and 54, based on a water concentration (including food) of 1.3 ppb. Therefore, although the mosquito larvae serves as a food source for the fish, there does not appear to be any biomagnification of TCDD. However, the experiments were short-term (4 to 7 days). Isensee and Jones (1975) determined bioconcentration factors on a dry weight basis for algae, snails, daphnids, and mosquito fish in model ecosystems. The aquatic organisms represented part of two food chains: algae —> snails, and daphnids (small freshwater crustaceans) —> mosquito fish. Thus, biomagnification potential was also determined. The experiments were run as follows. They applied C-labeled TCDD to soil, placed the soil in tanks and filled the tanks with approximately 4 liters of water. One day later, approximately 100 daphnids, eight snails, and algae were placed in the tanks along with a few ml of old aquarium water which contained diatoms, protozoa, and rotifers. After 30 days, two mosquito fish were added to each tank. After 33 days the organisms were removed and analyzed for C content. The tissues were dried and then combusted. The CO- that evolved during the combustion was trapped in solution and counted. The analysis of methanol extracts of the mosquito fish and snails by TLC indicated that approximately 87-93 percent of the C was probably TCDD. The calculation, of biocojacentration,factors was based primarily on the assumption that all C was C-TCDD. The C content of the water was determined by centrifuging the water to remove suspended sediment, evaporating the water, and analyzing the remaining residue by combustion (counting of evolved CO- in trapping solution). Final water concentrations for eight experiments ranged from 0.05 ppt to 239 ppt, which is approximately the aqueous solubility of TCDD. The bioconcentration factors for algae ranged from 2.0 x 10 to 1.9 x 10 , with an average of 9.5 x 10 . ft .The bioconcentration factors £L for the snails ranged from 5.4 x 10 to 4.7 x 10 , with an average of 2.2 x 10 . The bioconcentration factors for the snails were slightly higher (approximately 2x) than those for the algae but no significant biomagnification was observed. The bioconcentration factors for the daphnids ranged from 1.8 x 10 to 4.8 x 10 with an average of 2.9 x 10-. The bioconcentration factors for the mosquito fish ranged from 9.2 x 10 to 6.3 x 10 with an average of 2.8 x 10 . Again, no significant biomagnification was observed. However, the mosquito fish were in the tanks for only 3 days. Although the extrapolation from model ecosystems to the environment is uncertain, it appears that TCDD levels in aquatic organisms exposed to TCDD in water for several days will probably be significantly greater than the TCDD concentrations in the water. However, there is no evidence that significant biomagnification will occur during progression through different entropic levels. Several studies have been performed to determine TCDD levels in aquatic organisms from water that has received direct 2,4,5-T application or that contains runoff from areas treated with 2,4,5-T. Shadoff et al. (1977) failed to detect TCDD (detection limit 10 ppt) in 10 samples of bass or one sample each of the viscera and eggs of catfish from a pond in Arkansas, which received rainfall and irrigation drainage from rice fields treated with 1.25 pounds per acre of 2,4,5-T for at least 18 years and whose water was continually reused for irrigation. They also failed to detect TCDD in 3-25 �10 samples of catfish and walleyed pike from a Texas impoundment which received drainage from a large area treated with 0.5-4 pounds per acre and equivalent of 2,4,5-T for at least 20 years. Although the 2,4,5-T application per spraying was lighter in Arkansas and Texas than in Vietnam (0.5-4 pounds per acre compared to 13.2 pounds per acre in Vietnam), the number of repeated sprayings was generally greater and the duration of spraying was longer than in Vietnam. Also, the water in the pond in Arkansas was used repeatedly for irrigation of fields sprayed with 2,4,5-T 4 to 8 weeks previously. Young'(1975; Young et al., 1978) detected approximately 12 ppt TCDD in one body sample of mosquito fish and two viscera samples of sailfin fish taken from a stream adjacent to a 1-mile-square test area at Eglin AFB which had received approximately 160,000 Ibs of 2,4,5-T (250 pounds per acre) from 1962 to 1970. They detected 4, 18, 4, and 85 ppt TCDD in pooled samples of skin, gonad, muscle, and gut, respectively, from sunfish taken from a pond on the test area. Analysis of the gut of bluegill indicated that a major food source was terrestrial insects. They failed to detect TCDD in numerous other fish species taken from the stream and pond. Bartleson, Harrison, and Morgan (1975; Esposito, 1980) detected an average concentration of 150 ppt TCDD in the composite bodies of 20 mosquito fish taken from a pond next to a herbicide loading area at Eglin AFB (Florida). They also detected between 150 ppt and 740 ppt TCDD in the liver and fat of 18 sunfish taken from the pond. Dow (1978; Esposito, 1980) determined TCDD levels in fish from the Tittahanasee River, which receives treated wastes from a Dow plant. TCDD levels in catfish taken from various locations in the river and its tributaries ranged from 70 ppt to 230 ppt. Baughman and Messelson (1973) detected between 18 ppt and 810 ppt average TCDD concentrations in fish and crustaceans caught by Vietnamese fishermen in 1970. The fish and crustaceans analyzed were caught in or near areas which received heavy sprayings of herbicide from 1967 through 1970. The specimens were frozen and analyzed approximately 2 1/2 years later. The tissues were homogenized and TCDD was extracted from the tissues with various organic solvents. The extracts were analyzed for TCDD by gas chromatography-mass spectroscopy. Carp and two species of catfish caught in the Dong Nai River a few miles northeast of Saigon contained average TCDD concentrations of 540, 810, and 520 ppt, respectively. Catfish and river prawn caught in the Saigon River a few miles northwest of Saigon contained 70 ppt and 42 ppt TCDD respectively. Croaker and prawn caught off the coast near the Rung Sat region southeast of Saigon contained an average 79 ppt and 18 ppt TCDD respectively. The TCDD concentrations given were calculated on a wet tissue basis. 3.2.9 Bioaccumulation. Partitioning, and Monitoring in Terrestrial Animals and Plants Studies by Fanelli et al. (1980), the Air Force, and Young et al. (1978) on the levels of TCDD in animals collected from areas with high TCDD contamination indicate that the bioconcentration in terrestrial animals of TCDD from soil may be generally less than the concentration in aquatic organisms of TCDD from water. Fanelli et al. (1980) detected TCDD in field mice collected in a heavily contaminated area near Seveso, Italy. TCDD concentrations in the mice ranged from 0.07 ppb to 49 ppb and averaged 4.5 ppb, based on 14 samples. 3-26 �TCDD concentrations in the upper 7 cm of soil collected from the same area ranged from 0.01 ppb to 12 ppb with an average of 3.5 ppb. Young et al. (1978) summarized various Air Force determinations of TCDD levels in wildlife and soil collected from 1972 to 1978 in a 3-kilometer testing area at Eglin AFB (Florida) which had received approximately 73,000 kg of 2,4,5-T contained in Orange and Purple between 1962 and 1970. Concentrations of TCDD in wildlife included beach mice (300-1,500 ppt in liver), hispid cotton rat «10-210 ppt), meadowlark (100-1,020 ppt in liver), mourning dove (50 ppt in liver), Savannah sparrows (69 ppt in liver), and the six-lined racerunner lizard (360-430 ppt in muscle). TCDD levels in 54 soil samples collected from the same area ranged from <10 ppt to 1,500 ppt and averaged 165 ppt. Although it appears, based on the above studies, that bioconcentration factors for terrestrial animals are generally less than aquatic species, terrestrial animals were not necessarily confined to the test area (particularly the birds), contrary to the aquatic species which were confined to aquariums. Several groups have tried to determine TCDD levels in cow milk and in beef fat of animals grazing on land which was sprayed with 2,4,5-T or accidently contaminated with TCDD. Fanelli et al. (1980b; Esposito, 1980) determined TCDD levels in milk in the Seveso, Italy area shortly after an industrial accident had released large quantities of TCDD to the environment. Milk from cows less than 1 km to over 5 km south and southeast of the plant was analyzed. Concentrations of TCDD in the milk ranged from 59 ppt to 7,919 ppt, depending primarily upon the distance of the grazing pasture from the plant. However, the release of TCDD in the Seveso area was several orders of magnitude greater than releases of TCDD to areas in Vietnam. Mahle et al. (1977) failed to detect TCDD to 1 ppt in milk from cows in Missouri, Arkansas, and Oklahoma that grazed on lands that typically were sprayed with 2 pounds per acre of 2,4,5-T annually. The application of 2,4,5-T in Vietnam per spraying was typically much heavier (13.4 pounds per acre), but was generally not repeated on an annual basis. Kocher et al. (1978), Meselson et al. (1978), and Solch et al. (1978), all in Esposito (1980), have detected TCDD in beef fat from cattle which grazed on lands treated with 2,4,5-T. TCDD levels ranged from 4 ppt to 70 ppt. Studies by Isensee and Jones (1971) indicate that only small amounts of TCDD were bioaccumulated from soil by oats and soybeans. They applied C-labeled TCDD to soil at 0.06 ppm and 0.10 ppm. Oats and soybeans.grown to maturity in the soil contained less than 0.15 percent of the applied C. Other studies indicated that translocation of TCDD applied to the leaves and other parts of oat and soybean plants was negligible. Approximately 94 percent of the applied TCDD remained on the soybean leaves 21 days after application. Cocucci et al. (1979) studied the adsorption and translocation of TCDD in various garden vegetable plants and fruit trees grown in TCDD-contaminated soil in the Seveso, Italy area. The average TCDD levels in the aerial parts of carrot, potato, onion, and narcissus plants were generally somewhat lower than the TCDD levels in underground parts, but TCDD levels in both were generally comparable to TCDD levels in the surrounding soil. Studies on cherry, fig, pear, apricot, and peach trees indicated that TCDD levels were generally lower in fruits than in leaves and much lower in fruits than in twigs. �3.3 3.3.1 ENVIRONMENTAL FATE OF CACODYLIC ACID Physical and Chemical Properties Cacodylic acid is a colorless crystalline compound at 25°C (Midwest Research Institute, 1975). It is described as nonvolatile and has a very high aqueous solubility (NAS, 1974). The chemical structure of cacodylic acid is given in table 2-1. The compound does not decompose in sunlight (NRG, 1974). It undergoes various biological transformations in soil, including reductive methylation to volatile dimethyl and trimethyl arsines, andoxidative cleavage of the carbonarsenic bond to form CO- and arsenate (AsO, ) . 3.3.2 Biological Transformations, Transport and Persistence Woolson and Kearney (1973) determined the persistence of cacodylic acid in three soils under both aerobic and anaerobic conditions. They applied C-labeled cacodylic acid to the soils. After incubating for 24 days, the soils were analyzed for C content and arsenic (As) content. The aerobic soils contained an average of approximately 65 percent of the As initially applied and 24 percent of the C initially applied. The authors assume that the 35 percent loss of the As from the soil could have occurred only through the formation and subsequent evaporation of volatile organo-arsenic compounds. Therefore, 35 percent of the total 75 percent C loss from aerobic soils can be accounted for by evaporation of volatile organo-arsenic compounds. The loss of the other 41 percent C_was assumed to be due to the oxidation of cacodylic acid to C0« and AsO, . After 24 days' incubation, the anaerobic soils contained an average of approximately 39 percent As and 39 percent C. Therefore, all loss of C from the anaerobic soils was assumed to be due to the formation and subsequent evaporation of volatile organo-arsenic compounds. Woolson (1977), by gas chromatography-mass spectrometry, later identified volatile organo-arsenic compounds evolving from the biodegradation of cacodylic acid to be primarily dimethyl and trimethyl arsenic. 3.3.3 Bioconcentration of Cacodylic Acid Isensee et al. (1973) determined the bioconcentration of C-labeled cacodylic acid by various aquatic organisms from water in a model aquarium. Three fish, 30 daphnia, 100 snails, and algae were exposed to cacodylic acid for 3, 29, 32, and 32 days, respectively. The organisms exposed represented parts of two food chains: algae —> daphnids, and daphnids —> fj-gh. The bioconcentration ratios for the algae and daphnids, based on the C content of the water and wet tissues, were, respectively, 1,635 and 419. The bioconcentration ratios for daphnia and fish were, respectively, 1,658 and 21. Therefore, although aquatic organisms appear to bioconcentrate cacodylic acid and/or a metabolite of cacodylic acid, biomagnification does not appear to occur. 3-28 �3.4 CONCLUSION Phenoxy herbicides in water may undergo a number of photolytic changes to form chIorophenols, or through photoreduction may be dechlorinated to form phenoxyacetic acids. Under dry conditions, 2,4-D, 2,4,5-T, and picloram are readily photolyzed and substantial losses have been noted. Photolysis of TCDD is strongly dependent on the surrounding medium; rapid photolysis is noted of TCDD in Orange or organic solvents, as opposed to slower rates in aqueous suspensions or on soil. Photolysis of TCDD seems to produce less chlorinated and less toxic dioxins; continued irradiation by sunlight may result in decomposition of the dibenzo-p-dioxin structure. 2,4-D, 2,4,5-T, and picloram show little adsorption to soil, and they are all moderately soluble in water. However, leaching does not appear to be a primary mechanism for the environmental distribution of 2,4-D or 2,4,5-T. Picloram has a somewhat higher potential for vertical displacement or leaching in soils. TCDD has less aqueous solubility and high soil adsorption, and has a high soil or sediment/water partition coefficient, and thus has a relatively low potential for leaching. The n-butyl esters of 2,4-D and 2,4,5-T are much more volatile than 2,4-D, 2,4,5-T, picloram, or the dimethylamine salts. TCDD is essentially non-volatile. The primary difference between TCDD and other herbicides is in its environmental persistence. In both soils and water TCDD has a long environmental life, and there was. fairly high level of bioconcentration observed in aquatic organisms. 2,4,5-T has a negligible half-life in the environment, while 2,4-D and picloram had slightly longer persistence. Cacodylic acid also has a relatively short environmental life; it is postulated to form organoarsenic compounds which are subsequently volatized. Despite its high rate of bioconcentration, there seems little evidence of any biomagnification of TCDD in an aquatic environment. This appears to be true for 2,4-D, 2,4,5-T, picloram, and cacodylic acid, as well. The following chapter looks at the metabolism in humans and animals of the herbicides under study. 3-29 �CHAPTER 3. REFERENCES Akermark, B. Photochemical reactions of phenoxy acids and dioxins. In Chlorinated Phenoxy Acids and Their Dioxins. ed. C. Ramel (Ecol. Bull, no. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 75-81. Allebone, J. E., Hamilton, R. J., and Ravenscroft, B. (1975) Environmental organic chemistry of 2,4-dichlorophenoxyacetic acid. Environ. Chem. 1:160-190. Aly, O.M., and Faust, S.D. (1964) Studies on the fate of 2,4-D and ester derivatives in natural surface waters. J. Agric. Food Chem. 12:541-546. Altom, J. D., and Stritzke, J. F. (1973) Degradation of dicamba, picloram and four phenoxy herbicides in soils. Weed Sci. 21(6):556-560. Associate Committee on Scientific Criteria for Environmental Quality. (1978) Phenoxy Herbicides—Their effects on environmental quality. (Ottawa, Canada: National Research Council of Canada) 440 p. Barnett, A. P., Hauser, E. W., White, A. W., et al. (1967) Loss of 2,4-D in washoff from cultivated fallow land. Weeds 15:133-137. Baughman, R., and Meselson, M. (1973) An analytical method for detecting TCDD (Dioxin): Levels of TCDD in samples from Vietnam. Environ. Health Perspect. 5:27-35, Baur, J. R., Bovey, R. W., and McCall, H. G. (1973) Thermal and ultraviolet loss of herbicides. Arch. Environ. Contain. Toxicol. 1(4):289-302. Baur, J. R., and Bovey, R. W. (1974) Ultraviolet and volatility loss of herbicides. Arch. Environ. Contam. Toxicol. 2:275-288. Blackman, G. E., Fryer, J. D., Lang, A., and Newton, M. The Effects of Herbicides in South Vietnam. Part B: Working papers: Persistence and disappearance of herbicides in tropical soils. National Academy of Sciences—National Research Council. NTIS Publication No. AD779025. 3-30 �Bovey, R. W., Dowler, C. C., and Merkle, M. G. (1969) Pesticides in soil. The persistence and movement of picloram in Texas and Puerto Rican soils. Pestic. Monit. J. 3(3):177-l81. Bovey, R. W., and Young, A. L. (1980) The Science of 2,4,5-T and Associated Herbicides. (New York: John Wiley and Sons.) Byast, T. H., and Hance, R. J. (1975) Degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Bull. Environ. Contain. Toxicol. 14(l):71-76. Cocucci, S., DiGerolamo, F., Verderio, A., Covallaro, A., Colli, G. , et al. (1979) Absorption and translocation of tetrachlorodibenza-p-dioxin by plants from polluted soil. Experientia 35(4);482-484. Commoner, B., and Scott, R. E. (1976) US Air Force studies on the stability and ecological effects of TCDD (dioxin): An evaluation relative to the accidental dissemination of TCDD at Seveso, Italy. St. Louis, Mo.: Center for the Biology of Natural Systems, Washington University, 51 pp. Crosby, D. G., and Tutass, H. 0. (1966) Photodecomposition of 2,4-dichlorophenoxyacetic acid. J. Agric. Food Chem. 14:596-601. Crosby, D. G., and Wong, A. S. (1973) Photodecomposition of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in water. J. Agric. Food Chem. 21(6):1052-1054. Crosby, D. G, (1976) Nonbiological degradation of herbicides in the soil. In Herbicides: physiology, biochemistry, ecology. Vol. 2. ed., L. J. Audus,(New York:Academic Press) pp 65-97. Crosby, D. G., and Wong, A. S. (1977) Environmental degradation of TCDD. Science 195(4284):1337-1338. Crosby, D. G., Wong, A. S., Plimmer, J. R., and Woolson, E. A. (1971) Photodecomposition of chlorinated dibenzo-p-dioxins. Science 173(3998):748-749. Esposito, M. P., Tiernan, T. 0., and Dryden, F. E. (1980) Dioxins. U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory, Office of Research and Development. US-EPA, Cincinnati, Ohio. 351 pp. 3-31 �EPA suspends the major uses of two herbicides. (1979) Environ. Sci. and Tech. 13(6):640-641. Fanelli, R., Castelli, M. G., Martelli, G. P., Noseda, A., and Garattini, S. (1980) Presence of 2,3,7,8- tetrachlorodibenzo-p-dioxin in wildlife living near Seveso, Italy: A preliminary study. Bull. Environm. Contain. Toxicol. 24:460-462. Grover, R., Maybank, J., and Yoshida, K. (1972) Droplet and vapor drift from butyl ester and dimethylamine salt of 2,4-D. 20(4):320-324. Harrigan, E. T. (1970) Calibration Test of the UC-123K/A/A45Y-1 Spray System. Technical Report ADIG-TR-70-36. Armament Development and Test Center, Eglin AFB, Florida. 160 p. Helling, C. S., Isensee, A. R., Woolson, E. A., Ensor, P. D. J., Jones, J. R., Plimmer, J. R., and Kearney, P. C., (1973) Chlorodioxins in pesticides, soils, and plants. J. Environ. Qual. 2(2):171-178. Isensee, A. R., and Jones, G. E. (1971) Absorption and translocation of root and foliage applied 2,4-dichlorophenol, 2,7-dichlorodibenzo-p-dioxin, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Agric. Food Chem. 19(6):1210-1214. Isensee, A. R., and Jones, G. E. (1975) Distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic model ecosystems. Environ. Sci. Technol. 9(7):668-672. Isensee, A. R., Kearney, P. C., Woolson, E. A., Jones, G. E., and Williams, V. P. (1973) Distribution of alkyl arsenicals in model ecosystem. Environ. Sci. Technol. 7:841-845. Karickhoff, S.W., Brown, D.S., and Scott, T.A. (1979) Sorption of hydrophobic pollutants on natural sediments. Water Research 13:241-248. Kearney, P. C., Woolson, E. A., and Ellington, C. P., Jr. (1972) Persistence and metabolism of Chlorodioxins in soils . Environ. Sci. Technol. 6(12):1017-1019. Koschier, I. J., Girard, P. R., and Hong, S. K. (1978) Transport of 2,4-dichlorophenoxyacetate by rat renal cortical slices. Toxicol. Appl. Pharmacol. 45:883-894. 3-32 �Lawson, E. R. (1976) 2,4,5-T residues in storm runoff from small watersheds. J. Soil Water Conserv. 31:217-219. Lutz, J. F., Byers, G. E., and Sheets, T. J. (1973) The persistence and movement of picloram and 2,4,5-T in soils. J. Environ. Qual. 2(4):485-488. MacKay, D., and Leinonen, P. J. (1975) Rate of evaporation of low-solubility contaminants from water bodies to atmosphere. Environ. Sci. and Technol. 9:1178-1180. Mahle, N. H., Higgins, H. S., and Getzendaner, M. E. (1977) Search for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin in bovine milk. Bull. Environ. Contain. Toxicol. 18(2): 123-130. Matsumura, F., and Benezet, H. J. (1973) Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Environ. Health Perspec. 5:253-258. Midwest Research Institute. (1975c) Substitute chemical program. Initial scientific 'review of cacodylic acid. Prepared for Environmental Protection Agency, PB-251 541. 130 pp. Mill, T. (1980) Data need to predict the environmental fate of organic chemicals. In Dynamics, exposure and hazard assessment of toxic chemicals. Bizuanual Haque, ed. (Ann Arbor: Ann Arbor Science), pp. 297-322. Mosier, A. R., and Guenzi, W. D. (1973) Picloram photolytic decomposition. J. Agric. Food Chem. 21(5):835-837. Nash, R. G., and Beall, M. L., Jr. (1978) Environmental distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) applied with Silvex to turf in microagroecosystem. Final Report EPA-IAG-D6-0054, Agricultural Environmental Quality Institute, U.S. Department of Agriculture, Beltsville, Maryland. National Research Council. (1974) The Effects of Herbicides in South Vietnam: Part A. Summary and Conclusions. National Academy of Sciences, Washington, D.C. AD-774-749. Norris, L. A. (1966) Degradation of 2,4-D and 2,4,5-T in forest litter. Forestry 64:475-476. 3-33 £._ �Norris, L. A., Montgomery, M. L., and Johnson, E. R. (1977) The persistence of 2,4,5-T in a Pacific Northwest forest. Weed Sci. 25(5):417-422. O'Connor, G. A., and Anderson, J. U. (1974) Soil factors affecting the adsorption of 2,4,5-T. Soil Sci. Soc. Am. Pro. 38(3):433-436. Schultz, D. P., and Harmon, P. D. (1974) Residues of 2,4-D in pond waters, mud, and fish, 1971. Pestic. Monit. J. 8(3);173-179. Scifres, C. J. , McCall, H. G., Maxey, R., and Tai, H. (1977) Residual properties of 2,4,5-T and picloram in sandy rangeland soils. J. Environ. Qual. 6(l):36-42. Shadoff, L. A., Hummel, R. A., Lamparski, L., and Davidson, J. H. (1977) A search for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an environment exposed annually to 2,4,5-trichlorophenoxyacetic acid ester (2,4,5-T) herbicides. Bull. Environ. Contam. Toxicol. 18(4):478-485. Smith, A. E. (1976) The hydrolysis of herbicidal phenoxyalkanoic esters to phenoxyalkanoic acids in Saskatchewan soils. Weed Res. 16:19-22. Stumm, W., and Morgan, J. j". Wiley and Sons], p. 69. (1970) Aquatic chemistry. Tinsley, I. J. Chemical concepts in pollutant behavior. Wiley-Interscience, 1979), 265 p. [New York: John (New York: Ward, C. T., and Matsumura, F. (1978) Fate of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) in a model aquatic environment. Arch. Environ. Contam. Toxicol. 7:349-357. Weber, J. B. (1972) Interaction of organic pesticides with particulate matter in aquatic and soil systems. In: Fate of Organic Pesticide in the Aquatic Environment. Advan. Chem. Ser. No. Ill; Am. Chem. Soc., Washington, DC: pp: 55-120. Woolson, E. A. (1977b) Generation of alkylarsines from soil. Weed Sci. 25:412-416. Woolson, E. A., and Kearney, P. C. (1973) Persistence and reactions of C-cacodylic acid in soils. Environ. Sci. Technol. 7:47-50. 3-34 �Yoshida, T., and Castro, T. F. (1975) Degradation of 2,4-D, 2,4,5-T and picloram in two Philippine soils. Soil Sci. Plant Nutr. 21(4):397-404. Young, A. L., Thalken, C. E., Arnold, E. L., Cupello, J. M., and Cockerham, L. G. (1976) Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) in the environment; Summary and decontamination recommendation's"] Headquarters Air Force Logistics Command Report No. AFA-TR-76-18. 41 pp. Young, A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. Young, A. L., Thalken, C. E., Arnold, E. L., Cupello, J. M., and Cockerham, L. G. (1976) Fate of 2,3,7,8-tetrachlorodiben2o-p-dioxin (TCDD) in the environment: Summary and decontamination recommendations. Headquarters Air Force Logistics Command Report No. AFA-TR-76-18. 41 pp. Zepp, R. G., Wolfe, N. L., Gordon, J. A., and Baughman, G. L. (1975) Dynamics of 2,4-D esters in surface waters. Hydrolysis, photolysis and vaporization. Environ. Sci. and Technol. 9(13):1144-1150. 3-35 �CHAPTER 4 METABOLISM, ENZYME INDUCTION, AND MECHANISM OF ACTION In this chapter, several aspects of metabolism and for TCDD, receptormediated effects are considered. Patterns and rates of absorption, distribution, and excretion of the phenoxy acids and TCDD are described under Biodynamics. Sites and pathways for biotransformation of the phenoxy herbicides and TCDD are described under Biotransformation. Studies that evaluate the potency of TCDD in inducing enzymes are described in the section, Enzyme Induction; these inducible enzymes, in general, have not been shown to be responsible for the biotransformation of the herbicides that induce them. The mechanism of action of TCDD has been the subject of many studies, and these studies are discussed in the sections entitled Mechanisms of Action. For the remaining compounds, fewer studies have been published; and these studies are described by chemical without further subdivisions, for each subject category. 4.1 2,4-D Dermal absorption of 2,4-D, as well as the pharmacokinetics of plasma clearance and excretion of oral doses, have been determined in human studies. Animal studies have investigated the biodynamics and biotransformation of 2,4-D. No studies were found on the ability of 2,4-D to induce any enzyme system or on its mechanism of toxicity. Esposito et al. (1980), Leng (1977), Loos (1975), and Young (1978) have reviewed literature on phenoxy acid metabolism. 4.1.1 4.1.1.1 Human Studies Biodynamics The rate of dermal absorption of 2,4-D has been measured in human volunteers. When [ C]-2,4~D was administered intravenously to human volunteers, 100 percent of the dose was excreted in the urine during the subsequent five days. The biological half-life for excretion was 13 hours. [ C]-2,4-D was then applied to the forearm, and excretion of radioactivity was monitored over the subsequent eight days. Using this method, 5.8 percent of the dose was absorbed and excreted. This rate was increased to 14.7 percent by occluding the application site with plastic film for 24 hours after dosing (Feldman and Maiback, 1974; Maiback and Feldman, 1974). The kinetics of absorption and excretion of orally ingested 2,4-D have been studied in man. After six male volunteers each ingested a subtoxic dose of 5 mg 2,4-D per kg, urine and blood samples were collected and monitored for 2,4-D levels. From pharmacokinetic analysis of the data, the half-life of plasma clearance was determined to be 33 hours (Kohli et al., 1974). In a similar study, five men each ingested 5 mg 2,4-D per kg and urine and blood samples were collected and analyzed for 2,4-D. This dose was completely 4-1 �absorbed from the gastrointestinal tract and produced no clinical symptoms. 2,4-D was cleared from the blood with a half-life of 11.6 hours, and both plasma clearance and urinary execretion followed first-order kinetics. (Sauerhoff et al., 1977; Sauerhoff et al., n.d.). Blood and urine levels of 2,4-D in patients who intentionally ingested large quantities have'been analyzed pharmacokineticaliy. In one case, after ingestion of a sublethal dose of 2,4-D estimated to have been 400 mg/kg, plasma clearance was calculated to be 16.7 hours (Young and Haley, 1977; Young and Haley, n.d.). In another case, an estimated 70 ml of a 10 percent 2,4-D preparation had been ingested. Initially, the half-time of clearance was 219 hours and the urinary pH was below 6.5. After alkaline diuresis was started, the pH increased to 7.5 and the half-life decreased to 42 hours. Continued therapy elevated the pH to 8.8 and, at this pH, the half-life of plasma clearance of 2,4-D was 5 hours (Park et al., 1977). The amounts of 2,4-D excreted by workers occupationally exposed to 2,4-D has been used to estimate total doses absorbed by these workers. Aerial applicators excreted an average of 0.012 mg/kg body weight/day of 2,4-D after a 12-day exposure period; ground applicators excreted a mean of 0.013 mg/kg of 2,4-D over a 6-day period after a 1-day exposure period (Nash et al., in press: Nash et al., 1981). In another study (Shafik, et al., 1971), urine samples of workers were analyzed for 2,4-D levels. Levels ranged from 0.2-1.0 ppm for samples from farmers and spray operators, while no 2,4-D was detected in samples from herdsmen, farm laborers, and pesticide project officers. These pharmacokinetic studies incidate that ingested 2,4-D is absorbed rapidly and completely by the gastrointestinal tract, but that it is not absorbed extensively after dermal exposure to a limited area of the skin; by these routes and by intravenous injection, 2,4-D is rapidly and completely excreted by the kidney, with a biological half-life for clearance of about one-half day. Only in the case of severe poisoning and resultant acidosis is this rate considerably slower; upon alkaline diuresis, clearance is stimulated to a higher rate than observed in non-treated cases after lower doses were given. 4.1.1.2 Biotransformation In the pharmacokinetic studies described above, in which known quantities of 2,4-D were administered to volunteers, urine samples were analyzed for 2,4-D. Kohli et al. (1974) did not detect metabolites by gas chromatography. Sauerhoff et al. (1977) reported that 82 percent of the administered dose was excreted as the acid and 12 percent as conjugates. 4.1.2 Animal Studies 4.1.2.1 Biodynamics The rate of absorption of 2,4-D by the rat lung has been investigated. In 1.4 minutes, 50 percent of intratracheally instilled doses of 2,4-D were 4-2 �absorbed by the lung, indicating that alveolar transport was probably by diffusion (Burton et al., 1974). Retention of 2,4-D by cultured human embryonic lung cells was relatively low, compared to retention of other herbicides that are known to bioaccumulate and that are far less water-soluble than 2,4-D (Murakami and Fukami, 1978). In various species, 2,4-D as the acid, salt or ester, has been shown to be transported rapidly to tissues. In the mouse, clearance rates from the body were higher for the butyl ester than the octyl ester of 2,4-D, and both were higher than the rate for clearance of the acid (Zielinski and Fishbein, 1967). Plasma clearance in the rat was in turn more rapid than in the pig, calf, and chicken, although the half-lives in the same species for the triethanolamine or sodium-potassium salts were the same as for the butyl ester. In the rat, these half-lives were 3-6 hours, and in the other species they were 7.5-12 hours (Erne, 1966). In another study in the rat, (Buslovich et al., 1973) the sodium salt was cleared far more rapidly than the diethylamine salt, although both rates fell substantially with time, perhaps due to renal toxicity from the high doses used (405-555 mg/kg). Shafik et al. (1971) also observed decreases in the rate and total amount of 2,4-D excreted in urine by rats, as the total dosage was increased. 2,4-D was distributed evenly among various tissues in several species, after acute or chronic exposure. The plasma levels were usually slightly higher than other tissues, followed by renal levels, and then hepatic and pulmonary levels, although all were within the same order of magnitude. 2,4-D was further distributed to the plasma fraction of whole blood primarily, and to the cytosol subfraction in cells (Erne, 1966; Khanna and Fang, 1966; Buslovich et al., 1973). "2,4-D showed high affinity binding to the albumin fraction of serum (Mason, 1975; Haque et al., 1975). 2,4-D was not accumulated in fat or muscle of sheep or cattle during a 28-day feeding experiment (Clark et al., 1975). Although 2,4-D levels in the brain were lower than in other tissues, the distribution of 2,4-D to the brain has been investigated further in relation to 2,4-D1s activity as a neurotoxin. At a high dose (250 mg/kg) which produced myotonia and lethargy, the levels of 2,4-D in the rat brain and cerebrospinal fluid increased by 11- and 39-fold, respectively, over levels after a slightly toxic dose of 100 mg/kg; after 500 mg/kg, these increases were 18- and 67-fold, respectively. In contrast, hepatic levels increased only five- and six-fold, after 250 and 500 mg/kg doses, respectively, over the levels produced by the 100 mg/kg dose. The authors suggested that toxic symptoms were associated with proportionately higher neural levels than hepatic or other tissue levels (Elo and Ylitalo, 1979, 1977). The mechanism of uptake by the brain was studied in the rabbit. 2,4-D was taken up by the organic anion system of the rabbit brain (choroid plexus) in vitro. Transport was energy-dependent and was inhibited by other anions (Pritchard, 1980). In all species that were studied, 2,4-D was excreted almost exclusively by the kidney. At high doses or in animals that were maintained on low protein diets, in vitro renal clearance by goat kidney was decreased. At high doses, both plasma binding and renal tubular secretion mechanisms became saturated (Orber, 1980a; 1980b). 2,4-D was accumulated by the renal organic transport system in the rat, but in a different manner than the way other 4-3 �compounds (para-aminohippurate) are handled by this system in vitro (Koschier et al., 1978). 2,4-D uptake by the renal transport system was greater in the rabbit than in the rat (Berndt and Koschier, 1973). A small amount, (3-6 percent) of an oral dose was excreted in the feces by rats, and 2,4-D was detected in nursing offspring, indicating that transfer via milk occurred (Fedorova and Belova, 1974). 4.1.2.2 Biotransformation Hydrolysis of the ester of 2,4-D butyl ester by the rat and the pig was demonstrated by Erne (1966). In the pig, an average of 7 percent of the excreted 2,4-D was conjugated (i.e., released by acid hydrolysis). In other studies, no urinary metabolites were detected in any other species, including the rat (Khanna and Fang, 1966), the cow (Lisk et al., 1963), the sheep (Clark et al., 1964) or the goat (Orberg, 1980). 2,4-D metabolites were not detected in expired air of rats (Khanna and Fang, 1966). 4.2 2,4,5-T The biodynamics of 2,4,5-T and formation of urinary metabolites have been studied in man and in a number of mammalian species. As with 2,4-D, 2,4,5-T has not been studied as an inducer of microsomal enzymes, and its mechanism of action has not been investigated. 4.2.1 Human Studies 4.2.1.1 Biodynamics Dermal absorption of 2,4,5-T was estimated in man (Newton, 1978, cited in RPAR, 1979). A 144-inch denim cloth soaked with 40 ml of 2,4,5-T solution was placed on the upper thighs of four volunteers, wrapped in plastic, and kept in contact with the skin for 2 hours. The concentrations of 2,4,5-T used for the four volunteers were different. Urine samples over the subsequent 5 days were collected and analyzed for 2,4,5-T levels. Total urinary excretion levels were then estimated by extrapolating the data to the time-point when no further excretion occurred. The rate of absorption was not directly proportional to the concentration sprayed. The rates of absorption were 0.22, 0.42, 0.57, and 1.13 mg/sq. ft. per 1 hr. for spray concentrations of 2, 4, 16 and 32 Ib. of 2,4,5-T acid per 100 gallons, respectively. Three studies have investigated the pharmacokinetics of 2,4,5-T in man, after known doses were administered. In all three studies 2,4,5-T was administered orally to healthy male volunteers between 31-58 years old. In two of the studies, doses of 2-5 mg/kg were ingested. No adverse effects were noted in physical exams or clinical laboratory tests in one study (Gehring et al., 1973) and adverse effects were were not noted by the subjects in the second study (Kohli et al., 1974). In the third study, doses of 100-150 rag/kg were ingested but the subsequent conditions of the volunteers were not mentioned (Matsumura, 1970). In all three studies, �blood and urine samples were collected after ingestion and analyzed for 2,4,5-T. The data were then subjected to pharmacokinetic analysis. All three investigations found that 2,4,5-T was rapidly and completely absorbed from the gastrointestinal tract. Plasma clearance was also rapid, with approximately 80 percent of the dose excreted in the urine within 4 days. The half-life for absorption was estimated at 0.75 hour; the plasma half-life for plasma clearance was 19-23 hours at low doses, and 11 hours at high doses. Plasma clearance followed first-order kinetics in all three experiments. All of the 2,4,5-T in the plasma was reversibly bound to protein (Gehring et al., 1973). The amount of 2,4,5-T absorbed by workers from occupational exposure has been estimated from the total amounts excreted in the urine. The amounts excreted correlated with the occupation of the worker and the extent of protected clothing worn. One study reported that an average of 1 mg of 2,4,5-T was excreted over a 24-hour period by four workers who spryaed 2,4,5-T from tractors for 2-4 hours period. (Kolmodin-Hedman and Erne, 1980). Lavy (1978) reported that excretion of 2,4,5-T was highest for workers involved in mixing 2,4,5-T formulation and lowest for flagmen involved in aerial spraying. After two exposures the total amounts excreted were 0.131 and 0.003 mg/kg body weight, respectively for these two groups of workers. No special precautions were made during this study to minimize exposure. Ramsey et al. (no date) estimated total excretion levels using several methods of pharmacokinetic analysis of urinary excretion data. The values reported ranged from 0.002 mg/kg for helicopter flagmen to 0.073 mg/kg for mixers. In another study, levels of I'.l- 3.6 ppm of 2,4,5-T were detected in the urine of spray operators while 2,4,5-T was not detected in the urine of farmers (Shafik et al., 1971) 4.2.1.2 Biotransformat ion In pharraacokinetic studies described above, in which known doses of 2,4,5-T were administered to man, urine was analyzed for metabolites (Matsumura 1970, and Gehring et al., 1973). None were detected by gas chromatography and conjugates were not detected in ether extracts of acidhydrolyzed samples. 4.2.2 Animal Studies 4.2.2.1 Biodynamics 2,4,5-T was absorbed rapidly by the rat lung. Of a tracheally instilled dose of 2,4,5-T in solution, 50 percent was absorbed in 1.7 minutes, a rate that implied that pulmonary absorption occurred by diffusion across a membrane (Burton et al., 1974). Cultured human lung cells were found to take up 2,4,5-T minimally, compared to other chemicals which are known to bioaccumulate in animals (Murakami and Fukami, 1978). The authors noted tha't 2,4,5-T was more water-soluble than the other persistent pesticides studied, 4-5 �and concluded that uptake was high for relatively insoluble, bioaccumulated compounds, and low for soluble, non-persistent compounds. However, retention of herbicide by cultured cells is determined by influx and efflux, and these results may have reflected a difference in the efflux of these chemicals out of the cell, rather than their rate of uptake. Plasma half-lives for 2,4,5-T were determined in several species. In the male rat, the half-life for a 100 mg/kg oral dose was 3 hours (Erne, 1966). In other studies, the half-life was dependent upon the dose, with half-lives of 4-5 hours for interavenous or oral doses in the order of 5 mg/kg, and 19-23 hours for 100 rag/kg doses (Sauerhoff et al., 1976; Piper et al., 1973). These results are not in agreement with the rates for human exposures. Compared to 2,4-D, 2,4,5-T was cleared at a slower rate by the mouse (Zielinski and Fishbein, 1967). Pharmacokinetic analysis of the data on plasma clearance in the rat revealed that the nonlinear pattern of the data from high doses was substantially altered when a component for enterohepatic recycling was introduced into the pharmacokinetic model (Colburn, 1978). Contributions from both significant enterohepatic recycling and saturation of the renal transport mechanism were suggested as causes of the slower clearance at higher doses. Administration of ion exchange resins in cases of 2,4,5-T intoxication was recommended as a measure potentially to reduce the 2,4,5-T tissue and plasma levels, as well as the toxicity. Evidence that the renal mechanism is saturable.has also been produced in the chicken (Erne and Sperber, 1974) and the rat (Hook et al., 1974; Shafik et al., 1971). Other factors that have been shown to influence the clearance of 2,4,5-T are related to the species and age of the animals tested. Newborn rats showed clearance rates of 97 hours, compared to 3.4 hours in adults (Fang et al., 1973). The half-life for clearance of a 5 mg/kg oral dose in dogs was 77 hours (Piper et al., 1973), compared to 4 hours in the rat. Clearance of 2,4,5-T by cattle and sheep was rapid, however, and within the same order as observed in the rat (St. John et al., 1964; Clark and Palmer, 1971). Tissue distribution in cattle also followed patterns in other species, with high levels in the kidney and low levels in fat (Clark et al., 1975). In the dog, the slow rate of 2,4,5-T clearance has been considered a likely explanation for the dog's higher sensitivity to the toxic effects of 2,4,5-T. In vitro studies have demonstrated that 2,4,5-T was higher for the adult rat than for the newborn rat or the dog (Hook et al., 1974), providing evidence that different rates of renal excretion were responsible for the differences in plasma clearance. Renal transport was also reduced in vitro by addition of plasma, suggesting that binding of 2,4,5-T to plasma protein decreases its availability for renal clearance in the dog in vivo, in addition to slower renal secretion into the urine (Hook et al., 1976). In vitro renal accumulation of 2,4,5-T in the rabbit was higher than in the rat (Berndt and Koschier, 1973). The rate of clearance in the rabbit has not been determined, but its toxicity is lower than in the rat (see chapter 6). Renal tubular transport by the organic anion system is probably the mechanism of renal handling of 2,4,5-T (Koschier and Berndt, 1976). In vitro accumulation by this system was shown to be energy-dependent (Berndt and Koschier, 1973). At high doses, 2,4,5-T produced nephrotoxicity, which 4-6 �reduced the rate of clearance by the perfused rat kidney (Koschier and Acara, 1979). 2S4,5-T was found to bind to renal microsomes and the binding was suggested to result in retention of high levels of 2,4,5-T that produce nephrotoxicity following administration of a large dose (Koschier et al., 1979). High-affinity 2,4,5-T binding to bovine serum albumin (probably at tryptophan residues) has been demonstrated (Haque et al., 1975; Mason, 1975) as well as binding to protein in human plasma (Gehring et al., 1973). The binding affinities for various species have not been compared, which would determine whether higher plasma binding in the dog may contribute to the lower renal clearance in this species. Along with its high-affinity binding to renal tissue and plasma proteins, 2,4,5-T was distributed among various other tissues and then was cleared rapidly from these tissues. Following oral administration to rats, the kidney had from 10- to 100-fold higher levels than those of other tissues (except the stomach at early times after dosing); plasma levels were also substantially higher than other tissues, but lower than renal levels. Tissue clearance rates averaged 3.4 hours, following a low dose of 2,4,5-T (Fang et al., 1973). The same relative distribution of 2,4,5-T to the kidney, compared to other tissues, was observed in sheep (Clark and Palmer, 1971). 2,4,5-T did not accumulate in the fat of cattle during a 32-week exposure period (Clark and Palmer, 1971) or in fat or muscle of sheep or cattle during a 28-day exposure period (Clark et al., 1975). 2,4,5-T was not excreted in expired air by rats, while 3-14 percent of oral doses were excreted in feces at higher doses; transfer of 2,4,5-T to offspring via milk has been shown in the rat (Fang et al., 1973; Piper et al., 1973; Sauerhoff et al., 1976). 4.2.2.2 Biotransforraation Although at least 94 percent at intravenous dose was identified as unchanged 2,4,5-T in the urine in one study (Sauerhoff et al., 1976), as many as three minor metabolites have been isolated (but not identified) in the urine in other studies after oral administration, which were more likely to be detected following larger doses (Fang et al., 1973; Piper et al., 1973). In another study, 10-20 percent of excreted 2,4,5-T was in bound form (susceptible to acid hydrolysis) and N-(2,4,5-trichlorophenoxyacetyl) glycine were identified in the urine of rats given 50 mg/kg of 2,4,5-T orally (Grunow et al., 1971). 4.3 TCDD A large number of studies have investigated the metabolism of TCDD and its biological effects on a cellular level. The only studies that used human tissues were studies that determined TCDD residue levels or enzyme induction in blood cells or in cell lines derived from human tissues. Animal studies have investigated the biodynamics, biotransformation, enzyme induction, and mechanism of action of TCDD. The role of receptors in the biological effects elicited by TCDD has been considered in some studies. The role of biotransformation in explaining species differences has also been considered in several recent studies presented in this section. 4-7 �4.3.1 Human Studies 4.3.1.1 Biodynamics Human tissue samples that have been analyzed for TCDD were all taken from people with exposure to TCDD from unknown sources, with unknown levels and durations of exposure and by unknown routes. The possible exception to this was the case of a woman who lived in the Seveso area (zone A) for 2 weeks. When the woman died of pancreatic carcinoma 3 months after the accident at the ICMESA plant, tissues removed at autopsy were analyzed for TCDD, Tissue levels were 1.84 ppb in fat, 1.04 ppb in pancreas, 0.15 ppb in liver, and 0.04-0.06 ppb in lung, kidney, and brain (Reggiani, 1979). Gross (1980) described the results of TCDD analyses in human tissues from three sources. No TCDD was detected in 44 adipose and liver samples removed at autopsy in hospitals serving agricultural areas. These areas were in the Southern U.S., in principal rice-growing regions which were considered likely to use 2,4,5-T. The detection limit for 43 of the samples was 1-10 ppt. In 103 milk samples including 72 mothers in the northwestern U.S. where 2,4,5-T use was considered likely, no TCDD was detected (1 ppt, average limit of detection). Adipose tissue from 22 veterans were also analyzed. Ten samples have detectable levels of TCDD, but the levels were not reported and whether the positive samples were from Vietnam veterans was not reported. Nisbet (1980) cited studies in which 5-16 ppt of TCDD was detected in 1 adipose sample (the donor was not described) and 11-31 ppt was detected in milk samples in 4 of 17 samples (3 samples were from women from Kansas and Texas, and 1 was from Italy). Without any information on exposure levels, these data cannot be used to assess the patterns of TCDD biodynamics in man, compared to animal data. 4.3.1.2 Enzyme Induction Aryl hydrocarbon hydroxylase (AHH) activity in human cells has been induced by TCDD treatment in vitro. Niva et al., (1975) showed that AHH activity in human lymphoyctes and in human Chang liver cells responded to TCDD treatment, although the dose required to produce half-maximal induction in lymphocytes was higher than the doses required for half-maximal induction in cells from other species. Kouri et al., (1974) also reported responsiveness of lymphocyte AHH activity to TCDD. The induced levels were low (approximately 3 times the control levels). 4.3.2 Animal Studies 4.3.2.1 Biodynamics TCDD clearance from plasma has been shown to be_slow. In the 7-day period following intraperitoneal administration of [ H]-TCDD to monkeys and rats, only 1 percent of the dose was excreted in the urine and 4-5 percent in 4-8 �the feces. Radioactivity in the monkey was highest in tissues with high lipid content, including the skin and fat, and in muscle. In the rat, 40 percent of the radioactivity remained in the liver 1 week after dosing (Van Miller et al. , 1976). Male rats retained an even higher proportion of a dose of TCDD in the liver than females. Half-lives for clearance of TCDD from the whole body were shorter for males (12 days) than females (15 days) (Fries and Marrow, 1975). In another study in the rat, the half-life for body clearance of a single oral dose was calculated at 17 days. The liver retained 3, 4, and 1 percent (per gram of tissue) of the dose, 3, 7, and 21 days, respectively, after dosing, and fat levels with 53, 13, and 3 percent in the feces, urine, and expired air respectively (Piper et al., 1971; Piper et al., 1973). After repeated doses were administered orally to rats, the feces remained as the major route of excretion. Radioactivity in the liver and fat reached steady state levels at the same rate as for the whole body, with a half-life of 7 weeks (Rose et al., 1976). The validity of the pharmacokinetic constants in the rat has been challenged, however, because the constants were calculated assuming first-order kinetics for TCDD in the rat; the data have since been shown to be compatible with a zero-order kinetic model as well (Hiles and Bruce, 1976). In the guinea pig, intraperitoneal administration of a single dose of TCDD resulted in high TCDD levels in the fat after 1 day (Gasiewicz and Neal, 1978; 1979). After 15 days, lower levels of TCDD were found in the fat, while the levels in the liver had increased 3 fold and increases occurred in the adrenal, kidney, and lung levels, during this period. These increases, the authors suggested, resulted from mobilization of fat stores. The liver contained 11 percent of the injected dose in this study, similar to the proportion of an intraperitoneal dose administered to the monkey (Van Miller et al., 1976) but in contrast to the level reported for the rat of 40 percent. In accordance with these findings, only the rat shows severe hepatotoxicity after TCDD treatment. Teitelbaum and Poland (1978) demonstrated that hepatic uptake of TCDD was increased by TCDD pretreatment and that the non-cytosol fractions showed enhanced capacity for TCDD binding in vitro, after the pretreatment. Gasiewicz and Neal (1978; 1979) reported that only 5 and 1 percent of an intraperitoneal dose of TCDD was excreted in the feces and urine, respectively, by guinea pigs in 15 days. Excretion was linear for 23 days. Assuming that the rate continued to remain linear, the half-life for excretion by both routes was calculated at 30 days. Nolan et al. (1979) reported that only half of an oral dose of TCDD was absorbed by guinea pigs and that the fat, thymus, liver, and adrenals retained the highest levels 22 days after dosing. The half-life for clearance from these tissues was estimated to be between 22 and 43 days. Recently, the hamster has been shown to be less sensitive than other species to the acute effects of TCDD (Olson et al., 1980). In this species, the highest tissue levels of TCDD, administered intraperitoneally or orally, were recovered in the liver, fat, and adrenals, during the 35-day period after administration (Olson et al., 1980). The urine and feces contained 35 and 4-9 �50 percent respectively, of the dose and the half-lives for elimination by these two routes were 11 to 15 days for oral and injected doses. The authors concluded that the rapid excretion in the species contributed to its lower sensitivity to TCDD. 4.3.2.2 Biotransformat ion Early studies reported that radioactivity in the rat liver remained as TCDD, which was not metabolized (Rose et al., 1976). TCDD radioactivity was located specifically in the microsomal fraction of the liver cells (Allen et al. , 1975). In the mouse, similarly, TCDD was localized in the microsomal fraction of liver cells and remained intact, without undergoing biotransformation (Vinopal and Casida, 1973). Beatty (1977), Beatty et al. (1978), Beatty and Neal, (1976) produced indirect evidence that TCDD was metabolized, by demonstrating a correlation between elevated mixed function oxidase levels and decreased toxicity. Tulp and Hutzinger (1978) showed that a series of polychlorinated dioxins were all metabolized by hydroxylation at the 2,3,7, or 8-position, except for octachlorodibenzo-p-dioxin, which is chlorinated at each of these positions, blocking hydroxylation. Although TCDD was not included in this study, the authors pointed out that it would not be likely to be susceptible to hydroxylation, since all four sites are blocked. Recently, several studies have examined bile samples for TCDD metabolites. Ramsey et al. (1979) collected 24-hour bile samples of rats after they received 2-6 oral doses of TCDD. Five metabolites were isolated by liquid chromatography. The metabolites were more polar than TCDD, based on their extraction into selective solvents, and included glucuronide conjugates, based on susceptibility to beta-glucuronidase. The rate of biliary excretion was comparable to the rate of fecal excretion. Matthews and Kato (1979) reported that 90 percent of the TCDD excreted in bile was in the form of metabolites. Poiger and Schlatter (1979) also cited in Donzel et al., 1980, also reported that rats that were administered TCDD orally, excreted the metabolized compound in bile at a rate that accounted for fecal excretion. Only unmetabolized TCDD was recovered from the liver. Bile samples, collected over 3-4 days, were subjected to various chromatographic, enzymatic, and extraction procedures. The isolated metabolites were not completely identified, but their chemical characteristics were consistent with watersoluble conjugates, were labile to glucuronidase-arylsulphatase digestion, and possibly contained phenolic hydroxyl groups. Olson et al. (1980) isolated one major metabolite and several minor metabolites, by high pressure-liquid chromatography, from the bile of hamsters after TCDD was administered intraperitoneally. These authors suggested that the faster excretion rate in this species reflected a faster rate of TCDD metabolism, which led to the formation of polar metabolites that were rapidly excreted in bile. Guenther et al. (1979) proposed that TCDD was metabolized to arene oxides by the cytochrome P-450 system, although the proposed, highly-reactive metabolite was not isolated. 4-10 �4.3.2.3 Enzyme Induction TCDD has been shown to induce various hepatic and extraphepatic enzymes. Table 4-1 lists enzymes that were shown to be induced by TCDD, as well as enzymes that did not show increased activity following TCDD treatment. In these studies, TCDD induction of hepatic microsomal enzymes involved protein synthesis. Inhibitors of protein synthesis, like actinomycin-D, prevented induction (Beatty and Neal, 1978; Niwa et al., 1975; Hook et al., 1975a; Lucier et al., 1975a), and addition of TCDD to the enzyme preparation in vitro did not stimulate enzyme activity (Lucier et al., 1975a). TCDD also produced increased levels of cytochrome P-450, P-448, and b. (Hook et al., 1975b; Lucier et al., 1973; Guenther and Nebert, 1978). The a, b, and c forms of cytochrome P-450 that were induced in rabbit liver were shown to correspond to specific enzyme activities (Johnson and Muller-Eberhard, 1977). TCDD induced a spectrally distinct type of P-450 whose spectral characteristics resembled methylcholanthrene (MC)-induced cytochrome. TCDD also induced select enzymes, similar to the pattern of MC induction and in contrast to the non-specific pattern of phenobarbital (PB) induction (Poland and Glover, 1974; Aitio and Parkki, 1978; Pohl et al. 1976; Greig and DeMatteis, 1973). A combination of maximally inducing doses of 3-MC and TCDD did not produce additional enzyme induction, indicating that both inducers may act by the same mechanisms (Poland and Glover, 1974; Niwa et al., 1975). TCDD appeared to induce a kinetically different glutathione S-transferase than either MC or PB induced (Baars et al., 1978). TCDD was found to induce the same aldehyde dehydrogenase that was produced by hepatoma cells, but was distinct from the PB-induced isozyme (Lindahl et al., 1978). The TCDD-induced increase in aryl hydrocarbon hydrdxylase (AHH) activity, the enzyme that catalyzes the hydroxylation of bertzo(a)pyrene to 3-hydroxy-benzo(a)pyrene, was higher than for most other enzymes studied. TCDD was about 30,000 times more potent at stimulating AHH activity than 3-MC, making it one of the most potent enzyme inducers known (Poland and Glover, 1974). Likewise, DT-Diaphorase induction by TCDD was greater and lasted longer than induction by a. 200-fold greater dose (by weight) of 3-MC (Beatty and Neal, 1976). TCDD-stimulated enzyme induction was remarkably long-lasting. A single dose of TCDD has resulted in enzyme activities that were still significantly elevated 35 days after 31 nanomoles of TCDD per kg was administered. In contrast, the effect of a single dose of 75 micromoles of 3-MC per kg, which produced the same magnitude of response on AHH activity, was no longer evident after 8 days (Poland and Glover, 1974). Other enzymes remained elevated for a long time after TCDD treatment. Rat liver DT-diaphorase activity was substantially elevated 21 days after a single intraperitoneal dose of 25 ug TCDD per kg (Beatty and Neal, 1978); rat liver glucuronyltransferase was elevated 30 days after a dose of 5 ug/kg and 73 days after 25 ug/kg was given (Lucier et al., 1975a); biphenyl hydroxylase was significantly elevated 73 days after a 25 ug/kg dose (Hooke et al., 1975a); and aniline hydroxylase was elevated, 28 days after a 25 ug/kg dose; corresponding elevations in microsomal protein levels and cytochrome P-450 and b,. were observed at this time point, as well (Lucier et al., 1973). Renal 4-11 �TABLE 4-1: ENZYMES RESPONSIVE TO TCDD INDUCTION Enzyme Source Enzymes Tested: Indue ible Non-Inducible Species Tissue Reference AHH8 human lymphocytes Kouri et al, 1974 AHH 6 spp. cell lines Niwa et al, 1975 AHH rat hepatoma BradLaw et al, 1975 AHH aoinopyrine-DH N cyt. C reductase rat, mouse chicken liver Poland & Glover, 1974 AHH, GTC, cyt.d P-450 and b,, aniline-He 3 aminopyrine-DM benzphetamine-DM ethylmorphine-DM rat liver iucier et al, 1973 AHH, cyt. P-450 (3 in above listing) AHH rat guinea pig, rabbit rat guinea pig, rabbit guinea pig, rabbit liver liver lung, kidney, gut lung , kidney , 1 iver lung Hook et al, 1975 rat rat liver, kidney, lung liver, kidney, lung Aitio & Parkki, 1978 GT, AHH rat kidney Fowler et al, 1977 Glutathione-S-T rat liver Bears et al, 1978 steroid-GT rat liver Luc let et al, 1975 rat guinea pig 8 tissues liver, others Beatty & Neal, 1978 DT-diaphorase chicken embryo Poland & Glover, 1973 rat liver Woods, 1973 UDP-CT Biphenyl-He AHH, cyt. C reductase UDP-GT p-nitrophenol-GT UDP-CT epoxide hydrataje glutathione-S-T DT-diaphorase ALA* ALA 4-12 �TABLE 4-1: ENZYMES RESPONSIVE TO TCDD INDUCTION (Continued) Enzyme Source Enzymes Tested: Species Tissue Reference rat rat liver liver Guenther & Nebert, 1978 rat liver Hook et al, 1975 Aid. dehydrogenase rat liver Lindahl et al, 1978 AHH, aniline-H 7 -ethoxy coumar in deethylase mouse mouse skin skin Pohl et al, 1976 Hexabarbital mouse, rat liver Greig & De Matteis, 1973 Zoxazolamine mouse, rat liver Greig, 1972 pregnant rat rat fetal rat liver liver liver Lucier et al, 1975 fetal rat extra-hepatic Berry et al, 1977 Inducible Non-Inducible AHH, oyt. P-448, 450 Acetanilide-4-H Biphenyl-H, cyt. P-450 Cyt., AHH steroid-H p-aminophenol-GT steroid-GT p-aminopheno 1-GT AHH FAAJ, AHH FAA, AHH Cyt. P-450 a rabbit Norman et al, 1978 liver DM «• demethylation enzyme c Berry et al, 1976 Aryl hydrocarbon hydroxylase b rat (fetal, maternal) liver placenta, adrenal rat GT * glucuronyl transferase d cyt. " cytochrome e H » hydroxylase f T » transferase g ALA • delta-aminolevulinic acid h (relevant metabolic enzymes for this compound) j FAA * fluorenyl acetamide hydroxylation 4-13 �benzpyrene hydroxylase and glucuronyl transferase activities were also substantially elevated 16 days after a single oral dose of 25 ug/kg (Fowler et al., 1977), indicating that hepatic sequestration of TCDD may not necessarily explain the long duration of this effect. On day 5 of gestation, pregnant rats were administered 3 ug TCDD per kg; 73 days after exposure, the 56-day-old offspring had substantially elevated para-nitrophenol glucuronidase levels (Lucier et al., 1975b). This result, as well as results for other hepatic and non-hepatic enzymes in young rats exposed to TCDD only prior to birth, indicates that TCDD induction can be transmitted transplacentally. In the rabbit, TCDD transplacentally-induced hepatic cytochrorae b but not cytochrome c, while both were induced in the adult (Norman et al., 1978). This difference in response probably reflects differences in the hepatic cytochrome system with age. Doses of TCDD that produced half-maximal induction in AHH activity varied between 0.4 and 1.2 nmole/kg for the chicken, rat, and several strains^of mice. In the chicken, ALA synthetase was induced by a dose of 4.7 x 10 mole per egg (Poland and Glover, 1973). A single dose of 0.2 ug/kg to rats produced a significant induction of biphenyl hydroxylase activity (Hook et al., 1975a) and of aniline hydroxylase and AHH (Lucier et al., 1973). In several experiments that compared the effects of TCDD induction in male and female rats, females were more sensitive than males, to low doses of TCDD which induced AHH, para-nitrophenol glucuronyltranferase, and biphenyl hydroxylase activities (Lucier et al., 1975a; Hook et al., 1975a; Lucier et al. , 1973). TCDD induction was not limited to hepatic enzymes: skin, kidney, lymphocyte, and lung enzymes responded to TCDD induction (Berry et al., 1977; Aitio and Parkki, 1978; Fowler et al., 1977; Pohl et al., 1976); these enzymes in the-testes as in the intestine were not induced (Aitio and Parkki, 1978). TCDD also has not been shown to induce enzymes in a variety of cell lines and primary cell cultures 48 hours after it was added to culture medium (Bradlaw et al.: 1975, 1976, 1980; Bradlaw and Casterline, 1979; Kouri et al., 1974; Niwa et al., 1975). These culture systems have been suggested as potential TCDD assay systems because extremely low levels of TCDD were able to elicit positive responses. The kinetics of enzyme induction in vitro and the relative responsiveness of cells from various strains of mice followed the same patterns as were observed in vitro (Niwa et al., 1975). The strikingly low doses of TCDD that produce enzyme induction and the long-lasting inductive effect that TCDD elicits are analogous to the high potency of low doses of TCDD in producing toxicity and death and the delayed pattern of this toxicity. However, the wide range in sensitivity to TCDD toxicity among different species contrasts with the narrow range of doses that produce comparable enzyme induction in various species (Poland and Glover, 1974). In fact, the guinea pig, the species most sensitive to acute TCDD toxicity, showed no inductive response to TCDD at doses that elevated enzyme levels in the rat, a species that shows relatively low sensitivity to acute TCDD toxicity (Beatty and Neal, 1978). The extent of in vitro toxicity of various cell lines after TCDD treatment also did not correlate with sensitivity to AHH induction for the same cell lines (Niwa et al., 1975; Knutson and Poland, 1980) or to the sensitivity to TCDD in vivo (Beatty et al., 1975; Knutson and Poland, 1980). Although the possibility exists that 4-14 �the toxic effects of TCDD on lipid metabolism and nutrient utilization may be derived from TCDD's effects on activities of specific enzymes, this relationship and the potential enzymes involved in this effect have not been demonstrated. 4.3.2.4 Mechanism of Toxicity The remarkable potency of TCDD, its unusual temporal pattern of toxicity, and its severe dermal and metabolic effects have led to various studies on the mechanisms of TCDD toxicity. The relationship between enzyme inducibility and the toxicity of TCDD has been the subject of a series of elegant and thorough studies from several laboratories. Cell surface receptors for TCDD have been identified (Greenlee and Poland, 1979; Poland et al., 1979; Carlstedt-Duke, 1979; Carlstedt-Duke et al., 1979). This receptor has been shown to be a gene product of the Ah locus (Nebert et al., 1973; Okey et al., 1979; 1980; Nebert and Jensen, 1979). Binding of an enzyme inducer, including TCDD or 3-MC, to this receptor is a necessary requirement before enzyme induction can occur. However, receptor binding does not guarantee that enzyme induction will occur (Okey et al., 1980). Nuclear transfer of the receptor-inducer complex has also been demonstrated (Okey et al., 1979; 1980; Greenlee and Poland, 1979). Strains of mice that show low responsiveness to AHH induction by TCDD (Poland et al., 1974; Kumaki et al., 1979) were also shown to have lower hepatic binding affinity for TCDD. A genetic mutation at the Ah locus has been suggested to have caused an alteration in the structure of the receptor that diminishes its binding affinity to TCDD (Poland and Glover, 1975). Enzyme induction results from changes that occur after TCDD binds to hepatic cytosol receptors and the complex is subsequently transported to the nucleus (Greenlee and Poland, 1979; Poland et al., 1976). The role of the Ah gene product in cytochrome P-450 induction is the subject of several reviews (Nebert and Jensen, 1979; Fox, 1979; Poland and Kende, 1976; Poland and Glover, 1978). These receptors may play a role in eliciting the toxic effects of TCDD, in addition to the inductive effects (Poland and Glover, 1973; Poland, 1979; Neal et al., 1979). Poland and Glover (1980) have demonstrated that strains of mice with high-affinity receptors for TCDD, and high sensitivity to TCDD enzyme induction, also showed greater sensitivity to toxic effects of TCDD, including thymic atrophy and cleft palate teratogenicity than strains with low affinity binding and low inductive responsiveness. The thymus, an organ that shows severe atrophy after TCDD is administered, was shown to contain a high affinity receptor for TCDD, relative to other tissues (Carlstedt-Duke, 1979). Strains of mice that showed decreased AHH responsiveness also lost their susceptibility to the porphyrogenic effect of TCDD (Jones and Sweeney, 1980), both of which may require TCDD-receptor binding in order to be elicited. The porphyrogenic effects of TCDD are only elicited in animals with sufficient (normal) levels of iron. The mechanism by which iron deficiency protects against TCDD-induced liver toxicity and porphyria is unknown (Jones and Sweeney, 1979; Sweeney et al., 1979). 4-15 �Toxicity and teratogenicity of TCDD have also been suggested to result from the binding of TCDD metabolites to protein or from an effect on thymidine utilization. In one study, TCDD metabolite binding to protein in vitro was highly favored over metabolite binding to DNA (Guenther et al., 1979). Cellular utilization of thymidine, measured in vitro as nuclear incorporation of thymidine, was stimulated by TCDD (Conaway and Matsumura, 1977). The relevance of these in vitro findings to mechanisms of enzyme induction and toxicity awaits further studies. 4.4 DIQUAT The metabolism of diquat has been reviewed (Calderbank and Slade, 1976; Rose and Smith, 1977); diquat was absorbed by the lung. In the rat, the process of pulmonary absorption was biphasic and energy-dependent, probably related to uptake by alveolar membranes on the airway side (Charles et al., 1978). Diquat was poorly absorbed from the gastrointestinal tract. Only 10-20 percent of a 12 ug/kg oral dose was absorbed 6 hours after being administered to the dog (Bennett et al., 1976). Less than 20 percent of oral doses administered to rats was excreted in the urine, with the remainder recovered in feces. Since bile contained only 5 percent of the dose after 24 hours, and subcutaneous doses of diquat did not appear in feces, the fecal levels probably represented unabsorbed compound. About 70 percent of the oral dose of diquat was recovered as metabolites, which was attributed to degradation by gut microbes (Daniel and Gage, 1966). Paraquat, a structural analog of diquat and a pulmonary toxin, has been shown to concentrate in th'e lung. Diquat, which does not produce toxicity in the lung, also does not concentrate in the lung. These results have been confirmed by experiments in the rat in vivo (Sharp et al., 1972; Kurisake and Sato, 1979) and in vitro (Rose et al., 1976; Rose et al., 1974; Abou-Donia et al., 1976) and in the mouse (Litchfield et al., 1973). In the rat, diquat was distributed to the liver and cartilage at higher concentrations than other tissues, and did not reach the brain. Distribution to all tissues, as well as clearance from the tissues, was rapid. Diquat was cleared from the mouse in 24 hours after intravenous injection, primarily via feces. Only about 6 percent of the dose was recovered in the urine of rats in the 2-week period after a single oral dose was administered. In an 8-week feeding study, no evidence of tissue accumulation of diquat was produced (Litchfield et al., 1973). Less than 10 percent of the doses of diquat administered to the rat, rabbit, and guinea pig was excreted in the bile, while most of the remainder was recovered in urine. Only 1-5 percent.of the doses given to rabbits was metabolized, to an unidentified compound (Hughes et al., 1973). Diquat metabolism to two unidentified metabolites has also been reported for the embryonic chicken (Leakey and Hemingway, 1975). Free radical formation has been proposed as the reactive form of diquat in eliciting toxic effects. The potentiation of diquat's toxicity in the presence of oxygen (Kehrer et al., 1979) has been suggested to result from stimulation of free radical conversion by oxygen, while carbon monoxide inhibits free radical formation (Baldwin et al., 1974). This subject is �discussed further in chapter 6, Acute Toxicity. Diquat binding to melanin was demonstrated to be ionic in nature and was proposed to lead to diquat uptake in the eye, the target organ for diquat 1 s chronic toxicity (Larsson et al., 1977). 4.5 DIURON AND MONURON Diuron undergoes biotransformation in all species that have been studied. After oral ingestion of 38 mg/kg by a 39-year-old woman, diuron was recovered in the urine in the form of two metabolites. These metabolites were l-(3,4dichlorophenyl)-3-methylurea and l-(3,4-dichlorophenyl)-urea. No unaltered diuron was recovered (Geldmacher, Mallenckrodt, and Schussler, 1971). In the rat and the dog, diuron was metabolized and excreted in the urine and feces. Urinary metabolites were identified as N-(3,4-dichlorophenyl) urea (the prominent metabolite), N-^3,4-dichlorophenyl)-N -methylurea, 3-4-dichloraniline, and 3,4-dichlorophenol. Unmetabolized diuron was also detected. Chronic administration did not lead to tissue accumulation (Hodge et al., 1967). In the cow, diuron was excreted in urine and to a lesser extent in feces, but not in milk. Urinary metabolites were identified as 3-(3,4-dichlorophenyl)-l-methylurea and 3-(3,4-dichlorophenyl)urea (Kalra and Chahal, 1979). Monuron is also metabolized prior to urinary excretion (Midwest Research Institute, 1975). Diuron activity as an inducer of hepatic microsomal enzymes has been observed in the rat. Several diuron metabolites elicited this effect as well. The enzymes that were indueible were p-nitroanisole o-demethylase, aminopyrine N-demethylase, and o-p-nitrophenyl phenylphosphonothionate detoxification enzyme (Kenoshita and DuBois, 1970; Corthay et al., 1977). Diuron also produced type I spectral changes in cytochrome P-450 in vitro (Mailman and Hodgson, 1972). Monuron also induced hepatic enzymes in the rat. Midwest Research Institute (1975) and Geissbuhler et al., (1975) have reviewed the metabolism of monuron and diuron. Although little is known about the metabolism of monuron, the results of one rat study by Ernst (1969; cited in Midwest Research Institute, 1975) suggested that monuron underwent demethylation of both methyl groups and underwent hydroxylation at one of the ring carbon positions. 4.6 BROMACIL Bromacil biotransformation has been demonstrated in man and in the rat, with 5-bromo-3-sec-butyl-6-hydroxymethyluracil as the principal metabolite in both species. 5-Bromouracil was not recovered in the urine of either species (Midwest Research Institute, 1975). Dairy cows that were administered bromacil by oral intubation for 4 days excreted the herbicide in milk, but not in urine or feces. Bromacil was not metabolized in vitro by rumen fluid or the supernatant fraction of homogenized liver (after 10,000 G centrifugation) (Gutenmann and Lisk, 1970). 4-17 �4.7 PICLORAM In the dog, 90 percent of an oral dose of picloram was absorbed and excreted within 48 hours. No metabolites were detected in urine. In cattle, 0.02 percent of picloram, administered by gavage, was excreted in milk. Three days after administration ceased, picloram was not retained in any tissues (Foy, 1976). 4.8 CACODYLIC ACID Workers that applied cacodylic acid over an 11-week period were found to excrete arsenic in urine. Average arsenic levels ranged from 24 to 172 ug per 24 hours. Urine levels, but not blood levels, of arsenic correlated with the estimated exposure levels of the individual workers. The average exposure was estimated at 817 gm per man per week. The authors noted that some workers noticed the odor of garlic in areas they had treated with cacodylic acid and the possibility that workers were exposed to arsine gas was suggested (Wagner and Weswig, 1974). Another study reported average levels of 0.56 ppm arsenic in the urine of forest workers that applied cacodylic acid over a 2-raonth exposure period and noted that urine levels fell quickly when exposure ended (Tarrant et al., 1972). Wagner and Weswig (1974) suggested that the lower urine levels found in their study reflected more careful handling of the herbicide by the workers in their study. In these human studies, the source of arsenic was not identified. The possibility that the workers were exposed to other forms of arsenic, such as arsine gas, was alluded to in one study, but was not determined. Therefore, the urine levels may not have reflected absorption of cacodylic acid exclusively. Assuming that arsenic was derived from cacodylic acid, the differences in urine levels may have reflected different rates of metabolism, rather than different levels of exposure among the workers. Hwang and Schanker (1973) studied the absorption of cacodylic acid by the small intestine of the rat, in vivo. The half-time for absorption of cacodylic acid was 201 minutes for concentrations between 1 and 100 mM. Cacodylic acid appeared to be absorbed by diffusion, based on the rates of absorption of other organic arsenical compounds and the correlation of these rates with chloroform-to-water partition coefficients, but not with molecular weights. Stevens et al. (1977) studied the metabolism of cacodylic acid in the rat. The half-lives for absorption by inhalation and oral routes were 2.2 and 248 minutes, respectively. The latter half-time was close to the value of 209 minutes reported by Hwang and Schanker (1973). After intravenous administration, three kinetic components were observed for plasma clearance, with half-times of 0.01, 0.22, and 3.42 hours. After all doses were administered by three routes, the half-time for clearance of cacodylic acid from whole blood was 76-92 days. Cacodylic acid was cleared from all other tissues rapidly. Cacodylic acid also cleared from tissues of cattle and chickens �after exposure terminated, although whole blood levels were not determined (Boggen, 1968; Peoples, 1963: both cited in Midwest Research Institute, 1975). The tissue distribution of [ C]- cacodylic acid in the rat was the same as for [ As]- cacodylic acid (Stevens et al., 1977), which the authors concluded was an indication that cacodylic acid was probably not metabolized to inorganic arsenic. Further observation that only trace amounts of [ CjCO- were recovered in expired air following [ C]- cacodylic acid administration indicated that demethylation was a minor metabolic pathway. Cacodylic acid was excreted primarily in urine. When administered 1 day prior to parturition, cacodylic acid readily crossed the placenta. The extent of degradation of cacodylic acid to inorganic arsenic by mammalian species is unknown. Indirect evidence suggests that exposure to inorganic arsenic that is derived from cacodylic acid, following absorption of the organic compound, is limited. First, cacodylic acid is far more toxic than inorganic arsenic. The oral LD5Q for cacodylic acid is approximately 830 mg/kg, compared to 75 mg/kg for sodium arsenite and 15 mg/kg for arsenic trioxide (Palm, 1968). In addition, dimethylated arsenicals have been recovered from human urine samples, sometimes in large amounts relative to the levels of inorganic arsenic in the samples (Yamauchi and Yakamura, 1979; Braman and Foreback, 1973; Braman, 1975). The source of the dimethylated arsenicals was unknown in each of these studies and could have resulted from methylation of arsenic, absorbed as inorganic or monomethylated arsenic, or from excretion of absorbed cacodylic acid that did not undergo biotransformat ion. However, the observed excretion of methylated arsenic compounds argues against biotransformation of all absorbed cacodylic acid to arsenic. 4.9 SUMMARY AND CONCLUSIONS Both 2,4-D and 2,4,5-T are rapidly and completely absorbed from the gastrointestinal tract of man although dermal absorption in man is limited. Absorption from the lung is rapid in animals. Both phenoxy acids are cleared rapidly from blood. The half-life plasma clearance in man ranges from 12 to 23 hours. Almost all of the dose is excreted by the kidney. Differences in the rate of excretion among different species correlate with the relative toxicity of phenoxy acids in the same species, with a relatively low rate of excretion, and high toxicity in the dog. At high doses, saturation of the renal transport mechanism has been observed, along with nephrotoxicity, both leading to a decrease in clearance rates (which theoretically would increase apparent toxicity). The clearance rate of 2,4-D is also very low in humans when the urinary pH is low. Indirect evidence for enterohepatic recycling of 2,4,5-T based on kinetic modeling has been put forth and may provide a means of removing phenoxy acids in cases of acute poisoning. After phenoxy acids are administered, tissue levels are highest for the plasma and kidney; both compounds are excreted in milk and are distributed to fetal tissues (see chapter 8), indicating the potential for embryo-toxic 4-19 �effects. Neither compound is sequestered in fat or other tissues, indicating that they are unlikely to produce cumulative toxicity. 2,4-D is preferentially distributed to the brain following high doses (250 mg/kg), but not after lower doses (100 mg/kg) that produce no apparent neurotoxicity. Few studies have succeeded in isolating metabolites of phenoxyacids. In human urine 12 percent of 2,4-D was recovered as conjugates in one study; in the rat and pig, a few studies have recovered up to 20 percent of doses of 2,4-D and 2,4,5-T as metabolites. TCDD is cleared slowly from the body, with a half-life of 2-3 weeks in animals. TCDD is not sequestered in fat, although the liver retains a large portion of the administered dose. TCDD is excreted primarily in feces, which is the result of biliary excretion. Biotransformation to water-soluble, polar conjugates has been demonstrated recently. Once these metabolites are formed, they are rapidly excreted in bile. Metabolites have not been detected in liver or fat. TCDD induces various hepatic and extrahepatic enzymes. Extraordinarily low doses of TCDD are capable of eliciting induction and enzyme levels remain elevated for extended periods of time, in some cases for several months. The pattern of TCDD induction more closely resembles methylcholanthrene induction than phenobarbital induction. Transplacental induction by TCDD has been demonstrated. The mechanism of toxicity of TCDD remains unknown. TCDD binds to cytosol receptors; this binding which is thought to elicit its enzyme-inductive effects and separately may mediate its toxic effects; a role of enzyme induction in manifesting toxic effects has not been demonstrated. Three possible mechanisms for the differences in susceptibility among species can be proposed from the data that is currently available on TCDD metabolism and receptor studies: (1) The rate of metabolism of TCDD to less toxic metabolites that are rapidly excreted in bile will influence the extent of toxicity for each species. The strongest evidence for this mechanism are the studies in the hamster, by Gasiewicz and Neal (1979) and Olson et al., (1980) which indicate that the rates of metabolism and excretion for this species are higher, and the toxicity is lower than for other species. (2) The presence of high-affinity receptors is essential for enzyme induction by TCDD and differences in the amounts or affinity of these receptors may explain the differences in susceptibility of different species, and of different tissues. Segregation of sensitivity of toxic effects with enzyme induction and the receptor product of the Ah locus, reported by Poland and Glover (1980), supports this hypothesis. (3) Differences in the distribution of TCDD to different organs may affect the degree of toxicity in that organ. In the rat, a species that shows hepatotoxicity from TCDD, 40 percent of a dose of TCDD 4-20 �was sequestered in the liver, compared to 10 percent in the monkey and guinea pig, two species that are not susceptible to hepatotoxicity (Van Miller et al., 1976; Gasiewicz and Neal, 1979). Diquat is absorbed by the lung, but is poorly absorbed from the gut. Diquat is not retained by the lung and is cleared from the body rapidly. Diquat is not bioaccumulated in fat after subacute exposure. Diquat binding to melanin has been demonstrated in vitro and may potentially lead to diquat accumulation in the eye and the resultant production of cataracts after chronic exposure. The formation of diquat free radicals has been proposed as the active form of diquat in producing toxic effects, although under conditions of normal oxygen levels little evidence exists to support this theory. Diuron is extensively metabolized in man and in other animal species. The kidney is the major route of diuron excretion. Diuron has been shown to induce several microsomal enzymes. Bromacil is metabolized in man and in the rat, while picloram is not metabolized and is excreted rapidly. Arsenic has been detected in the urine of forest workers exposed to cacodylic acid, although the possibility of concomitant exposure to arsine gas, which could also result in detectable levels of inorganic arsenic in humans, was suggested. In animals, indirect.evidence, based on similar tissue distribution of radioactivity after either [ C]-labeled or [ As]-labeled cacodylic acid were given, suggests that cacodylic acid is not metabolized to inorganic arsenic. Cacodylic acid is rapidly absorbed from the lung and is absorbed from the gastrointestinal tract. Cacodylic acid is retained in whole blood. Excretion is primarily by the kidney and cacodylic acid crosses the placenta late in gestation. �CHAPTER 4. REFERENCES Abou-Donia, M. B., Charles, J.. M., and Menzel, D. B. 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Chemosphere 9:761-768. Van Miller, J. P., Marlar, R. J., and Allen, J. R. (1976) Tissue distribution and excretion of tritiated tetrachlorodibenzo-p-dioxin in non-human primates and rats. Fd. Cosmet. Toxicol. 14:31-34. Vinopal, J. H., and Casida, J. E. (1973) Metabolic stability of 2,3,7,8tetrachlorodibenzo-p-dioxin in mammalian liver microsomal systems and in living mice. Arch. Environ. Contain. Toxicol. 1(2):122-132. Wagner, S. L., and Weswig, P. (1974) Arsenic in blood and urine of forest workers as indices of exposure to cacodylic acid. Arch. Environ. Health 28(2):77-79. 4-36 �Woods, J. S. (1973) Studies on the effects of 2,3,7,8-tetrachlorodibenzo-pdioxin on mammalian hepatic -aminolevalinic acid synthetase. Environ. Health Perspec. 5:221-225. Woolson, E. A. Organoarsenical herbicides. In Herbicides: Chemistry, degradation and mode of action. Vol 2 eds., P. C. Kearney and D. D. Kaufman (New York:Marcel Dekker Inc., 1976) pp. 741-776. Yamauchi, H., and Yakamura, Y. (1979) Urinary inorganic arsenic and methylarsenic excretion following arsenate-rich seaweed ingestion. Jap. J. Ind. Health 21:47-54. Young, A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. Young, J. F., and Haley, T. J. (1977) Pharmacokinetic study of a patient intoxicated with 2,4-dichlorophenoxyacetic acid and 2-methoxy-3,6dichlorobenzoic acid. Clin. Toxicol. 11(5):489-500. Young, J. F., and Haley, T. J. (no date) Simultaneous human pharmacokinetics of 2,4-dichlorophenoxyacetic acid and 2~methoxy-3,6-dichlorobenzoic acid, Fed. Proc. 38:679. Zielinski, W. L., Jr., and Fishbein, L. (1967) Gas chromatographic measurements of disppearance rates of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice. J. Agr. Fd. Chem. 15:841-844. 4-37 �CHAPTER 5 HUMAN EXPOSURE TO TCDD FROM INDUSTRIAL AMD MILITARY USES TCDD (2,3,7,8-tetrachlorodibenzo-para-dioxin) is formed as an unintentional byproduct during the manufacture of 2,4,5-trichlorophenol (TCP), when certain reaction conditions are met. TCP is a precursor in the manufacture of 2,4,5-T, and TCDD is introduced into preparations of 2,4,5-T during this precursor step. TCDD is formed when two molecules of 2,4,5-trichlorophenol undergo a condensation reaction. Several conditions favor the formation of dioxins, including heat, pressure, photostimulation, and catalytic action. No definitive study has determined the temperature required to form TCDD from TCP (Esposito et al., 1980). Excessive temperature during the manufacture of 2,4,5-T is generally considered to be a major factor responsible for the presence of TCDD in 2,4,5-T. At reaction temperatures above 225°C, the reaction of 1,2,4,5-tetrachloro benzene and ethylene glycol to form trichlorophenol becomes exothermic (May, 1973) and unless the temperature is reduced an explosion can ensue. Seven explosions in factories that were manufacturing trichlorophenol have been reported and are listed in table 5-1. An eighth incident, which occurred in France in 1956 and involved acute exposure during an operation to steam-clean trichlorophenol processing equipment, is also listed and described with the seven explosions. Large quantities of TCDD have been released in these accidents. » Occupational exposure to TCDD has occurred in factories when TCDD was formed in excessive amounts because the reaction temperatures were high enough to favor dioxin formation, but not high enough to become exothermic and produce an explosion. These incidents are listed in table 5-2. Two additional incidents of human exposure to TCDD are considered in this section and listed in table 5-3. In one incident, TCDD-contaminated salvage oil was sprayed on horse arenas in Missouri in 1971, resulting in human and animal exposures. In the second incident, three laboratory workers in England were exposed while they were heating trichlorophenol or potassium trichlorophenate, or handling the products. Military exposure to TCDD resulted from the use of herbicides that contained contaminated 2,4,5-T. In this chapter, information on the exposure levels of TCDD from explosions and industrial exposures are summarized. The human effects of TCDD exposure reported after industrial accidents, occupational exposures, and military exposure are then compared. 5.1 INDUSTRIAL EXPLOSIONS Exposure conditions and symptoms reported for the 8 accidents listed in table 5-1 are described in this section. 5-1 �TABLE 5-1. Table 5-la. YEAR 1949 INDUSTRIAL EXPLOSIONS References on Industrial Explosions That Involved TCDD LOCATION REFERENCES Monsanto Suskind (1973); VA (1980) Nitro, WV Zack and Suskind (1980) 1953 BASF Ludwigshafen, W. Germany Goldmann (1972); Goldraann (1973) Thiess and Frentzel-Beyme (1977) 1956 Rhone Poulenc Grenoble, France Dugois et al. (1958) 1962 Italy Joint NIESH/IARC Working Group (1978) Young et al. (1978) 1963 Philips-Duphar Amsterdam, Netherlands Dalderup (1974); Berlin et al. (1976) Rawls and Sullivan (1976); Hay (1977) 1966 Rhone Poulenc Grenoble, France Dugois et al. (1968) Rawls and Sullivan (1976) 1968 Coalite & Chemicals Products Jensen and Walker (1972) Bolsover, Derbyshire, U.K. 1976 Jensen et al. (1972; May (1973) ICMESA Seveso, Italy Commoner and Scott (1976; Hay (1976) Rawls and Sullivan (1976); Fara (1977) Gianotti (1977); Walsh (1977) Bonaccorsi, et al. (1978); Greim (1978) Laporte (1978); Reggiani (1978) Homberger, et al. (1979) Malizia, et al. (1979) Pocchiari, et al. (1979) Reggiani (1979); Strik (1979) Reggiani (1980); VA (1980); VA (1981) Table 5-lb. References Which Review Industrial Exposures to TCDD Forth (1977) IARC (1977) IARC (Joint NIESH/IARC-1978) Hay (1979) Moses and Moore (1979) Esposito et al. (1980) Hay (1977) Firestone (1978) Young et al. (1978) Malizia et al. (1979) Crow (1980) Suskind (1980) 5-2 �TABLE 5-2. INDUSTRIAL EXPOSURES Table 5-2a. References on Industrial Exposures to TCDD YEAR LOCATION REFERENCES 1949 Nordheim, Westfallen, West Germany Baader & Bauer (1951) 1951-52 2 factories in Middle Rhein, W. Germany Bauer et al. (1961) 1954 Boehringer, Ingelheim Hamburg, West Germany Kiramig & Schulz (1957) Kimmig & Schulz (1957) Schulz (1957); Bauer et al. (1961) 1956 Diamond Alkali Newark, N.J. Bleiberg et al. (1964) Poland et al. (1971) 1964 U.S.S.R. IARC (1977) 1964 Dow Chemical (2,4,5-trichlorophenol production) Midland, Michigan Firestone (1978) Cook et al. (1980) Rowe (1980) 1965-9 Spolana Czechoslovakia Jirasek et al. (1973) Jirasek et al. (1974) Pazderova et al. (1974) Jirasek et al. (1976) Pazderova-Vejlupkova et al. (1980) 1970 Japan Miura et al. (1974) 1972 U.S.S.R. Zelikov and Danilov (1974) 1950-1975 Dow Chemical (2,4,5-T production) Midland, Michigan Kramer (1974)* Ott et al. (1980) Townsend et al. (1980)* 1955 - 1977 Monsanto Nitro, West Virginia VA (1980) *unpublished reports 5-3 �Table 5-2b Other Industrial Exposures YEAR LOCATION 1952-3 CHEMICAL MANUFACTURED Boehringer 1 NUMBER OF WORKERS EXPOSED Trichlorophenol 37 59 West Germany 1953-1971 Rhone Poulenc Grenoble, France Trichlorophenol 1956 Hooker U.S.A. Trichlorophenol 1960 Diamond Shamrock U.S.A. Trichlorophenol 1970 (?) Bayer West Germany Trichlorophenol 1973 Linz Nitrogen Works Austria Trichlorophenol 1974 Bayer Verdingen, West Germany Trichlorophenol Thorapson-Hayward Kansas City, Kansas Trichlorophenol Vertek Chemical Plant Jacksonville, Arkansas 2,4,5-T 1975 1979 2 50 190 1These incidents were listed first in Hay (1977) and later in subsequent lists; no reports that provide exposure levels or symptoms have been identified; the 1979 incident was mentioned in Suskind, 1980. "This incident was listed as an explosion by Hay (1977) and as an occupational overexposure in subsequent lists by Young et al. (1978) and Esposito et al. (1980). 5-4 �Table 5-3. REFERENCES ON OTHER HUMAN EXPOSURES TO TCDD YEAR LOCATION POPULATION DESCRIBED 1971 Missouri Farm residents Carter et al. (1975) Case (1976) Beale et al. (1977) Kimbrough et al. (1977) Crow (1980) 1970 England 3 laboratory workers Oliver (1975) 1967-1970 Vietnam Vietnamese residents NRC (1974) Vietnamese refugees Tung et al. (1971) Rose and Rose (1972) Tung et al. (1980) Vietnam veterans Bogen (1979) Halprin (1980) Stellman & Stellman (1980) VA (1980) Vietnam military Harmon (1971) Allen (1980) 5-5 REFERENCES �5.1.1 Exposure Conditions Several variables in the eight accidents that are relevant for a comparison of the data on health effects from these incidents are considered below. These variables include: • Dosage • Duration and routes of exposure • Concomitant exposure • Latency period • Unexposed controls. 5.1.1.1 Dosage The types and severity of health effects observed after TCDD exposure are presumably related to the doses of TCDD received. Unfortunately, almost no information on exposure levels following explosions or during occupational exposure has been published and only a very limited comparison of dosage levels and resultant health effects can be made. In two early accidents, TCDD was not known to be a contaminant released during the accidents or a potential health hazard. In 1949, when the Nitro, West Virginia accident occurred, TCDD had not been identified as a potential contaminant of the manufacture of trichlorophenol. Only after TCDD was identified following similar explosions elsewhere was the etiologic role of TCDD in the Nitro incident assumed. An incident in 1956 in a French factory manufacturing 2,4,5-T resulted in numerous cases of chloracne. The report that described these cases (Dugois et al., 1958) also mentioned that the causative agent could not be identified at the time, although any measures taken to identify the cause were not mentioned. A new manufacturing procedure, begun 2 years before the incident, introduced steam into a process that had been performed at colder temperatures. This steam process was stopped following the accident and other precautions were instituted to prevent worker exposure to released vapors. Subsequently, no new cases were reported. After the 1953 accident at the BASF factory, an extensive search for the agent responsible for the workers' health effects was made (Goldmann, 1972; 1973). Symptoms of chloracne that resembled the condition observed in the workers were reproduced in rabbits treated with crude extracts of the TCP distillate. None of the chemicals known to be present in the distillate were acnegenic when tested individually. A series of chemicals that were considered potential contaminants were synthesized, and several of these compounds, including TCDD and several chlorinated dibenzofurans, produced potent acnegenic responses. Analysis of the distillate for its dioxin content led to confirmation of the presence of TCDD. The concentration of TCDD the workers encountered remained unknown. TCDD levels were measured after three explosions. After the 1963 explosion in Amsterdam, a soot-like substance was released into the factory. 5-6 �Analysis of this substance revealed that it contained 1,000 ppm TCDD. Estimates of the total amount of TCDD released into the factory hall ranged from 30 to 200 g (Crow, 1980,). After the 1968 explosion in England, levels of up to 40 mg of TCDD per m were detected on wall surfaces (Crow, 1980). These levels are considered to correspond roughly to the amounts released in the Amsterdam factory. Three years after the explosion in England, two workers who worked with one of the few salvaged pieces of equipment came down with severe cases of chloracne within 3-4 weeks after they were exposed. TCDD could not be detected on the apparatus by either chemical or biological assays (May, 1973). Assays of the sebum discharged" from the skin of one of the affected men 1 year after exposure did not contain measurable amounts of TCDD by the gas-liquid chromatographic method used, which had a lower detection limit of 0.1 ppm; the sebum flow rate was normal (Jensen et al., 1972). After the explosion in Italy in 1976, a cloud that contained TCDD was released over the surrounding countryside. Extensive measurements of TCDD were made of the affected areas, which resulted in establishing three zones of contamination: zones A, B, and R had areas with up to 270, 44, and 15 ug of TCDD per nu , respectively. Mean values for various parts of zone A were 15-29 ug/ra and for zone B were 4 ug/m . Soil samples from zones A, B, and R contained greater than 10, 0,1-10, and less than 10 ug TCDD per kg, respectively (Reggiani, 1978). In the previous accidents, vapors released during the explosions were contained in limited areas of the factories. In Seveso, vapors were released into the environment, diluting the TCDD concentration substantially. Only one estimate of human TCDD body burdens based on measured tissue levels of TCDD was found in the published literature. A woman who lived in Seveso's zone A for 2 weeks (between the accident and the time when the evacuation was ordered) died in October 1976 of pancreatic cancer. This death was not attributed to TCDD exposure. Tissues removed at autopsy were analyzed for TCDD; the levels found led to an estimate of 0.5 mg of TCDD per kg body weight absorbed at the time of the incident, assuming a half-life of 30 days for TCDD (Reggiani, in VA, 1981). Information to compare relative exposures is inadequate. Even when the information on TCDD levels in the vicinity several months or years after the accident is available, only two attempts to measure it in people have been recorded. One attempt was successful, but was dependent on several pharmacokinetic assumptions and was based on measurements from one person at only one point in time. The actual levels workers were exposed to, the length of the exposure period, or the amounts absorbed remain unknown. 5.1.1.2 Duration and Routes of Exposure The eight industrial accidents listed in table 5-la are being considered together because they resulted in acute exposure of workers (or residents, in the Seveso incident) to TCDD during a short period of time. However, inadequate decontamination measures or lack of knowledge regarding the presence of TCDD and its high order of toxicity led, in most cases, to continued exposures after the explosions occurred. No specific decontamination procedures were described after the Nitro accident. Extensive efforts were made to decontaminate the BASF factory after the explosion, and manufacturing of other products 5-7 �was eventually resumed. Two years after the decontaminated facility was reopened, the first new case of illness occurred. The facility was closed again and in 1969 the autoclave and contaminated equipment were demolished (Goldmann, 1972). Decontamination measures in the English facility were also inadequate, and 79 cases of chloracne were reported in workers who were not present during the explosion but were exposed only to the "decontaminated" facility. Consequently, the contaminated equipment was dismantled and buried (May, 1973). Despite extensive measures taken to decontaminate the Amsterdam facility, residual TCDD remained and the factory Was dismantled and parts were encased in concrete and sunk in the Atlantic Ocean (Berlin, 1976; Dalderup, 1974). Decontamination efforts in zone A in Seveso have not been completed and the factory that released TCDD is not being used, even though TCDD was not released inside the facility. Cases of chloracne occurred in people who were never in the factories where the explosions took place. People whose only exposure to TCDD was from contact with affected Nitro workers developed chloracne; in one case a man who drove a truck that had been parked near the factory developed chloracne (Suskind, 1978). The health effects of Nitro workers who were not present when the explosion occurred was the subject of a separate investigation, which is described in the next section. After the BASF and British explosions, family members of workers who never entered the facility developed chloracne (Goldmann, 1972; Jensen et al., 1972). Workers involved only in the cleanup operation in Amsterdam contracted chloracne (Rawls and Sullivan, 1976). The Seveso incident resulted in high exposures of zone A residents over a 2-week period (before evacuation was ordered), and low, continuous exposures of residents of zones B and R, based on TCDD analyses of soil samples. Residents of one zone ate food from other zones and worked and traveled between the zones, complicating exposure estimates for these people (Bonaccorsi et al., 1978). After the explosion in France in 1956, chloracne was reported only in workers (Dugois et al., 1958); however, not knowing that TCDD was the causative agent may have prevented investigators from relating other cases among family members to the factory incident. In conclusion, exposure to TCDD probably was not limited to acute exposure during the explosions, but in many cases involved long-term exposures to low levels of TCDD that were not removed during the decontamination process, and in some cases involved indirect exposure of people who were never in contact with the contaminated factories. The routes of exposure of TCDD are speculative. TCDD exposure by the workers was probably by pulmonary and dermal routes. The Seveso residents were probably exposed by the oral route, in addition, since food supplies were contaminated. Children who played outdoors had potential for much higher inhalation and dermal exposures than adults who remained inside. 5.1.1.3 Concomitant Exposures Chemicals other than TCDD were present in the factories or were released during the explosions. The types and amounts of chemicals released are subject to speculation, as no results of quantitative or qualitative analyses of vapors during explosions were made. However, trichlorophenol was likely to be present in most cases and the early skin burns observed within the first two days of the Seveso accident have been attributed to trichlorophenol 5-8 �(Walsh, 1977). Sodium hydroxide, sodium chlorophenate, other phenates, and ethylene glycol were probably present in Stack emissions of the T.CMESA plant (Melvin, personal communication). The phase of the trichlorophenol reaction that was taking place when the accidents occurred probably affected the types and amounts of polychlorinated chemicals that were released. In the 1963 accident in Amsterdam, the accident occurred during the beginning of the reaction when mainly tetrachlorobenzene was likely to be present and polychlorinated dibenzofurans were likely to be produced. The accidents in 1968 in the United Kingdom and in 1976 in Seveso occurred near the end of the reaction when mainly trichlorophenol was likely to be present, which produces TCDD (Joint NIEHS/IARC Working Group, 1978). Some of the symptoms that were not observed in all accidents may have been the result of concomitant exposure to chemicals found only in specific incidents. However, different levels and durations of TCDD exposure may also be responsible for effects seen only after certain accidents. Some symptoms were not mentioned in descriptions of patients from some of the accidents because they were associated with conditions that probably were not suspected and could not be diagnosed without performing specific tests. For example, diagnosis of porphyria cutanea tarda requires that urine samples be analyzed for porphyrin content. This analysis is not routine and other symptoms of porphyria cutanea tarda, such as hirsutism and hyperpigmentation, may have gone unnoticed if porphyria was not suspected. 5.1.1.4 Latency Period In comparing the consequences of these industrial accidents on human health, the duration of time that has elapsed between the accident and examination of patients is significant. Hepatotoxicity, measured by liver enzyme tests, was observed in the United Kingdom and Seveso workers, in tests performed several days or weeks after the accidents, but in both groups of patients these effects were transient and were not observed in subsequent tests (May, 1973; Reggiani, 1978). The onset of early symptoms of chloracne, including redness and itching, was evident several days after exposure. Comedones that are characteristic of chloracne, on the other hand, may take 2 months to develop, but in severe cases such as occurred at the Nitro plant, lesions were still evident 28 years later (Crow, 1980). The results are complicated even when the time interval between the accident and examination are known, because exposure may have continued during an unknown part of this interval and the duration of symptc in some cases correlates to the severity of exposure, an unknown variable. The time between exposure and the onset of symptoms other than chloracne usually was not mentioned in reports of the accidents and was understandably more difficult to identify, as in the case of asthenia. 5.1.1.5 Unexposed Controls Most reports and studies of workers exposed to TCDD are descriptive. They usually have not included data from control groups, as a basis for 5-9 �comparison of experimental data. Two exceptions are the control group of Italian children who were compared to the TCDD-exposed children in Seveso for immunologic characteristics (Pocchiari et al., 1979), and several control groups in Germany used to evaluate the incidence of mortality in the BASF workers (Thiess and Frentzel-Beyme, 1977). 5.1.2 Symptoms Experienced After Exposure Table 5-4 lists some of the symptoms reported after the industrial explosions. Except for Seveso data, all symptoms were observed in workers. 5.1.2.1 Chloracne Chloracne occurred in exposed people after all eight accidents and is considered characteristic of TCDD exposure. The clinical symptoms that developed in all of these workers appear to have been similar in nature, varying primarily in severity and duration. These symptoms have been described clearly in most reports and have been the subject of several reviews (Birmingham, 1964; Schulz, 1968; Crow, 1970; Braun, 1970; Taylor, 1974; Crow, 1978a; Crow, I978b; Taylor, 1979). After some accidents, chloracne was severe, resulting in lesions still apparent many years after exposure ended. After the Nitro accident, 14 of the 122 people originally affected still had skin lesions 28 years later (Crow, 1980). The most severe cases involved workers considered most likely to have received high exposure to TCDD. After the explosion in Amsterdam, about 50 cases of chloracne developed. Thirteen years later, 10 cases remained (Hay, 1977). Seventy-nine workers in the factory in England developed chloracne; however, only seven cases remained 4 years later. Chloracne did not develop in any of the 13 men who survived the original explosion (May, 1973). The lower doses of TCDD likely to have been encountered in the decontaminated British factory probably explains the lower severity, compared to workers in the Nitro and Amsterdam accidents. Most of the 134 cases of chloracne from the Seveso accident occurred in children; however, only school-aged children, and not adults, were systematically screened for chloracne (Bonaccorsi et al., 1978). Children were not directly exposed in any of the other incidents but sometimes developed chloracne after contact with an exposed parent (Goldmann, 1972; Jensen et al., 1972). The Seveso data suggest that children may have a greater sensitivity to the acnegenic effects of TCDD than adults. The potential differences in exposure, between children (who were likely to have played outside) and adults, may have contributed to the differences in symptoms between children and adults. Chloracne has appeared in many cases without any other apparent health effects. Half of the 42 chloracne cases from the BASF accidents had no other symptoms (Goldmann, 1972), and only two or three of the 44 workers in the 5-10 �TABLE 5-4: Year Location No. Affected HEALTH EFFECTS FROM INDUSTRIAL ACCIDENTS INVOLVING TCDD Derma] Renal Organ Systems Affected: Lipid Blood GI Metab . Immuno . CV Hepatic Neuro .Asthenia 1949 + BASF, W. Ger. 55 + 1956 H» 228 1953 I I-1 Nitro, W.Va. + Grenoble, Fr. 17 + + + + + + 1963 Amsterdam, Neth 44 + - + 1968 Bolsover, Derbyshire U.K. 79 + - + + - 1976 Seveso, Italy 134 + + + + + Code: (+) An effect was observed; + + + + + + + + + (-) An effect was not observed in patients examined (or questioned) about the effect; ( ) Patients were not examined or questioned, regarding any effects in the indicated organ system. Pulm. + - + �Amsterdam accident (Crow, 1980) and the 79 workers in England (May, 1973; Hay, 1977) who developed chloracne had any other adverse effects; these effects were limited to asthenia. These trends have led to the conclusion that chloracne is the hallmark of TCDD poisoning (Grow, 1980). After most accidents, only patients that developed chloracne were examined further for other symptoms. 5.1.2.2 Porphyria Cutanea Tarda This condition results from a disturbance in the capacity to break down hemoglobin, which leads to high levels of porphyrins in the liver and urine. Like chloracne, it is a relatively unusual condition which is not frequently encountered in other occupational situations; some cases have been shown to be inherited, rather than acquired. Porphyria cutanea tarda was observed after the Nitro incident, and symptoms of this condition, including hirsutism and hyperpigmentation, were observed along with urine and liver changes (Suskind, 1978). After the Seveso accident, several cases of increased porphyria excretion and hyperpigmentation were observed; chromatographic examination of urine samples for levels of porphyrins excreted revealed patterns resembling mild chronic hepatic porphyria (Strik, 1979). These effects were observed only in a small portion of the exposed people in Seveso and were transient. Porphyria cutanea tarda was not mentioned in the accounts from the other accidents. High doses o.f TCDD or repeated exposures may be required before this condition is observed. The possibility that concomitant exposures may have produced this condition in the Nitro and Seveso patients cannot be ruled out. 5.1.2.3 Hepatotoxicity Aside from the liver function tests mentioned above, which showed transient effects immediately after exposure in workers exposed in England (May, 1973) and Seveso (Reggiani, 1978), hepatotoxicity was reported after other accidents. Hepatomegaly was observed in Nitro workers (Suskind, 1978) and Seveso residents (Bpnaccorsi et al., 1978); hepatitis occurred in four of the 42 affected workers in the BASF factory (Goldmann, 1973) and hepatic disorders were mentioned (but not described) as a consequence of the accident in France in 1956 (Dugois, 1958). Unlike chloracne, hepatotoxicity has not been reported consistently after TCDD accidents. When it is reported, it is absent in a large proportion of the people that develop chloracne. Furthermore, the different manifestations of hepatotoxicity, including hepatitis, hepatomegaly, and transient abnormal liver function tests may have resulted from different causes or as secondary or compensatory effects of TCDD exposure. It is not clear whether parameters of hepatic structure or function were even examined in the incidents that do not mention them. 5-12 �5.1.2.4 .Neurological Effects Neurological effects were reported after every accident. These effects always included asthenia and in particular, fatigue. The incidence of fatigue varied. The frequency of this complaint was low among the affected workers in the Amsterdam and United Kingdom accidents (Crow, 1980; May, 1973; Hay, 1977), but was one of the few symptoms other than chloracne that was mentioned. Other neurologic symptoms that were described after the Nitro, BASF, French, and Seveso accidents included headaches, sleep disturbances, irritability, and confusion (Suskind, 1978; Goldmann, 1972; Dugois et al., 1958; Walsh, 1977). The subjective nature of these symptoms decreases the reliability of identifying their frequency or severity. Peripheral neural damage involving the sensory organs and polyneuritis were mentioned in the Nitro and BASF workers. Pain in the extremities was mentioned by Nitro workers (Suskind, 1978) and encephalomyelitis occurred in some BASF workers. Seven of the 42 BASF workers with chloracne developed problems related to the central nervous system (Goldmann, 1972; 1973). Delayed peripheral nerve conduction was observed by one examiner, but not by another examiner who studied the same population from Seveso (see VA; 1981). Seveso workers were examined for neurological function; 4 percent (8 subjects) were diagnosed as having polyneuropathy of peripheral nerve fibers. Three of these people were hospitalized and diagnosed as having polyneuropathy of the lower extremities. Other common causes of polyneuropathy were ruled out in these patients (Pocchiari et al., 1979). 5.1.2.5 Other Effects Gastrointestinal effects were observed after the Seveso accident (Bonaccorsi et al., 1978) and the accident in France in 1956, although no other details were provided from the latter incident (Dugois et al., 1958). The delay in evacuation following the Seveso accident may have led to ingestion of TCDD-contaminated food. Oral exposure was unlikely after the other incidents and may explain why gastrointestinal effects occurred only after the Seveso incident. Effects on the kidneys, heart, lung, and spleen each were reported in only the BASF workers (Goldmann, 1972; 1973) and, therefore, do not appear to be consistent effects of acute TCDD exposure. Different types of blood effects were reported for three of the accidents and were transient in one case (Suskind, 1978; May, 1973). Reproductive effects were examined after the incidents in Amsterdam (Rawls and Sullivan, 1976) and Seveso (Homberger et al., 1979; Rehder et al., 1978; Reggiani, 1978), but no effects were demonstrated. Data on reproductive effects from the Seveso incident are considered in another section of this report. Immune function was evaluated after only the Seveso incident (Reggiani, in VA, 1981; Homberger et al., 1979). In vitro tests produced evidence of decreased immune competence, although the affected children at Seveso did not 5-13 �experience a higher incidence or severity of childhood diseases than children from other parts of Italy. 5.1.2.6 Mortality No deaths were attributed directly to TCDD exposure immediately after the accidents. Long-term mortality studies are being carried out on people exposed to or affected by the accidents at Nitro, BASF, Amsterdam, the United Kingdom, and Seveso. The incidence of death from cancers and from cardiovascular causes was not elevated in a study of the Nitro workers (Zack and Suskind, 1980). Of the 17 deaths among the BASF workers that occurred in the 24-year period after the accident, six were attributed to cancers, including four gastrointestinal cancers. The first death was caused by pancreatic necrosis and involved the first worker who entered the "decontaminated" facility; he was not present when the accident occurred (Thiess and Frentzel-Beyme, 1977). A higher-than-expected incidence of death from myocardial infarction has been observed in the Amsterdam workers (Zack and Suskind, 1980; Joint NIEHS/IARC Working Group, 1978). Only one death has occurred in the workers from England, and was caused by coronary thrombosis (Joint NIEHS/IARC Working Group, 1978). The Seveso data on mortality rates indicate that the accident did not produce an observable increase in the death rate (Homberger et al., 1979). The long latency period expected for cancers to develop requires that mortality studies should be conducted on a long-term basis; IARC is coordinating efforts in this area. In general, the number of deaths among the approximately 500 people affected in these incidents is very small. Trends have emerged in the data that exist today to indicate that TCDD exposure may increase the likelihood of death from cancer or cardiovascular reasons, but these trends were found in only one of the groups of workers and were not duplicated in any other. 5.1.3 Conclusions In conclusion, chloracne and asthenia are consistently reported in the accidents described above. The incidence of asthenia is low compared to the incidence of chloracne. The subjective nature of asthenia complicates evaluating its significance as a primary effect of TCDD or a secondary effect of patients suffering from the discomfort and disfigurement of chloracne and the fears associated with exposure to toxic agents. Other neurological disorders and hepatic disorders were commonly observed after these accidents. They were usually studied in chloracne patients and were more likely to occur in groups of workers with incidences of asthenia, although the specific symptoms involving these organ systems varied after the accidents. Peripheral neuritis and hepatomegaly were the most commonly encountered of these conditions. 5-14 �Porphyria cutanea tarda occurred in the Nitro workers, suffered more types of effects and more severe effects than of the other accidents, except for the BASF workers. These been exposed to the highest level of TCDD, as was suggested 5.2 who in general workers from most workers may have above. OCCUPATIONAL EXPOSURE THAT DID NOT INVOLVE EXPLOSIONS Twenty incidents other than explosions have been reported that resulted in exposure of workers to TCDD. These incidents are listed in table 5-2. Nine incidents, listed in part 5-2b of the table, were listed in a table by Hay (1977) and have appeared on several subsequent lists of accidental exposures, but no other information on the circumstances of exposure or clinical manifestations of TCDD exposure have been reported and the incidents are not described in this section. The incident in 1975 in Kansas was described by Hay as an explosion, but was listed as an industrial exposure in more recent lists by Young et al. (1978) and Esposito et al. (1980). Three incidences of industrial exposure occurred in West Germany: in Westfallen in 1949, in Middle Rhein in 1951-52, and in Hamburg in 1954 (table 5-2). 5.2.1 Exposure Conditions TCDD was identified as the likely cause of chloracne in several incidents, although the work environment was not sampled in any of the factories to determine actual levels of contamination to which workers were exposed. Workers in Westfallen who developed symptoms in 1949 were working with pentachlorophenol. Trichlorophenol was used only briefly in this facility and at the time was not considered to be the source of the intoxications (Baader and Bauer, 1951). The relationship of TCDD to adverse health effects was determined for several incidents. TCDD was identified in the byproduct material of the Hamburg factory and one of the investigators confirmed that it was acnegenic after he applied it on his own skin and developed lesions resembling those seen in the workers. Other materials, including the purified trichlorophenol preparation and its trichlorophenol precursor, were not acnegenic in animal studies (Bauer et al, 1961). TCDD was identified on walls and in some of the products manufactured at the Czechoslovakian factory. The air was not analyzed and no quantitative analysis of any samples was reported- (PazderovaVejlupkova et al., 1980). Levels of TCDD were determined in the workplace for products made at several factories. Diamond Alkali had produced 2,4,5-T with a TCDD contamination level of 10-25 mg/kg at the time chloracne was identified among workers at this plant. Manufacturing procedures were changed to decrease this contamination level, and 7 years later a reexamination of the workers revealed marked improvement in their clinical picture as well as a reduction in the TCDD level to 1 mg/kg (Poland et al., 1971). At the Dow Chemical facility, air sampling revealed 2,4,5-T concentrations up to 0.8 mg/m . The TCDD levels were not measured, but between 1966 and 1972 the TCDD levels were required to be no higher than 1 ppm (Ott et al., 1980). 5-15 �5.2.1.1 Duration and Routes of Exposure In general, the appearance of symptoms of chloracne in workers has been interpreted as an indication that toxic chemicals were being released, and usually has resulted in changes in manufacturing conditions, improvements in industrial hygiene, and the elimination of new cases of chloracne. However, the intervals between the start of exposure, the appearance of symptoms, and the decrease or elimination of exposure have not been identified in most of the accounts, precluding any estimates of latency periods between exposure and symptoms, or threshold doses, or time periods below which symptoms do not occur. The health of Dow workers 6 years after chloracne was first diagnosed was reported, but no data were presented on their health status when the problem was first diagnosed (Firestone, 1978; Rowe, 1980). Unlike the incidents involving accidents, the occupational exposures offered a reasonable risk for oral exposure in cases where food was stored or consumed in the workplace. The relative importance of exposure by various routes of exposure was seldom mentioned in reports and is not known. 5.2.1.2 Concomitant Exposures Other than trichlorophenol, 2,4,5-T, and TCDD, chemicals to which workers were exposed were rarely mentioned in the reports discussed in this sectio.n. In two cases, however, concomitant exposures were mentioned and in both of these cases certain adverse health effects observed in the workers could be attributed to these chemicals. Seven of 10 workers exposed to pentachlorophenol in the Westfallen factory in 1949 developed bronchitis. This symptom was not reported in the other incidents (except for one case in the Hamburg workers) and may have been caused by pentachlorophenol (Baader and Bauer, 1951). A mortality study of the Monsanto workers revealed a ninefold higher incidence of bladder cancer than expected. However, exposure to pararaenobithenol (PAB), a known bladder carcinogen, was confirmed in at least 80 percent of these cases (Gaffey in VA, 1980). The impact of concomitant exposure to other chemicals on the health of workers exposed to TCDD is impossible to evaluate, as many of these people worked in the chemical industry for several decades (see VA 1981) and were exposed to a wide variety of chemicals. 5.2.1.3 Unexposed Controls Most reports provided descriptions of health of workers, without any comparison to control groups. Control groups were used in evaluating the results of the second study of Diamond Alkali workers (Poland et al., 1971) and in the Czechoslovakian study to establish an expected range of values for the metabolic tests performed on the workers (Pazderova-Vejlupkova et al., 1980). 5-16 �5.2.2 Symptoms Experienced After Exposure Table 5-5 lists organ systems that were affected in workers who were occupationally exposed to TCDD. Symptoms discussed in this section are related to: • Chloracne • Porphyria cutanea tarda • Hepatotoxicity • Neurological effects • Other effects. 5.2.2.1 Chloracne As observed after the industrial accidents, almost every industrial exposure in table 5-2 has included cases of Chloracne and, in fact, the appearance of chloracne has usually initiated the suspicion of TGDD exposure. Frequently, other clinical symptoms are reported only for patients that developed chloracne. Severe cases of chloracne occurred from the early exposures, including ten cases in 1949, nine of which remained 1 1 / 2 years after exposure ended. Seventeen workers Were involved in the pentachlorophenol operation, but the skin conditions of the seven men who were not examined were not stated (Baader and Bauer, 1951). Twenty-nine workers in the Diamond Alkali plant were diagnosed with chloracne, although the total number of workers exposed to potential TCDDcontaminated operations was not reported (Bleiberg et al., 1964). Seven years later, 18 percent of 73 workers at this facility had chloracne (Poland et al., 1971). In 1964, chloracne was reported in 69 of 83 workers in the USSR (IARC, 1977) and in 49 of 60 workers at Dow in the U.S. (Cook et al., 1980). Other incidents of chloracne involving 78 workers in Czechoslovakia (Jirasek et al., 1973), 14 in Japan (Miura et al., 1974), and one in 1972 in the USSR (Zelikov and Danilov, 1974) were reported without giving the total numbers exposed. In these last two incidents, chloracne was the only clinical symptom observed. However, a 20-year study of Dow workers involved in the manufacture of 2,4,5-T revealed no chloracne or any other health effects among these workers (Kramer, 1974; Ott et al., 1980; Townsend et al., 1980). 5.2.2.2 Porphyria Cutanea Tarda Porphyria cutanea tarda was observed in workers from two factories. Among 78 Czechoslovakian workers examined, 76 had chloracne and 11 had hepatic lesions and disorders of porphyrin metabolism; one of the 11 did not have chloracne (Jirasek et al., 1973; 1974; 1976; Pazderova et al., 1974; 1980). Eleven of the 29 Diamond Alkali workers examined had porphyria cutanea tarda (Bleiberg et al., 1964). In the study 7 years later, none of the 73 workers had porphyria (13 had chloracne) and the authors of this report (Poland et al., 1971) concluded that chloracne and porphyria cutanea tarda were two independent disease entities in these workers. 5-17 �TABLE 5-5: HEALTH EFFECTS OF OCCUPATIONAL EXPOSURE TO TCDD Organ Systems Affected: Dermal Year Location No. Affected 1949 Nordheim,W.Ger. 17 + 1952 Middle Rhein, W. Ger. 60 + 1954 Boehringer Hamburg, W. Ger. 31 + Renal Hepatic Neuro Asthenia Blood Lipid GI Metab . Immuno CV + Pulm. + + + 1956 Diamond Alkali Newark, N.J. 1964 USSR 1964 Dow Chemical Midland, Mich. 60 1965- Spolana 69 Czecko Slovakia 78 + + 1970 Japan 14 + USSR 1 + + 29 + + 128 + + + - + + - 1972 + + + + + + + + + oo Code: iH + + m + - - (+) An effect was observed; (-) An effect was not observed in patients examined (or questioned) about the effect; ( ) Patients were not examined or questioned, regarding any effects in the indicated organ system. �The Japanese workers did not have porphyria cutanea tarda (Miura et al., 1974) and no other mention of porphyrin analyses or symptoms of this disease were mentioned in the accounts of the other industrial exposures. Hyperpigmentation occurred in all of the workers in Westfallen (Baader and Bauer, 1951), and was observed in the Hamburg workers (Kimraig and Schulz, 1957) and in both studies of the Diamond Alkali workers (Bleiberg et al., 1964; Poland et al., 1971), it was not observed in the 1972 Soviet exposure (Zelikov and Danilov, 1974). Descriptions of this symptom were inadequate to classify it as a manifestation of chloracne or porphyria. 5,2.2.3 Hepatotoxicity Liver biopsies from Hamburg workers revealed hepatic disorders in a few cases, which involved fatty infiltration of iron deposits. Liver impairment was observed in some patients with severe chloracne who were exposed in 1964 in the Russian factory (IARC, 1977). Liver enlargement and abnormal liver function tests were observed in the Czechoslovakian workers (Jirasek, 1974; Pazderova-Vejlupkova et al., 1980), although the Dow workers had normal liver function tests (Firestone, 1978). Hepatotoxicity observed after the industrial accidents is manifested in different ways. Reports rarely mention whether a patient was examined for a palpable liver and liver function tests appear not to have been done in other workers. 5.2.2.4 Neurological Effects Neurological disorders were observed in workers from all of the early industrial exposures (the first seven are listed in table 5-5). Asthenia was observed in the Middle. Rhein and Hamburg workers listed in table 5-2 (Bauer et al., 1961), in the Russian workers exposed in 1964 (IARC, 1977), in the Czechoslovakian workers (Pazderova-Vejlupkova et al., 1980) and in one Dow worker who experienced depression (Firestone, 1978). The Diamond Alkali workers were administered the Minnesota Multiphasic Personality Inventory (Poland et al., 1971). Scores for one part of the test were found to correlate with the severity of chloracne of the workers. Headaches, fatigue, sweating, dizziness, and sleep disturbances were the most common asthenic symptoms reported. Weakness of the lower extremities was reported in the same groups of workers that experienced asthenia. Joint pain, peripheral neuropathy or loss of sensory function was also reported for each of these groups of workers. 5.2.2.5 Other Effects Gastrointestinal problems were common among the same groups of workers that experienced asthenic symptoms. Abdominal pains were reported in 30 percent of the Diamond Alkali workers (Poland et al., 1971) and in some of the Hamburg workers (Bauer et al., 1961; Kimmig and Schulz, 1957) and in the Russian workers exposed in 1964 (IARC, 1977). Nausea, vomiting, and diarrhea 5-19 �in one group of workers (Poland, 1971), and decreased appetite and weight loss in another (Bauer et al., 1961), were reported. One of the Dow workers continued to experience difficulty in swallowing 6 years after TCDD exposure had ceased (Firestone, 1978). Increased serum cholesterol levels were observed in 10 percent of the Diamond Alkali workers (Poland et al., 1971) and in the Japanese workers (Miura et al., 1974); there is no evidence that serum cholesterol was analyzed in other workers. No abnormal hematology results were observed for the Russian workers exposed in 1972 (T.ARC, 1977), although 10 percent of the Diamond Alkali workers had decreased white blood cell ccunts (Poland et al., 1971). The only cardiovascular problems reported were in the Hamburg workers and included orthostatic hypotension (Bauer et al., 1961). Chronic bronchitis observed in West German workers might have been attributable to pentachlorophenol exposure, as mentioned above. 5.2.2.6 Mortality A mortality study of workers suspected of industrial exposure was performed on workers at Monsanto who were employed between 1949 and 1969, the year Monsanto ceased production of trichlorophenol (Gaffey, in VA, 1980). The study included an identification of the causes of the 164 deaths that occurred among a total of 885 workers. Higher than normal incidences of deaths from bladder cancer, lung cancer, and arteriosclerotic heart disease were reported. The ninefold increase over the expected number of deaths from bladder cancer was attributed to exposure of these workers to paramenobitherol, a bladder carcinogen. The other two causes of death were increased both in fCDD-exposed workers and in all other workers as well, and was attributed to exposure to the industrial environment, with no increased risk attributed to TCDD exposure. Mortality studies were also performed on Dow workers involved in the 1964 outbreak of chloracne (Ott et al., 1980) and in 2,4,5-T production (Cook et al., 1980). Neither study identified an increased mortality with industrial exposure; however, only 4 and 11 deaths, respectively, have occurred in the two study populations. 5.2.3 Conclusions In general, the signs of toxicity in workers exposed to TCDD were similar whether or not the exposure involved an explosion. Chloracne was by far the most common condition recognized in workers. Earlier incidents of occupational exposure, which occurred prior to 1970 or accidents before 1960, in general produced more symptoms, higher frequencies of affected workers among those exposed, and worse cases of chloracne than reported for later incidents. If workers experienced only one condition, it is was almost always chloracne (partly biased by the way the studies were conducted); if they had two, the second one was asthenia. These trends are general impressions; as described in the foregoing discussion, inadequate descriptions of worker health, lack of analyses, and 5-20 �lack of reporting results in quantitative terms precludes making strong statements regarding these trends. These trends also suggest that workers involved in the earlier incidents were exposed to higher TCDD levels than other workers. A logical hypothesis that emerges from this soft evidence is that hepatic and neurological effects require a higher threshold dose tha'n chloracne. Porphyria cutanea tarda requires specialized tests to detect; it may also require high or extended TCDD exposure, or may have a slightly different etiology than the other symptoms, or a different time course to explain its relative infrequency. Animal studies have produced evidence that porphyria cutanea tarda results from chronic exposure and not acute exposure to TCDD. Since no decontamination followed the Nitro accident, these workers probably were exposed over a long period of time; these were the only workers who had symptoms of porphyria after an industrial explosion. Human data are in agreement with animal studies in this instance. Two human health effects which were common after industrial exposure but not after the explosions were gastrointestinal disorders and elevated serum cholesterol levels. The actual extent of the consequences of TCDD accidents and occupational exposures on human health cannot yet be stated with confidence because: 1) most reports did not identify the number of workers who were exposed but not affected; 2) the descriptions of the health status of workers who did not develop chloracne were not provided; and 3) the incidence and severity of health effects were rare.ly compared to appropriate control groups. 5.3 HUMAN EXPOSURE TO TCDD FROM INDUSTRIAL WASTE DISPOSAL, AND LABORATORY EXPOSURE Two incidents are considered in this section and are listed in tables 5-3 and 5-6. One incident considered in this section involved human exposure to TCDD in industrial waste that was combined with salvage oil and sprayed on three horse arenas in Missouri to control dust. The second incident involved three scientists in two government laboratories in England. Two men had heated trichlorophenol (or its potassium salt,, in one case) to synthesize TCDD for use as an analysis standard, and the third handled one of the samples after it was diluted. Despite their awareness of potential hazards and precautions to avoid personal exposure, they developed chloracne and other health effects. 5.3.1 Exposure Conditions The dosage, route, and duration of exposure, and concomitant exposures and control groups for the incidents involving TCDD exposure at the Missouri horse arenas and the British laboratory, are described in this section. 5-21 �TABLE 5-6: HEALTH EFFECTS OF HUMAN EXPOSURE TO TCDD Dermal Renal Hepatic Location United Kingdom + Hiss our i + Q Organ Systems Affected: Neuro . Asthenia Blood GI Lipid Metab. + + + - + - + + - + + + + Plum. + + CV + + + Immuno Vietnam Veterans + Vietnam Veterans + i/ietnam Veterans6 + + + + Vietnam Refugees + + + + + + + CM CM I m Code: (+) An effect was observed; (-) An effect was not observed in patients examined (or questioned) about the effect; ( ) Patients were not examined or questioned, regarding any effects in the indicated organ system. Reference Reference Reference Reference Reference Reference a b c d e f - United Kingdom, Oliver, 1975 Missouri, Beale et aL, 1977 Vietnam Veterans, VA, 1980 Vietnam Veterans, Bogen, 1979 Stellman and Stellman, 1980 Rose and Rose, 1972 �5.3.1.1 Dosage Investigation into the cause of a high incidence of animal mortality and human illness at three horse arenas in Missouri led to the identification of levels of 31.8-33.0 ug of TCDD per gram soil at the farm where human illness occurred (Carter et al., 1975). The date of sample collection and analyses was not reported. The source of TCDD was traced to an industrial hexachlorophene producer in Missouri who disposed of distillate residues from its trichlorophenol production with the same company that sprayed the arenas. The waste residue that remained in the storage tank at the industrial site in 1974 was found to contain 306-456 ug of TCDD per gram residue. Estimates of the potential amounts of TCDD synthesized by the British scientists, or of amounts of trichlorophenol or potassium trichlorophenate initially used to synthesize TCDD, were not reported. One of the workers handled only a diluted sample of TCDD. This worker developed symptoms of asthenia, but not chloracne. The other two men worked with the concentrated lots of TCDD they synthesized, and both developed chloracne (Oliver, 1975). 5.3.1.2 Route and Duration of Exposure Human illness from the Missouri incident occurred in four children and in the mother of two of the children (Carter et al., 1975). These children played in the contaminated arena. The child most severely affected played in the arena daily between the time of spraying and recognition of symptoms. The major exposure was probably dermal. The arena was sprayed on May 26, 1971 and hemorrhagic cystitis was diagnosed in one child on August 21, 1971. The time of appearance of other symptoms in the patients, including skin lesions, was not stated. Since the laboratory workers took precautions to avoid contact by skin or inhalation, their routes of absorption are unknown. Although symptoms did not develop in two of the three scientists until two years after the day they synthesized or handled TCDD, no intervening instances of potential exposure could be identified (Oliver, 1975). 5.3.1.3 Concomitant Exposures and Controls The chemical compositions of the salvage oils sprayed on the arenas were not reported. Potentiating or synergistic effects of combinations of hydrocarbon compounds, including the effects of solvents on the rate of dermal absorption of TCDD, are presently only matters for speculation. The laboratory workers were also exposed to many other chemicals. This factor was considered when a control group was established to compare the results of serum cholesterol values. This group was comprised of laboratory workers who were exposed to the same chemicals as the patients, except that they had no known exposure to TCDD (Oliver, 1975). 5-23 �5.3.2 Symptoms Experienced After Exposure Symptoms reported in the children that played in the sprayed horse arenas (and one parent) and in the British scientist are described here, under the categories: • Chloracne • Porphyria cutanea tarda • Hepatotoxicity • Neurological effects • Other effects. 5.3.2.1 Chloracne The original reports of the health effects of people exposed to the TCDDcontaminated horse arenas did not mention the presence of chloracne. Later communications by an interested dermatologist with the physicians who examined the patients revealed that the 6-year-old girl who was diagnosed as having hetnorrhagic cystitis, as well as her mother and 10-year-old sister, had skin conditions which included blackheads and were consistent with chloracne. The inquiring dermatologist concluded that the conditions were probably mild and went unnoticed by clinicians who were inexperienced in diagnosing chloracne or did not expect to see this condition (Crow, 1980). Twin 2-year-old boys from the second arena that was sprayed also developed skin conditions consistent with chloracne (Crow, 1980). No accounts of this Missouri incident ever clarified the number of people who resided on the farms that were sprayed or the proportion of exposed people who were affected. As observed in the Seveso incident, children were more affected than adults, but their exposure, was likely to be higher from play than adult exposure. One of the three British laboratory workers developed chloracne, with no other symptoms. The other two workers were from a second laboratory. Only one of these two workers developed chloracne. Other symptoms that developed in the workers from the second laboratory were very similar (Oliver, 1975). 5.3.2.2 Porphyria Cutanea Tarda There is no indication that urinary porphyrins were measured after the Missouri incident. This analysis was performed after the laboratory workers contracted chloracne, but no evidence of porphyria was produced. Two of the workers developed hirsutism and the third developed an unusual pigmentation which was attributed to chloracne (Oliver, 1975). 5.3.2.3 Hepatotoxicity Liver function tests performed on the 6-year-old girl from Missouri and on the three laboratory workers were all negative (Beale et al., 1977; Oliver, 5-24 �1975). Hepatomegaly or other evidence of hepatotoxicity were not mentioned in accounts of either incident. 5.3.2.4 Neurological Effects The mother and sister of the affected child complained of intermittent headaches at the time hemorrhagic cystitis was diagnosed in the younger child (Carter el al., 1975). No other evidence of neurological effects was produced at that time (within 4 months of the onset of exposure) or at a 5-year followup examination which included a detailed neurological examination of the younger child (Beale et al., 1977). Two of the British laboratory workers experienced several symptoms of asthenia, including, in particular, fatigue and irritability. Both reported experiencing transient visual problems. One worker reported having headaches and difficulty in muscular and mental coordination and the other worker experienced neurologic pain in one thigh. These symptoms emerged 2 years after the workers were exposed to TCDD (Oliver, 1975). 5.3.2.5 Other Effects Heraorrhagic cystitis in the 6-year-oid girl from the sprayed Missouri farm was accompanied by hematuria and proteinuria (Beale et al., 1977). These symptoms were resolved 1 week after they were recognized, although hemorrhagic areas of the bladder were still demonstrable by cystoscopy 3 months later. The mother and sister complained of abdominal pains and diarrhea. the laboratory workers also experienced abdominal pains and flatulence (Oliver, 1975). Two of One worker reported having lost about 6.5 kg in weight but experienced no loss in appetite. The second experienced a loss in appetite (but no weight loss) as well as diarrhea and indigestion. Three years after the exposure took place, all three workers had elevated serum cholesterol values (above 300 mg/100 ml). 5.3.2.6 Conclusions These two incidents reaffirm that chloracne is a likely consequence of TCDD exposure. They also demonstrate that chloracne may not be an obvious effect and can be overlooked by the examining physician. Furthermore, neurological symptoms can occur in workers with or without chloracne. The latency period for symptoms to develop can be on the order of 2 years. Gastrointestinal problems and elevated serum cholesterol levels were common results of these incidents, as well as of other occupational exposures described in the previous section. Hypercholesterolemia remained 3 years after exposure in all three laboratory workers, while only residual signs of hirsutism in one worker and indigestion and flatulence in the second worker remained. 5-25 �The level of TCDD in the soil in Missouri arenas also provides a comparison of exposure levels to levels from other incidents, although the source that reported this level (Carter et al., 1975) did not indicate whether this level was present shortly after the spraying when toxicity was recognized, or several years later, when the industrial storage tank was assayed for TCDD. 5.4 HUMAN EXPOSURE TO TCDD FROM MILITARY USE OF HERBICIDES This section addresses five populations whose alleged exposure to TCDD are thought to be related to military use of herbicides in Vietnam. 5.4.1 Exposure Conditions Health effects have been reported which the reporters suggested were associated with military use of chemicals in South Vietnam. Reports of health effects, listed in table 5-3, include conditions submitted as claims by Vietnam veterans (VA, 1980), symptoms observed by a physician who conducted a 10-month study of 78 Vietnam veterans (Bogen, 1970), the results of a nationwide study conducted by mail on 535 Vietnam veterans (Stellman and Stellman, 1980), a survey of 98 Vietnam refugees in Hanoi (Rose and Rose, 1972), and a survey of health effects of Vietnamese by the National Academy of Sciences (NRC, 1974). i 5.4.1.1 Dosage An estimate of 0.080 ug of TCDD per kg of topsoil in South Vietnam has been set forth based on the amount of TCDD in herbicides used in Vietnam, the size of the areas sprayed and the rates of spraying (Westing, 1978). Using the same methods of estimation, these authors estimated that contamination of the Missouri horse arenas was 110,000 ug TCDD/kg, compared to 32,000 ug TCDD/kg actually measured by Carter et al. (1975) and 5.9 ug TCDD/kg in Seveso soil. Estimates from another source were based on surface area (Reggiani, 1978); for South Vietnam an estimated .083 g/ha of TCDD was applied, compared to 5,900 g/ha in Missouri and 5.6 and 0.013 g/ha in zones A and B, respectively, in Seveso. A committee organized in 1971 to evaluate the effects of herbicide use in Vietnam set forth as one of its goals quantitative analyses of Vietnamese soil for herbicide residue levels, but security problems in postwar South Vietnam precluded obtaining any Vietnamese soil for analysis (NRC, 1974). 5.4.1.2 Route and Duration of Exposure The major routes of herbicide exposure of U.S. servicemen in South Vietnam were dermal and inhalation, assuming they consumed U.S. military food rations. Vietnamese citizens were also exposed by ingestion when their food sources were sprayed. 5-26 �The refugees whose symptoms were described by Rose and Rose (1972) all experienced alleged exposure to spray missions. They were requested to report the effects of the last spray mission they experienced; 95 percent had experienced at least two spray missions and 60 percent had experienced at least three missions. The survey was conducted between 1970 and 1971. All three of the reports of symptoms of Vietnam veterans in table 5-3 were published between 1979 and 1980, at least 10 years after herbicide use in Vietnam ceased. 5.4.1.3 Concomitant Exposures The health effects of herbicides used in war are difficult to distinguish from those caused by other elements of war. The skin rashes and eye problems that were reported by Vietnamese refugees immediately after some spray missions have been attributed to CS spray missions, for example (Rose and Rose, 1972). Pentachlorophenol, a wood preservative used in Vietnam, is produced by a process that is likely to introduce dioxins and chlorinated dibenzofuran contaminants, but not TCDD. Symptoms from these contaminants are likely to be indistinguishable from those of dioxin (Moses and Moore, 1979). 5.4.1.4 Controls The complaints submitted by Vietnam veterans or by Vietnamese refugees have not been compared to symptoms reported by other veterans or refugees not known to be exposed to TCDD. Only one systematic examination of people known to have been in Vietnam between 1966 and 1969 (the period of heavy use of defoliants) has been conducted by physicians and reported (Bogen, 1979). No details of study design or methods were reported, however, and no clinical biochemical analyses or standardized procedures were described which would provide objective parameters for comparison. Furthermore, no control group was employed in the study and the study population was self-selected. 5.4.2 Symptoms Experienced After Exposure Symptoms observed in Vietnamese or Vietnam veterans and suspected to be related to military use of herbicides are listed in table 5-6. 5.4.2.1 Chloracne Skin rashes or conditions were reported in each study. However, no study included a description of skin conditions that would allow comparisons to be made with chloracne. Among the Vietnamese refugees, 16 percent reported prolonged skin conditions that involved pustules, scabs, or eczema. In a book prepared by the U.S. Army (Harmon, 1971), skin diseases encountered in the military in Vietnam were described. Chloracne was not 5-27 �mentioned. A 1-year field study conducted in South Vietnam in 1966-67 and reported by Harman showed cystic acne to be the major dermatologic disease which resulted in medical evacuation. This condition occurred primarily in soldiers who previously had acne and experienced a worsening of the condition about 6 weeks after they arrived in Vietnam. Allen (1977) reported about 7 percent of the U.S. military hospital admissions in Vietnam from 1965-1972 were for skin diseases. About 10 percent of cases examined in a U.S. Army dermatology clinic between 1970-1971 were cases of acne. A recent report (Halprin, 1980) has suggested that chloracne has been uncommon among Vietnam veterans, even when examinations were conducted by dermatologists familiar with this condition and specifically seeking evidence for it. 5.4.2.2 Porphyria Gutanea Tarda and Hepatotoxicity Little evidence for these conditions has been reported. Hepatitis and jaundice were reported by 10 percent and 5 percent, respectively, of Vietnam veterans that filed claims (VA, 1980). 5.4.2.3 Neurologic Effects Veterans and refugees have reported fatigue, dizziness, and other symptoms of asthenia (VA, 1980; Bogen, 1979; Rose and Rose, 1972; Stellman and Stellman, 1980). Peripheral neuritis, joint pain, or numbness and sensory problems were also commonly reported (VA, 1980; Bogen, 1979; Stellman and Stellman, 1980). 5.4.2.4 Other Effects Gastrointestinal problems were reported by most groups and usually involved diarrhea, vomiting, and abdominal pains. Increased susceptibility to infections (Bogen, 1979), and pulmonary and cardiovascular problems (VA, 1980) were each reported by one group. Reproductive problems were reported in four of the reports and involved child deaths, miscarriages, or birth defects (Tung et al., 1971; Rose and Rose, 1972; Stellman and Stellman, 1980; NRC, 1974). However, the incidents were not documented, nor compared to expected rates for control groups. The types of birth defects and proportions of each were also not presented. A high incidence of carcinogenicity was also claimed, but the type of cancers and expected rates were not presented. 5.4.3 Conclusions The information on human health effects from exposure to TCDD in Vietnam suffers from lack of any systematic approach to collecting and documenting 5-28 �data by health professionals. Furthermore, exposure levels in Vietnam are unknown and the groups described do not seem to constitute those likely to have had higher than usual exposure to herbicides in particular (as would be expected of military personnel involved in spray missions, for example). The symptoms reported were non-specific and are logically associated with many components expected to be present in a war zone. A comparison of symptoms presented in the cited reports to those of control groups would be needed to establish higher than expected frequencies of adverse effects. 5-29 �CHAPTER 5. REFERENCES Allen, A. M. (1977) Skin Diseases in Vietnam, 1965-72. In Internal Medicine in Vietnam. U.S. Army, Washington, D.C. pp. 1-13, 29-51, 125-129. Baader, E. W., and Bauer, H. J. (1951) Industrial intoxication due to pentachlorophenol. Ind. Med. Surg. 20(6)-.286-290. Bauer, H., Schulz, K. H., and Spiegelberg, V. 18:538-555. (1961) Arch. Gewerbepath. Beale, M. G., Shearer, W. T., Karl, M. M., and Robson, A. M. effects of dioxin exposure. Lancet 1(8014):748. (1977) Long-term Berlin, A. (ed.) (1976) Proceedings of the expert meeting on the problems raised by TCDD pollution. Commission of the European Communities, Milan, 1976. 178 p. Birmingham, D. J. (1964) Occupational dermatology: current problems. Skin Feb. 1964:38-42. Bleiberg, J., Wallen, M., Brodkin, R., and Applebaum, I. L. (1964) Industrially acquired porphyria. Arch. Dermatol. 89:793-797. Bogen, G. (1979) Symptoms in Vietnam veterans exposed to Agent Orange. 242(22):2391. Bonaccorsi, A., Fanelli, R., and Tognoni, G. AMBIO 7(5-6):234-239. Braun, W. (1970) Chloracne. Therap. Umscham. JAMA (1978) In the wake of Seveso. 27(8):541-546. Carter, C. D., Kimbough, R. D., Liddle, J. A., Cline, R. E., Zack, M. M., Barthel, W. F., Koehler, R. E., and Phillips, P. E. (1975) Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188(4189):738-740. Case, A. A. (1976) Tetrachlorodibenzodioxin (TCDD) - clinical aspects of poisoning. Clin. Toxicol. 9(6):963-967. 5-30 �Commoner, B., and Scott, R. E. (1976) US Air Force studies on the stability and ecological effects of TCDD (dioxin): An evaluation relative to the accidental dissemination of TCDD at Seveso, Italy. St. Louis, Mo.: Center for the Biology of Natural Systems, Washington University, 51 pp. Cook, R. R., Townsend, J. C., Ott, M. G. (1980) Mortality experience of employees exposed to tetrachlorodibenzo-p-dioxin (TCDD). J. Occup. Med. 22:47-50. Crow, K. D. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA Docket No. 415 et al., Nov. 14. Crow, K. D. (1978a) Chloracne - an up to date assessment. Ann. Occup. Hyg. 21:297-298. Crow, K. D. 78-80. (1978b) Chloracne; the clinical disease. New Scientist pp. Crow, K. D. (1970) Chloracne, Transactions of the St. Johns Hospital Dermatological Society 56:79-99. Dalderup, L. M. (1974) Safety measures for taking down buildings contaminated with toxic material. J. Soc. Geneesk. 52:582-623. Dugois, M. M., P., Amblard, P., Aimard, M., and Deshors, G. (1968) Acne chlorique collective et accidentelle d'un type nouveau. Bull. Soc. Fr. Derm. Syph. 75:260-261. Dugois, P., Marechal, J., and Colorob, L. (1958) Chloracne due to 2,4,5trichlorophenol. Arach. Mai. Prof. 19:626-627. Espo.sito, M. P., Tiernan, T. 0., and Dryden, F. E. (1980) Dioxins. U.S. Environmental Protection Agency, Industrial Environmental Research Laboratory, Office of Research and Development. US-EPA, Cincinnati, Ohio. 351 pp. Fara, G. M. (1977a) Seveso: Studies on teratogenic and other chronic effects of chemical pollutants following an accident in a chemical plant. Teratology 16:365. 5-31 �Firestone, D. The 2,3,7,8-tetrachlorodibenzo-para-dioxin problem: a review. In Chlorinated Phenoxy Acids and their Dioxins; ed. C. Ramel, (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978) p. 39-52. Forth, W. (1977) 2,3,7,8-tetrachlorodibenzo-l,4-dioxin (TCDD): accident. Dentsches Arzteblatt. 44(3):2617-2628. the Seveso Gianotti, F. (1977) Chloracne in children from 2,3,7,8-tetrachlorodibenzop-dioxin. Ann. Dermatol. Venereol. (Paris) 104:825-829. Goldmann, P. J. (1973) Severe, acute chloracne, a mass intoxication due to 2,3,7,8-tetrachlorodibenzo-dioxin. Per Hausarzt 24(4):149-152. Goldmann, P. J. (1972) Extremely severe acute chloracne due to trichlorophenol decomposition products. A contribution to the perna problem. Arbeitsmedizin Socialmedizin Arbeitshygiene 7(1):12-18. Greira, H., and Loprieno, N. 2:53. (1978) No permanent injuries at Seveso? Umschau Halprin, K. M. (1980) Chloracne recognition and its significance. Presented at the 2d Continuing Education Conference on Herbicide Orange, Washington, DC, May 28-30. Harraan, L. E. (1971) "Chapter 21: Skin Diseases in United States Military Personnel Serving in Vietnam," In The Skin. (Amsterdam: International Academy of Pathology) pp. 423-434. Hay, A. (1979) Accidents in trichlorophenol plants: A need for realistic surveys to ascertain risks to health. Ann. N.Y. Acad. Sci. 320:321-324. Hay, A. (1977b) Tetrachlorodibenozo-p-dioxin release at Seveso. Disasters 1:289-308. Hay, A. (1976b) Toxic cloud over Seveso. Nature 262:636-638. Hofmann, H. T. (1957) Neure Erfahrungen mit hochtoxischin chlorkoleminasextoffin. Naumiun. Schnidebergys Arch. Exp. Path. Pharmak. 232:228-230. 5-32 �Homberger, E., Reggiani, G., Sambeth, J. , and Wipf, H. K. (no date) The Seveso accident: its nature, extent and consequences. Ann. Occup. Hyg. 22:327-366. International Agency for Research on Cancer. (1977) IARC monographs on the evaluation of the carcinogenic risk of chemicals to man: Some fumigants, the herbicides 2,4-D and 2,4,5-T, chlorinated dibenzodioxins and miscellaneous industrial chemicals. 15:41-299. Jensen, N. E. (1972) Chloracne: 3 cases. Proc. Roy. Soc. Med. 65:21-22. Jirasek, L., Kalensky, J., and Kubec, K. (1973) Acne chlorina and porphyria cutanea tarda during the manufacture of herbicides. Part I. Cesk. Dermatol. 48(5):306-315. Jirasek, L., Kalensky, J., Kubec, K., Pazderova, J., and Lukas, E. (1974) Acne clorina, porphyria cutanea tarda and other manifestations of general intoxication during the manufacture of herbicides, Part II. Cesk. Dermatol. 49(3):145-157. Jirasek, L., et al. (1976) Chloracne, porphyria cutanea tarda and other intoxications by herbicide. Per Hautarzt 27:328-333. Joint NIEHS/IARC Working Group Report. (1978) Long-Term Hazards of Polychlorinated Dibenzodioxins and Polychlorinated Dibenzofurans. World Health Organization - International Agency for Research on Cancer, Lyon. No. 78/001. 57 pp. Jensen, N. E., Sneddon, I. B., and Walker, A. E. (1972) Tetrachlorodibenzodioxin and chloracne. Trans. St. Johns Hosp. Derm. Soc. 58(2):172-177. Kimbrough, R. D., Carter, C. D., Liddle, J. A., Cline, R. E., and Phillips, P. E. (1977) Epidemiology and pathology of a tetrachlorodibenzodioxin poisoning episode. Arch. Environ. Health 32(2):77-86. Kimmig, J., and Schulz, K. H. (1957a) Occupational acne due to chlorinated aromatic cyclic esters. Dermatologica 115:540. Kimmig, J., and Schulz, K. H. (1957b) Chlorinated aromatic cyclic ethers as the cause of so-called chloracne. Naturwissenschaften 44:337-338. 5-33 �Kramer, C. G. (1974) Health of employees exposed to 2,4,5-T. Findings of the DOW Chemical Company, Corporate Medical Department, 2030 DOW Center, Midland, Mich. 19 pp. Laporte, J. R. (1978) Multinationals and health: Reflections on the Seveso catastrophe. Intern. J. Health Serv. 8(4)619-632. Malizia, E., Andreucci, G., Chiavarelli, M., Amato, A., and Gagliardi, L. (1979) A follow up of 20 months in Seveso, an environmental calamity. Vet. Hum. Toxicol. 21(Supplement):139-140. May, G. (1973) Chloracne from the accidental production of tetrachlorodibenzodioxin. Br. J. Ind. Med. 30:276-283. Miura, H., Omori, A., and Shibue, M. (1975) The effect of chlorophenols on the excretion of porphyrins in urine. Pestic. Abstr. 5(7):456-457. Moses, M. (1979) Effects of TCDD on human health. Testimony before the House Subcommittee on Oversight and Investigations, June 26, 1979. 6 pp. National Research Council. (1974) The Effects of Herbicides in South Vietnam: Part A. Summary and Conclusions.National Academy of Sciences, Washington, D.C. AD-774-749. Oliver, R. M. (1975) Toxic effects of 2,3,7,8- tetrachlorodibenzo 1,4-dioxin in laboratory workers. Br. J. Ind. Med. 32:49-53. Ott, M. G., Holder, B. B., and Olson, R. D. (1980) A mortality analysis of employees engaged in the manufacture of 2,4,5-trichlorophenoxyacetic acid. J. Occup. Med. 22(l):47-49. Pazderova, J., Lukas, E., Nemcova, M., Spacilova, M., Jirasek, L., Kalensky, J., John, J., Jirasek, A., and Pickova, J. (1974) Chronic intoxication by chlorinated hydrocarbons formed during the production of sodium 2,4,5-trichlorophenoxyacetate. Prac. Lek. 26C9):332-339. Pazderova-Vejlupkova, J., Lukas, E., Nemcova, M., Pickova, J., and Jirasek, L. (1980) Chronic poisoning by 2,3,7,8-tetrachlordibenzo-p-dioxin. Pracov. Lek. 32:204-209. Pocchiari, F., Silano, V., Zampieri, A. (1979) Human health effects from accidental release of tetrachlorodibenzo-p-dioxin (TCDD) at Seveso, Italy. Ann. N. Y. Acad. Sci. 320:311-320. 5-34 �Poland, A. P., Smith, D., Metter, G., and Possick, P. (1971) A health survey of workers in a 2,4-D and 2,4,5-T plant. With special attention to chloracne, porphyria cutanea tarda, and psychologic parameters. Arch. Environ. Health 22(3):316-327. Rawls, R. L., and O'Sullivan, D. A. (1976) Italy seeks answers following toxic release. Chem. Eng. News Aug 23:27-28,33-35. Reggiani, G. (1980) Acute human exposure to TCDD in Seveso, Italy. J_._ Toxicol. Environ. Health 6:27-43. Reggiani, G. (1979) Estimation of the TCDD toxic potential in the light of the Seveso accident. Arch. Toxicol. 2:291-302. Reggiani, G. (1978) Medical problems raised by the TCDD contamination. Toxicol. 40:161-188. Arch. Rose, H. A., and Rose, S. P. (1972) Chemical spraying as reported by refugees from South Vietnam. Science 177(4050):710-712. Rowe, V. K. (1980) Direct testimony before the U.S. Environmental Protection Agency, FIFRA Docket No. 415 et al., Nov 13. Schulz, K. H. (1968) Clinical picture and etiology of chloracne. Arbeitsmedizin-Sozialmedizin-Arbeitshygiene 3(2):25-29. Schulz, K. H. (1957) Arch. Klin. Exp. Derm. 206:589-596. Stellman, S., and Stellman, J. (1980) Health problems among 535 Vietnam veterans potentially exposed to toxic herbicides. Society for Epidemiological Research: Abstracts. 444. Strik, J. J. T. W. A. (1979) Porphyrins in urine as an indication of exposure to chlorinated hydrocarbons. Ann. N. Y. Acad. "Sci. 320:308-310. Suskind, R. R. (1980) TCDD contamination in the United States case study. Presented at the National Academy of Sciences International Workshop of Areawide Chemical Contamination, March 17. 13 p. 5-35 �Suskind, R. R. (1978) Chloracne and associated health problems in the manufacture of 2,4,5-T. Report to the Joint Conference, National Institute of "Environmental Health Sciences and International Agency for Research on Cancer, WHO, Lyon, France, January 11. 7 pp. Taylor, J. S. (1979) Environmental chloracne: Y. Acad. Sci. 320:295-307. Taylor, J.S. Update and overview. (1974) Chloracne—a continuing problem. Ann. N. Cutis 13:585-591. Telegina, K. S., and Bikbulatova, L. I. (1971) Affection of the follicular apparatus of the skin in workers occupied -in production of butyl ether of 2,4,5-trichlorophenoxyacetic acid. Vestn. Dermatol. Venerol. 44(12):35-39. Thiess, A. M., and Frentzel-Beyme, R. (1977) Mortality study of persons exposed to dioxin following an accident which occurred in the BASF on 13 November 1953. Presented at the Fifth International Conference of Medichem - Occupational Health in the Chemical Industry, 9 pp. Townsend, J. C., Bodner, K. M., VanPeene, P..F. D., Olson, K. D., and Cook, R. R. (1981) Survey of reproductive events of wives of employees exposed to chlorinated dioxins. Unpublished draft report of Dow Chemical Co., Inc. 39 p. Tung, T. T., Anh, T. K., Tuyen, B. Q., Tra, D. X., and Huyen, N. X. (1971) Clinical effects of massive and continuous utilization of defoliants on civilians. Vietnamese Studies 29:53-81. Tung, T. T., Lang, T. D., and Van, D. D. (1980) The problem of mutagen effects on the 2nd generation after exposure to herbicides. The Veterans Administration. (1981) Advisory Committee on Health-Related Effects of Herbicides - Transcript of Proceedings. Feb 4, 1981. 158 pp. The Veterans Administration. (1980e) Advisory Committee on Health-Related Effects of Herbicides - Transcript of Proceedings (Sixth Meeting, Nov. 6, 1980), 144 pp. The Veterans Administration. (1980h) Proceedings from the 2d Continuing Education Conference on Herbicide Orange. Washington, DC, May 28-30. 5-36 �Vos, J. G., et al. (1978) TCDD accident at a chemical factory in the Netherlands. Working Papers. IARC, Lyon - Joint NIEHS/IARC working group report. Walsh, J. (1977) Seveso: The questions persist where dioxin created a wasteland. Science 197:1064-1067. Westing, A. H. Ecological considerations regarding massive environmental contamination with 2,3,7,8-tetrachlorodibenzo-p-dioxin. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol Bull. No. 27, Stockholm:Swedish Natural Science Research Council, 1978) p. 285-294. Young, A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide-orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. Zack, J. A., and Suskind, R. R. (1980) The mortality experience of workers exposed to tetrachlorodibenzodioxin in a trichlorophenol process accident. J. Occup. Med. 1-4. Zelikov, A. K., and Danilov, L. N. (1974) Occupational dermatoses (acne) in workers engaged in production of 2,4,5-trichlorophenol. Sov. Med. 7:145-146. 5-37 �CHAPTER 6 ACUTE TOXICITY This chapter describes the effects of an acute (i.e., single) exposure of herbicide or TCDD in humans and animals. The first section of this chapter describes factors related to mortality produced by these compounds. For each compound, the potency of an acute dose is described in terms of its lethality, expressed as LD^ values, where these values are available. The LD_0 is the dose that is lethal to 50 percent of the treated animals. For inhalation studies, potency has been expressed in terms of the LC,-n, the concentration of. vapor which produced 50 percent mortality in the exposed animals. The pattern of toxicity in terms of the target organs affected by the compounds and the causes of death, are also discussed in the first section of this chapter. The second part of this chapter describes the acute effects observed for each organ system. Each section in this part describes the specific effects for a particular organ system. Within an organ system, these effects are organized by the chemicals that produced them. The only compounds that are described under each organ system are those which have been reported to cause acute effects in that system. 6;1 MORTALITY Acute toxicity is usually expressed in terms of LD5Q values, or the single dose that produces death in 50 percent of treated animals. These values are presented in this chapter for the herbicides under study, along with information on the latency period between treatment and death, the organs most severely affected, and the possible cause of death. Detailed descriptions of toxicological manifestations are provided in the annotations of the cited literature. 6.1.1 2,4-D Both 2,4-D and 2,4,5-T have been frequently described as moderately toxic (Edson, 1960; Way, 1969; Dalgaard-Mikkelsen and Poulsen, 1962; Council for Agricultural Science and Technology, 1978). This conclusion is based on the LD-0 values for these chemicals, which range between 300 and 1,000 mg/kg for a single oral dose to various species (see tables 6-1 and 6-2). Comprehensive reviews have been published on the acute toxicity of 2,4-D. and 2,4,5-T (Rowe and Hymas, 1954; Dalgaard-Mikkelsen and Poulsen, 1962; IARC, 1977; Young et al., 1978; Gehring and Betso, 1978; NRC, 1974; President's Science Advisory Committee, 1971). References on the acute toxicity of 2,4-D are listed in table 6-3. 6.1,1.1 LD5Q The oral LD,-« values for 2,4-D have been calculated for various species and are listed in table 6-1 according to the salt or ester of 2,4-D that was 6-1 �Table 6-1. LD Values (in mg/kg) for 2,4-D Chemical Form of 2 4 D Tested: , Species (Sex) Acid Sodium Salt Alkanolamine Salt Isopropyl Ester >214a Monkey Rowe and Hymas, 1 5 9 4 iood Dog Cat Rat Bjorn 6, Northern, 1 4 9 8 M F M,F Mouse Rabbit Drill and Hiratzka, 1 5 93 3753 a 805a 666C 200 .01 M F M,F 469a 620^ 150 ,0^ 90 0" a 54 l 551a 5503 848a 100 ,0C 8001" 541a 3 0 7 5 8 - 6 3 150-1886 Cow McLennan, 1974 Biorklund and Erne, 1 6 96 Konstantinova, 1974 860f 1050 0-08 140 .23 200 .03 50200 0-,0 CN VO Shillinger, 1960 (cited in Young.' 1978) 424a F M,F 5703 Shaygulidze et aL, 1 7 9 6 713^ 380»» 375C Chicken M,F Sheep 70 03 368 M F M,F Guinea Pig Hill & Carlisle, 1947 820h F Pig Mixed Butyl Ester mono-to tripropylene glyc-ol butyl ether esters �Table 6-2. LD Values (in mg/kg) for 2,4,5-T Chemical Form Tested: Species (Sex) Acid Butyl Ester Amyl Ester iooa Dog Rat Isopropyl Ester 500 M F M,F 481 495 389; Mouse 67 4b 778b M F 551 Guinea Pig M F Rabbit Chicken 940 449 750 381 712 M M,F 310 from Drill and Hiratzka (1953) from Roll ( 9 1 , for NMRI mice; all 17) other values from Rowe and Hymas (1954) 6-3 750 �Table 6-3. References on the Acute Toxicity of 2,4-D Organs Affected or Studied: Species Route Monkey oral oral oral Rat in vitro in vitro skin liver kidney neural muscle X Reference X Abo-Khatwa & Hollingsworth, 1974 X X X X Guinea Fig oral X Dog oral oral Pig X body wt. X oral oral Sheep gut X X Rabbit lungs Hill & Carlisle, 1947 Hill & Carlisle, 1947 Szocs et al, 1970 Olson et al, 1974 X oral oral Mouse heart X blood X X X X Hill & Carlisle, 1947 Bucher, 1946 X X X X X X K X Hill & Carlisle, 1947 Bucher, 1 4 96 X X Hill & Carlisle, 1947 X X X X X X X X X oral X X X X X Bucher, 1 4 96 Drill & Hiratzka, 1953 Shavgulidze et al, 1976 X X X X X X Bjorklund & Erne, 1966 �tested. These data suggest that the pig and the dog show higher sensitivity to acute doses of 2,4-D than the other species examined, while the chicken shows the lowest sensitivity. A slower rate of metabolism of 2,4-D has been offered as a possible explanation of the higher sensitivity of the pig and dog (Bjorklund and Erne, 1966). Disregarding which salt or ester was administered, the LD,.« values for the rat, guinea pig, and mouse suggest that males are more sensitive to 2,4-D than females. The rabbit LD values suggest that female rabbits are more sensitive than males. However, 2,4-D has never been administered in the same chemical form to separate groups of males and females of the same species to validate this trend. The acid form appears to be the most toxic of the different chemical forms of 2,4-D. The LDcn values obtained for the other chemical forms from different laboratories show wider differences than those obtained from the same laboratory. Baker et al. (1953) orally administered 500 mg of 2,4-D butyl ester per kg to two dogs and observed no clinical effects or histopathology, 4 or 82 days after dosing. Whether the lack of sensitivity in this study compared to those in table 6-1 reflects differences in effects of the acid and butyl esters or other factors, such as type of dog used, cannot be resolved from the limited data for this species. Most of the LD,-n values for 2,4-D were published prior to 1967. It is not known whether manufacturing operations have changed since that time, altering the levels or types of contaminants in the preparations. The LD,Q values obtained for rats by Russian and Czechoslovakian investigators are substantially higher than those obtained by investigators who used preparations manufactured in the U.S. The reasons for these discrepancies in LD..Q values are unknown. For other species, LD,-n values which were obtained from different laboratories varied by an order or two. LDcQ values were similar for doses of 2,4-D administered by gavage, by intubation, or intraperitoneally to the rat (Rowe and Hymas, 1954; Hill and Carlisle, 1947) and the guinea pig; subcutaneously to the mouse; or intravenously to the rabbit (Hill and Carlisle, 1947; Rowe and Hymas, 1954; Bucher, 1946; Young et al., 1978; Guseva, 1956). The latency period between the time that 2,4-D was administered and death occurred is on the order of 2-9 days (Shavgulidze et al., 1976; Drill and Hiratzka, 1953) for most species. In some cases, death occurs rapidly after 2,4-D is administered; in these cases, ventricular fibrillation has been the cause of death. 6.1.1.2 Cause of Death and Target Organs Lethal doses of 2,4-D produced the same symptoms in man as observed in other mammalian species. Neurotoxicity is the predominant effect of 2,4-D. Symptoms include stiffness of arms and legs, incoordination, lethargy, anorexia, stupor, and coma. Myotonia has been observed in man, as well as in the dog (Drill and Hiratzka, 1953), rabbit (Hill and Carlisle, 1947), and mouse (Bucher, 1946). Decreased body temperatures were observed in sheep (Shavgulidze et al., 1976) and mice (Bucher, 1946), but not cattle (McLennan, 1974) after a lethal dose of 2,4-D. Congestion of the viscera and renal 6-5 �swelling, localized to the proximal convoluted tubules, has been observed in rats and guinea pigs. Gastrointestinal disorders have been observed in the pig, mouse, dog, and monkey (Bjorklund and Erne, 1966; Bucher, 1946) indicating that 2,4-D is an irritant to the gastrointestinal tract and is likely to induce vomiting in man (Young et al., 1978). In the dog (Drill and Hiratzka, 1953) and pig (Bjorklund and Erne, 1966), death has been attributed to hepatic congestion and to pneumonia. In the mouse, rabbit, guinea pig, and rat, ventricular fibrillation has been the cause of death (Hill and Carlisle, 1947). Paralysis of respiratory muscles may have been a contributing cause of death in mice (Bucher, 1946) and sheep (Shavgulidze et al., 1976). Increased heart rates were noted in cows that ingested lethal or near lethal doses of 2,4-D (McLennan, 1974). 6.1.2 2,4,5-T The potency of 2,4,5-T in producing lethal effects and the patterns of toxicity it produces are described in this section. Most of the data were published before 1970, when the dioxin contaminant of 2,4,5-T was first recognized. 6.1.2.1 LD5Q The LD values for 2,4,5-T for various species are listed in table 6-2, according to the chemical form that was administered. Except for the LD,0 in the dog and two of the values for the mouse, all of the values were obtained from the same laboratory. As was observed for 2,4-D, the acid form of 2,4,5-T appears to be more toxic than the salts or esters. As with 2,4-D, of the species tested, the dog is most sensitive to 2,4,5-T. Male mice appear to be more sensitive to the lethal effect of 2,4-D than female mice. The paucity of data prevents any further analysis of differences in sensitivity between males and females for other species or differences in toxicity among various esters. The validity of the LD,fl values in table 6-2 has not been challenged or confirmed since they were published in 1953-1954 except for the data by Roll, who administered 2,4,5-T with less than 0.1 ppm TCDD. TCDD was not recognized as a contaminant of 2,4,5-T at the time the other studies were published, and there is no way to evaluate the contribution of potential TCDD contamination to the acute lethal toxicity of 2,4,5-T. The species differences that are observed for acute toxicity of TCDD are not apparent in table 6-2, but the LDcQ values for 2,4,5-T may generally be lower than those for 2,4,5-T with no detectable TCDD present. The data by Roll suggest this trend. Differences in sensitivity among different strains of mice may also have contributed to the differences in mouse LD,-n values in table 6-2, although the strain used by Rowe and Hymas (1954) was not stated. The effect of the route of administration of 2,4,5-T on its toxicity does not appear to have been studied. 6-6 �6.1.2.2 Cause of Death and Target Organs No cases of deaths from 2,4,5-T alone have been reported in humans. One case of suicide from a mixture of 20 percent 2,4-D and 40 percent 2,4,5-T was reported (Verhulst and Grotty, 1968). Symptoms typical of the neurotoxic effects of 2,4-D were described, along with vomiting, fever, hyperventilation, low blood pressure, and sinus tachycardia. Death occurred about 46 hours after the ingestion and was attributed to cardiac standstill. This report also mentioned another instance of hallucinations that occurred after inhalation of 2,4,5-T, but no other information was given. References of the acute toxicity of 2,4,5-T in mammalian species are listed in table 6-4. High doses of 2,4,5-T have produced anorexia, ataxia, and mild to moderate stiffness in the hind legs in dogs (Drill and Hiratzka, 1953). No marked effects were observed in these dogs and one dog that died from 2,4,5-T exhibited no symptoms. Histopathologic changes in these animals were considered nonspecific and pneumonia was suggested as the cause of death. Rowe and Hymas (1954) described the effects of all of the phenoxy acids they tested (including 2,4-D, 2,4,5-T, Silvex, and 2-methyl-4-chlorophenoxyacetic acid) together. These effects included anorexia, weight loss, depression, tenseness, muscular weakness, and histological evidence of stomach irritation, minor kidney and liver injury, and occasional cases of congestion of the lungs. The cause of death was not reported. Amounts of 2,4-D and 2,4,5-T that produce toxicity in cattle vary depending on the chemical form administered. A mixture of equal amounts of esters of 2,4-D and 2,4,5-T was administered to cows. A single dose of 1,000 mg/kg (33 percent 2,'4,5-T and 35 percent 2,4-D) had no toxic effect. A single oral dose of 100 mg 2,4,5-T (amine salt)/kg administered to two pigs produced anorexia, vomiting, diarrhea, and locomotive disturbances. The same dose of 2,4-D (amine) produced diarrhea, stilted gait, and depression in two pigs, while 100 mg 2,4-D (ester)/kg was ineffective. After dosing, blood levels of 2,4,5-T peaked at 250 mg/ml, while 2,4-D (amine) levels reached 230 mg/ml; blood levels after 2,4-D (ester) was administered never exceeded 40 mg/ml. The investigators attributed the differences in toxicity among the compounds to the differences in circulating phenoxy acid levels. Reports of accidental ingestion of lethal doses of 2,4,5-T by animals are rare. A case of fatal 2,4,5-T poisoning of a dog was surmised after levels of 2,000 ppm 2,4,5-T were measured in the stomach at autopsy. The likely cause of death was not stated by the veterinarian, who examined the dog two days after its death. Autopsy findings included hemorrhages of internal organs; evidence of vomiting was also reported (Leyland, 1975). 6.1.3 6.1.3.1 TCDD LD5Q TCDD is often referred to as the most toxic man-made chemical known. This conclusion is based on the observation that the oral LDcQ for TCDD in the 6-7 �Table 6-4. References on the Acute Toxicity of 2,4,5-T Organs Affected or Studied: Species Route Rat skin liver oral oral oral kidney neuro muse. blood heart lungs •gut body wt. Stroo et al, 1979 X Manis & Kim, 1979 X () x3 SC Reference Sjoeden et al, 1977 Koschier & Berndt, 1976 Koschier & Berndt, 1977 X in vitro in vitro Mouse Dog X Abo-Khatwa & Hollingsworth, 1974 in vitro X Olson et al, 1974 X oral oral measured indirectly X X X X Madge, 1977 X oral in vitro Guthrie et al, 1974 X X X Drill & Hiratzka, 1953 Leyland, 1975 oo �guinea pig is 0.6-2.1 ug/kg, one of the lowest LD values obtained in any species for any compound, except for several high molecular weight animal toxicants. In general, the oral LD values for TCDD for all species are low, although the values for various species vary by several magnitudes. Table 6-5 lists the LD,. values for TCDD administered by several routes to several species. In some cases LD,-n values have not been determined, but ineffective or marginally effective doses as well as doses lethal to over half of the treated animals are known. These values are given as the range of values that the LDcQ falls within. Few comparisons of TCDD either by different routes or in different sexes have been published. The LD,-n values for rabbits were of the same magnitude after oral, intraperitoneal, and dermal exposures to TCDD (Moore, 1978). In one instance when the effects of TCDD were compared between groups of male animals and female animals, male rats were more sensitive than females (Schwetz et al., 1973). However, Moore (1978) reported the opposite trend in other species. The reasons for the wide differences in LD-.- doses for various species is at present unknown. As discussed in Chapter 4, differences in the amounts of TCDD-binding receptors present in different species (as differences in affinities of these receptors for TCDD) and differences in the rates of TCDD metabolism and excretion probably contribute to these differences in toxicity among species. The low sensitivity of the hamster has been shown to correlate with rapid biotransforraation and excretion of TCDD in this species, compared to other species. An understanding of the mechanisms of toxicity and of the variability of TCDD tissue distribution among different species will be required to understand this species variability. A high correlation has been observed between the extent of TCDD binding to receptors in a given strain of mouse and its potency as an inducer of hepatic mixed function oxidases in the same strain. The relationship between TCDD toxicity and enzyme induction has recently been suggested (Poland and Glover, 1980). The rate of binding of TCDD differs among several strains of mice. Only strains with high rates of binding showed sensitivity to thymic atrophy and cleft palate effects, and only these strains showed enzyme induction responses to TCDD. 6.1.3.2 Cause of Death and Target Organs References on the acute toxicity of TCDD in mammalian species are listed in table 6-6. Death from TCDD usually occurs at least 1 week after an acute dose is administered and sometimes up to 7 weeks later. This latency period is very long, compared to the latency periods for other compounds. When higher doses of TCDD are administered, the time until death is not shortened appreciably, although the proportion of treated animals that die increases (Greig et al., 1973; Moore, 1978). This latency period indicates that TCDD is probably toxic at the cellular level, rather than causing death rapidly by impairing the physiologic function of an organ system such as the nervous system (Poland and Kende, 1976; McConnell et al., 1978a; 1978b). 6-9 �Table 6-5. LD5Q Values (in ug/kg for TCDD) Species (Sex) Route L D Monkey (F) oral <70 Rat - Spartan ( ) M () F oral oral 22 45 Rat - Portion (F) () F oral oral 190a 125-500 Mouse - C57BL () M () M oral oral oral oral ip 126 114 >130 <150 120 Guinea Pig (M) oral 0621 .-. <3 Guinea Pig (,) MF (,) MF oral ip 1157 >3000 Rabbit (,) MF (M,F) (M,F) oral dermal ip Dog (M) () F Frog in DMSO in arachis oil 50 Reference McConnell et al . , 1978 Schwetz et al., 1973 Schwetz et al., 1973 Greig et al., 1974 Greig et al., 1974 Jones and Greig, 1975 Vos et al., 1974 Schwetz et Goldstein et al., 1973 Vinopal and Casida, 1973 Schwetz et al., 1973 McConnell et al., 1973 Olson et al., 1978b Olson et al., 1980 115 275 252-500 Schwetz et al., 1980 Schwetz et al., 1973 Schwetz et al., 1973 oral oral 30-300 >100 Schwetz et al., 1973 Schwetz et al., 1973 oral 1,000 Vos et al, 1974 �Table 6-6. References on the Acute Toxicity of TCDD Organs Affected or Studied: Species Route Monkey oral oral Rat skin X oral oral oral oral, ip oral , Ip oral oral, Ip oral oral oral oral oral oral oral oral oral oral oral oral, Ip oral , Ip Guinea Pig Rabbit X X X X X X X X X X X X X X oral Hamster oral, ip lymph. X blood heart lungs gut X body wt. X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X oral oral oral oral Dog neural X X X X oral oral oral oral oral oral oral , ip dermal, eye X X kidney X X X X iP IP iP Mouse liver X X X Seefeld et al., 1979 McConnell et al., 1978a Greig et al., 1973 Harris et al. , 1973 Schwetz et al. , 1973 Manis & Apap, 1979 Manis & Kim, 197 9 a Madge, 1977 Manis & Kim, 1979b Bastomsky, 1977 Van Logten et al., 1980 Thunbert et al., 1979 Cumminghara & Williams, 1972 Courtney et al., 1978 Jones, 1975 Guiney et al., 1978 Jones & Butler, 1974 Fowler et al., 1973 Hamada & Peterson, 1978 Peterson et al. , 1979 Greig et al., 1974 Pegg et al., 1976 Hook et al., 1978 Buu-Hol et al., 1977 Buu-Hoi et al., 1972a McConnell et al., 1978b Harris et al., 1973 Vos et al., 1974 Drill & Hiratzka, 1953 Luster et al., 1978 Jones & Greig, 1975 X X X X Reference X X X Greig et al., 1973 McConnell et al., 1978b Harris et al., 1973 Schwetz et al., 1973 X X X X X X X Schwetz et al., 1973 Schwetz et al., 1973 X X X Schwetz et al., 1973 X Olson et al., 1980 X X �The actual cause of death in TCDD-poisoned animals is often difficult to identify. Animals become emaciated and all fat reserves are depleted, even though food intake is not appreciably depressed (McConnell et al., 1978a; Harris et al., 1973) and absorption through the gut is normal (Greig et al., 1973). Death has been attributed to severe hepatic lesions observed in the rat, although this effect is often absent in other species such as the mouse and the guinea pig. In the hamster, oral doses showed higher toxicity than intraperitoneal doses, and ileitis and peritonitis were observed only after oral doses (Olson et al, 1980). The intestinal lesions were moderate to severe, and may have been responsible for the increased lethality of oral doses. Severe thymic atrophy from lethal doses of TCDD has been observed consistently among different species (Harris et al., 1973; Schwetz et al., 1973; Vos et al., 1974; Olson et al., 1980). In the guinea pig and mouse, the change in thymus weight is the most sensitive indicator of TCDD exposure (Harris et al., 1973). The corresponding loss in immune function is more difficult to demonstrate and seems to be significant primarily in animals that were treated with TCDD before they reached adulthood. Deaths from pulmonary infections have been reported for TCDD-treated rats, but rats bred under sterile conditions also suffer the lethal effects of TCDD (Greig et al., 1973), indicating that the lethal effects of TCDD are not dependent on an active immune system. Hydropericardium, ascites, and subcutaneous edema occur in chickens that were fed toxic fat containing TCDD and other dioxins, and death has been attributed to pulmonary edema. Edema occurs in other species including rats, pigs, dogs, and monkeys, but to a limited extent (Firestone, 1973; Vos, 1978; Huff et al., 1980). 6.1.4 Diquat The toxic effects of diquat are described in several reviews (Manzo et al., 1979; Witschi, 1977; Smith and Heath, 1976; Conning et al., 1969). 6.1.4.1 LD5Q LD.ft values for diquat have been reported for oral and subcutaneous doses (Clark and Hurst, 1970). The oral LD values range from 30 mg/kg in cattle to 200-400 mg/kg in the hen. Values ror the rat (231 mg/kg), the mouse (125 mg/kg), the guinea pig (100 mg/kg), and the dog (100-200 mg/kg) were all similar, indicating little variation among species. However, most of these values are based on results from groups of only three animals per species. Only groups of rabbits, rats, and mice were large enough in this study to produce reliable LD,-n values. In another study, an acute oral dose of 100 mg/kg was lethal to one of two treated monkeys, while a dose of 400 mg/kg was lethal to both monkeys that received it (Cobb and Grimshaw, 1979). For both the dibromide and dichloride salts of diquat, subcutaneous LD values were 10-11 mg/kg in male and female rats. Acute inhalation and dermal exposures to diquat have been reported that did not elicit any toxicity. Exposure to 23 mg of diquat aerosol per liter air for 15-30 minutes caused no adverse effects in male rats (Gage, 1968). In rabbits, a dermal dose of 400 mg diquat/kg produced no toxicity (Clark and Hurst, 1970). 6-12 �Several studies have demonstrated that animals exposed to high oxygen levels show high susceptibility to the acute toxicity of diquat. In one study, 20 rag diquat/kg produced 50 percent mortality in rats, while 10 mg/kg was not lethal. Half of the deaths occurred within 2 days of treatment. Other rats, exposed to 85 percent oxygen, were more susceptible to the lethal effects of diquat: a dose of 10 mg/kg produced 50 percent mortality, with a mean latency period to death of 41 hours; 20 mg/kg produced 100 percent mortality, with a mean latency period of 17 hours. Lesions observed in diquat-treated rats in 85 percent oxygen, but not in air, were type II cellular damage and lung edema. The authors implied that increased oxygen levels may facilitate generation of superoxide anions by diquat, which in turn may induce pulmonary toxicity (Pratt et al., 1980). In another study, rats exposed to 100 percent oxygen showed reduced latency periods between intravenous administration of diquat and death, compared to rats in room air. Oxygen exposure also induced respiratory distress in diquat-treated rats, a symptom which was not observed in rats treated with diquat and exposed to room air. Since tissue distribution was not altered by high oxygen exposure, the authors concluded that increased oxygen facilitated diquat oxidation that might lead to the production of toxic oxygen radicals (Kehrer et al., 1979). 6.1.4.2 Cause of Death and Target Organs Cases of human intoxication after exposure to diquat have been reported. One fatal case of diquat poisoning has been reported (Okonek and Hofmann, 1975). A 43-year-old woman intentionally ingested two unknown doses of diquat, 2 days apart. After the second dose, the woman was admitted to the hospital with severe ulceration of the mouth, throat, and esophagus, and anuria, and was in shock. Extracorporeal dialysis was performed twice, but was not effective in removing a significant amount of diquat and the patient died 46 hours after admission, of protracted cardiovascular collapse. Symptoms reported in cases of nonlethal diquat poisoning included ulcerations of the mouth and pharynx (Oreopoulos and McEvoy, 1969) after ingestion; high fever, cough, jaundice, and skin rash in one case of exposure by inhalation (Wood et al., 1970); and fever, dizziness, headaches, eye pain, and temporary disturbances in liver and kidney function tests in another case of inhalation exposure (Weirich, 1969). Direct contact with concentrated formulation of diquat and paraquat mixtures have been reported to cause ocular damage (Cant and Lewis, 1968) and nail damage (Sammon and Johnston, 1969). The toxicity of diquat has been evaluated in several animal species. Clark and Hurst (1970) reported that the pattern of diquat poisoning was similar in all species they studied. One day after an oral dose was administered, animals became lethargic, developed some respiratory difficulty, and died within 2 weeks. Histopathological changes were limited to the gastrointestinal tract. Additional symptoms observed after subcutaneous exposure were pupillary dilation within several hours of the injection, which remained until death (within 2 weeks), and distended abdomens with bile-stained contents in the digestive tract. Reduction of lymphocytes and in the sizes of the spleen and thymus were observed several days after treatment. Injection 6-13 �of high doses (four to five times the LD-Q) produced lethargy within minutes, labored respiration within 1 hour, and muscle twitches, convulsions, and death within several hours. Symptoms of diquat poisoning in the monkey include diarrhea and lethargy, and, in severe cases, coma. In addition, histopathological changes in the gastrointestinal tract and kidney occur in this species (Cobb and Grimshaw, 1979). In cattle, dehydration, rapid respiration and pulse, unsteady gait, and coma were observed prior to death; all were attributed to diquat poisoning. Histopathological changes involved the gastrointestinal tract, heart, liver, brain, and lungs (Thomas and Amor, 1968). The cause of death in diquat poisoning of animals remains unknown. 6.1.5 Cacodylic Acid Peoples et al. (1979) reported the effects observed in 34 cases of accidental exposure of workers to organoarsenical herbicides. All cases involved exposure to cacodylic acid, sodium cacodylate, methanearsonic acid, its salts, or combinations of these compounds. The systemic effects of cacodylic acid and its salt involved gastrointestinal irritation. Direct contact of the eyes or skin with the herbicides resulted in mild injuries. None of the clinical symptoms persisted. The authors suggested that all of the accidents could have been avoided by proper use and maintenance of the applicator equipment. Stevens et al. (1979; Stevens et al., 1976) studied the acute inhalation toxicity of cacodylic acid in the rat and mouse. Mortality was higher after exposure to a low concentration than after a high concentration in male rats, and was low in both male rats and in mice, precluding calculation of LC (the concentration causing 50 percent mortality) values. Two of 10 male rats and no mice died after exposure to particulate concentrations of cacodylic acid of 4.1 mg/liter air for 2 hours, while none of the rats and one mouse exposed to a higher concentration of 6.9 mg/liter died during the subsequent 2 weeks. The LC for female rats was calculated at 3.9 mg/liter, after a 2-hour inhalation exposure. Symptoms observed in rats and mice from inhalation exposure were respiratory distress, rhinorrhea, porphyrin-like encrustation of the eyes, erythemia, and histopathological changes of the lung and gastrointestinal tract. Cacodylic acid also elicited a weak respiratory irritant response. LD,-0 values after intraperitoneal injection of cacodylic acid were 720 and 520 mg/kg in male and female rats, respectively, and 520 and .600 mg/kg in male and female mice, respectively. The intravenous LDr~ in female rats was 470 mg/kg. Symptoms observed after cacodylic acid injections included loss of the righting reflex, rigidity, decreased body temperature, decreased thymic size, and dark adrenals and livers, in addition to the symptoms that resulted from inhalation exposure (Stevens et al., 1979). LD... values for cacodylic acid have been published in several reviews. Oral LD values for the rat were reported by Weakley (1977) to range from 600-3,200 mg/kg, by Woolson (1976) to range from 700-3,200 mg/kg, by Peoples 6-14 �(1975) to range from 1,200-1,600 rag/kg, by Dietz and Moore (1978) to be about 700 mg/kg, and by Palm (1968) to be 830 mg/kg. Details of the methods used to determined these LD s Or reasons for the wide variation in reported values were not presented in these review articles. Dietz and Moore (1978) reported that the dermal toxicity was far less than the oral toxicity of cacodylic acid in the rat. Woolson (1976) reported that the oral and intravenous LD,.- in mice were 184 and 316 mg/kg and the subcutaneous LD,-n in the dog was 1,000 mg/kg. Midwest Research Institute (1975) also reviewed the acute toxicity data for laboratory and domestic species. 6.1.6 Picloram The LD values for picloram have been determined in several species. Based on the number of deaths that occurred within 14 days of an oral dose of picloram, the LD values were calculated to be 8,200 mg/kg in the female rat, 2,000-4,000 mg/kg in the female mouse, about 3,000 mg/kg in the female guinea pig, about 2,000 mg/kg in the rabbit, and about 6,000 mg/kg in the male chicken. Single oral doses of 650 mg/kg to sheep and 488 mg/kg to cattle produced no ill effects (Lynn, 1965). Another study also found that single oral doses of 720 mg/kg to sheep or 540 mg/kg to calves produced no ill effects (Jackson, 1966, cited in McCbllister and Leng, 1969). Dermal application of 4,000 mg/kg to rabbits for 24 hours produced local edema and pigmentation, but no other effect. An undiluted preparation and a 10 percent slurry in water were both slightly irritating to the rabbit eye, producing conjunctivitis that lasted 2 days (Lynn, 1965). Adverse symptoms and cause of death after fatal doses of picloram were not described. No cases of human poisoning from picloram have been reported. 6.1.7 Monuron The toxicological data on monuron have been reviewed (IARC, 1976; Midwest Research Institute, 1975). The acute toxicity of raonuron was studied in the rat. The oral LD,.- for the rat was 1,480 mg/kg and the mean time to death was 27 hours (Boyd and Dobos, 1969). Symptoms observed 1 to 8 hours after monuron treatment were primarily neural disorders, including ataxia, drowsiness, hyporeflexia, pallor, piloerection, exophthalmos, and tachypnea. After 24 hours, irritability, hyperreflexia, and diarrhea were observed. Prior to death, anorexia, dyspnea, hypothermia, loss of body weight, epistaxis, bliguria, and prostration occurred. Deaths were attributed to respiratory or cardiorespiratory failure. Histopathologic changes included gastric ulcers, gastroenteritis, hepatic necrosis, renal lesions, pneumonitis, a stress reaction in lymphatic tissues, and congestion in the brain, heart, and lung (Boyd and Dobos, 1969). Rats fed low protein diets were more susceptible to the lethal effects of monuron than rats fed normal diets. This susceptibility seemed to be associated with their inability to tolerate weight loss from raonuron in addition to the weight loss from the protein deficient diet (Boyd and Dobos, 1969). 6-15 �6.1.8 Diuron The LD,- for diuron was calculated at 1,017 mg/kg in the rat after a single oral dose. The average time to death was 24 hours. Gastritis, enteritis, dehydration of the cecum, congestion of the brain and lungs, and a yellowish color of the kidney resulted from diuron administration. Hypoflexia, hypothermia, loss of body weight, drowsiness, and bradypnea were also observed. Irritability and hyperreflexia occurred in survivors a day after treatment. Death was caused by respiratory failure. Histopathological changes were observed in the gastrointestinal tract, adrenals, thymus, spleen, kidney, and liver. Protein deficient rats showed higher susceptibility to diuron (with LD,-Q as low as 437 mg/kg) than rats fed standard diets (Boyd and Krupa, 1970). Diuron in vitro has been shown to reduce transport of glucose across the mouse intestine (Guthrie et al., 1974). 6.1.9 Dalapon Dalapon has been reported to cause contact dermatitis. Seven forest workers were diagnosed with this condition, having known contact with this herbicide and having developed a rash that cleared rapidly. Dermatitis, which was observed in 21 other workers, was suspected to be caused by dalapon or sodium chlorate, although exposure to either chemical could not be confirmed. In response to a questionnaire on exposure to dalapon, workers indicated having frequently experienced such subjective symptoms as nausea, skin lesions, anorexia, and pain in the throat. The results of clinical chemistry tests were found to be similar for a group of dalapon sprayers and a control group (Matsushita et al., 1975). Two animal studies considered the acute toxicity of dalapon. The acute oral LD of dalapon in the rat was 7,570 mg/kg in females and 9,300 mg/kg in males; values for female mice, female guinea pigs, female rabbits, and chickens were 4,600, 3,860, 3,860, and 5,660 mg/kg, respectively. Death occurred within one day of treatment. No gross pathological changes were observed and the only histopathological finding was an accumulation of fluid and gas in the gastrointestinal tract. When dalapon, as dry powder, was applied to the eye of a rabbit, it produced conjunctivitis and corneal injury within an hour; these lesions healed in several days (Paynter et al., 1960). Norris obtained lower LD,0 values for dalapon, which were 5,660 mg/kg in female rats, 2,830-4,930 mg/kg in female guinea pigs, and 2,830-2,140 in female rabbits (published by Kenaga, 1974). 6.1.10 Bromacil Sherman and Kaplan (1975) reported an LD-,, value for male rats of 5,200 mg/kg. A dermal dose of 5,000 mg/kg produced no clinical signs or gross pathology in rabbits and the LCSf. was greater than 4.8 mg/liter for the rat. Toxicological data on bromacil nave been reviewed (Midwest Research Institute, 1975). 6-16 �6.1.11 Agent White Dermal application of a 5 percent solution of Agent White, a mixture of 10.2 percent picloram (triisopropanolamine salt) and 39.6 percent of 2,4-D (triisopropanolamine salt), produced no skin irritation of sensitization in man (Weimer et al., 1970). The oral LD for Herbicide White in rats was 3,080 mg/kg. In comparison, 5,000 mg/kg of an 11.6 percent preparation of the potassium salt of picloram produced no deaths. Exposure of rats to air bubbled through the mixture produced no toxicities, following a 7-hour exposure. A dose of 2,000 mg/kg of Agent White administered dermally to rabbits produced slight hyperemia and slight necrosis, but no systemic toxicity (Weimer et al., 1970; Lynn, 1965). Single ocular applications of 0.01 ml of Agent White to rabbits caused blepharitis, iritis, and conjunctivitis; higher doses (0.05-0.2 ml) produced corneal opacity as well. These lesions cleared within 28 days (Weimer et al., 1970). 6.2 DERMAL LESIONS None of the herbicides considered in this report produce significant dermal lesions, other than the local irritation described in section 6.1. TCDD, a potential contaminant of the herbicide 2,4,5-T, produces chloracne. Chloracne occurred in workers after every reported instance of occupational exposure to TCDD during the manufacture of trichlorophenol and after other industrial exposures that released TCDD (see chapter 5). TCDD has been shown to be responsible for chloracne: an investigator applied a 0.01 percent solution to his forearm and developed lesions characteristic of chloracne (Bauer et al., 1961). Mild dermatitis developed in 2 days, followed by comedones several days later. The results of experiments in which TCDD was applied to the skin of human volunteers were presented in testimony in the Environmental Protection Agency is 2,4,5-T/Silvex Cancellation Hearing (Rowe, 1980). A 1 percent suspension of TCDD in alcohol chloroform was applied to the backs of 10 subjects on alternate days for 1 month. This protocol resulted in the application of a cumulative dose of 7,500 ug of TCDD. Eight of the 10 subjects developed chloracne, which lasted four to seven months. No other adverse health effects were noted from urinalysis or chemical tests monitoring blood, liver, and kidney function. A cumulative dose of 16 ug applied in the same manner was ineffective; no intermediate doses were tested. TCDD applied to the rabbit ear produces local lesions resembling human chloracne. Inflammation and hyperkeratosis were produced after 0.01-0.005 percent TCDD in polyglycol was brushed on a rabbit ear (Kimmig and Schulz, 1957a; 1957b). Dermal doses above 0.002 percent, however, resulted in death from liver lesions before dermal lesions appeared (Kimmig and Schulz, 1957b). The rabbit ear bioassay was used to identify the presence of TCDD, through its acnegenic potential, after industrial accidents. A dermal dose of 0.3 ug of TCDD in acetone, applied in three applications over three days, was sufficient to cause hyperkeratosis in 14 days; higher doses produced keratinous masses in 6-17 �the follicular epithelium that resembled comedones, and caused a reduction in the number of sebaceous cells that could be observed by histology (Jones and Kizek, 1962). In agreement with these results, another laboratory (Rowe, 1980) reported that a dose of 0.2 ug of TCDD did not cause acne in the rabbit ear bioassay, 0.5 ug caused a marginal effect, and 4-8 ug caused a severe acnegenic response. From these results, man appears to be much less sensitive than the rabbit to the acnegenic effects of dermally applied TCDD. In man, lesions usually do not occur in the follicles of beard hair, possibly because beard hair facilitates drainage of sebum and keratinaceous debris (Greig, 1979). The only strain of rodent that develops chloracne after TCDD exposure is a hairless strain of mouse. Acne-like lesions also develop on the lips of monkeys, along with hyperkeratosis of glands of the eyelids after oral administration of TCDD. Facial alopecia, blepharitis, and loss of fingernails and eyelashes also occur in monkeys after acute exposure (McConnell et al., 1978a). After horses were exposed to salvage oil which contained TCDD as well as many other chemicals, hyperkeratosis developed. This condition involved hair loss and the development of a thick layer of keratin covering the epidermis. Ulcerative dermatitis and hair loss were also observed in dogs, cats, and mice that were exposed to the sprayed salvage oil (Case and Coffraan, 1973). In man, as in the monkey and rabbit, histological appearance of skin lesions progresses from dilated hair follicles filled with keratin to the disappearance of sebaceous glands and the replacement of follicles with keratin (VA, 1980). Blepharitis and conjunctivitis have been observed in cases of TCDD-induced chloracne in man (Moore, 1978) and in the monkey (McConnell et al., 1978a). Delayed conjunctival chemosis has resulted from the direct application of 2 mg of TCDD to the conjunctival sac of the rabbit eye. A latency period of 13-22 days preceded the appearance of symptoms (Van Miller et al., 1976). The mechanism of development of chloracne after TCDD exposure is unknown. Observations that lipid metabolism is disturbed by TCDD, that oil accumulates on the skin prior to the development of chloracne, and that certain fatty acids can cause follicular keratosis has led to the suggestion that fatty acid metabolism may play a role in the etiology of chloracne caused by TCDD (Vos, 1978). Vitamin A deficiency also can cause hyperkeratosis, but use of this vitamin as a therapeutic agent has not been very effective against TCDD-caused chloracne. 6.3 PULMONARY LESIONS Pulmonary lesions are rare in animals exposed to the herbicides in this study. The effects of diquat treatment on the lung have been studied in detail. Diquat does not produce significant pulmonary toxicity. Acute intratracheally instilled and subcutaneously injected doses of diquat caused decreased body weights in rats, but only temporary decreases in vital capacity and static lung compliance 24 hours after administration (Lam et al., 1979). 6-18 �Pulmonary lesions, including type II alveolar cell damage and edema, have been induced in diquat-treated rats by exposure to 85 percent oxygen but were not present in rats exposed to ambient oxygen and treated with the same dose of diquat (Pratt et al., 1980). Various studies have focused on the biochemical changes that diquat elicits in the lung in attempts to identify the mechanism of action of paraquat, a structural analogue of diquat that produces lethal pulmonary lesions including edema and fibrosis. Intermediary metabolites of high energy, tricarboxylic acid cycle, and glycolytic pathways in the lung were decreased, and pulmonary levels of adenosine triphosphate (ATP) and adenosine monophosphate were increased, following an intraperitoneal dose of 50 mg diquat/kg to mice. Microsomal adenosine triphosphatase activity was not altered after diquat treatment. The authors suggested that increased ATP synthesis provided energy to repair cellular damage, although they did not describe any damage in diquat-treated lungs (Hawkins, 1980; Hawkins et al., 1979). Diquat was also shown to inhibit acetylcholinesterase in the rat lung (Brown and Maling, 1975). Whether this inhibition occurs at the neuromuscular junction, where it would be expected to cause paralysis, was not evaluated. At 10 M, diquat produced a significant stimulation of CO production from glucose in slices of rat lungs, with a maximum effect observed at 10 M. This effect was also observed in lung slices from rats that were treated intravenously with diquat. Intravenous doses of paraquat were less potent than diquat in producing this effect (Rose et al., 1976). Both compounds also produced rapid decreases in pulmonary NADPH/NADP ratios that were sustained for 24 hours following an intravenous dose of 140-156 uM/kg of herbicide. Diquat produced damage to type I alveolar cells, while paraquat damaged type II cells in this experiment (Witschi et al., 1977). Lung lesions were evaluated in rats after a lethal intraperitoneal dose of diquat was administered. No increase in water content (indicative of edema) or in thymidine incorporation into lung DNA (indicative of lung fibrosis) was detected. The mortality rate was 70 percent over 14 days, with 20 percent of the deaths occurring within three days. Diuresis and dehydration were the only effects of diquat that were mentioned (Smith and Rose, 1977). The effects of diquat on alveolar macrophage viability were comparable to those of paraquat, producing irreversible damage after 30 minutes at concentrations of 10 M/liter. Diquat was 10 times more toxic than paraquat to fibroblasts in vitro, indicating that in vitro toxicities of these compounds do not reflect their in vivo toxicities (Styles, 1974). The distribution of paraquat to the lung has been shown to be four times higher than that of diquat; this difference in distribution between the two compounds probably explains the difference in pulmonary toxicities. This difference in distribution may also explain the ability of paraquat, but not diquat, to produce large, dose-dependent increases in albumin content of the lung (a measure of edema), following subcutaneous administration to rats (Shu et al., 1979). 6-19 �6.4 HEPATOTOXICITY 6.4.1 2,4-D One report considers the effects of 2,4-D on serum and hepatic liver enzymes (Szocs et al., 1970). A single oral dose of 625 rag/kg to the rat produced decreases in serum and liver aldolase activities; a 30 percent decrease in serum cholinesterase; increased serum and liver glutamic oxaloacetic transaminase and acid phosphatase levels; and fluctuations in the serum and liver glutamic pyruvic transaminase, alkaline phosphatase, succinic dehydrogenase, and catalase activities. These changes were monitored for two days after treatment. Centrilobular fat deposits were observed in the liver at this time. 6.4.2 TCDD Several types of hepatotoxic effects have been described for TCDD. Structural alterations, including changes observed by gross or histopathological examination of the liver, are described first. Necrosis, fatty degenerative changes, cellular infiltration, multinucleation of parenchyma! cells, and increased size of the liver are the types of changes considered in this section as structural alterations. Changes in serum enzymes that are released from the liver and are used to evaluate liver function in some situations are described next. Finally, changes in the structure of bile ducts and the rate of biliary excretion of exogenously added compounds are described. Distribution of TCDD to the liver and biliary excretion of TCDD are described in chapter 4. 6.4.2.1 Structural Alterations Changes in the histological appearance of the liver after TCDD treatment are common. The types of changes that occur and the severity of these changes show wide variability among species. In the rabbit, death occurred 8-20 days after a large dose (0.05-0.1 mg/kg) of TCDD was administered; death was attributed to hepatic lesions (Kimmig and Schultz, 1957a; 1957b), Lesions in the rat have been observed that were severe and potentially contributed to death (Huff et al., 1980). Reproducible hepatic changes were observed in the mouse, but were usually not severe enough to cause death; in the monkey and guinea pig, hepatic changes were minimal or absent in animals that died of TCDD poisoning (McConnell et al., 1978a; 1978b). After acute doses of TCDD are given, the types of hepatic changes observed vary among different species. In the rabbit, necrosis and diffuse fatty degenerative changes have been reported (Kimmig and Schultz, 1957a; 1957b). Centrilobular necrosis occurs in the dog, rabbit, rat, and mouse (Schwetz et al., 1973). Both extensive (Jones and Greig, 1975) and single-cell necrosis (McConnell et al., 1978b) have been reported in the mouse, while the lesion in the rat is usually focal or scattered (Vos et al., 1974; van Logten et al., 1980; Greig et al., 1973; Jones and Greig, 1975). 6-20 �Cellular infiltration has been observed in livers of both rats and mice after TCDD treatment (Greig et al., 1973; Vos et al., 1974; McConnell et al., 1978b; Jones and Butler, 1974; Jones and Greig, 1975). Accumulation of lipids occurs in parenchymal cells of the mouse after TCDD poisoning, but not in the rat or monkey. Lipid accumulation has also been observed in mice that were starved (Jones and Greig, 1975; Jones and Butler, 1974; McConnell et al., 1978a; 1978b; Vos et al., 1974). Multinucleated parenchymal cells have been observed in the monkey (McConnell et al., 1978a) and rat. These multinucleated cells result from the fusion of parenchymal cell membranes (Jones and Butler, 1974; Jones, 1975-1254). Proliferation of the rough and smooth endoplasmic reticulum in rat hepatocytes has been demonstrated and is probably related to increased synthesis of microsomal proteins, induced by TCDD (Fowler et al., 1973). Increased liver weight relative to body weight has been reported for rats and mice after a single dose of at least 50 ug TCDD/kg (McConnell et al., 1978a; 1978b; Greig et al., 1973). Removal of the adrenal or pituitary gland does not block this increase in liver weight in the rat (van Logten et al., 1980). In the mouse, the increase in liver weight was shown to correlate with increased hepatic levels of lipids, esterified fatty acids, and cholesterol, at the same time that liver protein and water contents were decreased and DNA content was unchanged (Jones and Greig, 1975). TCDD also has been shown to have no effect on DNA synthesis of hepatocytes after partial hepatectomy (Greig et al., 1974) or on mitochondrial respiration of hepatocytes (Courtney et al., 1978). 6.4.2.2 Serum Enzyme Activity The evaluation of liver function by measuring serum enzymes that are assumed to be released from the liver as a response to toxic agents is dubious in TCDD poisoning. TCDD has a direct inductive effect on hepatic enzymes, and increased serum levels of these enzymes may not necessarily reflect altered liver function. Altered serum enzyme activities were reported after a massive dose of TCDD was administered to the rat (Buu-Hoi et al., 1972b), and were correlated with histological changes in the liver (Buu-Hoi et al., 1972a). Histological and tissue weight changes in the liver have been reported in the absence of changes in serum enzyme levels (Greig et al., 1973). Changes in serum levels in humans have been transient in individual workers who were free of any other symptoms (see chapter 5). In other workers these changes have been associated with altered lipid metabolism and cardiovascular disorders (Walker and Martin, 1979). 6.4.2.3 Biliary Excretion Bile duct proliferation has been observed in the mouse, but not in the rat (Jones and Greig, 1975; McConnell et al., 1978b; Vos et al., 1974). Hyperplasia and thickening of the bile duct has been observed in the rat from a single dose of 10-25 mg TCDD/kg. Other hepatic changes caused by this dose were less obvious than the effect on the bile duct (Croft et al., 1977). 6-21 �A single dose of TCDD produced a long-lasting decrease in biliary excretion of ouabain (Young and Peterson, 1976). This effect is thought to be mediated by TCDD at the surface membrane of the hepatocyte (Peterson et al., 1979), and is reversed by treatment with repeated doses of pregnenolone 16 alpha-carbonitrile or spironolactone (Hamada and Peterson, 1978). Acute TCDD treatment also depressed biliary excretion of biphenyls in the rat (Guiney et al., 1978), of indocyanine green in the monkey prior to death (Seefeld et al., 1979), and in the rat (Hwang, 1973), and of thyroxine in the rat (Bastomsky, 1977). 6.4.3 Diquat Minimal liver changes involving focal necrosis and centrilobular fat droplets were observed in monkeys that were administered lethal doses of diquat. Serum glutamic oxaloacetic and pyruvic transaminase levels were also elevated (Cobb and Grimshaw, 1979). Liver changes attributable to diquat were not observed in laboratory species (Thomas and Amos, 1968); Clark and Hurst, 1970). Liver necrosis was not observed in rats that were administered lethal amounts of diquat, unless the rats were also deficient in selenium. Selenium is an essential constituent of the enzyme glutathione peroxidase. This enzyme catalyzes reactions that break down hydrogen peroxide and polyunsaturated fatty acid hydroperoxides, compounds that promote lipid peroxidation. Rats maintained on a selenium-deficient diet have enhanced potential for lipid peroxidation. When diquat was administered to these rats, survival was very low compared to survival of diquat-treated rats with normal selenium levels. Both lipid peroxidation (measured by determining ethane production in expired air) and liver damage were' severe in the selenium-deficient rats after diquat treatment. Liver damage involved histopathologic evidence of necrosis and elevated serum glutamic pyruvic transaminase levels. Pretreatment with selenium 6-10 hours prior to diquat administration protected the rats from both the lethal and hepatic effects of diquat. However, selenium did not act by increasing glutathione peroxidase activity, as in several tissues this activity was not increased in selenium-pretreated rats (Burk et al., 1980; Burk et al., 1979). These results indicate that diquat can cause liver damage, probably through its ability to generate superoxide anions by lipid peroxidation. However, these processes do not seem to occur in diquat poisoning unless a situation such as selenium deficiency also exists which predisposes the animals to enhanced lipid peroxidation. Studies of the biochemical mechanism of diquat have focused on its action in subcellular hepatic preparations. Diquat does not penetrate liver mitochondrial membranes. The liberation of hydrogen peroxide, when diquat free radicals are oxidized by molecular oxygen, is considered to be important in plants but not in animals. The free radical, released by the microsome, has been suggested as the active component in animal tissues where cyclic reduction and reoxidation of diquat may lead to aromatic hydroxylation and an increase in thiobarbituric acid-reacting components of phospholipids (Gage, 1968). Another report also demonstrated diquat 1 s ability to inhibit microsomal oxidation (Kreiger et al., 1973). Kopaczyk-Locke (1973) suggested that diquat acts at the level of liver mitochondria by stimulating citric acid cycle dehydrogenase and uncoupling oxidative phosphorylation. 6-22 �6.5 NEUROTOXICITY Human and animal exposure to 2,4-D, 2,4,5-T, and TCDD that resulted in the development of various neuropathologic symptoms are discussed in this section. The majority of the cases involve exposure to 2,4-D herbicides; human exposure resulted mainly from inadvertent or accidental contact while spraying gardens or farm fields. Exposure in these cases was via cutaneous absorption and respiration. Several experiments that exposed rats, cats, and dogs to 2,4-D are also discussed. One experiment described in this section examines the neurologic effect of 2,4,5-T in rats, and one report describes the neurological effects on humans following exposure to TCDD. In addition, brief references are made to numerous industrial accidents, involving primarily 2,4-D and TCDD, that caused neuropathologic disorders in exposed humans. 6.5.1 2,4-D Several clinical studies describe the neurologic symptoms resulting from acute exposure to 2,4-D. These reports indicate that exposure occurred during mixing or spraying liquid 2,4-D herbicides. The liquid chemical was splashed onto the skin, or was deposited onto the skin or inhaled as a mist. Three humans exhibited pronounced neurologic disorders following cutaneous exposure to 2,4-D (Goldstein et al., 1959). Initial symptoms developed within two days of the exposure and included numbness in the fingers and toes, muscle aches and fatigue, tetany of the limb muscles, and muscular ataxia causing difficulty in walking. Clinical examination revealed partial or total hyporeflexia, absence of joint sensation, and fasciculations of the arm and leg muscles. Similar symptoms and clinical results were found in a 39-year-old farmer who had been exposed to 2,4-D while spraying. In addition, this individual exhibited paresthesia of the extremities; myokymia of facial, trunk and leg muscles; and loss of manual dexterity (Berkley et al., 1963). All of these neuromuscular disorders were apparent in a 21-year-old male exposed to 2,4-D while mixing herbicides (Wallis et al., 1970). Ulnar nerve conduction velocities in exposed individuals were measured (Goldstein et al., 1959; Wallis et al., 1970). All patients exhibiting the neurologic symptoms of 2,4-D poisoning had decreased conduction velocities. Electromyographic examinations, nerve block tests, and nerve biopsies indicated that 2,4-D inhibits the normal functioning and may even damage peripheral nerves (Wallis et al., 1970). The authors of the above studies all concluded that although the etiology was not known, 2,4-D was responsible for the peripheral neuropathy observed in their patients. In addition to peripheral neuropathy, 2,4-D exposure may also cause neurologic abnormalities in the central nervous system. Kontak et al. (1973) recorded the electroencephalographic (EEC) patterns of 17 farmers who had been spraying 2,4-D herbicide. More than half of these farmers displayed aberrations in the spontaneous electrical activity of the cerebral cortex and reticular formation. No irregularities were evident in the EEC of another patient exposed to 2,4-D (Berkley et al., 1963). Since only one individual was examined, however, this result should not be compared directly to the findings of Kontak et al. (1973). Although the study of Kontak et al. (1973) was not entirely conclusive, the findings indicate that 2,4-D may affect the normal functioning of the brain. 6-23 �Table 6-7 provides a brief overview of accidental exposures to 2,4-D, showing route of exposure, number of people examined, dose if known, and the main types of neuromuscular disorders that were evident in the individuals. The details of each accident, along with other health effects caused by the exposure, are described in the annotated bibliography. As seen in table 6-7, only two fatalities occurred. No clinical data were obtained prior to one of the deaths (Nielson et.al., 1965), but the other fatality was preceded by hyporeflexia and ataxia (Seabury, 1963). Histological examination of brain tissue from one of the fatalities revealed degenerative changes in the ganglionic cells of the pons (Nielsen et al., 1965). It is apparent that cutaneous, respiratory, or oral exposure to 2,4-D in humans produces a characteristic syndrome of neuromuscular disorders. The syndrome consists of hypesthesia and myotonia in the muscles of the extremities, hyporeflexia, and general muscular weakness leading to ataxia. These symptoms can appear singly or together, but usually at least one becomes evident within a few days of exposure. According to the reports, exposure to 2,4-D should not be fatal unless large quantities are absorbed cutaneously or the chemical is ingested. The exposed individual should regain neuromuscular control within a period of several months or a year (Wallis et al., 1970; Monnrca and Di Vito, 1961). The myotonic symptoms of 2,4-D poisoning in humans have been demonstrated to occur in experimental animals. Administration of various doses of 2,4-D produced myotonia in skeletal muscles of the rat (Danon, 1979; Eberstein and Goodgold, 1979; Ranish et al., 1977), dog (Drill and Hiratzka, 1953), and rabbit (Hill and Carlisle, 1947). Although the mechanism is not completely known, 2,4-D apparently increases the resting membrane potential in association with reducing chloride conductance (De Reuck et al., 1979). 2,4-D has produced the same neuromuscular disorders in humans as in animals. Pigs, calves, rats, and mice displayed symptoms of asthenia, lethargy, and ataxia when various 2,4-D compounds were administered intraperitoneally and orally (Hill and Carlisle, 1947; Bjorklund and Erne, 1966). Experiments have been conducted investigating the effect of 2,4-D on EEC patterns of the rat, cat, and dog. Several studies demonstrated that 2,4-D causes irregularities in the EEC of anesthetized and non-anesthetized animals (Desi et al. 1962 and 1962b; Desi and Sos, 1962a). The irregularities found were primarily a decrease in spontaneous cerebral electrical activity and a reduction or complete loss of desynchronization in the cerebral cortex and reticular formation. In addition, the animals experienced loss of a previously learned conditioned reflex. Histologic examination of the spinal cord of rats that had received doses of 2,4-D for 5 consecutive days revealed regions of demyelinization in the pyramidal tract (Desi et al., 1962a). The authors concluded that 2,4-D acts directly on the cerebral cells via the blood stream, to alter the electrical activity of the brain. They also stated that 2,4-D may cause demyelinization in the spinal cord, which could lead to serious neurologic complications. These studies are of limited value, as a complete description of the experimental procedures was not included in the reports. The use of control animals is not clearly stated and the purity and preparation of the 2,4-D 6-24 �Table 6-7: ACUTE HUMAN EXPOSURES TO 2,4-D Route Cutaneous Intravenous Cutaneous Oral Oral Oral Inhalation Dose t of people Asthenia Hyporeflexia Ataxia 1 X 1 X X — 1 X 1 X Rigidity, Myotonia Fatal X 3,600 mg Hypesthsia — 30 ml — 30 ml — Reference Todd (1962) X X Seabury (1963) X Foissac-Gegoux (1962) X X Berwick (1970) 1 X 1 X 1 X X X Brandt (1971) X Paggiaro et al. ( 9 4 17) X Prescott et al. (1974) X X Oral X X Dudley et al. (1972) Tsapko (1966) X Bezuglyi et al. (1979) N5 Cutaneous — 11 Cutaneous Cutaneous Cutaneous Cutaneous Cutaneous X several — — — — X X 1 X X 1 X X 3 X X 1 X X Monarca and DeVito (1961) X X X X X Wallis et al. (1970) X Goldstein et al. (1959) Berkley et al. (1963) �compounds are not detailed. In addition, the authors did not consider other physical, biological, and environmental factors that may have influenced the EEC readings from the animals. These factors include the age, sex, and health of the animals, species differences, diet, ambient temperature, and possible traumatic effects caused by surgical emplacement of the EEC electrodes. . In spite of these shortcomings, these experiments indicate that the EEC abnormalities observed in some humans exposed to 2,4-D (Kontak et al., 1973) may be similar to EEC patterns evoked in animals by administration of 2,4-D, It should b^ noted that 2,4-D may cause morphological damage in the central nervous system of humans and animals. Nielsen et al. (1965) found degenerative changes in ganglion cells of the cerebral cortex in a man who died from 2,4-D intoxication, and Desi et al. (1962a) found demyelinization in the spinal cord of animals. Both tentatively concluded that these morphological alterations may have been caused by 2,4-D. 6.5.2 2,4,5-T One study is available that discusses the neurologic and behavioral effects of 2,4,5-T in rats (Sjoden and Soderburg, 1978). Adult rats that received oral doses of 2,4,5-T displayed reduced learning abilities and decreased food and water intake. Neurochemical analysis of brain tissue showed a decrease in the concentration of several enzymes. Neonatal rats exposed prenatally to 2,4,5-T exhibited hyperactivity and impaired ability to learn a maze and a conditioned response. The authors concluded that exposure to 2,4,5-T produced significant behavioral and neurochemical effects. These conclusions, however, are not definitive, since the purity of the 2,4,5-T was not specified and control animals apparently were not used. The authors did not discuss other factors that may have influenced the results, particularly for the behavioral effects. They did not relate their findings to human health effects or to effects produced in animals by similar compounds such as 2,4-D. 6.5.3 TCDD One clinical study is available that discussed the neurological effects resulting from TCDD exposure in humans. Boeri et al. (1978) conducted neuro~ logic examinations of people exposed to TCDD as a result of the Seveso accident. Individuals from zone A (high risk of acute exposure) were examined and results were compared to individuals from zone R (low risk). The study populations included both adults and children. Individuals from both zones complained of numbness and weakness in their arms and legs, hyporeflexia, ataxia, and loss of coordination in the hands and fingers; nerve conduction velocities were also found to be reduced. Although people from both high- and low-risk zones exhibited these symptoms, a higher prevalence was observed in the high-risk population. The authors concluded that a higher prevalence of neuromuscular disorders was evident in the highrisk population because of a greater probable TCDD exposure. As described in chapter 5, other chemicals are likely to have been released in the Seveso accident, including chlorinated phenols and other components of the reaction mixture. 6-26 �Neuromuscular disorders described in the above report are similar to those described in reports of acute human exposure to 2,4-D. Hyporeflexia, general muscular weakness, ataxia, and loss of manual dexterity were the main symptoms of 2,4-D poisoning in humans (Goldstein et al., 1959; Berkley et al., 1963; Wallis et al., 1970). TCDD and 2,4-D apparently produce similar neuromuscular effects in humans, indicating that the mechanism of action may be similar. 6.5.4 Diquat Common symptoms of diquat poisoning include lethargy and coma (Thomas and Amor, 1968; Cobb and Grimshaw, 1979). Pupillary dilatation was also observed, in rats, along with a decrease in body temperature after subcutaneous administration of diquat (Clark and Hurst, 1970). 6.6 NUTRIENT ABSORPTION AND UTILIZATION This section considers herbicides that cause deficiencies in nutrient utilization manifested as decreased body weight. Effects on several other organ systems, whose functions are related to maintenance of body weight, are considered in this section. These systems include the gastrointestinal tract and the endocrine system. Molecular manifestations of altered nutrient utilization, including metabolism of lipids and proteins, are also discussed in this section. 6.6.1 2,4-D and 2,4.5-T Both of these compounds have been studied, sometimes in the same study, in terms of their effects on nutrient absorption and utilization. Since the amount of information on each compound is limited, the effects of both compounds will be described together in this section. 6.6.1.1 Body Weight and Food Consumption Anorexia has been described as a symptom of toxicity of 2,4-D and 2,4,5-T. Weight loss has been measured in only one study. After a single oral dose of 100 mg 2,4-D/kg was administered to dogs, weight losses of 1.7-2.5 kg were observed in four dogs; two of four dogs survived for 14 days, while two died at 9 days (the time after the dose that the weight losses were noted was not stated). Weight losses of 0.1 to 2.0 kg occurred in dogs fed 250-400 mg 2,4-D/kg, which produced 100 percent mortality in 8 days. A single dose of 100 mg 2,4,5-T/kg produced weight losses of 0.4-1.0 kg and 75 percent survival, while higher doses of 250-400 mg/kg produced 1.1-1.4 kg weight losses and 100 percent mortality. 6-27 �6.6.1.2 Effects on the Gastrointestinal Tract In a brief report, a single oral dose of 100 mg 2,4,5-T/kg was described as having a stimulatory effect 24 hours later on mucosal uptake of iron, increasing this uptake by 8 percent in duodenal gut sacs in vitro. The authors pointed out the potential for this effect to produce adverse effects on iron absorption and metabolism. The implications of this effect on nutrient transport by the gut was not discussed and the dioxin content of the 2,4,5-T preparation was not reported (Manis and Kim, 1979). 2,4,5-T had no effect on glucose transport by the mouse intestine in vitro. However, the amount of glucose associated with the gut tissue was increased in the presence of 2,4,5-T in the culture system (Guthrie et al., 1974). These results do not support the concept that weight loss in 2,4,5-T-treated animals results from an effect on nutrient uptake by the gastrointestinal tract. 6.6.1.3 Effects on the Endocrine System The uptake of I by various tissues was evaluated in rats after a single oral dose of 100 mg 2,4,5-T/kg (with less than 1 ppm TCDD) was administered. A marked decrease in serum radioactivity and increases in thyroid and liver levels were observed within 3 days of 2,4,5-T treatment, although the amount of tissue uptake was inadequate to explain the loss in serum radioactivity. The authors presented these data as evidence that 2,4,5-T alters the permeability of cellular membranes, and suggested the thyroid, brain, and kidney as potential sites where deleterious effects from these changes could occur (Sjoden et al., 1977). 6.6.1.4 Alterations in Lipid Biosynthesis and Mitochondrial Respiration Both 2,4-D and 2,4,5-T inhibited incorporation of radioactive precursors into cholesterol and fatty acids, in vitro. At levels of 4-9 mM,.2,4-D and 2,4,5-T (with less than 1 ppm dioxin) inhibited incorporation of C-mevalonate into cholesterol and. nonsaponifiable lipids, C-acetate into fatty acids and cholesterol, and C-isopentenyl pyrophosphate into cholesterol by rat liver homogenates. The authors indicated that these effects may not have any toxicological significance, however (Olson et al., 1974). -4 At 10 M, both 2,4-D and 2,4,5-T produced uncoupling effects on rat hepatic mitochondrial respiration and reduced the respiratory control index (the ratio of succinate oxidation in the presence of ADP to the rate obtained after the acceptor is exhausted). The authors suggested that these effects are related to potential mechanism of actions of these compounds in plants and animals, but did not indicate what types of lesions would be produced from these biochemical changes (Abo-Khatwa and Hollingsworth, 1974). 6.6.2 TCDD The effects of TCDD on nutrient utilization have been the focus of many studies. TCDD has been shown to produce severe weight loss in the animal 6-28 �studies described in this section. The mechanisms by which TCDD produces this effect are unknown. Changes that have been investigated in other organ systems which could ultimately produce this effect are considered below. These changes involve nutrient absorption by the gastrointestinal tracts and changes in endocrine organs that participate in regulating nutrient utilization. Finally, biochemical events related to nutrient utilization are considered here which relate to changes in circulating metabolites of protein or lipid catabolism. 6.6.2.1 Effects on Body Weight and Food Consumption An acute dose of TCDD produces weight loss in a variety of species, including the monkey, rat, mouse, guinea pig, rabbit, hamster, and chicken (Greig et al., 1973; Harris et al., 1973; McConnell et al., 1978a, 1978b; Luster et al., 1978; Vos et al., 1974, Olson et al, 1980). Weight loss is not a prominent feature in man and was seldom mentioned in workers after industrial exposure; in humans weight loss has sometimes been implied to be a consequence of anorexia, a neurologic disorder. In animals, weight loss, accompanied by depletion of fat deposits and the inability to utilize ingested nutrients, has been identified as the lesions responsible for death. In man, however, exposure to a lethal dose of TCDD has not been reported. Several investigators have observed a biphasic pattern to the weight loss that results from a single exposure to TCDD. In the monkey, guinea pig, and mouse, an initial period of weight loss occurred for 7-10 days after TCDD was given, followed by a recovery in non-lethal poisonings or a second period of weight loss in lethal poisoning (McConnell, 381). Initial weight loss for 7-10 days after 100 ug TCDD/kg was administered to rats resulted in a decrease in body weight of 15-30 percent (Courtney et al., 1978). In the next 4-6 days this trend was reversed and 10-15 percent of the initial body weight was gained back. In lethal poisoning a second period of weight loss ensued and terminated in death. Food and water consumption were decreased in these rats, but forced administration of a balanced liquid diet, water, or an electrolyte solution failed to reverse the weight loss or death. In another experiment, TCDD-treated rats lost more of their body weight than was lost by pair-fed control rats (van Logten et al., unpublished, mentioned by Moore, 1978). Lethal doses of TCDD have caused terminal weight losses of 15-50 percent in guinea pigs (McConnell et al., 1978b; Greig et al., 1973). At death, the gastrointestinal tract was empty and food consumption fell only in the terminal stages. Loss in body weight has been reported at doses that do not cause any other signs of toxicity in the guinea pig, indicating that it may be the most sensitive feature of toxicity in this species. Weight losses in the guinea pig, rat, and mouse are transient after nonlethal acute doses of TCDD (Harris et al., 1973; Vos et al., 1974). Weight losses of 13-38 percent in monkeys and in mice have been reported following a lethal dose of TCDD (McConnell et al., 1978a; Luster et al., 1978). Food consumption has been variable and occasionally has shown a trend toward decreased consumption. These trends have not been significantly different between TCDD-treated and control groups, however, and the decreases 6-29 �have not been large enough to account for the substantial weight losses observed (Greig et al., 1973; Harris et al., 1973; McConnell et al., 1978a; 1978b). Weight losses from TCDD treatment occur in rats after the adrenal or the pituitary gland is removed, indicating that these organs are not essential in mediating the toxic effects of TCDD (van Logten et al., 1980). 6.6.2.2 Effects on the Gastrointestinal Tract Studies of gastrointestinal function have been carried out in rodents after an acute dose of TCDD was administered. Transport of iron was shown to increase in vivo and in vitro (Mania and Ap*ap, 1979; Manis and Kim, 1979a; I979b). In another study glucose transport was depressed, while transport of other nutrients was unaltered (Madge, 1977). An increased sensitivity of crypt cells compared to tip cells of the intestine to TCDD induction of enzyme activity has led to the suggestion that undifferentiated cells have high sensitivity to TCDD-mediated effects (Schiller, 1979). However, decreased ability of these cells to transport nutrients was not evaluated. The importance of1 decreased glucose transport across the gut or nutrient utilization after TCDD treatment depends on whether this transport system is the limiting factor in supplying nutrients to organs. No evidence of this. limiting role has been reported, and no strong evidence exists at present to suggest the contention that TCDD prevents nutrient utilization by blocking intestinal absorption of nutrients. 6.6.2.3 Lipid and Protein Metabolism Several studies have considered the effect of TCDD treatment on protein and lipid biosynthesis. Radioactive precursors of lipids and protein were administered to TCDD-treated rats and the incorporation of these precursors into macromolecules in the liver was examined. Protein synthesis increased, while lipid biosynthesis appeared to decrease. However, concomitant changes in liver weights and specific activity of the radioactive precursors, as well as the use of inadequate techniques in quantitatively isolating pools of specific biochemicals, complicates the interpretation of these results. The authors concluded that TCDD caused an inhibition in the secretion of lipids from the liver (Cunningham and Williams, 1972). In an abstract, Lovati et al. (presented at Workshop, Impact of Chlorinated Dioxins and Related Compounds on the Environment, Rome, October 22-24, 1980) reported that an acute dose of TCDD in rats resulted in elevated total plasma and high-density lipoprotein cholesterol levels. Hypercholesterolemia has been reported in humans after industrial and laboratory exposures, as well (see chapter 5 of this report). Lovati et al. also reported that TCDD treatment resulted in increased triglyceride levels in rabbits. However, a group of rabbits that became hypercholesterolemic by chronic cholesterol administration did not have altered triglyceride levels after TCDD was administered, but had severe atheromatous lesions. 6-30 �Other investigators (Walker and Martin, 1979) have suggested that TCDD induces gamma-glutamyl transpeptidase activity which leads to abnormal lipid levels and predisposes exposed workers to ischemic vascular disease. They examined eight workers who were exposed to dioxins and found clinical signs of ischemic vascular disease in several workers, and raised triglyceride and transpeptidase levels in five workers. All eight workers had decreased levels of high-density lipoprotein cholesterol levels and elevated total cholesterol levels. Acute TCDD treatment has produced changes in serum protein and lipid levels, although these changes have not been reported frequently and occur with high doses of TCDD. Changes in serum protein levels have been observed in rats after an acute dose of 200 ug TCDD/kg was administered (Greig et al., 1973). In monkeys a decrease in serum protein 30 days after an acute lethal dose of TCDD was shown to be caused specifically by a decrease in albumin. Serum cholesterol levels progressively fell to half their normal value at death, while serum triglycerides increased (McConnell et al., 1978a). Increased serum protein and cholesterol levels, and decreased serum triglyceride levels have been observed in hamsters administered doses of 1,000 ug/kg orally or intraperitoneally (Olson, et al., 1980). In the guinea pig, increased plasma levels of albumin, total protein, cholesterol, and triglycerides were observed after a 1 ug/kg intraperitoneal dose. These levels were increased above pair-fed controls, suggesting that the effect is hot simply a manifestation of the effects of TCDD on fat depot utilization (Gasiewicz and Neal, 1979). Both the presence of fatty acids and a deficiency in vitamin A have been suggested as mechanisms by which TCDD produces chloracne (see Section 6.2 of this report). The relationship between alterations in lipid metabolism by TCDD and the severe weight loss in experimental animals is speculative at this point. There is no compelling evidence to suggest that animals eat less or fail to absorb nutrients. TCDD is known to produce substantial chronic induction of enzymes and to alter parameters related to lipid biosynthesis. Investigations into the nature of this relationship may provide a basis for our understanding of weight loss, which has been implicated as the fatal lesion in some of the species most sensitive to TCDD. 6.6.3 Diquat Loss in body weight has been reported following acute doses of diquat (Clark and Hurst, 1970). Food consumption has not been measured, although death does not occur in some animals for 14 days. Severe histopathologic changes of the gastrointestinal tracts, diarrhea, and alterations in corticosteroid levels all probably contribute to this weight loss. Neurological abnormalities, especially lethargy, that occur in these animals, could also contribute to the weight loss. 6.6.3.1 Effects on the Gastrointestinal Tract The major histopathological changes seen after fatal diquat poisoning involve the gastrointestinal tract. Distension of the gastrointestinal tract, 6-31 �with necrosis, exfoliation of the epithelium, and cellular infiltration were observed in monkeys (Cobb and Grimshaw, 1979). Distended abdomens were also observed in rats and other laboratory species after diquat poisoning (Clark and Hurst, 1970). In cattle, congestion, edema, and inflammation were observed in the gastrointestinal tract (Thomas and Amor, 1968). Alterations in gastrointestinal function have been observed after diquat administration to rats. A single oral dose of diquat of 900 uM/kg (the LD-Q) caused a rapid accumulation of water in the stomach and, associated with it, hemoconcentration. Rats that failed to gain weight in the 24-hour period after diquat administration usually died within 3 days of dosing, while survivors showed a 3 percent increase in body weight. Fluid accumulation in the stomach was delayed and less extensive after subcutaneous dosing. Prolonged inhibition of gastric emptying was observed after diquat administration by either route. Fluid accumulation was not considered to be a consequence of inhibition of gastric emptying because accumulation did not occur when diquat was administered subcutaneously to rats, following pyloric ligation (Crabtree, et al., 1977; Crabtree and Rose, 1978). 6.6.3.2 Effects on the Endocrine System Diquat administration causes alterations in circulating corticosteroid levels in the rat. A large increase in plasma corticosteroid levels and increased AMP levels in the adrenals were observed 24 hours after diquat was administered. The increased plasma corticosteroid levels were assumed to result from increased synthesis, because metabolism and excretion were unaltered by diquat treatment. These increases were not observed after diquat treatment to hypophysectomized rats. Increases in liver glycogen synthesis and blood glucose levels after diquat treatment were blocked by adrenalectomy. The authors concluded that diquat stimulated ACTH release from the pituitary, which led to increased synthesis of corticosteroids by the adrenal. Free radical generation was not considered the likely mechanism by which diquat elicited this effect. The high circulating corticosteroid levels that diquat elicits could be responsible for the changes in the thymus, spleen, and adrenals observed by other investigators (Rose et al., 1974; Crabtree and Rose, 1976). 6.7 HEMATOLOGICAL EFFECTS In this section, effects on blood components are described. Effects of herbicides and TCDD on the levels of circulating blood cells and bone marrow components as well as effects on thrombosis, incidents of hemorrhages, and changes in hemoglobin are considered here. 6.7.1 2,4-D Kuz'minskaya and Bersan (1975) reported that a single dose of 2,4-D (1/2 the LD ) produced enzyme changes in erythrocytes. The rate of glycolysis doublea and ATPase activity increased by 50 percent 5 days after dosing; the 6-32 �levels were normal 15 days after dosing. Corresponding compromise in the physiologic function of erythrocytes was not studied, raising the question as to the toxicologic significance of these findings. Other hematologic effects of either 2,4-D or 2,4,5-T have not been described. 6.7.2 TCDD Acute doses of TCDD have produced adverse effects on the hematopoietic system in the monkey, rat, guinea pig, and mouse. HypoceLlularity of the bone marrow has been observed in the monkey (McConnell et al., 1978a) and guinea pig, but not in the mouse (McConnell et al., 1978b). In the monkey, a relative increase in myeloid elements and a decrease in erythroid elements were observed in sternal bone marrow, while the lymphoid component was unchanged. Pancytopenic depletion was reported in the sternal bone marrow of guinea pigs, although cells were not counted. Increases in red blood cell count and in hematocrit and hemoglobin content of the blood were reported in female rats 2-3 weeks after a single dose of 200 ug/kg was administered (Greig et al., 1973). Leukocytosis was also reported in these animals and in monkeys, due to an increase in neutrophils (McConnell et al., 1978a). Hemorrhages have been observed in animals given an acute dose of TCDD. Death of a monkey 14 days after 70 ug TCDD/kg had been administered was attributed to bleeding (McConnell et al., 1978). Hemorrhages in the gastrointestinal tract have been observed in the mouse, rat, and guinea pig (McConnell et al., 1978b, Vos et al., 1974, Greig et al. , 1973). Pulmonary bleeding also occurred in the rat and intraorbital bleeding and severe splenic atrophy were observed in the mouse. 6.7.3. Diquat Acute administration of diquat to rats produced hemoconcentration. This effect was attributed to an accumulation of fluid in the stomach after oral treatment (Crabtree et al., 1977) and an alteration in renal hemodynamics (Lock, 1979). In monkeys an increase in the polymorphonuclear leukocyte count was observed. This effect was transient in monkeys that survived, but persisted in monkeys that died (Cobb and Grimshaw, 1979). Cardiac hemorrhages were observed in cattle that consumed a fatal dose of diquat (Thomas and Amor, 1968). 6.8 STRUCTURE AND FUNCTION OF LYMPHATIC TISSUES Effects on the structure of the thymus and other lymphatic tissues and on the immunologic function of these tissues are described in this section. Most of the studies in this section investigated effects of TCDD. 6-33 �6.8.1 TCDD Thymic atrophy has been reported in many species from administration of a single dose of TCDD. No changes in thymic function or structure have been reported in man. After a lethal dose of TCDD was administered to monkeys, almost complete loss of the cortex of the thymus was observed at necropsy. Other lymphoid tissue also showed losses of lymphocytes, and lymphopenia was observed in the circulating blood one month after TCDD treatment (McConnell et al., 1978a). In guinea pigs, early changes in the thymus included scattered necrosis of lymphocytes of the cortex during the first two weeks after treatment, but was no longer observed by the third week. The cortex and medulla were not differentiable by the second week. The thymus of guinea pigs that survived for one month was normal histologically, but reduced in size. Lymphocytes were reduced in other lymphoid tissue. Changes in mice resembled those observed in guinea pigs (McConnell et al., 1978b). A dose of 3 ug TCDD/kg caused severe thymic atrophy and death in the guinea pig, while a single dose of 10 or 50 ug/kg to mice produced a transient decrease in thymic weight (evident only at 3 weeks after dosing) and is not lethal (Harris et al., 1973). Thymic atrophy, loss of differentiation between the thymic cortex and medulla, and lymphocyte depletion were also reported in mice by other investigators after an acute dose of TCDD was administered (Vos et al., 1974). In the rat, a significant decrease in the weight of the thymus relative to body weight was observed 2 weeks after a single dose of 5 ug TCDD/kg was administered. This effect was transient and was not accompanied by a change in liver weight. A single dose of 25 ug/kg produced a more sustained decrease in thymic weight that was significant 3 days after dosing and was at its maximum 16 days after dosing (Harris et al., 1973). Thymic involution occurred in adrenalectomized rats after a single dose of 10 or 20 ug TCDD/kg was administered. Thymic involution from these doses of TCDD was more severe in hypophysectomized rats than in rats with intact pituitary glands. These results suggest that the adrenal and pituitary do not have essential roles in modulating the effect of TCDD on the thymus (van Logten et al., 1980). Several studies have investigated the effects of brief exposure to TCDD on lymphocyte function. In one study, splenic lymphocytes from mice that received a single dose of 10 ug TCDD/kg were evaluated in vitro for,their ability to incorporate H-thymidine. Two weeks after treatment, H-thymidine uptake was increased in the.absence of mitogens. With phytohemaglutanin or poke weed mitogen present, H-thymidine uptake was increased above nonmitogen-treated cultures. This response to mitogen was less for TCDD-treated lymphocytes than for control lymphocytes, partly because uptake with mitogens present was expressed relative to uptake without mitogens, and TCDD increased this last parameter. Other changes observed two weeks after treatment were a reduction in the size of the thymic cortex and an increase in liver weight. None of the in vivo or in vitro changes were observed four or eight weeks after treatment. Splenic lymphocytes from control mice incubated in the presence of TCDD failed to duplicate the effects of lymphocytes exposed in vivo; cells treated in vitro showed a cytotoxic response to TCDD and failed to respond to mitogens (Sharma and Gehring, 1979). 6-34 �A second study of lymphocyte function examined the effects of brief submersion of mouse spleens in TCDD on lymphocyte responsiveness to mitogens. Submersion for 10 seconds in 2 x 10 M TCDD in DMSO resulted in uptake of 0.2 ng of TCDD into the spleen. Lymphocytes from the submerged spleen showed decreased incorporation of radioactive precursors into RNA, DNA, and protein. TCDD treatment also diminished DNA, RNA, and protein synthesis of mitogenstimulated lymphocytes compared to DMSO-treated controls. The authors noted that these changes were more marked in the presence of T-lymphocyte specific mitogens (phytohemaglutanin and concanavalin A) than B-lymphocyte-specific mitogens (E_. coli lipopolysaccharide), indicating that T-lymphocytes were more susceptible to TCDD than B-lymphocytes (Luster et al., 1979). The differences between responses, after different mitogens were added, actually were not large or consistent, and further experimentation would be needed to validate this difference in responsiveness between B- and T-lymphocytes. The authors found no effect of TCDD on H-concanavalin A binding to lymphocytes or precursor incorporation in the absence of mitogens. Mantovani et al. (1979) examined the effect of TCDD treatment on the cytotoxic activity of macrophages and of natural killer cells in vitro. The natural host-defense mechanisms of these cells were not altered by TCDD pretreatment in vivo. The pretreatment did lead to a reduction in the total numbers of spleen and peritoneal cells recovered, and to marked hypocellularity of the bone marrow. The cell-mediated immunity was thus compromised in that fewer cells were on reserve to fight infectious agents. The circumstances which would require these reserves are unknown. In conclusion, TCDD produces severe thymic atrophy, and this effect can be produced from a single dose of TCDD in every laboratory species examined. In the rat decreased thymic weight was seen at a dose that produced no other effects. The effect is not mediated by hormones released from the adrenal or pituitary, and the mechanism of action remains unknown. The implications of these structural changes on functional compromise of the thymus are not obvious. Immunocompetence has not been evaluated after acute doses of TCDD, although TCDD had the same lethal effects on aseptic rats as it did on rats housed in a normal environment (Greig et al., 1973), indicating that decreased resistance to infection is not the only cause of death from TCDD. Death has been attributed to pulmonary infections in rats on occasion, but many other causes of death have been noted in TCDD-treated animals. Functional compromise of the thymus or lymphocytes derived from the thymus does not appear to be as severe as the structural changes seen in the thymus, but the in vitro tests used to evaluate lymphocyte function may not have evaluated the parameters of immune function that are most sensitive to TCDD. Other studies of immune function have been performed after subacute doses of TCDD were administered (see chapter 7). These studies have identified an increased susceptibility of immature mice to the effects of TCDD on the immune system and to bacterial endotoxin after TCDD treatment. These studies have not identified effects on the immune system that are comparable in severity to the effects on thymic structure, nor effects that would account for the lethal effects of TCDD. 6-35 �No observations of thymic involution after human exposure to TCDD have been reported. Human sensitivity to this effect may be less than in other species and may be age-dependent. At the present time, however, one cannot rule out the possibilities that thymic effects were too subtle or too difficult to diagnose in patients or that the doses of TCDD required to produce this effect were not reached in cases where thymic atrophy could be evaluated. 6.8.2 Diquat Acute subcutaneous doses of diquat to rats produced large reductions in organ weights and lymphocyte depletion in the cortex of the thymus and in the spleen (Clark and Hurst, 1970). Rose et al. (1974) suggested that these changes were a result of elevated plasma corticosteroid levels from diquat administration (see Section 6.6.3.2). 6.9 RENAL EFFECTS The effects of herbicides and TCDD on the structure and function of the kidney are described in this section. Renal excretion of the herbicides are described in chapter 4. 6.9.1 2,4.5-T The effects of 2,4,5-T on renal function have been reported. A single acute dose of 100 mg 2,4,5-T/kg has been shown in rat cortical slices to lead to a decrease in para-aminohippurate (PAH) accumulation (by 58 percent) and in tetraethylammonium (TEA) accumulation (by 34 percent) 24 hours later. A dose of 20 mg/kg had no effect after 24 hours, although after 4 hours it produced a 51 percent decrease in PAH accumulation into slices and a 24 percent decrease in PAH clearance in vivo (Stroo et al., 1979). 2,4,5-T also produced a decrease in renal transport of 2,4-D (Koschier and Berndt, 1976). 2,4,5-T did not alter N-methylnicotinamide efflux, however, and 2,4,5-T was not itself transported by the renal organic base secretory mechanism. The authors suggested that 2,4,5-T interference with organic transport is related to its high degree of binding to renal cortex tissue, and eliminated an effect of 2,4,5-T on tissue oxygen consumption (Koschier and Berndt, 1976; Koschier and Berndt, 1977). 6.9.2 TCDD Effects of TCDD on the kidney have been reported for the rat and have been attributed to a general toxic effect of TCDD, rather than a specific lesion on the kidney. Renal cortical slices from rats treated intraperitoneally with 25 ug TCDD/kg 1 week prior to the in vitro experiment showed decreased uptake of para-aminohippurate and N-methylnicotinamide (Hook et al., 1978), but not of deoxyglucose (Pegg et al., 1976). Decreased ammoniogenesis and gluconeogenesis were also observed in renal slices from chronically acidic 6-36 �rats after TCDD treatment (Hewitt et al., 1976). In vivo renal clearance of para-aminohippurate and inulin were also decreased one week after an intraperitoneal dose of 25 ug/kg of TCDD was given. No changes in the fractional reabsorption of sodium or of the renal response to volume expanders was observed and the authors concluded that the observed changes were not specific for renal function (McCormack et al., 1976). Decreased renal DM synthesis was observed after TCDD treatment of rats that were given folate or lead to simulate DNA synthesis by the kidney. TCDD treatment reduced the effects of lead and folate on renal DNA synthesis. The authors considered this effect of TCDD to be indirect, resulting from altered levels of inducible renal and hepatic enzymes (Greig et al., 1974). 6.9.3 Diquat Severe renal changes have been observed in animals after diquat administration. In the monkey, exfoliation, vacuolation, and pycnotic nuclei were observed in the epithelial cells of the proximal and distal convoluted tubules, along with congestion of the glomeruli and hyperemic papillae. These changes were most severe in a monkey that died one day after diquat treatment. In animals that died after three to four days, these changes appeared to be regressing (Cobb and Grimshaw, 1979). No changes were observed in the kidneys of cows that died from diquat poisoning (Thomas and Amor, 1968) or in laboratory animals (Clark and Hurst, 1970; Burk et al., 1980), except in selenium deficient rats who also displayed hepatic lesions and high rates of peroxidation in response to diquat administration (see section 6.4.4). Renal function was diminished after diquat treatment to rats. Glomerular filtration rates and clearances of acidic and basic compounds were reduced 24 hours after 540 uM/kg was administered orally (Lock, 1979). A 680 uM/kg dose produced proteinuria and glucosuria, and minor biochemical changes (Lock and Ishmael, 1979). 6.10 CARDIOVASCULAR EFFECTS A low incidence of cardiovascular effects was observed after occupational exposure to TCDD in the early 1950s in Germany (Bauer et al., 1961). Acute exposure of the rat to a massive dose of TCDD also caused cardiovascular effects (Buu-Hoi et al., 1972). A clear cause-and-effect relationship between TCDD and cardiovascular disorders has not been convincingly demonstrated and these disorders may be elicited only after a substantial acute dose or after chronic doses are administered. 6;11 SUMMARY AND CONCLUSIONS Single oral doses of about 350-800 mg of either 2,4-D or 2,4,5-T per kg are lethal to most species. Few accidental lethal ingestions of 2,4,5-T by animals or humans have been reported. In experiments in which fatal single doses were administered, a variety of nonspecific symptoms were produced. The cause of death is not always apparent. LD,-0 values given for 2,4,5-T in the 6-37 �literature were published almost 30 years ago, when the possibility of TCDD contamination was not taken into account. The actual LD.,, values may be higher than the published values. About 15 accounts of human ingestion of 2,4-D have been reported. These ingestions involved accidental or suicidal use; cases of injection for therapeutic use have also been reported. The principal effect of these acute doses has been neurotoxicity. Acute toxicity in animals also involves neurotoxicity. Death is delayed, usually occurring within a week of administration. The cause of death is not usually apparent, as symptoms of neurotoxicity are usually accompanied by many non-specific effects. The acute toxicity of TCDD is characterized by: • Extremely low LDcn values (between 1-300 ug/kg) • Large variation in LD5f)s among species • Long latency period to death, usually about 3 weeks • Cellular toxicity, with no clear cause of death and different target organs for different species. The LD for diquat is between 30 and 200 mg/kg for all mammalian species studied. Doses in this range produce severe gastrointestinal lesions and death occurs within 2 weeks. Doses four- to fivefold higher produce neurotoxicity, and death occurs in several hours. Toxicity data for the remaining compounds are sparse and for tandex have not been published. Reported oral LD,Q values for cacodylic acid range from 600-3,200 mg/kg in rats and 200 mg/kg in mice; reasons for the wide variation in reported values for the same compound and species have not been identified. Various organs are affected by cacodylic acid and death occurs within 2 weeks. The oral LD_ n s for both monuron and diuron are about 1,000 mg/kg. Both compounds produce neurotoxicity, including central nervous system depression followed by stimulation. Death occurs 1 day after exposure from respiratory or cardiac failure. Picloram and dalapon have low toxicities, with oral LD^s ranging from 2,000-8,000 mg/kg. Fatal doses of dalapon cause death within several hours. The latency for picloram, and the cause of death and target organs for both compounds have not been described. TCDD is the only compound of those covered in this report that produces dermal toxicity. Chloracne has been demonstrated in man and in several animal species including the rabbit, which provides a suitable animal model. Man appears to be less susceptible to the acnegenic effects of TCDD than the rabbit. Chloracne is not necessarily a local response to TCDD, as chloracne is produced following systemic administration. Pulmonary lesions are not produced by any of the herbicides under study. Differences in distribution to 6-38 �the lung of diquat and its structural analogue, paraquat, explain the failure of diquat to produce pulmonary lesions characteristic of paraquat poisoning. TCDD produces hepatic necrosis, lipid accumulation, hepatitis, and cholestasis. The nature and frequencies of these effects following lethal doses vary for different species, from being of minor importance to being lethal. Weight loss is a common symptom for most of the compounds studied but its cause cannot be explained for any of the compounds. In cases of TCDD poisoning, biochemical lesions involving lipid metabolism may cause this effect, and in diquat poisoning severe gastrointestinal lesions may prevent nutrient utilization. Various hematologic effects of TCDD have been reported. These effects vary with dose, species, and time after dosing, suggesting the likelihood that they are secondary effects. Thymic atrophy is the only lesion of TCDD that is seen among all mammalian animal species studied. Despite the severity of this lesion, no correspondingly severe loss in immune function seems to occur. Diquat also produces atrophy of lymphatic tissue, which may be secondary to changes in ACTH and circulating corticosteroids. Renal effects after treatment with 2,4-D, 2,4,5-T, and TCDD involve decreased renal function. These effects have not been shown to have toxicological significance, except at very high doses when elimination of the administered herbicide is impaired. 6-39 �CHAPTER 6. REFERENCES Abo-Khatwa N., and Hollingworth, R. M. (1974) Pesticidal chemicals affecting some energy-linked functions of rat liver mitochondria in vitro. Bull. Environ, Contain, Toxicol. 12(4) :446-454. Backstrom, J. The phenoxy acid problem in Sweden. In Chlorinated Phenoxy Acidls and Their Dioxins, ed., C. Ramel (Ecol. Bull. no. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 108-121. Baker, D. L., Ramsey, F. K., and Sylvester, E. P. (1953) Suspecting poisoning of dogs from eating grasses treated with 2,4-D. North Am. Vet. 34:194. Bastomsky, C. H. (1977) Enhanced thyroxine metabolism and high uptake goiters in rats after a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Endocrinology 101:292-296. Bauer, H., Schulz, K. H., and Spiegelberg, V. 18:538-555. (1961) Arch. Gewerbepath. Berkley, M. 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(1976) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on plasma disappearance and biliary excretion of Ouabain in rats. Pharmacologist 18(2):246. Young, A. L., Calcagni, J. A., Thalken, C. E., and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. 6-54 �CHAPTER 7 SUBACUTE AND CHRONIC TOXICITIES Studies described in this chapter include reports of the effects of herbicides and TCDD exposure after more than one dose has been administered. In this chapter, subacute studies refer to studies that involved administration of more than one dose but less than chronic exposure, while chronic exposure refers to administration of compounds over a substantial proportion of the expected lifespan for that species. In animals, chronic studies usually involve exposure for at least 1 year, and in human studies industrial exposures for 20 years have been considered chronic exposures. In most of the subacute studies described below, the same parameters have been considered which were described for acute studies in chapter 6. For this reason, the present chapter is organized in the same format that was used for chapter 6. Oncogenicity has been addressed in most of the chronic studies, and these studies are considered in chapter 10 in detail and are mentioned here regarding other toxic effects. Descriptions of the potencies of herbicides and TCDD in producing mortality and the target organs affected are described in the first section of this chapter. In the remaining sections, effects of herbicide or TCDD exposure on specific organ systems are described. Only compounds that have been reported to affect a particular organ system are referred to in these sections. 7.1 MORTALITY In this section, the subacute and chronic dosage regimens that have produced lethal effects are described. Nontoxic dosage regimens are also mentioned. The organ systems affected by each compound and the causes of death are discussed here, as well. Reviews of the subacute and chronic toxicities of 2,4-D and 2,4,5-T have been published (Esposito et al., 1980; NRC, 1974; IARC, 1977; Young et al., 1978; Dalgaard, Mikkelsen, and Poulsen, 1962; President's Scientific Committee, 1972). 7.1.1 2,4-D Effects of human exposure to 2,4-D have been described and are considered in this chapter. These exposures resulted from occupational exposures in the manufacture or application of 2,4-D. Absorbed doses were not established in any of the studies. Subacute and chronic studies of 2,4-D have been carried out in several species. References on the subacute effects of 2,4-D are listed in table 7-1. 7.1.1.1 Ineffective and Lethal Dosages 2,4-D does not appear to be a cumulative toxicant in most species (Rowe and Hymas, 1954; Bjorn and Northen, 1948), although low doses may be cumulative in the dog. Oral doses of 100 mg/kg were lethal in the rat when administered daily for 10 consecutive days, but not when administered 5 days �TABLE 7-1. Route Species Dose REFERENCES ON THE SUBACUTE AND CHRONIC TOXICITIES OF 2,4-D Durat ion Organs Affected or Studied: Skin gavage .12 125 mg/kg 80 rag/kg/d 2-5 g/kg 1 rao. NS several wks 4-7 wk. Mouse NS 1/3-1/5 LD5Q oral, inh. Ig total 12 d. Rabb it dermal Dog iv oral 25 rag/kg 2-20 mg/kg oral oral Pig Cattle X X X 2x/d.; 3 wk-3 mo. Guinea Pig Liver Rat gavage NSa EC Sheep Kidney Neural Muse. Blood X X CV Reference Lungs Gut Body Wt X X Hill and Carlisle, 1947 Szocs et al., 1970 Florsheim and Veloff , 1962 Chang et al. , 1974 X Bucher, 1946 X X X X X X X X X 15x X X X X X 6x 13 wk. X X X X X X X X 18 mg/kg/d 120 d. 100-500 mg/kg/d 48 Ix X X X X X X oral 50 mg/kg 2-5 Ix X X X X X X X Biorklund and Erne, 1966 oral 50-250 mg/kg/d 1-112 X X X X X Palmer, 1963; and Palmer and Radeleff, 1964 NS, not stated; X X X Hill and Carlisle, 1947 X X X Kay et al., 1947 X Hill and Carlisle, 1947 Drill and Hiratzka, 1953 X x' X X X X Shavgulidze et al., 1976 Palmer and Radeleff, 1964 CM I �per week for 4 weeks. Daily doses of 75 mg/kg or lower produced no toxic effects. In one study (Hansen et al., 1971), rats fed diets containing 1,250 mg 2,4,-D per kg for 2 years exhibited no toxic effects, while in two other studies (Rowe and Hymas, 1954; Hill and Carlisle, 1947) slight toxicity was observed in rats fed 1,000 mg/kg feed for 30-113 days. In these studies, doses below 1,000 mg/kg feed were ineffective, and doses above 1,250 mg/kg feed or 1,000 tag/liter drinking water were toxic, but not lethal (Chang et al., 1974; Bjorklund and Erne, 1966). In most species studied, repeated oral doses of 100 mg/kg/day of 2,4-D at levels of 1,000 ppm or higher produce toxicity, while lower doses are generally ineffective. Oral doses of 2,4-D were also administered to the dog and guinea pig. Oral doses of 20 mg/kg 2,4-D 5 days per week for 13 weeks were lethal to the dog, while 10 mg/kg doses were ineffective (Drill and Hiratzka, 1953). Dogs fed 500 mg 2,4-D per kg feed for 2 years also showed no adverse effects (Hansen et al., 1971). The effect of 50-100 mg of 2,4-D administered orally to guinea pigs for 10 days was difficult to evaluate because of unexpected mortality in the control groups (Hill and Carlisle, 1947). In sheep, oral doses of 18 mg/kg/day for 120 days produced subtle changes on serum and liver enzymes (Shavgulidze et al., 1976). Doses of 100 mg/kg/day for 481 days produced no overt signs of toxicity, while doses of 250 mg/kg/day were lethal to sheep (Palmer and Radeleff, 1964). Cattle showed no effects from 112 doses of 50 mg/kg/day, slight toxicity from 88 doses of 100 mg/kg/day and lethality at 200 mg/kg/day after 44 doses (Palmer, 1963; Palmer and Radeleff, 1964). Only histopathological signs of toxicity were observed in pigs that were administered 300 mg/kg or diets containing 500 ppm 2,4-D for up to 12 months (Bjorklund and Erne, 1966). Other routes of administration that have been employed in subacute 2,4-D studies include subcutaneous injections to mice, dermal applications to rabbits, intravenous injections to dogs, and inhalation exposure of guinea pigs (see table 7-1.) The effects of these experiments were not compared to effects of similar doses that were administered orally, precluding the determination of effects of route on 2,4-D toxicity. 7.1.1.2 Cause of Death and Target Organs The health effects of occupational exposure to 2,4-D have been addressed in several studies. Festisov (1966) reported on effects of 2,4-D in workers. A group of 150 Russian workers was examined. These workers were between 18 and 47 years old, and were occupationally exposed to 2,4-D manufacture or application for 2-10 years. Symptoms reported by the workers were fatigue, headaches, abdominal pain, poor appetite, and impairment of sensory perception. The frequency of each complaint was not reported. Physical examinations were performed on another group of 292 Russian workers, comprised primarily of men between 21 and 40 years of age, exposed for up to 10 years (Bashirov, 1969). Complaints of fatigue, headache, dizziness, sweating, irritability, insomnia, and digestive disorders were reported by workers. Hypertension was detected in 20 percent of the workers and functional (asthenic) disorders of the autonomic nervous system in 61 percent. In a selected group of 50 workers examined, abnormalities in electrocardiograms, 7-3 �and in gastric and liver function tests, were observed, compared to a control group of 20 workers. In another study, serum samples of workers exposed chronically to 2,4-D and 2,4,5-T during their manufacture were analyzed for cholinesterase, acetylcholinesterase, and tributyrinase activities. No differences in these levels were observed, compared to a group of paired controls (Bonderman et al, 1971). In another study, blood pressure of chlorophenoxy herbicide workers in Oregon was measured. A modest increase in the incidence of hypertension was observed in this group, compared to another group of pesticide workers not exposed to chlorophenoxy herbicides and to a group of control workers. This increase was attributed to a predisposition to hypertension, based on a high incidence of hypertension reported in family histories. Subacute or chronic administration of 2,4-D to animals does not produce a clear syndrome of effects. At low doses, decreased weight gain and slight effects on the liver and gastrointestinal tract have been observed. Death has been attributed to infections and severe anorexia in some studies, (Palmer, 1963; Bucher, 1946; Rowe and Hymas, 1954), but in most studies no lethal lesions were mentioned. Renal changes, neurological changes, gastrointestinal problems, bleeding and hematologic disorders and, in the dog, hepatic changes have been reported (Hill and Carlisle, 1947). In dogs, bleeding of the gums was observed after subacute doses of 2,4-D were administered, but myotonia was not observed (Drill and Hiratzka, 1953). 7.1.2 2,4.5-T Effects of occupational exposure to 2,4,5-T are considered in chapter 5 and, where reproductive effects were evaluated, in chapter 8. Animal studies of 2,4,5-T have been carried out, using several laboratory and domestic species. References on the subacute toxicity of 2,4,5-T are listed in table 7-2. 7.1.2.1 Ineffective and Lethal Dosages Lethal effects have not been observed in rats or mice given subacute doses of 2,4,5-T. Oral doses of 334 mg/kg for 2 days or administration of 3,000 mg 2,4,5-T per kg feed for 90 days were not lethal to rats. Doses of 0.1 mg/kg per day to pregnant rats throughout gestation or 100 mg of 2,4,5-T per kg feed for 90 days produced slight toxicity in rats (Konstantinova, 1974; Coulston, 1970). Oral doses of 2,4,5-T administered to pregnant mice produced varied effects, depending on the strain (Highman et al., 1976a; 1976b). Doses of 120 mg/kg on days 6-14 of gestation had no effect on CRBL mice, while doses of 60 mg/kg to NCTR mice on the same days produced severe maternal toxicity. Whether pregnant mice and rats show the same response to 2,4,5-T as their non-pregnant counterparts is not clear, although maternal toxicity was not considered to be the cause of teratologic effects observed in mice (Highman et al., 1976a). Comparing subacute doses to the LD for 2,4,5-T in these species (in tables 6-2 and 7-2) indicates that 2,4,5,-T is not a cumulative toxicant. Analogous to species differences in response to acute effects of 2,4,5-T, the dog seems to have a higher sensitivity to subacute doses of 2,4,5-T than other species. Oral doses of 20 mg/kg/day for 90 days were 7-4 �TABLE 7-2. REFERENCES ON THE SUBACUTE AND CHRONIC TOXICITIES OF 2,4,5-T Species Route Dose Organs Affected or Studied Durat ion Skin Liver Kidney Neural Muse . Blood Lungs Gut Body Wt. X gavage oral gavage 10 mg/6g 20-100 mg/kg 2-5 g/kg 6 d. 2 wk. 4-7 wk. X House oral 30-140 mg/kg 10 d. X Dog oral 2-20 mg/kg 13 wk. X X Sheep oral 100-250 mg/kg/d X Cattle oral 25-250 mg/kg/d X Rat I —I cv Reference 369 to 7d , max X X X X X X Rip and Cherry, 1976 Stroo et al., 1979 Chang et al., 1974 Highman et al., 1976a X X Drill and Hlratzka, 1953 X X X Palmer and Radeleff , 1964 X X X Palmer and Radeleff, 1964 �lethal in the dog, while doses of 10 mg/kg/day for 90 days were ineffective (Drill and Hiratzka, 1953). In cattle, two to three doses of a mixture of 2,4-D and 2,4,5-T at 500 mg/kg/day was not toxic, while three days of 1,000 mg/kg/day was lethal (Palmer and Radeleff, 1964). Seven daily doses of 250 mg 2,4,5-T/kg/day was fatal to sheep and c.attle. Although 481 doses of 100 mg/kg of the salt were nontoxic to sheep, the ester form of 2,4,5-T was lethal at this dosage after 369 days. The differences in toxicity from different chemical forms of 2,4,5-T was not compared in other studies. 7.1.2.2 Cause of Death and Target Organs As with 2,4-D, subacute doses of 2,4,5-T do not syndrome of effects. The causes of death in animals 2,4,5-T overlap with those of 2,4-D and TCDD. Often stated or could not be determined. Moribund animals ing blood changes and weight loss have been observed treatment. 7.1.3 produce a characteristic subacutely exposed to the cause of death is not as well as animals showafter subacute 2,4,5-T TCDD The subacute and chronic toxicities of TCDD after human exposure are described in chapter 5 and reproductive effects from subacute and chronic exposures are described in chapter 8. Subacute and chronic animal studies of TCDD have been performed in a number of species and are described in this section. References on the subacute effects of TCDD are listed in table 7-3. 7.1.3.1 Ineffective and Lethal Dosages Subacute and chronic administration of TCDD has been shown to be lethal in the monkey, rat, and mouse. Subacute doses of TCDD are cumulative in the monkey and the rat. Daily doses of 10 ug TCDD per kg for 30 days produced almost 100 percent mortality in the rat, and reduced weight gains were observed after 1 ug/kg was administered for 30 days (Gupta et al., 1973). The single oral LD-n for this strain of rat is between 50 and 100 ug/kg (Harris et al., 1973). 50 In monkeys, subacute doses of TCDD are far more effective than administering the cumulative dose as a single dose. TCDD levels of 500 ppt in feed—a cumulative dose of 3 ug/kg over 9 months—was lethal to five of eight monkeys (Allen et al., 1977); the estimated single-dose LD5Q is about 70 ug/kg. Subacute doses of 6 ug/kg body weight, administered in feed over 61 days, or less than 10 ug/kg fed over 10 days, were also lethal to monkeys. The authors of this study predicted that only 1 ug/kg administered subacutely in food would probably be lethal to monkeys (McNultey, 1977). However, 0.9 ug/kg TCDD was not fatal to any of a group of eight monkeys when fed over 20 months at a level of 50 ppt in their feed (Schantz et al., 1979). This cumulative effect is limited and is not seen in rats when TCDD is administered over a period longer than a few weeks (Moore, 1978). The half-life of TCDD is 7-6 �TABLE 7-3. Route Species Dose REFERENCES ON THE SUBACUTE AND CHRONIC TOXICITIER OF TCDD Duration Organs Affected or Studied Skin Liver Rat gavage 2-20 gavage gavage Monkey 500 ppt (toxic fat) oral oral 10 ug/kg/d oral oral oral oral Mouse oral oral oral oral ip Guinea pig oral oral oral ppb 10 ug/kg/d 1-10 ug/kg/d 5 ug/kg/wk 1-10 ug/kg/d .2-5 ug/kg/wk .001-1 ug/kg/d .1-1 ug/kg 58 d. 9 mo. 445 d. X X 31 d 14 31 d 6 wk 30 d 6 wk 13 wk (5/wk) X cv Gut X X X Body Wt. X X X McNulty, 1977 Allen et al., 1977 Allen and Cars tens, 1967 X X X Lungs X X X X X Harris et al., 1973 Weissberg and Zinkl, 1973 Gupta et al., 1973 Zinkel et al., 1973 X X X X 2/wk x 12 mo. X X X .2-10 ug/kg/wk 4-8 wk 8 wk 1 ug/kg/wk .008-1 ug/kg/wk 8 wk X X X X X X X X X X X X X 4 wk 4 wk 2-6 wk 4 wk 8 wk 11 wk 1.5-50 ug/kg/wk 25 ug/kg/wk .2-25 ug/kg/wk .5-20 ug/kg/wk .1-10 ug/kg/wk 25 ug/kg/wk Kidney Neural Lymph Blood Reference X X X X X X Voa et al., 1973 Kociba et al., 1976 King and Roesler, 1974 X X X Rabbit dermal .3-10 ug/kg/d 3d dermal salvage oil (1 yr) X Thigpen et al., 1975 Sharma & Gehring, 1979 Sweeney et al., 1979 X X X X Gupta et al., 1973 Harris et al., 1973 Zinkel et al., 1973 X X Horse Vos et al., 1978 Goldstein et al., 1973 Vos et al., 1974 Jones & Kizek, 1962 X X X X Case and Coffmann, 1973 �between 20-30 days; the cumulative effects seen within this period of time probably reflect the body burden of TCDD that is not cleared from the body in this time period. Daily oral doses of .001-0.1 ug/kg TCDD over 90 days were reported to produce no evidence of toxicity in the rat (Murray et al., 1979), and lifetime exposure of male and female beach mice to sand contaminated with a mean level of 164 ppt TCDD produced only a subtle increase in liver weights of pregnant females (VA, 1981). The Environmental Protection Agency found that the monkeys used in the studies by Allen and Schantz had been exposed to polyhalogenated biphenyls in a previous experiment and the results of the TCDD experiments have not been generally accepted by the scientific community. Crude industrial fats contaminated with dioxins (with TCDD comprising 64 percent of the dioxin content) were administered in feed to monkeys, rats, and chickens. The level of fat in feed that produced 50 percent mortality in chickens within 15 days was not lethal to monkeys within 100 days (the mortality rate of monkeys at 100 days was not stated). Rats were fed five times the dose of fat in feed that produced liver necrosis and ascites in the monkey and chicken. At 80 days, rats experienced 50 percent mortality from this level of feeding; the mortality rates were not reported for chickens and monkeys fed one-fifth the dose given to the rat (Norback and Allen, 1973). Lack of information on mortality rates for all three species given the same dose, and the quantitatively imprecise content and contaminated nature of the samples administered, limits the usefulness of these data, but these results imply that the chicken is most sensitive to subacute exposure to dioxins, the rat least sensitive, and the monkey intermediate between the two. Presumably the same order would apply for TCDD, which in most species is the most toxic of the dioxins. A dose-related effect of toxic fat on mean survival time in the monkey was observed. Monkeys fed diets of 0.125 percent toxic fat survived an average of 445 days while those fed 10 percent fat lived 91 days (Allen and Carstens, 1967). 7.1.3.2 Cause of Death and Target Organs In general, the symptoms observed after TCDD is administered acutely are also observed after subacute and chronic dosing (except for oncogenic effects of chronic dosing, which are discussed in chapter 10). Prophyria cutanea tarda appears to result in animals from long-term exposure, and not from acute exposure. The effects of TCDD on immune function have been studied after subacute doses were administered, although thymic atrophy and .altered responses of splenic lymphocytes to mitogens have also been demonstrated after acute doses of TCDD were administered (see chapter 6). 7.1.4 Diquat Two studies included experiments on the subacute toxicity of diquat. Twenty daily dermal applications of 20 mg/kg diquat to rabbits produced local erythema but no systemic effects, while four of six rabbits died after 8-20 daily applications of 40 mg/kg of diquat. Symptoms produced by this higher 7-8 �dose included weight loss, unsteadiness and muscular weakness (Clark and Hurst, 1970). Rats, mice, rabbits, guinea pigs, and a dog were exposed to 1.1 ug of diquat aerosol per liter air for 15 6-hour periods, with no adverse effects noted. Rats exposed to 2 ug/liter of diquat for 15 6-hour periods showed a small decrease in weight gain and female rats of this exposure group experienced difficult breathing during the first few sessions only (Gage, 1968). Chronic exposure to diquat results in cataracts. In rats, this effect was observed within 6 months in all rats fed diets containing 0.1 percent diquat, in 12 months in all rats fed 0.05 percent diquat, and in 18 months in all rats fed 0.025 percent diquat. One-quarter of the rats fed 0.01 percent and 0.005 percent diquat showed slight opacity in 12 months, while a diet of 0.001 percent diquat was ineffective in causing cataracts. Rats fed 0.05 percent diquat for only 8 weeks did not develop cataracts. In advanced conditions, opacity was accompanied by hemorrhages into the vitreous humor, synechiae, and retinal detachment. Rats fed ascorbic acid supplements or housed in darkness showed the same development of cataracts in response to diquat. The only other effect in these rats was reduced growth rates and food consumption in the highest dosage group (Clark and Hurst, 1970). Reduced glutathione levels remained high (Pirie et al., 1970) while ascorbic acid levels were low in eyes that developed diquat cataracts; distribution of diquat to the eye has been demonstrated (Pirie and Rees, 1970; Pirie et al., 1969). Cataracts also developed in dogs from chronic exposure to diquat. Daily oral doses of 15 mg/kg of diquat produced cataracts in all treated dogs in 10-11 months, and 5 mg/kg 'doses produced cataracts in all dogs in 15-17 months. Daily doses of 1.7 mg/kg for 4 years or 0.4 or 0.8 mg/kg for 3 years were ineffective. No other changes were seen in these dogs (Clark and Hurst, 1970). 7.1.5 P icloram The subacute doses of picloram administered to various species by several routes were usually too low to produce evidence of toxicity. Rats fed picloram (acid form) as 1,000 ppm in their diet for 90 days (equivalent to 75 mg/kg body weight per day) did not experience adverse effects. At levels of 3,000 and 10,000 ppm, moderate hepatic and renal changes^ were observed and females showed decreased weight gains. Picloram as the triisopropanolamine salt, at levels of 0.3 percent or less in feed, produced no alterations in rats over a 90-day feeding period. Doses of 100 mg of piclorara per kg per day for 31 days produced no ill effects in sheep, while doses of 72 and 154 mg/kg for 31 days caused no adverse effects in cattle. Chronic exposure of dogs and rats to daily doses of up to 150 mg/kg of body weight for two years produced no clinical, gross, or microscopic alterations (McCollister and Leng, 1969). Dermal applications of undiluted picloram to rabbits followed by bandaging the site for 11 days, or ten repeated applications of a 25 percent solution over 14 days, produced only local effects, involving slight exfoliation and hyperemia (Lynn, 1965). 7-9 �7.1.6 Dalapon Local or nonspecific systemic effects have been observed in dalapontreated animals. Ten consecutive dermal applications of dalapon to rabbits over 14 days produced local dermal effects, involving moderate hyperemia and slight necrosis, but no systemic toxicity. Doses of 1,000 mg/kg daily to dogs for 81 days caused gastrointestinal irritation (vomiting), but no other clinical or histopathological findings. Systemic effects of dalapon poisoning were produced in the rat and in cattle. Diets of .346 and 1.15 percent dalapon, fed to rats for 97 days, caused growth retardation, increased liver and kidney weights, and slight histopathologic changes in these organs. Ten oral administrations of 1,000 rag dalapon/kg to cattle caused anorexia, lassitude, diarrhea, weight loss, slowed pulse, and discharge from the eyes in one animal and no symptoms in the second animal. The only histopathological lesion that was observed involved the kidney of the second (unaffected) animal. Few effects from chronic dalapon exposure were observed in animals. The only change produced after chronic exposure of rats and dogs to dalapon was increased kidney weight. This effect followed administration of 100 mg/kg/day of dalapon to dogs for 1 year and 50 mg/kg/day to rats for 2 years. Daily doses of 50 mg/kg to dogs for one year or 15 mg/kg to rats for two years produced no toxic effects (Paynter et al., 1960). The only effect observed in one sheep was a 6 percent loss in body weight after 10 daily doses of 100 mg/kg dalapon and 10 percent loss after 86 doses were administered; after a total of 481 doses were given, no other changes were observed. Ten daily oral doses of 500 mg/kg produced no effects in cattle, but weight losses of 6-17 percent in sheep (Palmer and Radeleff, cited in Kenaga, 1974). 7.1.7 Monuron Monuron was administered to rats at levels of 0.25 percent, 0.025 percent and 0,0025 percent in their diets, for 2 years. Effects observed at the highest dosage were decreased growth rates, slight anemia, and increased liver and spleen weights; all tissues appeared normal, histologically, at autopsy. The mortality for all rats, including those in the control group, was 70-90 percent by the end of the experiment; this high rate was attributed to several epidemics of respiratory infections (Hodge et al., 1958). Monuron was administered at doses of 2.5, 12.5, and 25 mg/kg body weight per day to dogs for 1 year. No treatment-related effects were observed on body weights, organ weights or histology or by analyses of blood and urine samples (Hodge et al., 1958). 7.1.8 Bromacil Bromacil was administered in the diet at 1,250 ppm to rats for 2 years. Lower weight gains and decreased food consumption were observed in this group, compared to controls. Rats fed diets with 50 or 250 ppm bromacil for 2 years did not show these effects. None of the animals in the study showed any 7-10 �clinical signs of toxicity and results of their hematologic, urine, and biochemical analyses were normal. Dogs fed the same dosages, as diets with 501,250 ppm bromacil, also showed no effects in any of these parameters over a 2-year period (Sherman and Kaplan, 1975). Midwest Research Institute (1975) reviewed bromacil toxicity studies. 7.1.9 Cacodylic Acid No histopathological effects or clinical changes were observed from cacodylic acid, administered at dietary levels of 30 ppm for 90 days to dogs, or at dietary levels of up to 100 ppm, or at 280 mg/kg body weight for 20 days to rats. In rabbits & 77 percent cacodylic acid preparation was applied and exposure was continued for 12 hours per day, 5 days per week for 3 weeks. The effects from this exposure were local hyperemia, and other dermal lesions and rapid loss of conditioning, diarrhea, fluid accumulation in the gastrointestinal tract, congestion in the spleen, and distended bowels. Doses of 1 and 1.6 g/kg were not lethal to the single rabbit tested at each dose, while doses of 2.5 g/kg or higher were lethal (cited in Midwest Research Institute, 1975). 7.1.10 Herbicide White Repeated dermal applications of a 5 percent solution of Herbicide White to man produced no skin irritation or sensitization. Sheep were administered daily oral doses of 0.55 ml of Herbicide White per kg for 5 days. Four of 11 sheep died and losses in body weight were noted in the remaining sheep. Doses of 0.11 ml/kg per day for 30 days produced neither mortality nor effects on appearance, behavior, or weight gain in sheep. Rabbits were dermally administered 15 ml of a 5 percent solution of Herbicide White and the site of application was wrapped for 7 hours. After a total of 15 daily applications were made, no dermal or systemic effects were observed. Applications of undiluted herbicide in the same manner produced slight irritation (Lynn, 1965). Herbicide White was also applied to the skin and eyes of rabbits under conditions of high humidity and high temperature. Erythema from 0.02 ml of Herbicide White appeared more rapidly under adverse conditions than in temperate conditions, although the adverse conditions caused death to many of the control and treated rabbits (Weimer et al., 1970). 7.2 7.2.1 DERMAL LESIONS 2,4-D and 2,4,5-T Both phenoxy acids, 2,4-D and 2,4,5-T, produce mild degenerative changes in the conditions of the fur of rodents and muzzle of cattle after subacute oral doses are administered. Doses that produce these changes also produce general debilitation. Dermal lesions were described in rabbits that were administered 2,4-D dermally. These changes involved local skin reactions and inflammation. However, these changes were also observed in rabbits that were treated with vehicles only (Kay et al., 1965). 7-11 �7.2.2 TCDD Chloracne has been shown to result from a single exposue to TCDD (see chapter 6). One study evaluated the effect of repeated small dermal doses of TCDD in the rabbit. In the rabbit ear bioassay 0.004 ug of TCDD in benzene, applied 5 days a week for 4 weeks, produced comedones. The effect of administering the cumulative dose ( 0 0 ug of TCDD) as a single dose was not .8 tested. Application of one-tenth of the effective dose of 0.004 ug for the same 4-week period of time did not produce chloracne (Schwetz et al., 1973). Dermal lesions and chloracne were common,effects of subacute exposure of monkeys to TCDD. Adult female rhesus monkeys fed diets containing 50 ppt TCDD for 20 months ( . ug/kg cumulative dose) experienced hair loss and hyper09 keratosis, especially of the arms (Schantz et al., 1979). This exposure to TCDD was not lethal for any of the eight monkeys. Five of eight female rhesus monkeys died after 7 to 9 months of exposure to 500 ppt TCDD in feed (2-3 ug/kg cumulative dose). All eight monkeys developed acne, accentuated hair follicles, periorbital edema, swelling of the eyelids, loss of facial hair and eyelashes, and irregular nail growth. These changes appeared during the first 3 months of exposure. Hair loss and periorbital edema persisted in the survivors for 3 months after exposure ended. Keratinization of hair follicles, sebaceous glands, meibomian glands of the eyelids, and nails were observed at necropsy (Allen et al., 1977). One male rhesus monkey who was fed 2 ppb TCDD in feed for 61 days (6 ug/kg cumulative dose) experienced no hair loss or acne prior to death on day 76 (death may have been caused by unidentified post-operative complications, although signs of severe TCDD intoxication were also present). Another monkey was fed 20 ppb TCDD and died after 12 days; in both of these monkeys, squamous metaplasia of sebaceous glands was observed at necropsy (McNulty, 1977), Alopecia and subcutaneous edema, which appeared first in the eyelids and then the face and other parts of the body, were observed one month prior to death in monkeys that were fed lethal doses of toxic fat in their diets. Dermal edema and keratinization of hair follicles and sebaceous glands were observed microscopically (Allen and Carstens, 1967). Horses, dogs, cats, and mice at arenas that were sprayed with TCDDcontaminated salvage oils developed contact dermatitis and hair loss. Mild dermatitis of the face, limbs, and ventral surfaces became progressively severe, and eventually involved ulcerative lesions, as well as inflammation of the mucous membranes of the mouth and nasal passages. Hair loss in horses included extensive loss from the mane and tail (Case and Coffmann, 1973). The contribution of TCDD in the salvage oil to this condition, compared to other components which may have caused some of the deleterious effects, is unknown. Acute TCDD treatment has been reported to cause a depletion in vitamin A storage levels in the liver to 30 percent of control levels, 8 weeks after a 10 ug/kg dose of TCDD was administered to rats (Thunberg et al. 1979). These authors noted that chloracne seen in man after TCDD exposure resembles dermal lesions observed in hypovitaminosis A. 7-12 �7.3 HEPATOTOXICITY 7.3.1 2,4-D and 2.4.5-T Subacute doses of 2,4-D or 2,4,5-T produce degenerative histological changes in the liver, enzyme changes, and liver enlargement. Focal necrosis of the liver was observed in dogs that received lethal subacute doses of 2,4-D or 2,4,5-T, but the authors of this study concluded that these lesions were of no. toxicologic signficance, as their severity was not dose-related (Drill and Hiratzka, 1953). However, other investigators also observed centrilobular degeneration and atrophy in dogs that received lethal doses of 2,4-D (Hill and Carlisle, 1947). Multinucleated hepatocytes and degeneration of the liver parenchyma was observed in pigs that received 500 ppm 2,4-D in their feed for 12 months. This dose also produced locomotory disturbances and reduced growth rates, but was not fatal (Bjorklund and Erne, 1966). Degeneration of the liver was observed in sheep and cattle that received lethal subacute doses of 2,4-D or 2,4,5-T (Palmer and Radeleff, 1964). The same types of serum and hepatic enzyme changes were observed in the rat after a single dose of 625 mg of 2,4-D per kg, or subacute doses of 125 mg/kg. These changes included decreased aldolase levels and increased glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, catalase, cholinesterase, and acid phosphatase levels, 21 days after treatment ended (the duration of treatments was not stated). Cellular infiltration of the liver was observed after subacute treatment, but not after an acute dose. However, livers were examined only within 2 days of the acute dose. The authors concluded that 2,4-D produces subacute hepatitis (Szocs et al., 1970). Increased liver weights, increased cellular RNA and protein content, and decreased nuclear DNA content were observed in rats that were administered 2-5 g 2,4,5-T per kg (less than 0.05 ppm TCDD) over 4 to 7 weeks. The same dose of 2,4-D did not produce these changes, although both phenoxy acids caused a 50-100 percent elevation in liver glycogen content (Change et al., 1974). The effect on liver size was reversible, as liver size returned to normal after 2,4,5-T (less than 0.05 ppm TCDD) administration ended. Metabolism of 2,4,5-T was not ehanced in the enlarged livers and 2,4,5-T did not show strong hepatotoxic activity regarding enzyme induction (Rip and Cherry, 1976). In another study, sublethal doses of 2,4,5-T to rats produced liver enlargement accompanied by swelling parenchymal cells and slight centrilobular necrosis. Toxicity in these rats was mild, and included a small reduction in body weight and histological evidence of renal damage (Coulston, 1970). 7.3.2 TCDD 7.3.2.1 Structural Alterations Subacute doses of TCDD were administered to monkeys in four studies (Schantz et al., 1979; McNulty, 1977; Allen et al., 1977; Allen and Carstens, 1967). Only one study, in which TCDD was administered as toxic fat, reported histological changes in the liver other than in the bile duct. Allen and Carstens (1977) reported that fatal doses of toxic fat produced enlarged livers, with focal necrosis, fat deposits, and multinucleated hepatocytes. 7-13 �Proliferation of the smooth endoplasmic reticulum was observed in electron micrographs. Since these effects were not seen when lethal doses of purified TCDD were administered to monkeys, the possibility exists that other components of coxic fat caused the observed hepatotoxicity. Hepatotoxicity was the principal effect observed in rats that were administered 0.1 or 1.0 ug of TCDD per kg per week of TCDD for 28 weeks; decreased weight gain was also observed in these rats. The major hepatic change was the accumulation of centrilobular fat deposits, which the authors suggested could be caused by impaired lipid transport or metabolism. In no case were the observed fatty changes considered severe. Fatty changes were first observed 16 weeks after treatment and regressed but did not completely disappear by the end of the 12-week recovery period. Other hepatic changes were described as subtle or insignificant, and included focal changes, multinucleation, and altered hepatic architecture. Female rats had less severe lesions than male rats (King and Roesler, 1974). The severity of liver damage in rats is related to the dose of TCDD administered. Slight liver damage was observed in rats administered weekly doses of 5 ug/kg for 6 weeks, slight to moderate damage occurred from 31 daily doses of 1 ug/kg, while severe damage was observed after 16-31 daily doses of 10 ug/kg. A single oral dose of 2 ug/kg was ineffective in the same strain of rat, while 50 ug/kg produced severe damage. Marked necrosis of hepatocytes, multinucleation, and disturbance of the architecture of the liver were the hepatic lesions observed. In contrast to rats, liver damage was slight in guinea pigs that received four to five doses weekly of 1 ug TCDD per kg, which produced 100 percent mortality (Gupta et al., 1973). TCDD causes an increase in liver size relative to body size in the rat. Ten or more daily doses of 0.1 or 1 ug TCDD per kg to rats resulted in increased absolute liver weights, while daily doses of 10 ug/kg for more than 10 days produced a progressive fall in absolute liver weights and a mortality rate above 90 percent. Weekly sublethal doses of 0.04 or 0.2 ug/kg to female guinea pigs did not produce significant changes in liver weight, although thymic weights were adversely affected by both doses, and body weights by the higher dose (Harris et al., 1973). In another study, changes in liver weight was found to be the most sensitive response to TCDD. The only adverse effect observed in rats given 0.01 ug/kg/day for 13 weeks was a slight increase in liver weight relative to body weight, and the author associated no toxicological significance with this small change (Kociba et al., 1976). At 0.1 ug/kg/day, liver degeneration occurred which involved mild changes in liver architecture and slight lipid accumulation. These hepatic changes were accompanied by lymphoid depletion and the decreased body weights. At 1.0 ug/kg/day, alteration in liver architecture and hepatocyte sizes, focal necrosis, mild fat accumulation, cellular infiltration, brown pigmentation, and multinucleation were observed. This.dose produced 33 percent mortality and produced changes in hematologic and reproductive parameters as well as the effects seen at lower doses (Kociba et al., 1976). 7-14 �As observed after subacute administration, chronic administration of TCDD to rats also produced hepatic changes. Feed containing 0.05 ppm TCDD or higher was lethal to all rats within four weeks and produced severe liver necrosis. Feed with 0.001-5.0 ppb TCDD was administered to rats for 65 weeks. Mortality rates were about 8 percent and all rats that died showed moderate liver alterations which were not described further (Van Miller and Allen, 1977). However, the laboratory practices used for this study were later found to be inadequate by the Environmental Protection Agency; thus, these results are questionable. In another chronic study, proliferation of the smooth endoplasmic reticulum was observed in rats fed diets of 0.1 ug/kg/day (2,200 ppt) for 2 years (Kociba et al., 1978; 1979). TCDD also produces histopathological changes and increased liver weights in the mouse. Liver weights relative to body weights were significantly increased 2 and 6 weeks after weekly doses of 1, 5, or 25 ug/kg were administered to mice. Lipid accumulation was detected after six weekly doses of 0.2 ug TCDD/kg. The effect was more severe at higher doses and involved centrilobular and midzonal hepatocytes. Necrosis and cellular infiltration were observed after doses of at least 5 ug/kg. Degenerated liver cells and nuclear changes were seen in the 25 ug/kg dosage group (Vos et al., 1974). Young (VA, 1981) reported that the only toxic effect observed in field mice exposed chronically to sand containing a mean concentration of 164 ppt TCDD was increased liver-to-body weight ratios in pregnant females, compared to unexposed control field mice. This effect was seen consistently over a 5-year period of study. Mice on an iron-deficient diet, after TCDD treatment (25 ug/kg/week for 11 weeks intraperitoneally) did not develop porphyria or hepatocellular lesions that were observed in mice with normal iron levels. These changes included altered liver architecture and hepatocyte sizes, focal necrosis, cellular infiltration, and mid-zone and peripheral vacuolation. Other degenerative changes in the conditions of the mice after TCDD were also absent in iron-deficient mice. The only difference in general appearance between untreated mice and TCDD-treated iron-deficient mice was the smaller size of the latter. Subtle changes observed after TCDD treatment included increases in liver weight and induction of microsomal enzymes in both the iron-deficient mice and mice with normal iron levels. The authors suggested that tissue iron (probably in another form) plays an essential role in the toxic changes induced by TCDD (Sweeney et al., 1979; Jones and Sweeney, 1979). 7.3.2.2 Serum Enzyme Changes Changes in serum enzymes have not provided an accurate indication of TCDD hepatotoxicity, although increased serum glutamic pyruvic transaminase levels were detected in one study, in monkeys fed diets with a sub lethal dose of 50 ppt TCDD for 20 months (0.9 ug/kg cumulative dose) (Schantz et al., 1979). Serum enzyme assays that are often used to indicate liver malfunction found no change in horses or in rats after TCDD exposure (Case and Coffmann, 1973; Kociba et al., 1976). Assays for biliary function, however, were altered by 7-15 �TCDD treatment (see section 7.3.2.3). Rats fed diets with 2,200 ppt TCDD for 2 years showed increased serum enzyme levels as well as increased excretion of porphyrins and hepatic lesions (Kociba et al., 1978; 1979). 7.3.2.3 Biliary Excretion TCDD produces adverse structural and functional effects on the biliary system. Dilation of .the bile duct was observed at death in monkeys fed 500 ppt TCDD in feed'for nine months (Allen et al., 1977). Proliferation and stratification of bile duct epithelium were observed in monkeys that died after subacute doses of toxic fat were administered in their feed (Allen and Carstens, 1967). Prolonged bromsulphthalein clearance times were observed in horses after high and often lethal exposure to TCDD in salvage oil sprayed on arenas. Severe biliary cirrhosis was observed in all horses at autopsy (Case and Coffmann, 1973). Proliferation of bile ducts was observed in mice administered weekly doses of 25 ug TCDD/kg TCDD (Vos et al., 1974). In rats that were administered 1.0 ug/kg/day of TCDD for 13 weeks, biliary hyperplasia was observed. Elevated serum alkaline phosphatase and direct bilirubin levels were also detected in these rats, indicating impairment of biliary functions. At a dose of 0.1 ug/kg/day for 13 weeks these changes were observed only in females, and at lower doses (0.01 and 0.001 ug/kg/day) these changes did not occur (Kociba et al., 1976). Chronic administration of feed with 0.001-5.0 ppb TCDD for 65 weeks produced mortality rates that were proportional to dose. Hyperplasia of the bile ducts was observed in all animals that died (Van Miller and Allen, 1977). 7.3.2.4 Porphyria Porphyria is a disorder of hemoglobin metabolism. Porphyria cutanea tarda is a severe form of porphyria and has occurred in workers that were exposed to TCDD (see chapter 5). The suggested biochemical lesion in hepatic porphyria is an inhibition of the enzyme uroporphyrinogen decarboxylase. This enzyme converts uroporphyrinogen to coproporphyrinogen, intermediates in the hepatic conversion of delta-aminolevulinic acid to heme. Urinary porphyrin excretion is elevated in cases of hepatic porphyria. In man, a relative increase in 7- and 8-carboxy-containing porphyrin metabolites over 4- to 6-carboxy-porphyrin metabolites is a more sensitive indicator of porphyria than the level of total porphyria excretion. In cases of porphyria cutanea tarda, total urinary porphyrins can be comprised of 45-70 percent uroporphyrin and 25-35 percent heptacarboxylic porphyrin, compared to much lower proportions in normal urine. Hepatic accumulation of these porphyrins occurs before clinical symptoms are manifest or urinary levels are elevated (VA, 1980). Porphyria has been observed in male and female rats after 1 ug/kg/day of TCDD was administered for 13 weeks. Only females developed porphyria from 0.1 ug/kg/day doses for 13 weeks. Evidence of porphyria included elevated 7-16 �excretion of total porphyrins, uroporphyrin, and delta-aminolevulinic acid. Mortality for the higher and lower doses were 33 percent and none, respectively (Kociba et al., 1976). Porphyria was also observed in the rat after chronic dosing (Greig et al., 1979; Kociba et al., 1978; 1979). In another study, porphyria was not observed in any rats that died after 16-31 daily doses of 10 ug/kg of TCDD or guinea pigs that died from four to five weekly doses of 1 ug/kg TCDD. In this study, porphyria was evaluated by examining the liver under ultraviolet light for red fluorescence, an indication of excess amounts of porphyrins, and this method may have been too insensitive to detect the expected increase in porphyrins (Gupta et al., 1973). No changes in delta-aminolevulinic acid synthetase levels were detected in rat livers pretreated with up to 25 ug/kg TCDD 1 day prior to sacrifice, and induction of delta-aminolevulinic acid synthetase by allylisopropylacetamide (ALA) was not affected by TCDD over 28 days. Twenty-four-hour pretreatment of fetal or neonatal rats with TCDD also failed to alter ALA synthetase activity (Woods, 1973). In these experiments, TCDD was not administered chronically or at a high dose with a latency period greater than 1 day, which is probably necessary to allow enzyme changes to become manifest. In mice, porphyria has been observed after four weekly doses of 25 ug/kg TCDD were administered. This dosage produced a 2,000-fold increase in the hepatic levels of 7- and 8-carboxyporphyrins, which were analyzed by thin layer chromatography. Total iron content in the liver was elevated significantly (to 150 percent of the control level), delta-aminolevulinic acid synthetase activity was increased, and histopathological changes in the liver were noted. A single lethal dose of 150 ug/kg of TCDD produced a 4,000-fold increase in hepatic uroporphyrins 21-25 days after treatment. Hepatic iron levels were elevated to about 250 percent of the level of the control group (Goldstein et al., 1973). In another study, the porphyrinogenic effects of intraperitoneal doses to mice of 25 ug/kg/week for 11 weeks included a 10-fold elevation in urine porphyrin levels and a reduction to 20 percent of uroporphyrinogen carboxylase activity, compared to control mice. As described above (section 7.3.2.1), iron-deficient mice with hemoglobin levels of 5.5 g/dl showed no signs of porphyria after the same dosage of TCDD was administered (Sweeney et al., 1979; Jones and Sweeney, 1979). 7.4 NUTRIENT ABSORPTION AND UTILIZATION Changes in body weight and food consumption after herbicide and TCDD treatment are described here. Factors that can affect nutrient utilization are also considered in this section. These factors include absorption of nutrients from the gut, hormonal alterations that effect regulation of nutrient utilization, and biochemical factors that reflect levels of circulating protein and lipid precursors and metabolites. 7.4.1 2,4-D and 2,4,5-T Decreased weight gain has been observed in most species studied, after low subacute doses of phenoxy acids were administered. Higher doses produced complete cessation of eating in some animals (see tables 7-1 and 7-2). 7-17 �7.4.1.1 Body Weight and Food Consumption Actual measurements of body weight and food consumption have been reported. Reduced food consumption and body weights were demonstrated in chicks that consumed 5,000 mg of 2,4-D or 2,4,5-T per kg of feed for one week. Two weeks after the chicks were returned to normal diets, their normal growth rates resumed (Whitehead and Pettigrew, 1972). In another study, three doses of 280 mg 2,4-D/kg of 2,4-D were administered per week for 4 weeks to chicks and a significant decrease in body weight of 20 percent was observed. Lower doses did not produce significant reductions in weight (Bjorn and Northern, 1948). In the dog, lethal subacute doses of 2,4-D and 2,4,5-T produced weight losses of up to 4 kg, while lower sublethal doses of 2,4,5-T produced smaller weight changes and sublethal doses of 2,4-D produced no weight changes (Drill and Hiratzka, 1953). 7.4.1.2 Effects on the Endocrine System Florsheim and Velcoff (1962) described an effect of 2,4-D on the thyroid. Rats administered daily subcutaneous injections of 100 mg/kg 2,4-D showed decreased thyroid weights and body.weights, At 80 mg/kg, these changes were not observed, but an increase in I uptake by the thyroid was observed. This effect did not involve pituitary thyrotrophic hormone, and was observed in rats with normal thyroid function but not in hypophysectomized or iodinedepleted rats. 7.4.2 TCDD 7.4.2.1 Body Weight and Food Consumption Reduction in body weight has been observed in many species following subacute TCDD exposure. TCDD caused weight loss in monkeys fed 50 ppt TCDD in feed for 20 months, 500 ppt for 9 months, 2 ppb for 61 days, or 20 ppb for 12 days. At death the monkey that was fed 2 ppb TCDD had lost 33 percent of its body weight in 76 days. For the monkey fed 20 ppb TCDD, food consumption progressively decreased, from the third day of feeding until day 12 when its body weight was decreased by 30 percent and death occurred (McNulty, 1977). Monkeys fed 500 ppt TCDD in their diet lost an average of 13 percent of their initial body weight at death (which occurred following at least 7 months of exposure) or at 1 year, for the survivors. Food consumption did not change in these animals during the treatment period, but was not compared to food consumption of untreated monkeys (Allen et al., 1977). Monkeys fed lethal amounts of toxic fat in their diets lost up to 1 kg in body weight at death (about 20 percent of their body weight) and food consumption was observed to be decreased. The authors did not clarify several points, including whether they actually measured consumption, whether the amounts consumed prior to death were lower than those of controls or than those of treated monkeys at the start of the experiment, or whether the observed decrease in food consumption could account for the decrease in body weight (Allen and Gartens, 1967). Horses, cats, and dogs exposed to arenas that were sprayed with TCDDcontaminated salvage oil showed severe weight losses of up to 40 percent in 7-18 �some horses. Absence of demonstrable body fat was a common finding at autopsy. Loss of appetite and preference for sweets were observed in these animals as well as in children who played in the sprayed arena (Case and Coffmann, 1973). In the rat, body weight losses were observed in rats once the total dose administered exceeded 20 ug/kg; daily doses of 1 ug/kg for 1 month, weekly doses of 5 ug/kg, or a single dose of 25 ug/kg caused decreases in weight gains in the rat. This effect was reversible, as normal growth rates resumed several weeks after the dose regimens were terminated. Doses that failed to alter body weight included a single dose of TCDD of 5 ug/kg, weekly doses of 1 ug/kg over 6 weeks, or daily doses of 0.1 ug/kg over 30 days (Harris et al., 1973). In the guinea pig, a single dose of 1 ug TCDD/kg or weekly doses of 0.2 ug/kg over 8 weeks produced significant decreases in body weight, while doses of 0.04 ug/kg were ineffective. In the mouse, weekly doses of 25 ug/kg over 4 weeks were effective while weekly doses of 5 ug/kg or a single dose of 50 ug/kg were not (Harris et al., 1973). In another study, however, two weekly doses of 5 ug/kg caused significant weight reduction (Vos et al., 1974). These results indicate that effects on body weight, like the lethal effects, are cumulative, being produced when an effective amount of TCDD is administered as a single dose or in smaller doses over a period of about 1 month. Chronic doses of TCDD that cause body weight reduction have been described for the rat. Male rats administered 1 or 5 ppb TCDD in feed for 65 weeks showed significantly decreased weight gain as well as 40-50 percent mortality. Rats fed 0.001-0.5 ppb TCDD in their diets had a mortality rate of 8 percent; no disturbances' in body weight were mentioned for this group (Van Miller and Allen, 1977). Doses of 1.0 or 0.1 ug TCDD/kg, administered to rats for 13 weeks, caused reduction in weight gain and food consumption compared to controls, while doses of 0.01 ug/kg/day or less were ineffective (Kociba et al., 1976). Rats treated twice weekly for 28 weeks with 0.1 or 1.0 ug/kg/week had reduced body weights compared to controls. This effect was sustained in male rats, but not in females, during the subsequent recovery period of 12 weeks. Food consumption was not measured in this study (King and Roesler, 1974). 7.4.2.2 Effects on the Gastrointestinal Tract Hyperplasia and ulceration of the gastric mucosa have been observed in monkeys after lethal subacute doses of TCDD were administered (McNulty, 1977; Allen and Carstens, 1967). The affected monkeys also showed large changes in body weight. Lethal subacute doses of TCDD also produced ulceration in the stomach of rats (Gupta et al., 1973). Gastrointestinal transport of nutrients was not evaluated in these studies. However, most organs in these animals showed effects from TCDD and no attempt has been made by the investigators to relate the observed intestinal lesions to weight loss in these animals. 7.4.2.3 Lipid and Protein Biosynthesis No systematic study of protein or lipid levels following acute dosing of TCDD have been reported. Sporadic instances of altered protein or lipid 7-19 �levels have been mentioned. Decreased serum protein levels and altered albumin-to-globulin ratios were observed in monkeys that were fed lethal amounts of toxic fat (Allen and Carstens, 1967). Decreased serum cholesterol levels were observed in monkeys fed sub lethal amounts of TCDD (50 ppt in the diet for 20 months, Schantz et al. , 1979). No connections between these altered serum levels and disturbed protein or lipid metabolism by TCDD were drawn by the authors. Lowered serum gamma globulin levels was one of the only abnormal results of clinical chemistry tests of horses exposed to arenas sprayed with TCDD-contaminated salvage oil. Serum lipid levels were not assayed, however (Case and Coffmann, 1973). Serum protein levels and globulin levels were reduced in mice that received weekly doses of 25 ug TCDD/kg for 6 weeks, while weekly doses of 5 ug/kg were not effective (Vos et al., 1974). 7.5 7.5.1 HEMATOLOGICAL EFFECTS 2,4-D and 2,4,5-T Bleeding disorders, including increased bleeding of the gums in dogs and hemorrhages in the heart and gastrointestinal tract, have been observed in many species after phenoxy acids were administered subacutely. 2,4-D and 2,4,5-T have produced various hematological changes which are difficult to categorize. In sheep that were administered 18 mg/kg daily for 120 days, hematologic changes included increases in erythrocyte, hemoglobin, and leukocyte levels within the first months, followed by decreases in all of these parameters later in the exposure period. Biochemical changes in blood components and erythrocyte enzyme levels were also noted; these changes also showed bimodal patterns (Shavgulidze et al., 1976; Kuzminskaya and Bersan, 1975). None of these changes in blood cell counts were observed in dogs that received lethal doses of 2,4-D or 2,4,5-T, although a terminal fall in the relative proportion of lymphocytes to other blood cell types was noted (Drill and Hiratzka, 1953). In another study large decreases in white blood cell counts were observed in only some of the dogs that received lethal doses of 2,4-D. Mild decreases in red blood cell counts and hematocrits were also observed in these dogs (Hill and Carlisle, 1947). Hematocrit and hemoglobin levels were lowered in pigs that were fed 2,4-D at 500 ppm for 12 months (Bjorklund and Erne, 1966). Slight decreases in red cell counts and hemoglobin levels were observed in rats that were administered sublethal doses of 2,4,5-T orally (Coulston, 1970). 7.5.2 TCDD Depletion of various types of circulating blood cells from high doses of TCDD in monkeys has been observed. TCDD at a sublethal level of 50 ppt in feed, administered to monkeys for 20 months, produced decreases in the hematocrit and white blood cell count (McNulty, 1977). At a level of 500 ppt in feed for 9 months, TCDD was lethal to five of eight monkeys. All five fatal cases developed pancytopenia prior to their deaths, which occurred 7-10 months after the experiment started. One of the survivors developed severe leukopenia and thrombocytopenia by the 12th month of the experiment. Hematocrits and hemoglobin levels began to fall by the sixth month in all monkeys and 7-20 �subsequently showed a progressive fall. Hypocellularity of bone marrow was observed at necropsy (Allen et al., 1977). In monkeys that consumed toxic fat peripheral red and white blood cell counts were depressed; bone marrow hematopoiesis and activity of lymphoid tissue were also depressed in these animals as well (Allen and Carstens, 1967). However, neither hemoglobin levels nor hematocrit values were found to be useful parameters in monitoring the condition of horses exposed to TCDD in salvage oil (Case and Coffmann, 1973). Rats that were administered 10 ug/kg TCDD daily were bled on days 10 and 14, and various hematologic parameters were evaluated. Hemoconcentration, as evidenced by elevated packed cell volumes and erythrocyte counts, observed, along with neutrophilia, lymphocytesis, and eosinopenia. These changes were considered to be nonspecific, reflecting the widespread toxicity of TCDD on various organs. Thrombocytopenia was found but no evidence of decreased platelet synthesis (based on normal marrow megakaryocyte levels), or losses by incorporation into microthrombi (since the level of serum fibrinogen degradation products was not elevated). Most platelet function tests were normal as well (Weissberg and Zinkl, 1973). In another study, hemoconcentration was observed terminally in rats given 10 ug/kg/day of TGDD for 30 days but not in rats that received 1 ug/kg/day or less, and was attributed to shock and dehydration. Thrombocytopenia occurred in all three groups while no significant changes occurred in leukocyte counts or in lymphocyte counts. Thrombocytopenia and decreased lymphocyte counts were observed in guinea pigs following eight weekly doses of 0.08-0.2 ug TCDD/kg (Zinkl et al., 1973). Differences in hematologic effects of TCDD between males and females have been noted. Thrombocytopenia was observed in male and female rats fed 1.0 ug/ kg/day of TCDD. Packed cell volume, red blood cell counts, and hemoglobin concentrations were elevated significantly for females only and decreased in males, compared to controls. White blood cell counts also showed these trends, although the decrease in males was not significant. At doses of 0.1 ug/kg/day or less, most changes in blood cell counts were not significant (Kociba et al., 1976). Lethal hematologic changes have resulted from chronic exposure to TCDD. Death was attributed to aplastic anemia in 25 percent of the rats that succumbed to dosing at a level of .001-0.5 ppb TCDD in feed for 65 weeks (Van Miller and Allen, 1977). Hemoglobin and mean corpuscular hemoglobin levels were decreased in mice given 25 ug/kg/week of TCDD for 6 weeks, while leukocyte, lymphocyte, and erythrocyte counts were the same as controls (Vos et al., 1974). Hemorrhages were observed in animals that were treated with high levels of TCDD. Monkeys fed 500 ppt TCDD in their diets for 9 months were found to have hemorrhages in many organs (Allen et al., 1977). Hemorrhages which proved lethal in 4 weeks were observed in the gastrointestinal tract of rats fed diets containing 0.05-1 ppm TCDD (Van Miller and Allen, 1977). 7-21 �7.6 STRUCTURE AND FUNCTION OF LYMPHATIC TISSUES 7.6.1 2.4-D and 2.4.5-T Highman et al. (1976a) have described changes in the structure of lymphatic tissues in mice that were administered 2,4,5-T (TCDD content less than .05 ppra). These changes involved atrophy of the thymus and spleen and hypocellularity of the bone marrow and lymph nodes. They resemble the changes that are usually seen in mice after TCDD treatment, but not after 2,4,5-T treatment. 7.6.2 7.6.2.1 TCDD Structure of Lymphatic Tissues Atrophy of lymphoid structures, especially the thymus and spleen, has been observed after subacute doses of TCDD were administered to various species. Lymph node atrophy, with loss of distinct germinal centers and sparse lymphocytes, was observed in monkeys fed lethal doses of TCDD (500 ppt in feed for 9 months) (Allen et al., 1977). At necropsy, the spleens of horses exposed to TCDD in salvage oil were found to be reduced to one-third the normal size, and most lymph nodes were small and inactive (Case and Coffmann, 1973). The thymuses of these horses and monkeys were not described in these reports. Lymphoid depletion of the thymus and other lymphoid tissues resulted from doses to rats of 0.1 ug/kg/day for 13 weeks. These changes were more severe in rats given 1.0 ug/kg/day for 13 weeks. The cortical region of the thymus was involuted from a decrease in the number of cortical thymocytes (Kociba et al., 1976). In mice that received 1 ug/kg/week of TCDD for 6 weeks, significant reduction of absolute thymus weights and thymus-to-body weight ratios were observed. At higher doses the reduction was more severe, and at 25 ug/kg/week, mean thymus weights were 20 percent of controls after 2 weeks. Microscopic examination indicated that the cortex of the thymus was depleted (Vos et al., 1974). 7.6.2.2 Immune Function The effects of TCDD on immune function have been the subject of several reviews (Luster and Faith, 1979; Luster et al., 1979; Vos, 1977; 1978). In the guinea pig, TCDD suppressed cell-mediated immunity determined by measuring delayed hypersensitivity to tubercutin. Humoral immunity, determined by . measuring the levels of antibodies produced against tetanus toxoid, was only slightly effected. TCDD did not produce an indirect immunosuppression by stimulating adrenocortical activity. In the mouse, cell-mediated immunity, determined by measuring graft versus host activity of donor spleen cells, was suppressed. In the rat however, no cell-mediated immunosuppression (based on delayed hypersensitivity response to tuberculin) occurred (Vos et al., 1973). TCDD at oral doses of 0.01 ug/kg/week for 2 weeks, stimulated splenic lymphocyte transformation; this effect reversed in a short period of time (Sharraa and Gehring, 1979). 7-22 �Doses of TCDD that produced no other clinical or pathological effects have been able to decrease the capacity for hose defense in mice, following salmonella infection; defense to pseudo-rabies virus was not altered, however (Thigpen et al., 1975; 1977). Hinsdall et al., (1979) also reported immunosuppression from low doses of TCDD in the ppb range. Vos et al., (1978) reported that the susceptibility to salmonella was the result of increased sensitivity to bacterial endotoxin in TCDD-treated animals. Thymic alrophy by TCDD in mice was not blocked by administering thymosin and serum zinc levels were not depressed in TCDD-treated mice. TCDD treatment also did not impair non-specific killing by macrophages or specific killing of Listeria (Vos et al., 1978). 7.7 RENAL EFFECTS In some reports of subacute toxicity of 2,4-D and 2,4,5-T, renal lesions have been mentioned (see tables 7-1 and 7-2). Effects of 2,4,5-T on renal function were observed after acute administration to rats. However, the effects on inhibition of renal organic anion transport were not observed 24 hours after subacute doses of 2,4,5-T were administered and the authors concluded that chronic doses of 2,4,5-T were unlikely to produce a cumulative effect on renal function (Stroo et al., 1979). Histological changes of the kidney from subacute exposure to phenoxy acids have been described in the chicken (Bjorklund and Erne), but the relevance of these lesions to those observed in mammalian species has not yet been established. 7.8 CARDIOVASCULAR EFFECTS Gangrenous necrosis of fingers and toes and extensive edema and ascites were observed in monkeys that were fed 500 ppt TCDD in their diets for 9 months. These lesions may have been manifestations of inadequate blood flow and changes in vascular permeability. Other cardiovascular problems that were observed in these animals included bilateral ventricular dilatation and cardiac enlargement (Allen et al., 1977). Cardiovascular disorders were also observed in monkeys that consumed lethal amounts of toxic fat. These disorders included blood vessel degeneration and cardiac edema (Allen and Carstens, 1967). The extensive number and severity of lesions in the animals from both studies indicates that some of the lesions, especially cardiovascular lesions that are not commonly observed after TCDD treatment, were produced by other compounds that these animals were administered, as mentioned previously in this chapter. 7.9 SUMMARY AND CONCLUSION 2,4-D and 2,4,5-T are not cumulative toxicants. Subacute doses of 2,4-D of at least 100 mg/kg/day or 1,000 ppm in feed are required to elicit toxicity. Symptoms produced by both compounds are nonspecific. Myotonia is not produced by subacute doses of 2,4-D, while bleeding of the gums has been noted in dogs. Other symptoms for 2,4-D and 2,4,5-T resemble those of acute toxicity. The phenoxy acids (in the absence of TCDD) also produced hepatotoxicity, including necrosis, hepatitis, and increased liver-to-body weight ratios. 7-23 �Doses of TGDD administered within a month of each other show cumulative effects; doses administered more than 1 month apart do not. Monkeys show higher sensitivity to repeated doses of TCDD than to the cumulative dose when it is given in a single administration, Subchronic doses of TCDD have caused porphyria and lethal depletion of blood cells. Other symptoms of subacute toxicity resemble those observed after acute exposure. The cause of death from poisoning by the phenoxy acids or TCDD is usually not apparent. Dermal and thymic effects of TCDD after subacute exposure resembled those seen after acute exposure. TCDD produced degenerative changes in -the liver, which varied widely in intensity among the species studied. At doses producing no other effects, increased liver-to-body weight ratios were observed. Porphyria was observed in the mouse and the rat, although iron deficiency protects animals from this effect of TCDD. All three compounds produced reduction in body weights, although the mechanisms involved are unknown for any of the compounds. All three compounds also produced hemorrhaging at high doses. Data on the subacute and chronic toxicities of the remaining compounds are sparse and for some compounds nonexistent. Chronic exposure to diquat produces cataracts. Picloram in doses of 225 mg/kg/day (3,000 ppra in the diet) for 90 days produces only mild effects, including hepatic and liver changes. Daily doses of 1,000 mg/kg of dalapon also produced local or mild nonspecific effects. 7-24 �CHAPTER 7. REFERENCES Allen, J. R., Barsotti, D. A., Van Miller, J. P., Abrahamson, L. J., and Lalich, J. J. (1977) Morphological changes in monkeys consuming a diet containing low levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fd. Cosmet. Toxicol. 15:401-410. Allen, J. R., and Carstens, L. A. 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Contam. Toxicol. 6:33-46. Courtney, K. D. (1976) Mouse teratology studies with chlorodibenzo-p-dioxins, Bull. Environ. Contam. Toxicol. 16(6):674-681. Courtney, K. D., Ebron, M. T., and Tucker, A. W. (1977) Distribution of 2,4,5-trichlorophenoxyacetic acid in the mouse fetus. Toxicol. Letters 1(2):103-108. Courtney, K. D., Gaylor, D. W., Hogan, M. D., Falk, H. L., Bates, R. R., and Mitchell, I. (1970) Teratogenic evaluation of 2,4,5-T. Science 168:864-866. Courtney, K. D., and Moore, J. A. (1971) Teratology studies with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 20:396-403. Cutting, R. K., Phuoc, T. H., Ballo, J. M., Benenson, M. W., and Evans, C. H. (1970) Congenital malformations, hydatidiform moles and stillbirths in the Republic of Vietnam 1960-1969. (Washington, DC.: Department of Defense. U.S. Government Printing Office No. 903.233.) 29 pp. Carere, A., Cardamone, G., Ortali, V., Bruzzone, M. L., and Di Giuseppe, G. 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L., Habermann, R. T., and Fitzhugh, 0. G. (1971) Chronic toxicity of 2,4-dichlorophenoxyacetic acid in rats and dogs. Toxic. Appl. Pharmacol. 20:122-129. 7-27 �Harris, M. W., Moore, J. A., Vos, J. G., and Gupta, B. N. (1973) General biological effects of TCDD in laboratory animals. Environ. Health Perspect. 5:101-109. Highman, B., Gaines, T. B., Schumacher, H. J., and Haley, T. J. (1976) Strain differences in histopathologic, hematologic and blood chemistry changes induced in mice by a technical and purified preparation of 2,4,5-trichlorophenoxyacetic acid. J. Toxicol. Environ. Health 1(6):1041-1054. Hill, E. V., and Carlisle, H. (1947) Toxicity of 2,4-dichlorophenoxyacetic acid for experimental animals. J. Indust. Hyg. and Toxicol. 29(2):85-95. Hinsdill, R. D., Thomas, P. T., and Couch, D. Immunotoxicity assessment of dioxins. In: Inadvertent modification of the immune response. The effects of foods, drugs and environmental contaminants. Office of Health Affairs, FDA, Washington, B.C. pp. 76-79. Hodge, H. C., Maynard, E. A., Downs, W. L., and Coye, R. D. (1958) Chronic toxicity of 3-(p-chlorophenyl)-l,l-dimethylurea (Monuron). Am. Arch. Ind. Health 17:45-47. International Agency for Research on Cancer. (1977) IARC monographs on the evaluation of the carcinogenic risk of chemicals to man: Some fumigants, the herbicides 2,4-D and 2,4,5-T, chlorinated dibenzodioxins and miscellaneous industrial chemicals. 15:41-299. Jones, K. G., and Sweeney, G. D. (1979) Iron deficiency prevents liver toxicity of 2,3,7,8- tetrachlorodibenzo-p-dioxin. Fed. Proc. Fed. Am. Soc. Exp. Biol. 38(3):536. Kay, J. H., Palazzolo, R. J., .and Calandra, J. C. (1965) Subacute dermal toxicity of 2,4-D. Arch. Environ. Health 11:648-651. Kenaga, E. E. (1974b) Toxicological and residue data useful in the environmental safety evaluation of dalapon. Residue Rev. 53:109-151. King, M. E., and Roesler, A. R. (1974) Subacute intubation study on rats with the compound 2,3 ,7,8-tetrachlorodioxin. IIT Research Inst. Report to EPA No. IITRI-L6073-12. 60 pp. Kociba, R. J., Keeler, P. A., Park, C. N., and Gehring, P. J. (1976) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 35:553-574. 7-28 �Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Gehring, P. J. (1979a) Long-term toxicologic studies of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) in laboratory animals. Ann. N.Y. Acad. Sci. 320:397-404. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., et. al. (1978) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46:279-303. Ronstantinova, T. K. (1974) Experiments on the effects of 2,4,5-T butyl ester on pregnant animals and on the development of their offspring. Gig. Sanit. 39(8):101-102. Kuz'minskaya, U. H., and Bersan, L. V. (1975) The effects of the sodium salt of 2,4-dichlorophenoxy-acetic acid on the glycolysis, ATP-ase and transketolase activity of the erythrocytes. Farmakologiya i toksikologiya. 38(1):102-104. Luster, M. I., Clark, G., Lawson, L. D., and Faith, R. E. (1979a) Effects of brief in vitro exposure to 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) on mouse lymphocytes. J. Environ. Pathol. Toxicol. 2:965-977. Luster, M. I., and Faith, R. E. (1979) Assessment of immunologic alterations caused by halogenated aromatic hydrocarbons. Ann. N.Y. Acad. Sci. 320:572-578. Luster, M. I., Faith, R. E., and Clark, G. (1979b) Laboratory studies on the immune effects of halogenated aromatics. Ann. N.Y. Acad. Sci. 320:473-486. Lynn, G. E. (1965) A review of toxicological information on TORDON herbicides. Down to Earth 20(4):pp. 6-8. McCollister, D. D., and Leng, M. L. (1969) Toxicology of picloram and safety evaluation of TORDON herbicides. Down to Earth 25(2):5-10. McNulty, W. P. (1977) Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin for Rhesus monkeys: Brief report. Bull. Environ. Contain. Toxicol. 18(1):108-109. Midwest Research Institute. (1975c) Substitute chemical program. Initial scientific review of cacodylic acid. Prepared for Environmental Protection Agency, PB-251 541. 130 pp. 7-29 �Moore, J. A. (1978) Toxicity of 2,3,7,8-tetrachlorodibenzo-para-dioxin. In Chlorinated Phenoxy Acids and Their Dioxins. ed. C. Ramel (Ecol. Bull, No. 27, Stolkholtn: Swedish Natural Science Research Council, 1978) pp. 133-144. Morton, W. E., Crawford, E. D., Maricle, R. A., Douglas, D. D., and Freed, V. H. (1975) Hypertension in Oregon pesticide-formulating workers. J. Occ. Med. 17(3):182-185. Murray, F. J., Smith, F.F., Nitschke, K. D_., Humiston, C. G., Kociba, R. J., and Schwetz, B. A. (1979) Three generation reproduction study of rats given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the diet. Toxicol. Appl. Pharmacol. 50(2):241-252. National Research Council. (1974) The Effects of Herbicides in South Vietnam: Part A. Summary and Conclusions. National Academy of Sciences, Washington, B.C. AD-774-749. Norback, D. H., and Allen, J. R. (1973) Biological responses of the nonhuman primate, chicken, and rat to chlorinated dibenzo-p-dioxin ingestion. Environ. Health Persp. 5:233-240. Palmer, J. S. (1963) Chronic toxicity of 2,4-D alkanolamine salts to cattle. J.A.V.M.A. 143(4):398-399. Palmer, J. S., and Radeleff, R. D. (1964) The toxicologic effects of certain fungicides and herbicides on sheep and cattle. Ann. N. Y. Acad. Sci. 111:729-736. Paynter, 0. E., Tusing, T. W., McCollister, D. D., and Rowe, V. K. (1960) Toxicology of dalapon sodium (2,2-dichloropropionic acid, sodium salt). J. Agric. Food Chem. 8(1):47-51. Pirie, A., and Rees, J. R. 9(2):198-203. (1970) Diquat cataract in the rat. Exp. Eye Res. Pirie, A., Rees, J. R., and Holmberg, N. J. (1970) Diquat cataract: formation of the free radical and its reaction with constituents of the eye, Exper. Eye Res. 9(2) .-204-218. Pirie, A., Rees, J. R., Holmberg, N. J. Biochem. J. 114(4): 89 pp. 7-30 (1969) Diquat cataract in the rat. �Rasmuson, B., and Svahlin, H. Mutagenicity tests of 2,4-dichlorophenoxyacetic acid and 2,4,5,-trichlorophenoxyacetic acid in genetically stable and unstable strains of Drosophila melanogaster. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, edT (Ecol. Bull. No. 27, Stolkholm: Swedish Natural Science Research Council, 1978) p. 190-192. Rip, J. W,, and Cherry, J. H. (1976) Liver enlargement induced by the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). J. Agric. Food Chem. 24(2):245-250. Rip, J. W., and Cherry, J. H. (1976) Liver enlargement induced by the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). J. Agric. Food Chem. 24(2):245-250. Rowe, V. K., and Hymas, T. A. (1954) Summary of toxicological information of 2,4-D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. Amer. J. Vet. Res. 15:622-629. Schantz, S. L., Barsott, D. A., and Allen, J. R. (1979) Toxicological effects produced in nonhuman primates chronically exposed to fifty parts per trillion 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Appl. Pharmacol. 48(1):A180. Schwetz, B. A., Norris, J. M., Sparschu, G. L., Rowe V. K., and Gehring, P. J. (1973) Toxicology of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5:87-99. Schwetz, B. A., Sparschu, G. L., and Gehring, P. J. (1971) The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and esters of 2,4-D on rat embryonal, foetal and neonatal growth and development. Fd. Cosmet. Toxicol. 9:801-817. Sharma, R. P., and Gehring, P. J. (1979) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on splenic lymphocyte transformation in mice after single and repeated exposures. Ann. N. Y. Acad. Sci. 320:487-497. Shavgulidze, M. M., Nanobashvili, V. I., and Mirianashvili, M. N. (1976) On the toxicity of the herbicide 2,4-D. Veterinaria (4):103-104. Sherman, H., and Kaplan, A. M. (1975) Toxicity studies with 5-bromo-3sec-butyl-6-methyluracil. Toxicol. Appl. Pharmacol. 34(2):189-196. 7-31 �Stroo, W. E., McCormack, K. M., and Hook, J. B. (1979) Renal functional effects of 2,4,5-trichlorophenoxyacetic acid and silvex after acute and prolonged exposure. J. Toxicol. Environ. Health 5(5):845-854. Sweeney, G. D., Jones, K. G., Cole, F. M., Basford, D., and Krestynski, F. (1979) Iron deficiency prevents liver toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Science 204(4390):332-335, Szocs, J. , Dr., Molnar, V., Balogh, E., Ajtay, M., and Fulop, I. (1970) Experimental data on the toxic effects of 2,4-D (diclordon) herbicide. Revista Medicala 16:91-93. Thigpen, J. E., McConnell, E. E., Moore, J. A., and Faith, R. E. (1977) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on host resistance to infectious agents. Environ. Health Perspec. 20:245. Thigpen, J. E., Faith, R. E., McConnell, E. E., and Moore, J. A. (1975) Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infection and Immunity 12(6):1319-1324. Thunberg, T., Ahlborg, U. G., and Johnsson, H. (1979) vitamin A (retinol) status in the rat after a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch. Toxicol. 42(4):265-274. Van Miller, J. P., and Allen, J. R. (1977) Chronic toxicity of 2,3,7.8tetrachlorodibenzo-p-dioxin in rats. Fed. Proc. Fed. Am. Soc. Exp. Biol, 36:396. The Veterans Administration. (1981) Advisory Committee on Health-Related Effects of Herbicides - Transcript of Proceedings. Feb 4, 1981. 158 pp. The Veterans Administration. (1980h) Proceedings from the 2d Continuing Education Conference on Herbicide Orange. Washington, DC, May 28-30. Vos, J. G. (1978) 2,3,7,8-Tetrachlorodibenzo-para-dioxin: Effects and mechanisms. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978) p. 165-176. Vos, J. G. (1977) Immune suppression as related to toxicology. CRC Critical Reviews in Toxicology 7-32 �Vos, J. G., Kreeftenberg, J. G., Engel, H. W. B., Minderhoud, A., and Van Noorle Jansen, L. M. (1978) Studies on 2,3,7,8-tetrachlorodibenzop-dioxin induced immune suppression and decreased resistance to infection: Endotoxin hypersensitivity, serum zinc concentrations and effect of thymosin treatment. Toxicology 9:75-86. Vos, J. G., Moore, J. A., and Zinkl, J. G. (1973) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals. Environ. Health Perspec. 5:149-162. Vos, J. G., Moore, J. A., and Zinkl, J. G. (1973) Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals. Environ. Health Perspec. 5:149-162. Weimer, J. T., Ballard, T. A., Owens, E. J., and McNamara, B. P. (1970) Toxicological studies on herbicide "White" in animals. US NTIS Technical Report No. AD 712 317. 28 pp. Weissberg, J. B., and Zinkl, J. G. (1973) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin upon hemostasis and hematologic function in the rat. Environ. Health Perspect. 5:119-123. Whitehead, C. C., and Pettigrew, R. J. (1972b) The subacute toxicity of 2,4-dichlorophenoxyactetic acid to chicks. Toxicol. Appl. Pharmacol. 21:348-354. Woods, J. S. (1973) Studies on the effects of 2,3,7,8-tetrachlorodibenzop-dioxin on mammalian hepatic -aminolevalinic acid synthetase. Environ. Health Perspec. 5:221-225. Young, A. L., Calcagni, J. A., Thalken, C. E. , and Tremblay, J. W. (1978) The toxicology, environmental fate, and human risk of herbicide orange and its associated dioxin. USAF Occupational and Environmental Health Laboratory Report No. USAF OEHL - 78 -92. 262 pp. Zinkl, J. G., Vos, J. G., Moore, J. A., and Gupta, B. N. (1973) Hematologic and clinical chemistry effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Perspec. 5:111-118. 7-33 �CHAPTER 8 REPRODUCTIVE TOXICITY This chapter describes the effects of herbicide and TCDD exposures on reproductive parameters. Studies that used 2,4,5-T preparations known to contain less than 1 ppm TCDD are described together. Effects produced by these preparations have been attributed* to 2,4,5-T. Studies that used 2,4,5-T preparations with unknown amounts of TCDD or that were known to have more than 1 ppm TCDD are described in a separate section, as the observed effects may have been caused by TCDD, 2,4,5-T, or both, in either an additive or synergistic manner. The effects of each herbicide on animals that grazed on sprayed pastures, resulting in ingestion of an unknown quantity of herbicide, are mentioned at the end of the section. Data from avian species are also mentioned only briefly, since their route of exposure and fetal isolation from maternal sources of metabolism render them to be inadequate models for mammalian teratogenicity. Aspects of reproductive effects of phenoxy acids and TCDD have been reviewed recently (Anonymous, 1980; Allen et al., 1979; Greig, 1979; Tschirley and Melvin, 1979; Gehring and Betso, 1978; Joint IARC/NIEHS Working Group, 1978; Nelson and Holson, 1978; Young et al., 1978; IARC, 1977; Wilson, 1977; Epstein, 1973; Johnson, 1971; Robson, 1970). 8.1 2,4-D One study evaluated the relationship between human male exposure to 2,4-D and the incidence of spontaneous abortions in their wives. Questionnaires pertaining to these two factors were mailed to almost 15,000 people who were employed in occupations involving the manufacture or use of herbicides. From the respondents, a group of 134 cases of miscarriages were selected and compared to a control group of 311 cases of live births. The cases were analyzed to determine the incidence of paternal exposure to 2,4-D prior to conception, and the incidence of other relevant confounding factors, such as smoking, illnesses, and drug use. The data were obtained or verified by telephone interviews. No positive association was established between 2,4-D exposure and abortions, for the entire study population. For a group of 21 cases of wives of young forest and commercial workers, an association was suggested, but was not found for any other subgroup. Further investigations were suggested to clarify whether this relationship resulted from biases in study design or reflected a real increase among this small subset (SRI International, 1981). The effects of 2,4-D administration during gestation have been studied in the rat, mouse, and hamster (see table 8-1). In the rat, 2,4-D at high doses produced a decrease in fetal weight in four separate studies. Malformations of the skeletal system, which were not severe or incompatible with life, were also reported in one of these studies (Khera and McKinley, 1972). In the second study, abnormal cranial development was noted after treatment with the butyl ester, but not the sodium salt, of 2,4-D (Buslovich et al., 1976). No �TABLE 8-1: Daily Dose REFERENCES ON THE REPRODUCTIVE EFFECTS OF 2,4-D IN MAMMALS Exposure Period3 Species Route Rat ( D S) oral 8.7mg/kg 6-15 (Wistar) oral lOOmg/kg Maternal Toxicity Fetal Death - - - 6-15 Decreased Malformations Growth Gross Visceral Skeletal Reference - - - Schwetz et al., 1971 - - + Khera and McKinley, 1972 - + all - - + (»50> 4-13° - + + + - + cp + - - (Osborne— gavage ISOOppm Mendel) Mouse oral oral, sc ImM/kg 12-15 Hamster oral lOOmg/kg 6-10 cranial Q Buslovich et al., 1976 Courtney, 1977 Bionetics, 19686 Collins and Williams, 1971 days of gestation ""level of 2,4-D added to the diet "on only one of these days CP, cleft palate a various strains of mice, routes and doses were used; effects were reported for some treatment groups Code: (+) a deleterious effect in this parameter was observed; (-) this indicated parameter was analyzed, but no effect was observed; ( ) this indicated parameter was not analyzed; �malformations were observed in the third and fourth studies (Schwetz et al., 1971; Hansen et al., 1971). In the study by Hansen, three consecutive generations of rats were continuously fed diets of 100, 500, or 1,500 ppm 2,4-D; in the highest group, decreased survival of neonates to 21 days of age was observed, although litter sizes were not decreased. A fifth study also reported embryotropic effects after 2,4-D administration, but neither the conditions of administration nor the effects were described (Ronstantinova et al., 1978). In the mouse a small increase in the incidence of cleft palate and a decrease in fetal growth were caused by 2,4-D, at a dose that also produced increased maternal liver-to-body weight ration (Courtney, 1977). A study by Bionetics (1968), found variable effects of 2,4-D in the mouse, depending on the chemical form administered, the route, the strain of mouse, and the dose and parameters studied. In the hamster, a small increase in fetal death was observed after 2,4-D administration, although this effect was not doserelated, and no malformations or effect on fetal growth occurred at doses of up to 100 mg 2,4-D/kg (Williams and Collins, 1971). These mammalian studies have shown that 2,4-D is capable of affecting fetal growth, but does not affect fetal survival and does not reproducibly cause malformations. Cattle and sheep have had deleterious reproductive effects after grazing pastures that were recently sprayed with 2,4-D (Sadykov et al., 1972; Bodai et al., 1974). Adverse teratological effects were observed in eggs that were treated with 2,4-D, although other lots of 2,4-D failed to reproduce these effects (Dunache and Fletcher, 1967; Whitehead and Pettigrew, 1972). In sum, the primary observed effect of 2,4-D was a decreased rate of fetal growth for exposed gestating mice. No link has been drawn conclusively between exposure of men to 2,4-D and either their fertility or the rate of spontaneous abortion in their wives. 8.2 2,4,5-T In this section only studies of 2,4,5-T preparations with less than 1 ppm TCDD contamination are discussed. Studies of 2,4,5-T with higher levels of TCDD and studies that used 2,4,5-T which were not analyzed for TCDD are described in section 8.4. None of the cases of suspected reproductive effects in humans from alleged exposure to 2,4,5-T involved situations in which concomitant TCDD exposure could be eliminated, so they are all discussed in section 8.4 as well. All reproductive studies of only male exposure to 2,4,5-T similarly involved 2,4,5-T preparations of unknown levels of TCDD contamination and are described in section 8.4. One mouse study involved exposure of males to 2,4-D, 2,4,5-T, and TCDD simultaneously. One group of mice was exposed to the phenoxy acids in the presence of 0.16 ppm TCDD, and no reproductive or teratogenic effects were observed (Lamb et al., 1980). The results from this entire study are discussed in section 8.4. Evidence that 2,4,5-T reaches the fetus after it is administered maternally has been produced in the mouse and guinea pig. Lindquist and Ullberg (1971) demonstrated by whole-body autoradiography that 2,4,5-T administered o-T �late in gestation is distributed evenly among fetal tissues and to the same levels as in maternal blood. 2,4,5-T was concentrated in the yolk-sac epithelium after administration either early or late in gestation. Dencker (1976) confirmed that 2,4,5-T uptake by the fetal mouse occurred only late in gestation (after day 12). No uptake of 2,4,5-T administered prior to day 12 was observed in the fetal hamster, and uptake in the hamster at later times was not studied. In the mouse, fetal concentrations of 2,4,5-T were highest eight hours after an oral dose was administered maternally on day 13 of gestation, although distribution to the palate could not be demonstrated (Courtney et al., 1977). In contrast, in the guinea pig, the maternal rate of 2,4,5-T excretion was so high that 2,4,5-T did not reach the fetus (Ebror and Courtney, 1976). All of the studies of the reproductive effects of 2,4,5-T (without significant TCDD contamination) involved administration only during gestation. Five mammalian species have been used for these studies: the monkey, rat, mouse, rabbit, and hamster. References on these experiments are listed in table 8-2. 2,4,5-T did not produce any malformations in rhesus monkeys that were administered up to 10 mg/kg doses during gestation (Dougherty et al., 1973; Dougherty et al., 1975; Dougherty et al., 1976). No maternal toxicity was observed in this experiment. The rate of fetal death, including abortions and stillborns, was 20 percent for groups of 10 monkeys given 10 mg/kg and 1 mg/kg doses, and 10 percent for the control group. This difference is probably not significant, because the number of animals per group was small (10 per group) and a spontaneous abortion rate of 15 percent is usually considered normal for untreated monkeys. In the rat, 2,4,5-T at doses of 50 mg/kg or less produced no adverse effects on maternal toxicity, or on fetal survival, growth, and development (Emerson et al., 1971; the same experiment was reported in abstracts by Emerson et al., 1970; Thompson et al., 1971; Khera et al., 1971; Sparschu, 1971; Khera and McKinley, 1972). Dilated renal pelvises were observed in 7-45 percent of Wistar rats that received 2,4,5-T, but the incidence was not treatment-related, as the control group also had a high incidence of this defect, with 20-30 percent of fetuses affected (Khera and McKinley, 1972). This defect was observed in mice and rats after TCDD treatment, as well (see section 8.3). Related doses of 100 mg/kg to Wistar rats during gestation produced increases in fetal deaths, growth retardation, and skeletal abnormalities, but no maternal toxicity (Khera and McKinley, 1972). This dosage produced severe maternal toxicity and fetal death in Sprague-Dawley rats. The incidence of dilated renal pelvis in the few fetuses that survived was two out of the six fetuses examined, compared to 21 percent of the 110 control fetuses that were examined (Sparschu, 1971; also reported in an abstract by Thompson et al., 1971). Behavioral effects, which were measured as alterations in open-field behavior, were observed in Wistar rats that were exposed to a single dose of 100 mg/kg 2,4,5-T in utero. This dose did not produce any other effects 8-4 �TABLE 8-2: Species Monkey Daily Dose Route og/kg oral 10 (SD) oral 24 (SD) Rat oral 50 REFERENCES ON THE REPRODUCTIVE EFFECTS OF 2,4,5-T (less Chan Ippm TCDD) ICDD Exposure Maternal Fetal Decreased Level _| Period Toxicity Death Growth Gross + .05 22-38 . . 6-15 .5 ,5 6-15 - - - _ _ + + + oral 100 .5 6-10 (Wistar) oral 50 .5 6-15 (Wistar) oral 100 .5 6-15 (Wistar) oral 100 1 7,8,9 (Wistar) oral 100 1 8,9,10 oral 35 50 .1 1 6-15 6-14 60 .05 6-15 - (SD) Mouse (NKRI) (NMRI) 9C (NMRI) - 4- - + + - 4- (NMRI) oral 80 .1 6-15 + 4. (NMRI) inhal .1 6-15 4- 4. (CD-I) oral 216 mg/m 60 .05 6-14 (CD-I) (CD-I) (CD-I) oral 100 oral . ImM/kg oral 250-300 .5 .1 .01 6-15 12-15 7-15" 4- + + 4- Hamster oral 100 .5 6-10 Rabbit oral 40 . 5 •f _ Malformations Viscera] Skeletal Behavioral Reference . Dougherty et al., 1975 . . Emerson «t al., 1971 Sparschu, 1971 renal • Thompson et al., 1971 _ Khera & _ McKinley, 1972 4. Khera f, McKinley, 1972 4, SJoden & Soderberg, 1972 + Sjoden & Soderberg, 1975 cp Roll, 1971 + cp Bage et al., 1973 cp Meubert & Dillman, 1972 4 . Frohberg et al.,1975 4Frohberg et al., 1975 cp renal Highman et al.,1977 + Beck, 1981 cp Courtney, 1977 _ . exancephHood ec al., 1979 aly short 6-18 4. + _ 4+ 4. + _ _ exenceph- GI hen. aly abnormal ayes _ _ _ Sheep oral 100 1 14-36 - 4, Collins & Williams, 1971 _ Emerson et al., 1971 Binns and Sails dose given for 3 consecutive days between d 7-15; other mica were administered 800-900 mg/kg on a single day between d. 8 and 15. J 8-5 �(although visceral and skeletal examinations were not mentioned). The significance of these transient behavioral defects in relation to effects in man has not been demonstrated. The authors suggested that these effects resulted from an effect of 2,4,5-T on the thyroid but did not attempt to demonstrate any thyroid deficiency in these animals (Sjoden and Soderberg, 1972, and 1975). 2,4,5-T produces cleft palate in NMR1 and CD-I mice. This effect was produced by doses of 35 rag/kg, administered during days 6-15 of gestation, which did not produce obvious maternal toxicity (Roll, 1971). Decreased fetal growth occurred in groups given lower doses (20 mg/kg), while higher doses (60-90 mg/kg) produced maternal toxicity and fetal deaths. Skeletal malformations (table 8-2) involved decreased rates of ossification of the skeleton and were observed along with decreased fetal body weights. In another experiment in NMRI mice, higher doses of 2,4,5-T than the 10 mg/kg dose that caused retarded fetal growth (20 mg/kg) were also needed to cause an increase in cleft palate; neither dose produced maternal toxicity (Neubert and Dillman, 1972). Subcutaneous and inhalation exposures to 2,4,5-T during gestation also produced cleft palate in NMRI mice (Bage et al. , 1973; Frohberg et al., 1975). In CD-I mice, exposure to 2,4,5-T on day 11 of gestation was the most likely day to result in cleft palate, while exposure on days 14-15 were the most likely days to cause decreased fetal growth (Hood et al., 1979). Since days 11 and 14-15 are closest to the days when the palate closes and rapid growth occurs, respectively, these results are in accord with the rapid distribution and clearance of 2,4,5-T. In the CD-I mouse, the isobutyl and isooctyl esters were found to be equivalent in producing cleft palate, fetal growth retardation, and fetal deaths. Maternal weights were not altered at oral doses (100 mM/kg) that caused these adverse fetal effects, but the ratio of maternal liver-to-body weights was elevated (Courtney, 1977). Since most teratology studies evaluated maternal body weights but not maternal liver weights, this subtle maternal effect was usually missed. Few malformations other than cleft palate were observed in mice after 2,4,5-T treatment. Beck (1981) reported that mice exposed to 2,4,5-T in utero showed a high incidence of skeletal anomalies in adulthood. Seventeen of 88 traits examined were more frequently encountered in rats treated with 100 mg 2,4,5-T/kg before birth than in controls. However, this dose produced high maternal toxicity, and a lower dose of 20 mg/kg did not result in skeletal anomalies in adulthood. Highman et al. (1977) observed a delay in renal development in CD-I mice following administration of 60 mg 2,4,5-T/kg. This dose also produced cleft palate, which was not observed in the control group. Doses of 100 mg 2,4,5-T/kg administered to hamsters on days 6-10 of gestation were embryotoxic, and malformations and decreased fetal weights were observed in the surviving offspring. The conditions of the mothers were not described. In the hamster, malformations involved abnormal development of the head, but not the cleft palate which was observed in the mouse (Collins and Williams, 1971). However, 2,4,5-T administration after day 10 in gestation may be required to provide an effective dose at the palate just prior to closing (which is complete on day 12 in the hamster). In the rabbit, the highest dose tested ( 0 mg/kg) was ineffective in altering fetal growth or 4 8-6 �development (Emerson et al., 1971). These mammalian studies indicate that 2,4,5-T is teratogenic, producing cleft palate in several strains of mice at doses that elicit no maternal toxicity (or only a subtle decrease in liverto-body weight ratios). 2,4,5-T causes fetal growth retardation at even lower doses. The effects were not observed in any other species. Studies of 2,4,5-T effects on chicken development have shown an effect on behavioral development (Sanderson and Rogers, 1981), but not on other parameters of structural development of eggs sprayed with 2,4,5-T (Somers et al., 1978a; 1978b; 1978c,), although decreased viability was observed in eggs that were immersed in 2,4,5-T (Gyrd-Hansen and Dalgaard-Mikelsson, 1974). Cardiovascular abnormalities were observed in fish, following exposure of eggs to 2,4,5-T (Schreiveirs and Murray, 1976). Significant effects of mammalian exposure to 2,4,5-T included increased incidence of cleft palate in mice at doses that caused no maternal toxicity, and decreases in fetal growth at lower doses. Teratogenicity and other embryotoxic or lethal effects were not observed in other species studied. 8.3 TCDD In this section, studies of the reproductive effects of TCDD are described. Human exposure to TCDD resulted from industrial accidents and occupational exposures, which have been described in chapter 5. Only reproductive data from these incidents are described in this section. The reproductive toxicity of TCDD has been studied in several laboratory species, as described below. 8.3.1 Human Exposure Evaluation of reproductive effects of TCDD have been made after TCDD exposure of male workers in Czechoslovakia and after TCDD exposures of residents near Seveso. The reproductive performance and frequency of abortions among wives of the Czechoslovakian workers were reported to be normal (Pazderova-Vejlupkova et al., 1980), although no systematic study of reproductive parameters among these workers or expected values of parameters for a matched control group were reported. The reproductive parameters of the residents near Seveso were never systematically studied, either. The total number of abortions were reported, but they were from zones A, B, and R, and the expected or actual incidence of abortions for each exposure group was not presented. Paternal exposure and the estimated length of exposure prior to mating were not described. An estimated 150 women in the area near Seveso were in the first trimester of pregnancy when the explosion occurred. Most of the 125 abortions were elected, after the mothers became aware of the potential for having a child with birth defects. This knowledge alone, and not necessarily exposure to TCDD, would be likely to cause a large decrease in the birth rate and increase in the rate of abortions. No study design has taken these factors into account. 8-7 �Information on the teratology of 34 fetuses that were spontaneously or therapeutically aborted by women in the Seveso area was published by Rehder et al. (1978). (The remaining abortions were not officially approved and had to be performed elsewhere.) Only three of the 34 mothers were reported to be residents of zone A where they would be likely to have received a relatively high exposure to TCDD. Five were from zone B, 13 from zone R, and nine from outside the contaminated areas. The report by Rehder et al. states that the ages of the fetuses were 4-20 weeks and that they were removed within 2 1/2 months of the accident. There is no way to know how many cases involved exposure which occurred only after conception or before and after conception, and whether one or both parents were exposed prior to conception. Although no structural abnormalities attributable to TCDD exposure were reported, the authors (Rehder et al., 1978) indicated that many of the fetuses were mutilated from the procedures used to perform the abortions. The authors did not state how many fetuses were suitable, based on age and condition, for evaluation of cleft palate, renal abnormalities, or other suspected malformations. Therefore, the teratologic data from Seveso do not provide enough information to know whether the fetuses from mothers that were exposed to TCDD were old enough and adequately preserved to detect any specific birth defects. Unless all of the known aborted fetuses (numbering 125) were examined, it seems unlikely that enough well-preserved fetuses from parents exposed to TCDD (during organogenesis, 3-8 weeks after conception) could be examined to identify any nonspontaneous malformations. Three of the four mothers who experienced spontaneous abortions were from zone R. The report did not indicate the time period between the Seveso accident and the spontaneous abortions and found no evidence for or against any causal relationship. 8.3.2 Animal Exposure Prior to Mating An unpublished report by Townsend et al., (1981) described a study conducted in workers at Dow Chemical Company who were involved in manufacturing processes that could potentially have resulted in their exposure to TCDD. These employees were identified by company records and their wives were interviewed to determine incidences of various reproductive parameters. A control group of wives of workers who were not likely to have been exposed to TCDD or other dioxins was also interviewed. No increases in the incidences of spontaneous abortions, stillbirths, infant deaths, or congenital malformations were identified in wives of the exposed group. The exposed group included pregnancies that occurred at any time after the father was exposed. Only male exposures that occurred near the time of conception are relevant to the outcome of a particular pregnancy (based on the male reproductive cycle). Thus, data limited to cases of male exposure close to conception would have provided more relevant information, although the study group used is more analogous to American veterans, who may have been exposed to dioxins many years before they were ready to become fathers. One animal study of reproductive effects from male exposure to TCDD prior to mating has been reported (Murray et al., 1979). Male rats that were fed 0.01 ug/kg body weight per day for 1 year were mated with younger, untreated 8-8 �females. Sixty percent of the 20 females became pregnant, with an average of 10 implants per female and a 9 percent rate of resorption of implants. Male rats that were fed a control diet for 1 year were mated with untreated, younger females; 58 percent of the females became pregnant with nine implants per female and a 10 percent resorption rate. Matings between untreated young male and female rats resulted in a 90 percent pregnancy rate, and matings of male and females fed control diets for one year produced a 30 percent pregnancy rate. These results indicate that the older rats showed diminished fertility, precluding any evaluation of the effects of TCDD on reproduction in these rats. In another study, male mice were exposed to a combination of 2,4-D, 2,4,5-T, and TCDD (0.16-2.4 ug/kg) prior to mating, and no adverse reproductive effects were observed (see section 8.3). Other studies have considered the effects of exposure of both males and females to TCDD prior to mating. In a three-generation reproductive study in rats, TCDD exposure started 90 days prior to mating and was maintained for three generations. Decreased fertility, increased numbers of stillbirths and neonatal deaths, and smaller litter sizes resulted from exposure to 0.1 or 0.01 ug TCDD/kg/day while exposure to .001 ug/kg/day produced no effects. Malformations were not studied. The litter sizes of beach mice that were progeny of an estimated 50 generations of male and female mice that lived in TCDD-contaminated sand were the same as those of control beach mice (VA, 1981). Alterations in the male reproductive system by TCDD have been described. Induction of aryl hydrocarbon hydroxylase activity and of cytochrome P-448 has been shown in rat testicular and prostate tissue (Lee and Dixon, 1978) and decreased catabolism of testosterone after TCDD treatment has been demonstrated in the rat (Nienstedt et al., 1979). The effects of these alterations on male reproductive potential or on the DNA of germ cells have not yet been demonstrated. Structural changes in male reproductive organs have been observed in the guinea pig and mouse, but were observed only in cases of lethal poisoning for both species. In the guinea pig, the changes involved reduction in the size of the testicles and loss of seminiferous components, while in the mice necrotic spermatocytes and reduced spermatogenesis occurred (McConnell et al., 1978). The effects of female exposure to TCDD prior to mating have been studied in the monkey and the mouse. Monkeys were fed diets with 500 ppt TCDD for 6 months prior to mating and during gestation. This dosage resulted in substantial maternal toxicity, including altered steroid hormone levels consistent with reduced fertility. A decreased number of conceptions and an increased rate of abortions were observed after mating. No gross malformations were mentioned in the aborted fetuses or in the few offspring (Barsotti et al., 1979). Female mice fed diets containing 5 ppb TCDD for 4 weeks prior to mating, during gestation, and after birth did not produce maternal toxicity, reduced fertility, fetal deaths, or decreased fetal growth. After 3 to 4 weeks of nursing, facial alopecia, periorbital edema, decreased spleen 8-9 �size, and immune suppression were observed in the offspring (Thomas and Hinsdill, 1979). These changes are consistent with toxicity observed in adult mice and suggest a higher suseptibility of newborns to TCDD toxicity, compared to adults. 8.3,3 Animal Exposure During Gestation Only The remaining studies of the reproductive toxicity of TCDD all involve administration of TCDD to females after conception has taken place. These studies are listed in table 8-3. Maternal toxicity was observed in monkeys that received a total of 1 ug TCDD/kg between days 20-40 of gestation. This dosage also produced cleft palate and an increased incidence of abortions. Only brief reports of these studies have been published (Zingeser, 1979; Colby, 1978). TCDD has been shown to reach the fetus after it was orally administered to pregnant rats on day 14, 18 or 21 of gestation (Moore et al., 1976). Teratologic effects of rats were reported in one study. A high frequency of kidney abnormalities was observed in fetuses of CD rats given 0.5 ug/kg/day of TCDD subcutaneously on days 6-15 of gestation. No maternal toxicity, or fetal death or decreased growth occurred at this dosage (Courtney and Moore, 1971). In another study, a lower dose (0.125 ug/kg/day) administered to rats orally during day 6-15 caused decreased fetal growth and signs of toxicity in the fetuses, but no malformations or increased fetal death (Sparschu et al., 1971; Sparschu et al., 1970). The remaining rat studies describe toxic effects in fetuses that are not considered teratogenic. The studies of immune function included TCDD exposure on 'day 18 of gestation and after birth. The effects observed in these rats were the same as in other rats that were exposed only postnatally, suggesting that prenatal exposure was not necessary for the observed decrease in cellular immunity (Faith and Moore, 1977; Faith et al., 1978; Faith and Luster, 1979; Vos and Moore, 1974). Few teratogenic effects would be expected from exposure on day 18, when most developmental processes and all organogenesis are complete. Teratology experiments in the mouse indicate that TCDD produces cleft palate and renal abnormalities after oral or subcutaneous administration on days 6-15 of gestation. Cleft palate was produced by daily doses of only 1 ug TCDD/kg a dose which produced no evidence of maternal toxicity, or fetal death or growth retardation (Smith et al., 1976). However, the incidence of cleft palate was low at this dose and a higher dose of 3 ug/kg/day not only increased the frequency of cleft palate, but also caused an increase in fetal death and evidence of maternal toxicity (Courtney and Moore, 1971; Neubert and Dillman, 1972; Smith et al., 1976). Cleft palate was also produced in strains of mice that have been shown to respond to the enzyme inductive effects of TCDD and to have a high capacity for TCDD-receptor binding, but was not produced in other strains that did not show induction and binding (Poland and Glover, 1980). Renal abnormalities have also been noted in mouse fetuses following TCDD exposure. These changes have been described as hydronephrosis, and resemble a slowed rate of renal development. Some evidence indicates that these changes 8-10 �TABLE 8-3: REFERENCES ON THE REPRODUCTIVE EFFECTS OF TCDD EXPOSURE TO FEMALES Species Route Dose us/kR/d oral 1 ug/kg total 20-40 oral _1 ug/kg (SD) Fetal Death _ Decreased Growth Gross _ Malformations Visceral _ Skeletal oral .2 15 6-15 B.C. .5 6-15 Reference Render et al.. 1978 + + 20-40 gavage 's'OOppt* pre, all Rat Maternal Toxicity 1-140 Han Monkey Days of Exposure Colby, 1978 cp + - Zingeser, 1979 Barsotti et al.. 1979 - + - - - (GI;edema)b - renal Sparschu et al., 1971 Courtney & Moore, 1971 + (hepatic)0 (spleen) 18, post + (spleen)d Faith 4 Luster, 1979 5 18, post + (spleen) Faith & Moore, 1977 oral 1 10-13 - renal Moore et al., 1973 (C57B1/6) oral 3 10-13 cp renal Moore et al., oral (CF-1) oral (C57B1/6) s.c. 1 6-15 3 6-15 6-15 cp cp cp renal - cp renal ( I e G) Courtney & Moore, 1971 cp renal ( I e G) Courtney & Moore, 1971 13-15 5 Mouse (C57B1/6) + 11, 18 5 - - Becker, 1973 Vos & Moore, 1974 1973 (F1 C-) 3 + - - - + - - - - - - - _ (DBA/2J) s.c. 3 6-15 + (B1 C-) s.c. 3 6-15 - (NMRI) oral 3 6-15 (NMRI) oral 9 9-13 - (NMRI) oral 9 6-15 + (CD-I) oral 25 7-16 + - (DI C-) s.c. 25 7-16 + + oral gavage a b c d - Smith et al.,1976 Courtney & Moore, 1971 cp Neubert & Dillman, 1972 cp Neubert & Dillman, 1972 - cp Neubert & Dillman, 1972 - - - cp. Level in feed GI hemarrhages; subcutaneous edema fatty degeneration decreased size of spleen and decreased cellular immunity e GI hemarrhages f facial alopecia; periorbital edema 8-11 Courtney, 1976 (spleen) (facial)1 pre, all, post renal renal Vos & Moore, 1974 (spleen)d Thomas & Hinsdall, 1979 Courtney, 1976 clubfoot + 14, 17, post 5ppb renal ( I e G) Smith et al., 1976 �are transient, as renal abnormalities do not persist in young mice who have had no exposure to TCDD after birth (Moore et al., 1972). Both cleft palate and renal abnormalities were produced in every strain of mouse that has been studied for these changes (see table 8-4). TCDD administered as toxic fat or as the isolated chemical in acetone, caused fetal deaths in chicken eggs. Structural malformations were not examined (Higginbotham et al., 1968; Allred and Strange, 1977). TCDD Reproductive or teratogenic effects of TCDD exposure in humans have not been substantiated, in part due to inadequate study designs. Chronic exposure of three generations of male and female rats has shown adverse reproductive effects that include low fertility; this may be the result of using animals with low fertility prior to exposure, or to doses that produced severe maternal toxicity. Reproducible teratologic effects in maternally exposed mice were primarily cleft palate, with some incidence of renal abnormalities. 8.4 COMBINATIONS OF 2,4-D, 2,4,5-T, AND TCDD In this section, the effects of 2,4,5-T with unknown levels of TCDD contamination and 2,4,5-T samples known to have at least 1 ppra TCDD are discussed. Studies of other combinations of 2,4-D, 2,4,5-T and TCDD are also included in this section. 8.4.1 Human Exposure Several instances of birth defects, abortions, and impotence in the human population have been purported to be associated with herbicide use. No quantitative assessments of herbicide exposure of the individuals involved has been made in any of the cases. Except for the cases of male impotence described below, either or both parents may have been exposed to herbicides, although the exposure periods, in relation to the time of conception, are unknown, or in some cases chronic exposure is assumed. Although 2,4,5-T, and in some cases its TCDD contaminant as well, have been the suspected teratogenic agents, concomitant exposures to many other substances were likely in each instance. In fact, unusually high exposure to 2,4,5-T of parents of affected children has never been demonstrated. In summary, all of the human cases described below involve reproduction abnormalities which retrospectively have been suggested to be caused by exposure to herbicides. Cutting et al. (1970) conducted a study of the incidences of abnormal births among the Vietnamese population relative to the extent of herbicide spraying during the same period of time. During the period of low military use of herbicides, between 1961 and 1965, the incidences of stillbirths, hydatidiform moles (spontaneous abortion of resorbed placental tissue), and malformations were 36.1, 6.6, and 5.5 per 1,000 live births, respectively. 8-12 �TABLE 8-4: REFERENCES ON THE REPRODUCTIVE EFFECTS OF 2,4,5-T (with unknown levels of TCDD) Species Route Dally Dose Exposure Maternal Fetal Decreasec (rag/kg) Period Toxicity Death Growth Monkey oral 1(/k 2-8 03w) 04 Rat oral + all 0.1 + + - + - (D S) oral 4.6 10-15 oral (SD) ioa 10-15 Lu 12-24 12-16 - - Mouse (C57Bl/b)oral (C57Bl/b)oral oral oral 46.4 - - 6-14 9-17 + - a 61 -5 + + 13 1 d (NMRI) sral 60 sc 10 Bionetics, 1968 Courtney et al. , 1970 King et a . 1973 l, - Hall, 1972 - renal Bionetics, 1968 cp renal + + 6-15 Courtney et al., 1970 cp renal Courtney et al., 1970 cp + 6-15 (GDI) renal (ic g) + a 113 Konstantinova, 1974 cp + a 113 Wilson, 1972 renal (iC g) + gavage 1000ppm all + - + b (AKR) Malformations 3ross Visceral Skeletal Reference - - cp - Courtney et al., 1970 Neubert & Dlllman, 1972 - - Hart & Valerio, 1972 CD-1,AJAX,C3H gavage 30 6-14 (DI C-) gavage 60 6-14 (DI C-) oral 100 (DI C-) oral 100 all IV 2 8 C57Bl/b, BALE Hamster cp renal + 30 ppm TCDD level in feed hemorrhages in GI tract 5 ppm TCDD = 0.3 ug/kg body weight Gaines et al., 1974 - + - Highman et al., 1976 renal Courtney and Moore, 1971 [enzymes) cp Courtney, 1979 Gale and Ferm, 1973 �During the period of high military use of herbicides, between 1965 and 1969, all of these rates fell, to 32.0, 5.6, and 4.5 per 1,000 live births, respectively. Despite difficulties in data collection and biases based on the sampling procedures, these results do not support the premise that herbicide use is correlated with elevated rates of fetal death or malformations in the exposed population. Tung et al. (1980) reported that birth defects were frequent among Vietnamese children whose fathers had been soldiers in South Vietnam. However, exposure specifically to herbicides was never established; neither experimental nor control groups were defined clearly (even in terms of military service) and other relevant factors, such as maternal health (or exposure), family predisposition, or drug use, were not considered in the study. A consultative council was appointed in Australia to determine whether a series of premature births and malformations in the Yarram district of Australia was higher than expected and whether these abnormal events could be attributed to exposure to 2,4-D or 2,4,5-T (Allred et al., 1978). Statistical analysis of these reported incidences of birth defects, which included three defects in neural tube development (two cases of spina bifida and one of anencephalus) and one case of renal agenesis, revealed that their rates per total number of births were not significantly different from the incidences in other regions of Australia which have lower usage of 2,4-D and 2,4,5-T. Other abnormal births were eliminated from the study because the mothers had not resided in the Yarram district prior to the births. Specific exposure to herbicides could not be established in any of the eight cases that were brought to the attention o'f the committee. The committee also presented estimates of the amount of contaminated food or water consumption required in a pregnant woman to reach the lowest dose of 2,4-D, 2,4,5-T, or TCDD that was teratogenic in animals (assuming contamination at the highest reported levels). These values ranged from 15,000-900,000 liters of water per day or 6,000-90,000 kg of meat per day. Another study of the incidence of neural tube defects among births in Australia, compared with 2,4,5-T usage during the previous year, showed a positive correlation between the two parameters. In the brief report of these data, the authors avoided drawing a causal relationship between these two factors (Field and Kerr, 1979). Cases of neural tube defects in New Zealand were also suggested to be caused by exposure to 2,4,5-T (Sare and Forbes, 1972). The New Zealand Department of Health reviewed a total of 20 cases of neural tube defects that were referred to them (McQueen et al., 1977). The Department was unable to demonstrate exposure to 2,4,5-T prior to neural tube closure in most of the cases or even a higher than expected incidence of these birth defects for the three regions where these cases occurred. In some cases, familial predisposition to these types of birth defects was established, although in most cases the etiology of the malformations could not be identified. Another study that compared the incidence of various birth defects in New Zealand with the rate of 2,4,5-T spraying found no association between 2,4,5-T usage and malformations in general, or neural tube defects, cleft palate, or other 8-14 �specific defects in particular. The only exception was a correlation between 2,4,5-T usage and talipes (malformations of the foot), a malformation that has not been associated with 2,4,5-T or TCDD administration in other studies (Hanify et al., 1981). Two studies were conducted in the U.S. to evaluate the relationship between herbicides spraying and reproductive events. The incidence of spontaneous abortions over a 6-year period in Alsea, Oregon, was found to be higher than the rates in two other regions of Oregon that had lower rates of 2,4,5-T usage (EPA, 1979). A cyclic pattern in the incidence of abortions in the Alsea group correlated positively with the pattern of annual 2,4,5-T spray usage. In another study, however, the incidence of cleft palate over a period of 32 years in Arkansas was not related to the usage of 2,4,5-T. The incidence of cleft palate for regions with a low proportion of rice acreage (indicating low exposure to 2,4,5-T) and for regions with high 2,4,5-T exposure increased over the years studied. This trend was attributed to better detection of cleft palate and not to herbicide usage (Nelson et al., 1979). Several cases of male impotence were identified among farm workers in England (Espir et al., 1970). Four out of a crew of five male workers experienced symptoms of impotence, which in each case began in April or August of 1967. No other symptoms were present in any of the men. The authors of this report were unable to identify the cause of these cases of impotence. All men were treated with methyltestosterone and terminated exposure to the chemicals they had been using in their work, which included 2,4-D as well as other phenoxy acids and various other pesticides. Symptoms cleared within 2 to 3 months in three cases and in 1 year in the fourth case. The authors suggested that exposure to pesticides caused the symptoms in these men, but they were unable to substantiate this claim with evidence from any other study of impotence as a sole symptom of exposure to any pesticide. 8.4.2 Animal Exposure Prior to Mating The exposure situation relevant to a consideration of reproductive effects of herbicides in Vietnam on American veterans is the situation in which exposure is limited to male exposure. Animal studies of male exposure to herbicides are considered in this section. 8.4.2.1 2,4,5-T With Known or Possible TCDD Contamination Buselmaier et al. (1972) studied the effect of male exposure to 2,4,5-T on reproductive parameters in the mouse (NMRI strain). Mice were administered 100 mg 2,4,5-T/kg of intraperitoneally at 10 weeks of age. They were then mated with unexposed females over the subsequent 6 weeks. Females were examined for evidence of pregnancy and, on day 14 of gestation, for numbers of fetuses, implantation sites, and corpora lutea. No increase in losses prior to or after implantation were observed in mice that were mated with 2,4,5-Texposed males, compared to mice mated with unexposed males. 8-15 �Changes in testosterone metabolism have been observed in 2,4,5-T-treated mice. Corresponding alterations in reproductive function have not been tested, however. Treated mice showed decreased accumulation of testosterone by the prostate gland, although hepatic metabolism of testosterone and androgenic responses of the testes and accessory sex organs were not altered (Lloyd et al., 1973; Thomas, 1974). 8.4.2.2 Combinations of 2,4-D and 2,4,5-T Lamb et al., (1980) exposed male C57BL/6 mice to feed with various combinations of 2,4-D, 2,4,5-T, and TCDD for 8 weeks. The levels of these components resulted in daily doses of 40 mg 2,4-D/kg, 40 mg 2,4,5-T/kg, and 2.4 ug TCDD/kg to one group; 40 mg/kg of each phenoxy acid and 0.16 ug TCDD/kg to the second group; and 20 mg/kg of each phenoxy acid and 1.2 ug TCDD/kg to the third group. The levels of 2,4-D, 2,4,5-T, and TCDD were not periodically analyzed to confirm the concentrations being administered. When these mice were mated with unexposed females, mating frequency, fertility, fetal deaths, and malformations were found to be the same as for matings of male mice administered diets with vehicle only. Toxicity in the treated males, which involved decreased thyraus and body weights and increased liver weights, was temporary, with recovery after termination of exposure. Sperm concentration, motility, and abnormalities were not adversely affected by treatment. This study protocol is relevant to the question of potential for reproductive risk from military use of these compounds. The negative result leaves open the possibility that the doses were too low to be effective through male exposure or that other species might be more sensitive to these compounds after male exposure than this strain 'of mouse (which is sensitive to the teratogenic effects). 8.4.3 Animal Exposure During Gestation Only The majority of reproductive studies of herbicides and TCDD in animals involved exposure only of the female after conception. This protocol is not relevant to an assessment of reproductive effects in American servicemen in Vietnam. The findings in the studies in this section have been used to design a number of epidemiological studies in which the incidence of cleft palate and of spontaneous abortions were examined in human populations. 8.4.3.1 2,4,5-T with Known or Possible TCDD Contamination Fetuses of monkeys that were treated with 10 mg/kg of 2,4,5-T three times per week between days 20 and 48 of gestation were smaller than control fetuses. Delayed ossification and small bones were noted upon examining their skeletons (Wilson, .1972). At doses of 40 mg/kg one of five monkeys aborted, compared to none of the five control monkeys. However, the 20 percent rate of abortion is probably not significantly higher than the normal abortion rate of about 10-15 percent given the small sample size. No malformations were observed in the 2,4,5-T-treated groups. 8-16 �In the rat, 2,4,5-T at low doses (4.6-10 mg/kg) produced cystic kidney and intestinal hemorrhage (Courtney et al., 1970; Bionetics, 1968). Only one study reported that 2,4,5-T produced cleft palate in the rat, but the incidence was very low (9 of 2,231 fetuses) and was produced by intrauterine administration of 2,4,5-T (King et al., 1973). In other studies, 2,4,5-Tadministered throughout gestation produced maternal toxicity and either fetal deaths or decreased fetal growth. In both experiments, lower doses produced no maternal or fetal effects (Konstantinova, 1974; Hall, 1972). In the mouse, 2,4,5-T produced cleft palate and cystic kidney. Two studies reported that both malformations occurred in C57BL/6 mice from doses of 113 mg/kg, while a dose of 46 mg/kg produced cystic kidney, but not cleft palate. In AKR mice, doses of 113 mg/kg produced cleft palate without cystic kidney (Bionetics, 1968; Courtney et al., 1970). In another study that used large quantities of animals, cleft palate incidence was elevated by 2,4,5-T doses of only 30 mg/kg to C57BL/6, BALB, C3H, and CD-I mice on days 6-14, and of 15 mg/kg to A/JAX mice (Gaines et al., 1974). A lower dose of 10 mg/kg administered subcutaneously to CD-I mice, however, was ineffective (Hart and Valeric, 1972). Combinations of 100 rag 2,4,5-T/kg and 1 ug TCDD/kg produced higher incidences of cleft palate and cystic kidney than either compound alone, but the effect was less than additive (Courtney and Moore, 1971). Neubert and Dillman (1972) found that TCDD levels of 1.5 ppm or less (0.1 ug/kg) were inadequate to potentiate the effect of cleft palate caused by doses of 60 mg 2,4,5-T/kg while higher TCDD doses of 0.3 ug/kg (5 ppm) did cause potentiation. In the hamster, the incidence of malformations, which involved abnormal cranial development, increased with increasing levels of TCDD. Doses of 80 mg 2,4,5-T/kg, with TCDD levels of 0, 0.5, and 45 ppm, caused 0, 40, and 100 percent, respectively, of the exposed litters to be abnormal. The protocol did not permit an evaluation of potentiation between the two compounds (Collins and Williams, 1971). Cleft palate was rarely encountered in 2,4,5-T-treated hamsters in this or in another study by Gale and Ferm (1973) in which only a single, low dose of 2,4,5-T was administered. In avian species, 2,4,5-T has been shown to be embryolethal, but not teratogenic (Lutz and Lutz-Ostertag, 1973; Strange et al., 1976; Strange and Kerr, 1976). 8.4.3.2 Combinations of 2,4-D and 2,4,5-T Mixtures of 2,4-D and 2,4,5-T have been administered to pregnant mice. Herbicide Orange, comprised of the butyl esters of 2,4-D and 2,4,5-T, was administered to CD-I mice at a dose of 1 mM/kg on days 12-15 of gestation. This combination (which had 0.4 ppm TCDD) produced a lower incidence of cleft palate than 1.0 mM/kg doses of either compound alone. All three preparations produced maternal toxicity and decreased fetal weights to the same extent while only 2,4,5-T produced fetal mortality (Courtney, 1977). Another study found that a 2:1 mixture of 2,4-D:2,4,5-T produced almost as high an incidence of cleft palate and dilated renal pelvis, and the same effect of fetal weight and fetal mortality, as the same dose of 2,4,5-T alone (Bage et al., 1973). Another study, which was described briefly in an abstract, indicated that 8-17 �2,4,5-T, whether administered alone or combined with 2,4-D, produced a very low incidence of cleft palate (9 of 2,231 fetuses) in the rat (King et al., 1973). 8.5 DIQUAT The reproductive effects of male exposure to diquat have been studied. Anderson et al. (1976) administered 10 mg diquat/kg orally for 5 days to male CD-I mice. This dosage produced no alterations in fertility, or in pre- or post-implantation deaths. A higher dose corresponding to the LD_ n was given as a single intraperitoneal dose to male Swiss-Webster mice (Pasi et al., 1974). These mice were then mated with new females weekly for 8 weeks. This treatment did not affect pre- or post-implantation deaths, but caused a reduction in fertility. The temporal pattern of this antifertility effect indicated that spermatogenesis was disrupted in premeiotic early and late spermatocytes and in all postmeiotic maturation. An increased incidence of fetal deaths has been observed in rats, following diquat administration during gestation. Single intravenous doses of 15 mg diquat/kg were administered on a single day, between day 7-21 of gestation (Bus et al., 1975). This dose produced 20 percent maternal mortality and 57 percent fetal mortality. Distribution of radioactivity to the fetus was demonstrated after C-diquat was administered intravenously on days 13, 16, or 21 of gestation. In a brief abstract, Khera and Whitta (1968) reported skeletal and total fetal growth retardation after a dose of 7 mg diquat/kg was administered on a single dose between day 6 and 14 of gestation. These effects were more severe, and were accompanied by increased fetal mortality at a dose of 14 rag/kg. 8.6 PICLORAM Administration of doses of 500, 750, or 1,000 mg pic loram/kg/day on days 8-17 of gestation did not cause adverse effects on litter size, fetal deaths, or fetal weights. The two highest doses produced maternal deaths and retardation of skeletal development in fetuses (Thompson et al., 1972). Picloram, at levels up to 3,000 ppm in the diet, was also reported to cause no adverse effects on fertility, fetal survival, fetal growth, structural development, or lactation in a three-generation study. Exposure to a diet with 0.01 percent picloram for 4 days prior to and 14 days after mating also had no effect on fertility. These experiments were not described further and whether one or both parents were treated prior to mating was not indicated (McCollister and Leng, 1969). 8.7 DALAPON Administration of 30-300 mg/kg/day doses of dalapon by gavage 110 days to each of three consecutive generations of rats produced on fertility, or on fetal or offspring survival or growth (Paynter 1960). Doses of 250-2,000 mg/kg/day were administered to pregnant 8-18 for no effect et al., rats on �days 6 to 15 of gestation. Maternal toxicity was produced at 1,500-2,000 mg/kg and reduced fetal viability was also observed. No effects, including gross malformations, were detected at doses of 1,000 mg/kg or less (Thompson et al., 1971, cited in Renaga, 1974). In another study, doses of 5001,500 rag/kg/day were administered on days 6-15 to rats (Emerson et al., 1971, cited in Kenaga, 1974). At doses of 1,000 mg/kg or above, maternal and fetal weights were depressed, but no visceral or significant skeletal malformations were observed in any group. 8.8 BROMACIL A three-generation study in rats showed no adverse reproductive effects in any generation from chronic feeding of diets containing 250 ppm bromacil (Sherman and Kaplan, 1975). Fertility, survival of fetuses and offspring, and growth were unaffected in the exposed group. No gross, visceral, or skeletal malformations were observed in fetuses. In a brief abstract, bromacil, included in the diets (level not indicated) of male mice for 7 weeks, produced no adverse effects on the fertility of these mice during the subsequent 8 weeks (Jorgensen et al., 1976). 8.9 DIURON Doses of 135-500 rag diuron/kg/day administered to rats on days 6-15 of gestation did not alter fetal viability, but caused a signficant decrease in fetal weight. The incidence of pregnancy was decreased from 19 to 20 in controls to between 14 and 15 of 20 rats given either 250 or 500 mg/kg doses. Both doses caused reductions in maternal weights, although this change was significant only at the higher dose. Skeletal abnormalities of questionable significance were observed at these doses, but no gross or visceral malformations were observed (Khera et al., 1979). 8.10 CACODYLIC ACID The effects of 1 and 10 ppt cacodylate on development of isolated fetal mice were investigated. Eighty-six percent of embryos exposed to the higher dose failed to undergo normal cell divisions, while neither dose produced a statistically significant reduction in DNA division, based on radioactive precursor incorporation (Williams et al., 1979). The validity of this novel system for testing teratogens awaits the results of in vivo tests to determine the reproducibility of these effects. 8.11 SUMMARY AND CONCLUSIONS 2,4-D has been studied for reproductive effects in man and in the rat, mouse, hamster, and in grazing animals and avian species. Industrial exposure in man was not associated with an increased rate of spontaneous abortions in the wives of the entire study group but in one subset of workers an increased risk was implicated. In other mammalian species, exposure was limited to the 8-19 �female during gestation, and the only effect of 2,4-D was a decreased rate of fetal growth. In mammalian studies administration of 2,4,5-T with less than 1 ppm TCDD was also limited to pregnant females. This exposure produced cleft palate in the mouse at doses that produced no significant maternal toxicity; a decrease in fetal growth was produced in mice at lower doses of 2,4,5-T than those causing cleft palate. Teratogenicity or other embryotoxic or lethal effects were not observed in the other mammalian species studied (monkey, rat, hamster, and rabbit). No evidence of reproductive or teratogenic effects has been observed after human exposure to TCDD. However, systematic testing of these parameters, in a manner that could evaluate these effects in a significant number of people with documented exposures, has not been carried out. In a three-generation reproductive study, chronic exposure of male and female rats to 0.1 or 0.01 ug TCDD/kg/day resulted in decreased fertility. Female mice fed 5 ppb TCDD in feed before and during gestation showed normal fertility and gave birth to normal offspring. Other experiments of TCDD exposure prior to mating either evaluated reproductive structures but not fertility, used animals with low fertility prior to exposure, or used doses that produced severe maternal toxicity. The only reproducible effects of TCDD administration during gestation were teratologic effects in the mouse. Cleft palate was observed in several strains of mice, and renal abnormalities that may be transient were also consistently produced. Cases of reproductive abnormalities in the human population have never been shown convincingly to correlate with usage of herbicide mixtures or of 2,4,5-T with possible TCDD contamination. One possible exception involved an increased incidence of abortions in Oregon at times and places where 2,4,5-T usage was high. However, this study did'not establish 2,4,5-T exposure of the individual who had abortions and did not investigate other factors that may have predisposed this group to an increased incidence of abortions. These factors were evaluated in the Australian and New Zealand cases of birth defects; exposure during the relevant time in gestation could not be established in most of these cases, while familial predisposition and other potential causes of birth defects were identified. Exposure of males only to 2,4,5-T with possible TCDD contamination alone or in combination with 2,4-D and known amounts of TCDD, has not been shown to produce antifertility effects. In the mouse, combinations of 2,4-D and 2,4,5-T, or 2,4,5-T and TCDD were not additive or synergistic in producing cleft palate, except possibly for 2,4,5-T doses above 60 mg/kg with TCDD levels above 5 ppm. Diquat exposure of males or pregnant females produced some adverse reproductive effects, but the doses causing these effects were also highly toxic to adult animals. 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(1978a) Influence of hen dietary calcium and phosphorous on the integrity of the egg shell as it would influence hatching success and the consequences of preincubation 2,4,5-T spraying with and without a high TCDD level. Bull. Environ. Contain. Toxicol. 19(6):648-654. Somers, J. D., Moran, E. T., and Reinhart, B. S. (1978b) Reproductive success of hens and cockerels originating from eggs sprayed with 2,4-D, 2,4,5-T and picloram followed by early performance of their progeny after a comparable in ovo exposure. Bull. Environ. Contain. Toxicol. 20(0:111-119. Somers, J. D., Moran, E. T., and Reinhart, B. S. (1978c) Hatching success and early performance of chicks from eggs sprayed with 2,4-D, 2,4,5-T and picloram at various stages of embryonic development. Bull. Environ. Contam. Toxicol. 20(3):289-293. Sparschu, G. L., Dunn, F. L., Lisowe, R. W., and Rowe, V. K. (1971) Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid on fetal development in the rat. Fd. Cosmet. 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The use of non-human primates in research on human reproduction. WHO Symposium, ed. E. Diezfolvsy and C. C. Standley. pp 261-292. 8-31 �CHAPTER 9 MUTAGENICITY This chapter discusses the cytogenetic and mutagenic effects of Vietnam herbicides in humans, mammals, and in vitro test systems. Major information gaps exist in the published literature on this subject. Only 2,4-D, 2,4,5-T, and TCDD have been studied for induction of aberrations in human or mammalian chromosomes. Only short-term in vitro testing has been performed on other Vietnam herbicides. The situation is further complicated by inconsistencies within and between test systems. Table 9-1 summarizes the results of shortterm tests of Vietnam herbicides. Table 9-1. SUMMARY OF SHORT-TERM TEST RESULTS Human Chromosome Effects Mammalian Chromosome Effects Host Mediated Assay Recessive LethalDrosophila 2,4-D 1/1 a 1/3 1/2 0/4 b 4/4 2,4,5-T 1/1 2/3 0/1 0/2 2/3 0/2 1/1 TCDD 0/1 1/2 N N 3/3 2/2 Pic lor am N N N N 2/5 0/6 Cacodylic Acid N N N N 0/2 0/1 Diquat N N N N 3/5 2/2 Monuron N N N N 0/1 Diuron N N N N 0/4 N 0/3 Dalapon N N N N 0/3 0/6 Bromacil N N N N 0/3 0/1 COMPOUND Bacterial S*ys terns Yeast Systems a = number of studies positive/number of studies performed b = assayed at pH 4.5 N = no information 9.1 HUMAN CYTOGENETIC STUDIES The information available on the effects of herbicides on human chromosomes is very limited. Table 9-2 lists the available studies concerning the clastogenic effects associated with 2,4-D, 2,4,5-T, TCDD, and herbicide mixtures. Five studies examined the effects of compounds under controlled 9-1 �Table 9-2: CYTOGENETIC EFFECTS OF HERBICIDES ON HUMAH CELLS END POINT RESULTS REFERENCE COMMENTS COMPOUND TEST SYSTEM 2,4-D Hunan peripheral lymphocytes Chromosome aberrations Increase in chromosome aberrations 2,4-D Human lymphocytes Chromosome aberrations Negative Wild ( 9 5 17) Cited by Seller ( 9 8 ) I7a 2,4-D dimethylamine salt Human embryonic fibroblasts Chromosome aberrations Increase in chromosome aberrations Berin et al. C1973) Cited by Seller ( 9 8 ) 17a 245T ,.- Human spleen fibroblasts Chromosome aberrations Not reported Hunan lymphocytes Chromosome aberrations ' Not reported 2,4,5-T ( . 9 ppm TCDD) 00 Human peripheral leukocytes Chromosome aberrations TCDD Hunan peripheral lymphocytes Pilinskaya et al. (1974) 2,4-D added to cells in culture at 0.002-50,000 ug/ml Occupational Increase in chromosome aberrat ions Fujita et al. (1975) 2,4,5-T ( . 9 ppm TCDD) 00 added to cells in culture at 10 M to 10 Chromosome aberrations Negative Tench in i et al. (99 17) Industrial accident Seveso, Italy Cultured human fetal tissue Chromosome aberrat ions Negative Human peripheral lymphocytes Sister chroma t id exchanges Human peripheral lymphocytes Herbicide mixtures3 Babitt et al. ( 9 3 17) Chromosome aberrations c Herbicide mixtures T » 1_— •» AT* *» * . C 1» 1 . _~J1 .*:_-_.-.. b Negative Increase in chromosome aberrations during spraying season exposure Industrial accident Seveso, Italy Crossen et al. ( 9 8 Occupational exposure 17) study; 3 individuals using no protection had elevated SCE's Yoder et al. (1973) Occupational exposure �conditions. Four other studies describe the clastogenic effects associated with accidental or occupational exposure to herbicides. Since these latter studies could not document the amount and type of herbicide exposure, they are of limited value in determining the clastogenic effects of the herbicides. The limited data available suggests that both 2,4-D and 2,4,5-T have the potential to induce clastogenetic effects in human cells in vitro. Whether these compounds will react similarly in vivo cannot be determined from the available data. TCDD has not been conclusively associated with any clastogenic effects in humans. Although studies which evaluated the effects of occupational exposure to a variety of herbicide sprays were generally unconsistent, there appears to be a disturbing trend in the data suggestive of a potential human genetic hazard. 9.1.1 2,4-D and 2.4,5-T In a review by Seiler (1978), it was noted that 2,4-D has been associated with both negative (Wild, 1975) and positive (Pilinskaya et al., 1974) results in human lymphocyte culture. Pilinskaya et al. (1974) demonstrated that 2,4-D concentrations of 0.02 and 0.2 ug/ml induced statistically significant increases in chromosome damage. Cytotoxicity was observed at 2,4-D doses of 2.0 ug/ml and above while no chromosome damage was detected at 0.002 ug/ml. Chromatid-type aberrations prevailed over chromosome-type aberrations in a ratio of 4:1. The authors concluded that 2,4-D was a potential genetic hazard 'to humans. According to Seiler (1978), 2,4-D dimethylaraine salt (0.4 mg/ml) has also been demonstrated (Berin et al., 1973) to induce an increased frequency of chromosome aberrations in human embryonic fibroblasts. It appears that 2,4-D has the potential to induce chromosome damage in human cells in vitro (Pilinskaya, 1974; Seiler, 1978). However, these results do not indicate that 2,4-D is a potential genetic hazard in vivo. Fujita et al. (1975) described the effects of 2,4,5-T on human chromosomes. The herbicide appeared to induce increasing single chromatid breaks with concentrations of 2,4,5-T ranging from 10 to 10 M. Since the preparation used in the study contained 0.09 ppm TCDD as a contaminant, the authors noted that it was not clear whether the 2,4,5-T, the contaminating TCDD, or the combination of both was responsible for the results. However, if the 2,4,5-T presently in existence is always accompanied by TCDD, it may be irrelevant which of these possibilities is responsible for the results. Babitt et al., (1973) reported a study on the effects of 2,4,5-T on chromosomes of two human cell lines, spleen fibroblasts and lymphocytes. No experimental results were described in this abstract and no subsequent reports were published by these authors. 9.1.2 TCDD , Reggiani (1979) and others (Homberger et al., not date; Reggiani, 1980; and Pocchiari et al., 1979) described chromosome analyses performed on individuals exposed to TCDD in the Seveso accident and in ICMESA plant 9-3 �workers. Peripheral lymphocytes were examined from persons receiving acute and chronic exposure to TCDD at the ICMESA plant, and from Seveso children with and without chloracne. No increases in chromosome abnormalities were observed in the sample population. The frequency of gaps and breaks was reported to be within the normal range. Effects on human chromosomes from accidental exposure to TCDD was also described by Tenchini et al. (1979), and results are in agreement with Reggiani (1979) and others. The authors analyzed peripheral blood samples and aborted fetal tissues from women who were exposed to TCDD during the Seveso accident. No increase in chromosome aberrations were observed in the peripheral blood samples. In fetal tissues, large numbers of chromosome aberrations were observed. However, no unexposed tissues were included in the experiment to control for the effects of culturing these tissues in a laboratory. It is possible that culturing conditions were responsible for the large numbers of chromosome aberrations observed. The effect of TCDD on fetal chromosomes is therefore unclear. Based on these studies, there is no indication that accidental exposure to TCDD is associated with detectable clastogenic effects in the peripheral lymphocytes. Although fetal tissues appear to be more susceptible to chromosome aberrations following the accidental exposure to TCDD, the evidence is inconclusive. 9.1.3 Herbicide Spray Mixtures Yoder et al. (1973) performed chromosomal analyses on lymphocytes from persons occupationally exposed to herbicides and insecticides, including 2,4-D, 2,4,5-T, diquat, and picloram. No documentation of type and quantity of pesticide exposure was available. Lymphocyte chromosomes of 26 herbicideexposed individuals and 16 unexposed controls were analyzed.during the peak spraying season and during the midwinter off-season. The herbicide-exposed group had approximately twice the number of chromatid gaps and four times the number of chromatid breaks compared to controls. Surprisingly, during the midwinter sampling the herbicide-exposed group had about one-half the number of chromatid gaps and one-fourth the number of chromatid breaks compared to the unexposed group. The exposed group had nearly four times as many chromatid gaps per person and 25 times as many chromatid breaks per person during the peak spraying season as compared to the off-season sampling. The authors speculated that enhanced chromosome repair may be taking place at this time as compensatory protection. No statistical analysis of the results was presented and the ranges of chromatid gaps as well as chromatid breaks overlapped in all groups tested. The data presented do not permit any conclusions about effects specifically associated with the herbicides of interest. Another study on occupational exposure to herbicides and pesticides was described by Crossen et al. (1978). Documentation of type and quantity of chemical exposure was not possible. However, the chemical exposures most commonly encountered included 2,4-D, 2,4,5-T, bromacil, and diquat. Peripheral lymphocytes from 57 pesticide and herbicide sprayers were analyzed for sister chromatid exchanges (SCE). When the means of SCE in control and exposed subjects were compared, no statistically significant differences 9-4 �between the two groups were observed. However, five sprayers had a SCE rate three standard deviations outside the mean of the controls. Upon further analysis, it was noted that a high SCE rate seemed to be associated with lack of protection during spraying in those individuals who had been spraying for longer than one year. When the exposed group was divided into categories of no protective clothing, some protective clothing, and full protection, the group with no protection was statistically different than the unexposed group. Similarly, when the exposed group was divided into exposure categories of one year or less of spraying exposure or greater than one year of exposure, the group with greater than one year's exposure had a tendency toward elevated SCE rate (p=0.5) compared to controls. This observation supports the concept that the increased frequency of SCE was associated with herbicide exposures rather than the pesticide exposures. The authors concluded that there is a genetic hazard arising from the careless use of herbicides and pesticides. No difference in SCE rates were observed between the groups that sprayed only herbicide and the group that sprayed both herbicide and insecticide. Although there appears to be a genetic hazard associated with the careless use of herbicides, the hazard associated with any individual herbicide or herbicide combination cannot be evaluated with this study. Tung et al. (1971) described a survey of chromosome aberrations in Vietnamese people exposed to herbicide spraying. Peripheral leukocytes were cultured from three experimental groups: • Group 1: six individuals affected with asthenia who lived two to three years in sprayed areas • Group 2: five normal North Vietnamese • Group 3: five normal South Vietnamese. The type and extent of herbicide exposure that was received by Group 1 was not determined or approximated by the authors. In Group 1 the rate of chromosome aberrations was observed to be 5.88 percent, while in control Groups 2 and 3 only 1.14 percent chromosome aberrations were observed. Because each subject was examined for "hematologic, metabolic, medicinal, and radiological" factors in order to exclude other causes of chromosome aberrations, the authors concluded that the increased incidence of chromosome damage was due to herbicide spraying. However, sample size in this experiment was very limited and herbicide exposure was not documented; therefore, no firm conclusions can be made regarding the clastogenic effects of herbicide spraying on the Vietnamese people. The authors also examined chromosomes of children with congenital malformations whose mothers had been exposed to herbicide spraying. All the individuals examined had high rates of chromosome aberrations. This, however, is not sufficient evidence to link herbicide spraying with induction of chromosome aberrations. None of these studies provide information on the effects associated with exposure to a specific herbicide or herbicide combination. Although each study associates general herbicide exposure with human chromosome aberration, the evidence presented is inconclusive. However, the trend in the data 9-5 �indicates that occupational herbicide spray exposure may'present a potential genetic hazard to humans. 9.1.4 Other Vietnam Herbicides No information was found in the available literature concerning the effects of other Vietnam herbicides on human chromosomes. 9.2 CYTOGENETIC AND HOST-MEDIATED STUDIES IN MAMMALS Investigations of the cytogenetic effects of herbicides on mammals are sparse and the results are inconsistent. Table 9-3 lists the cytogenetic and host-mediated studies in mammals. No information was available in the literature describing the rautagenic effects of the other Vietnam herbicides other than 2,4-D, 2,4,5-T and TCDD in mammals. Without additional substantiating data, no firm conclusions regarding the clastogenetic potential of. 2,4-D, 2,4,5-T, or TCDD are appropriate. 9.2.1 2.4-D and 2.4.5-T Pilinskaya (1974) described the cytogenetic effects of 2,4-D in mouse bone marrow cells. Groups of six mice received a single oral dose of 10, 50, 100, or 300 mg of 2,4-D per kg body weight. Twenty hours later the animals were killed and bone marrow cells were examined for chromosome aberrations. Statistically significant 'increased frequencies of chromosome aberrations were observed at 100 and 300 mg/kg. These doses also caused pronounced symptoms of intoxication in the animals. The authors concluded that 2,4-D was weakly mutagenic. Jenssen and Renberg (1976) tested both 2,4-D and 2,4,5-T «lppm TCDD) in a micronucleus test in mice. The micronucleus test measures the chromosome damaging ability of a substance. Male CBA mice received a single 100 mg/kg intraperitoneal injection of either 2,4-D or 2,4,5-T. Animals were killed 24 hours or 7 days later and bone marrow cells were analyzed for numbers of polychromatic erythrocytes with micronuclei. No increase in polychromatic erythrocytes with micronuclei was observed in animals treated with 2,4-D or 2,4,5-T after either 24 hours or seven days. However, a weak toxic effect on mitotic activity was observed in treated animals. Both compounds appeared in peripheral blood and bone marrow within four hours after injection. 2,4-D and 2,4,5-T levels in blood plasma declined by 24 hours after injection. No more than 5 percent of the plasma levels of phenoxy acid were found in cell fractions. Because the 2,4-D and 2,4,5-T do not enter the target cells to an appreciable extent, the authors concluded that results of the test did not reliably indicate a lack of mutagenicity. However, since the data were in agreement with the known rapid excretion of these compounds, the authors also concluded that no cytogenetic hazard is connected with 2,4-D and 2,4,5-T. In an abstract, Bongso and Basrur (1973) also reported that 2,4-D did not increase chromosome aberrations in bovine peripheral blood cells. However, embryonic bovine kidney cells exposed to 2,4-D for longer than 48 hours showed 9-6 �Table 9-3: SUMMARY OF CYTOGENET1C AND MUTAGENIC EFFECTS OF HERBICIDES ON MAMMALS END POINT RESULTS REFERENCE COMMENTS COMPOUND TEST SYSTEM 2,4-D Mouse bone marrow cells Chromosome aberrations Increase in chromosome aberrations Pilinskaya ( 9 4 17) Oral doses of 100 & 300 mg/kg caused intoxication and chromosome aberration; doses of 10 & 50 mg/kg had no effect. 2,4-D Micronucleus test —mice Induction of micronuclei Negative Jenssen and Renberg (96 17) Intraperitoneal injection of 100 mg/kg 2,4-D Bovine peripheral blood cells Chromosome aberrations Negative Bongso and Basrur (93 17) Abstract; no data 2,4-D Host mediated assay — rats Induction of S. typhlmurium revertants Negative Styles ( 9 3 17) Mouse serum + S>. typhlmurium; abstract; no data 2,4,5-T(<rippm TCDD) Micronucleus test —mice Induction of micronuclei Negative 2,4,5-T Mongolian gerbil bone marrow cells Chromosome aberrations Increase in chromosome aberrations Majumdar and Hall (93 17) Clastogenic effects at > 250 mg/kg; results 'disputed (Ramel, 1978) 2 4 5 T butoxy ethyl ,,ester (commercial preparation, Hormoslyr 5 0 t ( C D 0 1 ppm) 0-) TD<. Mouse bone marrow cells Chromosome aberrations Increase in chromosome aberrrations Davring and Hultgren (97 17) Emulsifler & solvent in the commercial preparation also caused chromosome aberrations 2,4,5-T and 2,4,5-T n-butyl ester Host mediated assay — mice Induction of nutations in S. tvphimurium and S. marcescens Negative Buselmaier et al. (92 17) 2,4,5-T Host mediated rats Induction of S. typhimurium revertants Negative Styles ( 9 3 17) rCDD Rat (male) bone marrow cells Chromosome aberrations Negative Green and Moreland (95 17) ICDD Rat (male i, female bone marrow cells Chromosome aberrations Increase In chromosomeaberrations Green, Moreland, and Sheu ( 9 7 17) Mouse serum + S. typhlourlura; abstract; no data �an increase in nucleolar size and number, and an increase in multipolar spindles and polyploid mitotic stages. Although the authors speculated that 2,4-D has an effect on spindle protein synthesis, no data were presented. Davring and Hultgren (1977) tested a commercial 2,4,5-T ester preparation (Hormoslyr 500-T, TCDD <0.1ppm), its components, and 2,4,5-T acid for their ability to induce chromosome aberrations in mice. Animals received either a single intraperitoneal injection or five daily intraperitoneal injections of 2,4,5-T ester preparation, 2,4,5-T acid, the commercial solvent, or the commercial emulsifier. All of the substances tested, including the solvent and the emulsifier, caused increased frequencies of chromosome aberrations. The positive and negative controls were associated with appropriate results, (chromatid gaps, breaks, deficiencies, and fragments). Majuradar and Hall (1973) investigated the clastogenic effects of 2,4,5-T in Mongolian gerbil bone marrow cells. Male and female animals received daily intraperitoneal injections of 10, 30, 50, 70, or 100 mg of 2,4,5-T (no detectable TCDD) per kg, a total of 50, 150, 250, 350, or 500 mg/kg over the fiveday period. According to the authors, chromosome damage (chromatid gaps and breaks) increased significantly at 250 mg/kg total dose. However, Ramel (1978) reported that these results were questioned by Natarajan in a statement in the Swedish Products Control Board: "While I cannot dispute the quantitative aspects of the authors' data, their classification of aberrations as well as the types illustrated are very confusing. According to my judgment of the figures, what they call breaks (which incidentally are termed as breaks and fragments in the table, and I cannot say how they differentiate between these two types) are really gaps. The so called deletions in the figures are certainly artifacts due to the spreading of the chromosomes. It is difficult to say as to how many such misjudgments have gone into the making of the table, which shows a very high frequency of abnormal cells in animals treated with high doses." Styles (1973) tested both 2,4-D and 2,4,5-T in a host mediated assay in rats. Animals received a single oral dose (unspecified) of 2,4-D or 2,4,5-T. Serum samples from these animals were incubated with histidine requiring mutants of £. typhimurium; the frequency of revertants to prototrophy was measured as an indicator of mutagenicity. Neither compound caused an increase in revertant numbers. This result is in agreement with Jenssen and Renberg (1976), who observed a negative result in a micronucleus test in mice. However, Pilinskaya (1974) concluded that 2,4-D was a weak mutagen. Buselmaier et al. (1972) tested 2,4,5-T and 2,4,5-T n-butyl ester in a host mediated assay in mice using _S_. typhimurium G46 and S_. marcescens a21 Leu as the indicator organisms. NMRI mice were injected subcutaneously with 0.2 ml of the test substance immediately after being injected intraperitoneally with a suspension of the bacterial strains. Three hours later the animals were killed and intraperitoneal fluid collected and plated on selective media for detection of mutant colonies. Neither compound had a mutagenic effect. 9-8 �The data concerning the clastogenic potential of 2,4-D appear to be conflicting. Chromosome aberrations were observed in murine bone marrow cells following oral dosage as low as 100 mg/kg (Pilinskaya, 1974). However, two studies of clastogenic effects in peripheral blood cells (Jenssen and Renberg, 1976; Bongso and Basrur, 1977) were negative. Whether these negative results represent a difference in cell type sensitivity, differential absorption rates due to route of exposure, the study of an ineffective dose level, or simply unsubstantiated claims (abstract by Bongso and Basrur, 1977) cannot be determined at present. Therefore, the report by Pilinskaya (1974) provides the only data that associate 2,4-D exposure with clastogenic effects. Any firm conclusions regarding the clastogenetic nature of 2,4-D will require additional substantiating data. While 2,4,5-T butoxyethyl ester appears to induce a clastogenic effect in mouse bone marrow cells (Davring and Hultgren, 1977), there are no undisputed results which indicate that 2,4,5-T has similiar biological activity in vivo. Any firm conclusions regarding the clastogenic potential of 2,4,5-T also require additional substantiating data. The last mediated assays were included in their section due to the in vivo aspects of these bacterial mutagenicity tests. These assays were universally negative for both 2,4-D and 2,4,5-T. These data support the conclusion that these phenoxy acids lack mutagenic activity in vivo. 9.2.2 TCDD Green and Moreland (1975) investigated the effects of TCDD on chromosomes of bone marrow cells in male rats. Daily for 5 days, animals received 10 ug TCDD per kg by intubation; 5, 10, or 15 ug/kg intraperitoneally; or 20 ug/kg orally. No increases in chromosome aberrations were observed in any of the treatments. In a followup study, however, Green, Moreland, and Sheu (1977) administered 0.25, 0.5, 1, 2, or 4 ug TCDD/kg body weight by gavage twice a week for 13 weeks. No controls were included on the study. TCDD produced statistically significant increases in the number of cells with chromosome abnormalities at 2 and 4 ug/kg in male rats and at 4 ug/kg in female rats. However, results of both the 2 ug/kg dose level in males and the 4 ug/kg dose in females fell within the normal frequency range of abnormalities (2-3 percent). Only the 4 ug/kg dose in males appeared to be mutagenic. This study indicates that TCDD appears to be a weak clastogen, but substantiating evidence is lacking. 9.2.3 Other Vietnam Herbicides No information concerning the mutagenic effects of other Vietnam herbicides in mammals was found in the available literature. 9.3 MUTAGENICITY STUDIES IN DROSOPHILA Several studies have been conducted on 2,4-D and 2,4,5-T in Drosophila. Table 9-4 lists the mutagenicity studies on Drosophila found in the available 9-9 �Table 9-4: COMPOUND SUMMARY OF MUTAGENIC EFFECTS OF HERBICIDES ON DROSOPHILA MELANOGASTER END POINT RESULTS REFERENCE 2,4-D Nondlsjunction Chromosome loss Recessive lethal Negative Negative Positive Magnusson et al. (1977) 2,4-D Recessive lethals Negative Vogel and Chandler ( 9 4 17) 2,4-D Appearance of mutant male flies in genetically stable and genetically unstable strains of flies Positive, unstable strain; negative, stable strain Rasmuson and Svahlin ( 9 8 17) 2,4, 5-T Recessive lethals Positive Majumdar and Golia ( 9 4 17) Negative 2,4, 5-T Rasmuson. and Svahlin ( 9 8 17) Appearance of mutant male flies in genetically stable and genetically unstable strains of flies Negative, both strains 2,4, 5-T (TCDD<0.1) Recessive lethal Positive Magnusson et al. ( 9 7 17) 2, 4, 5-T butoxyethyl ester, coimercial preparation (TCDD<I ppm) Nondisj unction Chromosome loss Negative Negative Magnusson, et al. ( 9 7 17) 2,4, 5-T o l-l I �literature. Of the two reports of sex-linked recessive lethal studies with 2,4-D in Drosophila, one reported an increase in sex-linked recessive lethals. Of the three sex-linked recessive lethal studies with 2,4,5-1, two reported an increase in sex-linked recessive lethals. Although results among these studies were inconsistent, 2,4-D and 2,4,5-T appear to have the potential inducing mutational events in Drosophila. Substantiating data is necessary before any firm conclusion can be drawn. None of the other herbicides of interest have been reported to be tested in Drosophila. 9.3.1 2.4-D and 2,4.5-T Magnusson et al. (1977) tested 2,4-D, 2,4,5-T (TCDD<0.1ppm), and a commercial preparation of 2,4,5-T butoxyethyl ester (TCDD<lppm) for induction of nondisjunction, chromosome loss, and sex-linked recessive lethals in Drosophila melanogaster. In the chromosome loss and nondisjunction tests, males and females were treated during their entire larval period with 250 ppm 2,4,5-T butoxyethyl ester or 100 ppm 2,4-D. No increases in chromosome loss or nondisjunction were induced by 2,4,5-T or 2,4-D. In the recessive lethal test, adult wild type Karsnas 60 strain males were treated with 1,000 ppm 2,4-D or 2,4,5-T containing less than 0.1 ppm TCDD. After two weeks of treatment males were mated to Muller 5 strain females. When F, and F_ results were combined, both 2,4-D and 2,4,5-T induced significant increases (p<0.01) in recessive lethals. However, when data from each generation were tabulated separately, only 2,4-D induced a statistically significant increase in recessive lethals in F~• Majumdar and Golia ( ' 7 ) also studied the effect of 2,4,5-T in the 194 sex-linked recessive lethal test in Drosophila. Male Oregon K flies were fed 250 or 1,000 ppm 2,4,5-T for 15 days and then mated to untreated females. The authors reported that 2,4,5-T induced a statistically significant increase in recessive lethals at 1,000 ppm but not at 250 ppm. Vogel and Chandler (1973), however, obtained negative results with both 2,4-D and 2,4,5-T in a sex-linked recessive lethal test in Drosophila. Adult two-day-old Berlin K males were fed 2,4-D (4.5 or 9.0 mM) or 2,4,5-T (3.6 or 7.2 mM) for three days. Following treatment, the males were mated to two females for three days (Brood 1). Males were mated with new females for an additional three-day-brood (Brood 2) and a four-day-brood (Brood 3). Results of the study were analyzed by the X test with criteria for a positive result being p<0.01. Neither 2,4-D nor 2,4,5-T induced increased numbers of recessive lethals compared to controls. However, a decline in fertility in Broods 2 and 3 was observed in flies treated with 7.2 mM 2,4,5-T. Rasmuson and Svahlin (1978) used a sex-linked genetically unstable strain of Drosphila to test 2,4-D and 2,4,5-T for their ability to induce somatic mutations. The results in the genetically unstable strain were compared with results in a genetically stable wild-type strain. 2,4-D induced mutational events in the genetically unstable strain but not in the stable strain, while 2,4,5-T was negative in both strains. Based on these studies, both 2,4-D and 2,4,5-T appear to be capable of inducing mutational events in Drosophila. However, additional substantiating data is needed. 9-11 �9.3.2 TCDD No information on the mutagenic effects of TCDD in Drosophila was found in the available literature. 9.3.3 Other Vietnam Herbicides No information on the mutagenic effects of other Vietnam herbicides in Drosophila was found in the available literature. 9.4 MUTAGENICITY IN IN VITRO SYSTEMS Most of the mutagenicity testing of Vietnam herbicides occurred in short-term in vitro assays. Results of these assays are not clear-cut. The phenoxy acids 2,4-D and 2,4,5-T were demonstrated to be weakly mutagenic in a variety of short-term in vitro test systems. TCDD was associated with mutagenic activity in all the in vitro assays in which it has been tested. However, these data on TCDD are suspect and any firm conclusions concerning TCDD would be inappropriate. Of the other herbicides discussed in this report most gave negative results in a variety of test systems. The exceptions to this are diquat, which was mutagenic in most systems, and monuron, which affects DNA synthesis in mammalian test systems. Tables 9-5 and 9-6 summarize the results of in vitro testing of herbicides. 9.4.1 2,4-D and 2,4,5-T Zetterberg (1977) reported that 2,4-D induced mitotic gene conversion and mitotic crossing over in Saccharomyces cerevisiae. The herbicide was mutagenic in this organism at pH_< 4.5 only. At pH 4.6, 2,4-D was not mutagenic. From these results and the pKa of 2,4-D, the authors speculated that yeast cells take up only the undissociated form of 2,4-D, which occurs at pH 4.5 or lower. Zetterberg et al. (1978) also reported that in a test for reverse mutation in yeast, 2,4-D induced increased numbers of histidine revertants in the RAD18 strain of Saccharomyces cerevisiae. As in the previous study, mutagenicity was observed at pH 4.5 or less. However, 2,4-D was not mutagenic in four strains of Salmonella typhimurium in reverse gene mutation assays (Ames test) at either pH 4.3 or 6.8 (Zetterberg, 1977). Similarly, Anderson et al. (1972) reported that 2,4-D gave negative results in the Ames test, and Nagy et al. (1975) also reported negative results with 2,4-D in a reverse mutation assay in IS. coli. Ahmed et al. (1977) investigated the potential of 2,4-D, diquat, and 11 other pesticides for inducing unscheduled DNA synthesis in human cells in culture. The cell line VA-4, a human fibroblast cell line transformed by the virus SV-40 grown on cover slipps, was used as the cell culture system. Unscheduled DNA synthesis measured by [ HJ-TdR incorporation and autoradiographic analysis was determined in cell cultures with and without rat (S9) metabolic activation. Results of the assay were analyzed by a t-test 9-12 �Table 9-5: ASSAYS OF HERBICIDES ON IN VITRO MUTAGENICITY ASSAYS REFERENCE (1) Anderson et al. (1972) COMPOUND TESTED TEST SYSTEM END POINT Bromacil (a) S. typhimurium his v- his Cacodylic acid (b) 2,4-D (c) E. coli B + T4 phage Dalapon (d) E. coli B + phage reversion to mutants wild type Diquat (e) Diuron (f) Monuron (g) Picloram (h) 2,4,5-T (i) RESULT - a through i - a through i - a through i (2) Aulicino et al. (1976) Dalapon (a) Picloram (b) A. nidulans 8-aza »• 8-aza -a,b non-disjunction -a,b Mitotic crossingover (3) Bignami et al. (1977) Dalapon (a) Picloram (b) A. nidulans Baza8 • 8-azar -a,b Non-disjunction -a,b Mitotic crossingover (4) Bignami & Crebelli Diquat (1979) S. typhimurium 8-azas *- 8-azar (5) Carere et al. (1976) Dalapon (a) Picloram (b) S. typhimurium S. coelicolor his »• his . strep3 *- strep1 (6) Carere et al. (1978) Dalapon-NA (a) Picloram (b) S. typhimurium S. coelicolor his ^ his strep »- strep (7) Hussain et al. (1972) TCDD E. coli sd-4 S. typhimurium E. coli K-39 strep w stjep his •*• his Prophage activation (8) Nagy et al. (1975) 2,4-D E. cjli WP2 trp" (her and her trp f trp (9) Shirasu et al. (1976) Bromacil 2,4-D Diuron Monuron 2,4,5-T B. subtilus rec assay (10) Seiler (1973) TCDD S. typhimurium his (11) Seiler (1979) Monuron S. typh imur ium his »• his 9-13 •**• his + -a,b +b;-a -a,b +b;-a + + + �Table 9-5: CONTINUED REFERENCE COMPOUND TESTED (12) Siebert & Lemperle (1974) Diquat 2,4-D S. cerevisiae Mitotic gene conversion + + (13) Zetterberg et al. (1977) 2,4-D Na salt S. cerevisiae S. typhimurium Mitotic gene conversion Mitotic recombination his"*- his + pH 4.5 + pH 4.5 his" to- his* + pH 4.5 (14) Zetterberg et al. (1978) TEST SYSTEM S. cerevisiae 2,4-D END POINT RAD 18 + pH 4.5 2,4,5-T (15) Simmon et al. (1977) RESULT Human fibroblast Bromacil (a) Cacodylic acid (b) cells Monuron (c) S. typhimurium E. coli WP2 S. cerevisiae Induction of -a,b;+c unscheduled DNA synthesis his- »• his+ trp- »- trp+ Mitotic , recombination ~a,b,c -a,b,c -a,b,c (16) Bronzetti et al. TCDD S. cerevisiae Reverse mutation Mitotic gene conversion (17) Seiler (1978b) Diuron (a) Monuron (b) Testicular DNA Inhibition of DNA +a,b synthesis (DSI test) synthesis-mice + + his" v his +a,b S. typhimurium mouse bone marrow micronucleus test -a,b cells (18) Benigni et al. (1979) Diquat S. S. S. A. A. typhimurium typhimurium typhimurium nidulans nidulans A. nidulans his" " his* Repair assay 8 8-aza8 «• 8-azar 8-aza — 8-azar raeth *- meth + + + Induction of + recessive lethala (19) Sailer (1979b) (20) Ahmed et al. (1977) (21) Ahmed et al. (1977) +a,b,c 2,4-D (a) 2,4,5-T (b) 2,4,5-T isooctyl ester (c) Testicular DNA Inhibition of synthesis-mice DNA synthesis 2,4-D Chinese hamster cells Induction of gene + mutations Human fibroblaat cells Induction of un- +a,b 2,4-D (a) Diquat (b) scheduled DNA synthesis 9-13a �Table 9-6: >S. TEST ^SSYSTEM COMPOUND Bacteria Reverse Mutation Bacteria Forward Mutation RESULTS OF IN VITRO ASSAYS FOR DETECTING MUTAGENICITY OF HERBICIDES Rec. Assay Bacteria Repair Assay YeastGene Mutat ion X . 2,4-D -l,8,13a -9 +14 2,4, 5-T -1 -9 +7,10 Bromacil -1,15 Cacodylic Acid -1,15 Dalapon -1,5 -5 Diquat -1,18 +4,18 Diuron -1,17 -1,11,15,17 -9 Pic lor am -1,5,6 Prophage Activation +13 Mammalian Cells Mammalian Cells Unscheduled DNA Gene Mutatior Synthesis +20 +20 -9 Monuron YeastMitotic Crossing Over +14 TCDD YeastMitotic Gene Conversion +16 +7 +12,13 +7 +16 -15 -15 -15 -2,3 +18 +5,6 Number designate reference found in table 9-5 +19 +19 -15 -9 Inhibition of Mouse Testicular DNA Synthesis -2,3 +18 -2,3 +12 +18,20 +17 -15 -2,3 -2,3 +15 -2,3 +17 �comparing the mean number of silver grains in controls versus treated cells. Both diquat- and 2,4-D-treated cultures had statistically significant increases in mean numbers of silver grains at all concentrations tested. No metabolic activation was necessary for this effect. In addition, the authors also measured 2,4-D-induced 313 nm photolysis of BudR containing repaired regions of DNA to determine the form of DNA repair induced. The shape of the curve plotting induced breaks for 2,4-D resembled the shape of curves obtained in similar experiments using ionizing radiation and alkylating agents such as ethyl methane sulfonate. This result suggests a similarity between alkylating agents and 2,4-D in the mechanism of induction of unscheduled DNA synthesis. In another experiment, Ahmed et al. (I977b) studied the mutagenicity of 2,4-D in an assay for detection of forward mutations in Chinese hamster V79 cells. 2,4-D was tested in triplicate at a concentration of 19 uM, which yielded about 40% survival. The forward mutation frequency of 2,4-D-exposed cells was calculated to be 25.5 per,10 survivors, while the mutation frequency of control was 1.8 per 10 survivors. The authors concluded that 2,4-D was a weak mutagen in this test system. Anderson et al. (1972) evaluated the mutagenicity of bromacil, cacodylic acid, 2,4-D, dalapon, diquat, diuron, monuron, picloram, and 2,4,5-T in four in vitro mutagenicity assays in bacteria and viruses. The test systems used were: • Test 1 - eight strains (unspecified) of Salmonella typhimurium that revert from histidine dependence to histidine independence when exposed to a mutagenic chemical • Test 2 - T, bacteriophage in Escherichia coli B cells that form morphologically distinguishable mutant plaques when exposed to a mutagen • Tests 3 and 4 - mutants of T, bacteriophage, AP 72 and NI7, that revert to the wild type with mutagen treatment. The S. typhimurium assay was carried out as a "spot test" in which the herbicide to be tested was added as a liquid or as crystals to the surface of the agar plate. No metabolic activation system was used; therefore, only directly acting mutagens could be detected. In bacteriophage assays the test compound was incubated in liquid suspension followed by plating. All of the herbicides tested in the four assays gave negative results. Nagy et al. (1975) evaluated the mutagenicity of 2,4-D in Escherichia coli WP 2 try. Two bacterial strains which detect reversion from tryptophanrequiring to non-requiring'were used. One strain (her ) was repair-deficient in addition to requiring tryptophan; the other strain (her ) was repairproficient. 2,4-D was tested in a spot test without metabolic activation as either 1-3 mg crystals or 20-25 ul liquid. Both positive and negative controls were included in the assay. According to the authors, 2,4-D gave negative results in this assay. However, since numerical values of results were not reported the authors conclusions could not be evaluated. 9-15 �Seller (1979) tested 2,4-D and 2,4,5-T in a mouse testicular DNA synthesis inhibition test. One.hour before treatment, male mice (number unspecified) received 1 uCi of C-thymidine intraperitoneally. Herbicides were administered in a single dose: 200 mg of 2,4-D per kg; 200 mg of 2,4,5-T acid per kg; and 50, 100, 200, and 400 mg of 2,4,5-T isooctyl eater per kg. The animals were given intraperitoneal injections of 10 uCi of H-thymidine, from three to 96 hours later. The ratios of H counts per ug DNA and per C counts were then compared to controls. Criteria for a positive result was that thymidine uptake was depressed significantly (statistical test not specified). All three compounds showed statistically significant inhibition of testicular DNA synthesis. The author concluded that, in light of their indicator of mutagenic activity, the potential for carcinogenicity of these compounds should be investigated. Seibert and Lemperle (1974) tested commercial preparations of dalapon, bromacil, diuron, diquat, 2,4,5-T amyl ester, 2,4-D, and 24 other herbicides in a mitotic gene conversion assay in Saccharomyces cerevisiae using strain D-4. Neither positive nor negative controls were reported by the authors. However, according to the authors, diquat and 2,4-D induced a significant increase in mitotic gene conversion compared to controls. Both compounds were tested at pH 4.5. 2,4-D increased convertants fivefold at the ade 2 locus and 6 fold at the trp 5 locus, while diquat induced a 7-fold increase at the ade 2 locus and 4-5 fold at the trp 5 locus. Bromacil (ph 4.5) and 2,4,5-T (pH 7.0) amyl ester increased convertants slightly, but not significantly, over controls. Convertant frequencies induced by dalapon (pH 4.5) or diuron (pH 4.5) did not differ from controls. Shiraus et al. (1978)' studied the DNA damaging and mutagenic capabilities of bromacil, 2,4-D, diuron, raonuron, and 2,4,5-T, using three bacterial assays: a rec assay in Bacillus subtilus, and reverse mutation assays in Escherichia coli and Salmonella typhihmurium. All of the herbicides were negative for mutagenicity in all three assays. Although bacterial reverse mutation assays were universally negative with 2,4-D and 2,4,5-T, a variety of other in vitro assays were positive. There are sufficient data to associate 2,4-D and 2,4,5-T with mutagenic potential. 9.4.2 TCDD Hussain et al. (1972) evaluated the mutagenicity of TCDD in three bacterial systems: 1) reversion to streptomycin independence in Escherichia coli Sd-4; 2) reversion to histidine independence in Salmonella typhimurium; and 3) prophage induction in E^. coli K-39. TCDD (99 percent purity) in DMSO was added to suspension cultures of lag phase E. coli Sd-4 at 0.5, 1, 2, and 4 ug/ ml for one hour. Mutation frequencies appeared to increase at 2 ug/ml. However, the two replicate cultures reported had wide variation in frequencies of mutation (34 x 10 and 256 x 10 ), but not in survival (18 percent and 11 percent, respectively). Since further testing was done, this wide discrepancy in replicate makes it difficult to interpret the test. In addition, lag phase cells were exposed in this experiment. Other investigators have found that this is not the most sensitive stage for mutagenic effects to occur. �In Salmonella typhimurium TA1530, no increased numbers of revartants were observed at 1 and 10 ug/tnl TCDD for one hour. Survival values were 90 percent and less than one percent respectively. In strain TA1532, increased mutation frequency was observed when TCDD caused the survival rate to decrease to about one percent . No numerical data were presented to analyze the author's results. However, in this test a survival rate of less than 10 percent is not generally recognized as being a reliable indicator of a positive result. In the prophage induction experiment, TCDD in DMSO was incubated at 0, 0.5, 1, 1.5, and 2.5 ug/ml for 30 minutes with £_". coli K-39 cells. J£. coli K-49 cells were used as indicator cells and incubated with the treated, washed K-39 cells for two hours. Numbers of replicate cultures were not reported. The solvent DMSO appeared to have had an effect on prophage induction. In the controls without DMSO, 7.3 x 10 plaques/ml were observed while only^l.9 x 10 plaques/ml were observed with DMSO. The significance of this effect could not be calculated because of lack of data. At 0.5 ug/ml, TCDD induced 5.4 x 10 plaques/ml, about a twofold increase compared to DMSO controls. No other concentration of TCDD increased the numbers of plaques observed. The authors conclude that TCDD is mutagenic in Salmonella typhimurium TA1532 and is a weak inducer of prophage. However, this conclusion is suspect, due to insufficient data from replicate cultures, no statistical analysis of results, and poor presentation of experimental design. Seiler (1973) also studied 2,3,7,8-TCDD in Salmonella typhimurium strains his G-456, TA1530, TAA1531, TA1532, and TA1534. In this system, the tester strain reverts from histidine dependence to histidine independence when treated with a mutagen. According to criteria set by the author, a strong mutagenic response was defined as a relative mutagenicity of greater than 10 (number of revgrtants from treated plates per 10 bacteria/spontaneous reversions per 10 bacteria). A medium mutagenic response was defined as a relative mutagenicity of 5-10, a weak response was a relative mutagenicity value of 3-5, a doubtful response was a relative mutagenicity of 1-2, and a negative response was a relative mutagenicity of 1. 2,3,7,8-TCDD was a strong mutagen in strain TA1532, and a doubtful mutagen in strains TA1531 and TA1534, strains which are reverted by frameshift mutagens. The compound was a directly acting mutagen; that is, it did not require exogenous metabolic activation for a mutagenic effect. 2,3,7,8-TCDD was negative in G46 and TA1530, which are reverted through base pair substitutions. No numerical data for chemically-induced revertants or spontaneous revertants were presented by the authors. This prevents a proper evaluation of the test system in that laboratory. The author also did not include the numbers of replicate plates used in the experiment, the dose of TCDD used, or the number of times the experiment was repeated. Failure to include this information severely limits the value of the data since no quality of experimental design can be evaluated. The positive mutagenicity data concerning TCDD is weakened by deficiencies in the reported studies. Although it appears that TCDD is mutagenically active, a firm conclusion to that effect is inappropriate at present. �9.4.3 Other Herbicides Benigni et al. (1979) studied the mutagenicity of diquat in several in vitro mutagenicity assays, ranging from prokaryotic bacterial cells to eukaryotic mammalian cells. This battery included: • Ames test, Salmonella typhimurium strains TA98, TA98, TA100, TA1535, TA1537, and TA1538, with and without metabolic activation, measuring reversion from histidine dependence to histidine independence • Forward mutation test in £. typhimurium measuring induction of 8-azaguanine resistance in a sensitive strain • DNA repair test in S. typhimurium measuring preferential killing of DNA repair deficient cells compared to DNA repair proficient cells • Forward mutation test in Aspergillus nidulans measuring induction of 8-azaguanine resistance and methionine suppression • Induction of recessive lethals in:A. nidulans • Induction of unscheduled DNA synthesis in a human cell line. This study represents a thorough in vitro test battery to study the mutagenicity of diquat and reveals much valuable information. Diquat was mutagenic in forward mutation tests in bacteria and yeast, DNA repair tests in bacteria and mammalian cells, and a recessive lethal test in yeast. Reverse mutation tests in bacteria were negative. The authors suggested the reason for these results may be that diquat is unable to induce frameshift or base-pair substitutiontype mutations, but may be able to cause other damage, such as deletions, strand breaks, or cross links at the gene level. Diquat was shown to cause damage at the chromosomal level (recessive lethal test) and to damage DNA (unscheduled DNA synthesis). Rocchi et al. (1980) studied the action of diquat on scheduled and unscheduled DNA synthesis in rat thymocytes and human lymphocytes. Diquat (95 percent purity) in DMSO was added to rat thymocyte cell cultures to determine the concentration of diquat that gave 50-70 percent DNA synthesis inhibition. This dose was then used to determine the effect of diquat on DNA synthesis in human lymphocytes. For four hours, diquat (500 ug/ml) was added to cultures of human lymphocytes with and without hydroxyurea. During this culture period, the cells were labeled with tritiated thymidine to measure DNA synthesis. Cells treated with ultraviolet radiation were used to compare scheduled and unscheduled DNA synthesis. At 500 ug/ml, diquat produced approximately the same amount of inhibition of scheduled DNA synthesis in rat thymocytes as in human lymphocytes. In this system, inhibition of scheduled DNA synthesis was comparable to inhibition of unscheduled DNA synthesis. The authors did not discuss the relevance of these data. However, it appears from the data presented that diquat at the dose tested in human lymphocytes did not induce unscheduled DNA synthesis compared to controls. �Carere et al. tested dalapon and picloram for mutagenicity in Salmonella typhimurium and Streptomyces coelicolor. ju typhimurium strains TA1535, TA1536, TA1537, and TA1538 were used in the assay with and without rat liver S-9 metabolic activation. A streptomycin-sensitive strain of J3_. coelicolor A3(2), hisAl, was used in a forward mutation assay. (Mutagenic effects of a chemical are measured by induction of streptomycin resistance.) Both assays were performed as a spot test, i.e., absorbent paper saturated with solutions of the chemical to b.e tested were placed on agar seeded with bacteria. Neither compound induced increased numbers of revertants, with or without metabolic activation, in any of the four tester strains of S. typhimurium. In the j^. coelicolor forward mutation assay, picloram-treated plates had approximately 40 times the number of resistant colonies compared to controls, while dalapon treatment did not induce an increase in numbers of resistant colonies. The authors studied the mutagenicity dalapon, picloram, and tordon in Aspergillus nidulans. Three types of mutagenic events were studied: forward point mutation from 8-azaguanine sensitivity to 8-azaguanine resistance in haploid strain 35; mitotic crossing over in the diploid strain p; and mitotic nondisjuction in the diploid strain P. In all three, a spot test was employed. Dalapon and picloram were negative in all three mutagenicity assays. Seiler (1978) evaluated the mutagenicity of diuron and monuron in the mouse testicular DNA synthesis inhibition (DSl) test and the Ames test. In the DSI test, each herbicide was administered in a single oral aequeous dose given by stomach tube to four male mice (age and strain unspecified) per dose level. Both monuron and diuron produced a statistically significant inhibition in testicular DNA synthesis. Each compound was also screened for mutagenicity in the Ames test. Data for only one tester strain (S. typhimurium TA1535) were reported. Rat liver S9 was used as the metabolic activation system. Both compounds induced dose-dependent increases in numbers of revertants in the plate assay, and also in a number of tubes with microbial growth in the fluctuation assay. Testing presented in this paper cannot be viewed as reliable evidence of mutagenic effects, primarily because the author fails to present methodology, description of animals used and controls, and criteria used in evaluating the results of this study. Simmon et al. (1977) reported a study of twenty pesticides including bromacil, cacodylic acid, and monuron for mutagenic activity in several in vitro test systems. The test systems included a mammalian cell unscheduled DNA synthesis, Salmonella typhimurium reverse mutation Escherichia coli WP2, Escherichia coli W3100/p 3478, and Bacillus sublilis H17/M45, and Saccharomyces cerevisiae DE. All of the tests had appropriate controls and sufficient detail was presented to substantiate the author's conclusions. Bromacil and cacodylic acid were negative in all of,.the_assays. In the presence of metabolic activating enzymes, monuron (10 -10 M) was positive in the mammalian cell unscheduled DNA synthesis assay. It was negative in this test without metabolic activation. With respect to this positive data on monuron, the authors concluded that this compound should be tested more extensively in vivo to evaluate its carcinogenic potential. Mutagenicity tests on other Vietnam herbicides were presented by Anderson et al. (1972), Seibert and Leraperle (1974), and Shirasu et al. (1978). These studies are described in detail in Section 9.4.1. �Diquat and monuron are the only herbicides which have been studied, other than 2,4-D, 2,4,5-T, and TCDD, that have demonstrated positive results in at least two different in vitro test systems. Diquat was positive in the majority of the assays in which it was tested. These results are sufficient evidence to suggest in vivo evaluation of the biological activity of these compounds is appropriate. 9.5 CONCLUSIONS 2,4-D and 2,4,5-T appear to be weak mutagens based on positive results in Drosophila and yeast assays. Some test results in human and mammalian cells also showed these compounds to be weak mutagens. TCDD was reported to be mutagenic in all bacterial and yeast assays in which it was tested, however, there are serious deficiencies in this data. TCDD appears to be a weak clastogen in rats dosed by gavage, however substantiating evidence is lacking. A survey of subjects exposed to TCDD in the Seveso accident associate no increase in chromosome aberrations with that exposure. Of the other herbicides studied for mutagenic potential, only diquat and monuron were positive in at least two different in vitro assay systems. This suggests that in vivo testing of these two compounds is warranted. There is, however, a disturbing trend in the data on human exposure to a variety of herbicides which is suggestive that such exposure may present a potential genetic hazard to humans. 9-20 �CHAPTER 9. REFERENCES Ahmed, F. E., Hart, R. W., and Lewis, N.J. (1977) Pesticide induced DNA damage and its repair in cultured human cells. Mutat. Res. 42:161-174. Ahmed, F. E., Lewis, N. J., and Hart, R. W. (1977) Pesticide induced ouabain resistant mutants in Chinese hamster V79 cells. Chem.-Biol. Interactions 19:369-374. Andersen, K. J., Leightty, E. G., and Takahashi, M. T. (1972) Evaluation of herbicides for possible mutagenic properties. J. Agric. Food Chem. 20(3):649-656. Aulicino, F., Bignami, M., Carere, A., Conti, G., Morpurgo, G., and Velcich, A. (1976) Mutational studies with some pesticides in Aspergillus nidulans. Mutat. Res. 38(2):138. Babbitt, B., Risch, S., Chbffnes, E., Kalis, J., Zupanic, M., et al. (1973) Effects of 2,4,5-T on human chromosomes. Genetics 71(Suppl):53. Benigni, R., Bignami, M., Carere, A., Conti, G., Conti, L., Crebelli, R., Dogliotti, E., Gualandi, G., Novelletto, A., and Ortali, V. A. (1979) Mutational studies with diquat and paraquat in vitro. Mutat. Res. 68:183-193. Bignami, M., Aulicino, F., Velcich, A., Carere, A., and Mopurgo, G. (1977) Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans. Mutat. Res. 46:395-402. Bignami, M., and Crebelli, R. (1979) A simplified method for the induction of 8-azaguanine resistance in Salmonella typhimurium. Toxicol. Lett. 3:169-175. Berry, D. L., DiGiovanni, J., Juchau, M. R., Bracken, W. M., Gleason, G. L., and Slaga, T. J. (1978) Lack of tumor-promoting ability of certain environmental chemicals in a two-stage mouse skin tumorigenesis assay. Res. Commun. Chem. Pathol. Pharmacol. 20(1):101-108. 9-21 �Berry, D. L., Slaga, T. J., DiGiovanni, J., and Juchau, M. R. (1979) Studies with chlorinated dibenzo-p-dioxins, polybrominated biphenyls, and polychlorinated biphenyls in a two-stage system of mouse skin tumorigenesis: Potent anticarcinogenic effects. Ann. N.Y. Acad. Sci. 320:405-414. Bionetics Research Laboratories, Inc. (1968a) Evaluation of carcinogenic, teratogenic, and mutagenic activities of selected pesticides and industrial chemicals. Vol 1: Carcinogenic study. NTIS PB 223159. Bongso, T. A., and Basrur, P. K. (1973) In vitro response of bovine cells to 2,4-dichlorophenoxyacetic acid. In Vitro 8(5):416-417. Buselmaier, M. V., Rohrborn, G., and Propping, P. (1972) Pesticide mutagenicity investigations by the host mediated assay and the dominant lethal test in mice. Biol. Zentralbl. 91:311-325. Fujita, K., Fujita, H. M., and Funazaki, Z. (1975) Chromosomal abnormality caused by 2,4,5-T. J. Jpn. Assoc. Rural Med. 24(2):77-79. Green, S., and Moreland, F. S. (1975) Cytogenetic evaluation of several dioxins in the rat. Toxicol. Appl. Pharmacol. 33:161. Green, S., Moreland, F., and Sheu, C. (1977) Cytogenetic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on rat bone marrow cells. FDA By-Lines 6:242-294. Homberger, E., Reggiani, G., Sambeth, J., and Wipf, H. K. (no date) The Seveso accident: its nature, extent and consequences. Ann. Occup. Hyg. 22:327-366. Hussain, S., Ehrenberg, L., Lofroth, G,, and Gejvall, T. (1972) Mutagenic effects of TCDD on bacterial systems. Ambio 1:32-33. Jenssen, D., and Renberg, L. (1976) Distribution and Cytogenetic test of 2,4-D and 2,4,5-T phenoxyacetic acids in mouse blood tissues. Chem. Biol. Interact. 14:291-299. Magnusson, J., Ramel, C., and Eriksson, A. (1977) Mutagenic effects of chlorinated phenoxy acids in Drosophila melanogaster. Hereditas 87:121-123. 9-22 �Majumdar, S. K., and Golia, J. K. (1974) Mutation test of 2,4,5-trichlorophenoxyacetic acid on Drosophila melanogaster. Can. J. Genet. Cytol. 16:465-466. Majumdar, S. K. , and Hall, R. C. (1973) Cytogenetic effects of 2,4,5-T on in vivo bone marrow cells of Mongolian gerbils. J. Hered. 64:213-216. Nagy, Z. S., Mile, I., and Antoni, F. (1975) The mutagenic effect of pesticides on Escherichia coli WP2 try. (1975) Acta Microbiol. Acad. Sci. Hung. 22:309-314. Pilinskaya, M. A. (1974) Cytogenetic effect of the herbicide 2,4-D on human and animal chromosomes. Tsitologiya i Genetika 8(3):202-206. Pocchiari, F., Silano, V., Zampieri, A. (1979) Human health effects from accidental release of tetrachlorodibenzo-p-dioxin (TCDD) at Seveso, Italy. Ann. H. Y. Acad. Sci. 320:311-320. Ramel, C. Genetic effects of phenoxyacetic acids in animals. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stockholm: Swedish Natural Science Research Council, 1978a) p. 182-185. Rasmuson, B., and Svahlin, H. Mutagenicity tests of 2,4-dichlorophenoxyacetic acid and 2,4,5,-trichlorophenoxyacetic acid in genetically stable and unstable strains of Drosophila melanogaster. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull. No. 27, Stolkholm: Swedish Natural Science Research Council, 1978) p. 190-192. Reggiani, G. (1980) Acute human exposure to TCDD in Seveso, Italy. JL_ Toxicol. Environ. Health 6:27-43. Reggiani, G. (1979) Estimation of the TCDD toxic potential in the light of the Seveso accident. Arch. Toxicol. 2:291-302. Rocchi, P., Perocco, P., Alberghini, W., Fini, A., and Prodi, G. (1980) Effect of pesticides on scheduled and unscheduled DNA synthesis of rat thymocytes and human lymphocytes. Arch. Toxicol. 45:101-108. Seiler, J. P. (1979b) Phenoxyacids as inhibitors of testicular DNA synthesis in male mice. Bull. Environ. Contam. Toxicol. 21:89-92. 9-23 �Seller, J. P. 2,4,5-T. (1978a) The genetic toxicology of phenoxy acids other than Mutat. Res. 55:197-226. Seiler, J. P. (1978b) Herbicidal phenylalkylureas as possible mutagens. I. Mutagenicity test with some urea herbicides. Mutat. Res. 58:353-359. Seiler, J. P. (1973) A survey on the mutagenicity of various pesticides. Experientia 15(5):622-623. Shirasu, Y., Moriya, M., Kato, K., Furuhashi, A., and Kada, T. (1976) Mutagenicity screening of pesticides in the tnicrobial system. Mutat. Res. 40:19-30. Siebert, D., and Lemperle, E. (1974) Genetic effects of herbicides: Induction of mitotic gene conversion in Saccharomyces cerevisiae-. Res. 22:111-120. Mutat, Simmon, S. F., Poole, D. C., Riccio, E. S., Robinson, D. E., Mitchell, A. D., and Waters, M. D. (1979) In vitro mutagenicity and genotoxicity assays of 38 pesticides. Environ. Mutagen. 1:142-143. Seiler, J. P. 2,4,5-T. (1978a) The genetic toxicology of phenoxy acids other than Mutat. Res. 55:197-226. Styles, J. A. (1973) Cytotoxic effects of various pesticides in vivo and in vitro. Mutat. Res. 21:50-51. Tenchini, M. L., Crimaudo, C., Simoni, G., De Carli, L., Giorgi, R., and Nuzzo, F. (1979) Approaches to the evaluation of genetic damage after a major hazard in chemical industry: preliminary cytogenetic findings in TCDD exposed subjects after the Seveso accident. Part of a special project to investigate TCDD-exposed pregnancies. Task Force from Universita di Milano (Italy). Supported by the Assessorato alia Sanita, Regione Lombardia (Italy). Tung, T. T. (1973) Primary cancer of the liver in Viet Nam. Chirurgie 99(7):427-436. The Veterans Administration. (1981) Advisory Committee on Health-Related Effects of Herbicides - Transcript of Proceedings. Feb 4, 1981. 158 pp. 9-24 �Vos, J. G., and Moore, J. A. (1974) Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8 tetrachlorodibenzo-p-dioxin. Int. Arch. Allerg. Appl. Immunol. 47:777-794. Yoder, J., Watson, M., and Benson, W. W. (1973) Lymphocyte chromosome analysis of agricultural workers during extensive occupational exposure to pesticides. Mutat. Res. 21:335-340. Zetterberg, G., Busk, L., Elovson, R., Starec-Nordenhammar, I., and Ryttman, H. (1977) The influence of pH on the effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) on Saccharomyces cerevisiae and Salmonella typhimurium. Mutat. Res. 42:3-18. Zetterberg, G. (1978) Genetic effects of phenoxy acids on microorganisms. In Chlorinated Phenoxy Acids and Their Dioxins, C. Ramel, ed. (Ecol. Bull, No. 27, Stockholm: Swedish Natural Science Research Council, 1978) pp. 193-204. 9-25 �CHAPTER 10 CARCINOGENICITY This chapter reviews human and animal studies concerned with the carcinogenic potential of herbicides used in Vietnam by the armed forces. The primary focus of the chapter is the assessment of the carcinogenicity of 2,4-D and 2,4,5-T (the primary constituents of Herbicide Orange), the contaminant TCDD, and other herbicide formulations. Some limited data are also presented for picloram, monuron, and cacodylic acid. The chapter is divided into three main sections. The first deals with available epidemiologic data. This is followed by a discussion of animal bioassays that have been conducted on the individual components of the herbicide formulations. The final section summarizes the human and animal data and assesses the potential carcinogenic risk to humans posed by exposure to the various herbicides. 10.1 10.1.1 EPIDEMIOLOGIC STUDIES Study of Cancer Prevalence in Vietnam Tung et al. (1973) reported an increase in the number of persons with liver cancer in proportion to the number of cases of all cancers in North Vietnam during the period 1962 to 1968, during which time herbicides were used in South Vietnam. A total of 159 cases of liver cancer out of 5,492 cases were reported for the period 1955 to 1961, prior to the start of herbicide spraying in South Vietnam. Between 1961 to 1968, 791 cases of liver cancer were found among 7,911 total cancer cases. The authors attributed the increased prevalence of liver cancer to the herbicide application, explaining that the cases appeared in the North either by population mixing or by transport of the herbicide by contaminated vectors. No evidence was presented to support this suggestion, nor were other possible etiologies adequately ruled out. Serious limitations in the report make a definitive conclusion regarding any relationship between the prevalence of liver cancer and herbicide spraying impossible. 10.1.2 Studies of Industrial Exposures The incidence of cancer in workers accidentally exposed to high levels of trichlorophenol (TCP) and TCDD has been investigated in two studies. Thiess and Frentzel-Beyne (1977) reported six cancer deaths that had occurred during a 24-year period among a group of 75 men who were exposed following an accident at. a BASF factory in Germany in 1953. Three of these deaths were attributed to carcinoma of the stomach. The incidence of stomach cancer in the exposed group was found to be significantly higher than that in three different age-matched control groups; however, this finding was significant only for those persons between the ages of 65 and 69 years. The 10-1 �incidence of mortality from all malignant neoplasms in the study group was not found to be significantly different from any of the control groups. Tt was also reported that among a group of BASF workers who were not involved in the accident, four had died of cancer. None of these deaths were due to stomach cancer. Comparisons between this group and the study population were not subjected to statistical analysis. In 1980, Zack and Suskind investigated cancer deaths among 121 male workers who had developed chloracne following TCP and TCDD exposure in an accident in 1949 at the Monsanto plant in Nitro, West Virginia. Nine deaths due to cancer occurred during the 29-year observation period. Five of these deaths were from lung cancer, three from neoplasms of lymphatic or hematopoietic tissue, and one death was caused by a malignant fibrous histiocytoma presumed to be of dermal origin. No deaths from stomach or liver cancer were found. The incidence of death from cancer was not found to be significantly higher than among age-matched U.S. males. The authors concluded that there was no apparent excess of cancer deaths resulting from the accidental high exposures to TCP and TCDD. The results of this study do not support the findings of Thiess and Frentzel-Beyne (1977) in the 65 to 69-year-old cohort, despite the larger cohort and longer observation time used by Zack and Suskind (1980). 10.1.3 Studies of Swedish Railway Workers In a preliminary study, Sundell et al. (1973) found no excess number of cancer deaths among a group of 194 Swedish railway workers exposed to a variety of herbicides including phenoxyacetic acids. In a followup report by Axelson and Sundell (1974) on a cohort of 207 workers exposed to phenoxyacetic acids this finding was confirmed. Only workers who had been known to be exposed primarily to phenoxyacetic acids for a period greater than 45 days were included in the cohort. The authors also recorded eight cases of cancer among members of the cohort who were still alive at the time of the study. This was not found to be significantly higher than the five cases that were expected for the general Swedish population. The authors attributed the slight increase in tumor morbidity in phenoxyacetic acid-exposed individuals to concurrent exposure of these workers to other herbicides, including amitrol. The authors found significant increases in cancer mortality and morbidity among workers exposed primarily to amitrol. The preliminary report (Sundell et al., 1973) and the completed report (Axelson and Sundell, 1974) each listed the herbicide formulations known to be in use during the study period, 1957 to 1971. According to these listings, the phenoxyacetic acids 2,4-D and 2,4,5-T were always used in combination with other herbicides, including atrazin, mecoprop, and dichloropropionic acids. It is not known to what extent the 2,4,5-T was contaminated with TCDD, nor in what proportions these herbicides were used in the formulations. In 1977, Axelson and Sundell reevaluated the data presented in the 1974 study. The authors reported that two of the cancer cases were difficult to evaluate with respect to exposure; both were initially included in the amitrol-exposed cohort but it was possible that exposure to phenoxyacetic acids alone took place. No details supporting this suggestion were provided. 10-2 �After including these two cases in the phenoxyacetic acid-exposed cohort, Axelson and Sundell (1977) found a significant increase in tumor morbidity as compared to the general population. It was concluded that this reevaluation supported the suspicion that exposure to phenoxyacetic acids leads to an increase in the incidence of tumor morbidity. However, the authors recognized the difficulty in interpreting their findings presented by mixed exposures. Another shortcoming of the studies by Axelson and Sundell (1974, 1977) is the short latency period used by the authors. Calculations of'' significance were based on 3- and 5-year latency periods. In an unpublished report, Axelson et al. (undated) performed a follow-up investigation which allowed for the use of a 10-year latency period. Six deaths from cancer were found among the study cohort, compared with 3.14 expected cases. Tiis difference was not significant. Two of these deaths were reported to be from stomach cancer, the incidence of which was found to be significantly higher than the 0.33 cases expected in the general population. Data on the incidence of tumor morbidity were not presented in the report. As with the previous reports by Axelson and Sundell (1974, 1977) it is impossible to draw any conclusions regarding a causal relationship between exposure to particular phenoxyacetic acids and cancer incidence, because of the confounding factor of mixed and multiple exposures to several herbicides among members of the study cohort. 10.1.4 Studies of Swedish Forestry and Agricultural Workers The first indication that forestry and agricultural workers in Sweden may be at increased risk from.soft-tissue tumors arose following a report by Hardell (1977), in which he described the employment histories of seven patients hospitalized in Umea and diagnosed with malignant mesenchymal tumors. Five of the seven patients were forestry workers with confirmed exposure to phenoxy acids. The remaining two were believed to be indirectly exposed through their work, which involved the clearing of phenoxy acid-treated areas. Although Hardell (1977) stated that this report was not conclusive, he believed that there were several reasons for suspecting that exposure to phenoxy acids might have been a factor in the development of the tumors. The reasons cited by the author included the observations that the tumor type is relatively rare and the latent period of 10 to 20 years agreed with the suspicion of chemical involvement. Additionally, without providing details, the author reported that the sex ratio of patients diagnosed as having mesenchymal tumors in Umea, a heavily wooded ares, deviated from national statistics. The report by Hardell (1977) prompted a formal investigation into the occurrence of malignant mesenchymal tumors among forestry workers exposed to phenoxy acids. Results of a case-control study of patients in northern areas of Sweden who were diagnosed as having malignant mesenchymal soft-tissue sarcomas during the period 1970-1977 appeared in two publications (Hardell and Sandstrom, 1978; Hardell and Sandstrom, 1979). The patients selected for study were all males between the ages of 26-80. For each patient alive at the time of the study, eight controls matched for age, sex, and place of residence were selected. Ten matched controls were selected for each deceased patient. Exposure of the patients to phenoxy acids was assessed by means of a questionnaire sent to the patients or their next of kin and persons selected 10-3 �as controls. Employers of the patients were also contacted for information regarding the patients' exposure to phenoxy acids. The authors reported that phenoxy acids in wide use during the study period included 2,4-D, 2,4,5-T, and 4-chloro-2-methylphenoxyacetic acid (MCPA). However, exposure to phenoxy acids was difficult to evaluate; replies from employers were obtained for less than half of the patients involved in the study. It was also reported that employers did not keep records of individual employees, and the answers received were based on memory. While conducting a parallel study on chlorophenol exposure within the study group, the authors received a 97 percent response from the patients' employers and good agreement between the employers' responses and patients' responses. Based on this finding, Hardell and Sandstrom (1978, 1979) concluded that patients' responses to questibns regarding exposure to phenoxy acids were probably adequate. It was found that 13 of a total of 45 patients had been exposed to phenoxy acids. Exposure times varied from 3 days to 49 months and latency periods varied from 3 to 27 years. Of 201 controls, 14 had indicated past exposure to phenoxy acids of a duration of at least I day. The relative risk of exposure to phenoxy acids was calculated to be 5.3. A similar finding was reported for patients exposed to chlorophenols either alone or in combination with phenoxy acids. According to the authors, confounding factors, such as smoking habits or exposure to DDT or chain saw exhaust fumes, did not contribute to the increase in relative risk, but the data supporting this conclusion were not reported. The influence of exposure to diesel oil or other herbicides could not be assessed due to a lack of data. The authors concluded that the use of phenoxy acids and chlorophenols contributed to an increased risk of soft-tissue sarcomas, although no evaluation of the effect of specific substances could be made. In 1979, Eriksson et al. reported the results of a similar study involving soft-tissue sarcoma patients who had worked in southern Sweden. The study included men in the age group of 25-75 years with soft-tissue sarcomas diagnosed between 1974-1978. Phenoxy acids used in the southern areas included 2,4-D, 2,4,5-T, MCPA, and phenoxypropionic acids. Control subjects were selected and exposure information was acquired in the same manner as by Hardell and Sandstrom (1978, 1979). Ninety-nine patients were included in the analysis, 14 of whom reportedly were exposed to phenoxy acids. Five of 211 subjects in the control group had indicated a history of exposure. The relative risk for developing soft-tissue sarcomas was calculated to be 6.1, The risk was found to be greater for test subjects exposed for more than 30 days. This change in relative risk with respect to length of exposure was not found to be significant. In an attempt to investigate the influence of TCDD contamination of the herbicides on relative risk for developing sarcomas, Eriksson et al. (1979) excluded patients with known exposure to 2,4,5-T from the calculations. When this was done, they reported that exposure to phenoxy acids not contaminated with TCDD led to a relative risk of 4.2, less than that reported for persons exposed to all phenoxy acids but still significantly higher than controls. The authors concluded that the data showed that exposure to phenoxy acids may 10-4 �be a contributing factor in the development of soft tissue sarcomas and that the risk is not limited to phenoxy acids that may contain TCDD. Eriksson et al. (1979) also presented data that failed to show any significant contributing effects from smoking histories or exposures to asbestos, glass fiber, power saw exhaust, other pesticides, or organic solvents. However, as with the reports by Hardell and Sandstrom (1978, 1979) it is impossible to derive any conclusions regarding exposure to any single agent as related to the risk of developing soft-tissue sarcomas. In 1979, Hardell described a case in which a patient with a tumor in the soft tissue of the left femur was found to have had massive exposure to phenoxyacetic acids. Histological examination showed that the tumor was a malignant lymphoma. In a pilot study, Hardell (1979) reported that 14 of 17 lymphoma patients questioned about their occupations responded that they had been exposed to phenoxyacetic acids or chlorophenols. A more detailed survey of 149 lymphoma patients with either Hodgkins disease or non-Hodgkins lymphoraa, of a similar design to the previous case-control studies described in this section, was reported by Hardell et al. (1980). Forty-one of the 149 patients were found to have had a history of past exposure to phenoxyacetic acids, as compared to 24 of 327 control subjects. The relative risk was found to be 4.8. No significant difference existed between cases and controls with respect to smoking history or exposure to a variety of other agents. The authors did note, however, that exposure to DDT and mercurycontaining seed dressings did correlate with exposure to phenoxyacetic acids. 10.1.5 Cohort Studies of American Workers Two cohort studies have been published examining the cancer mortality experience of American workers in 2,4,5-T or trichlorophenol production plants. In the first report, a cohort of 204 employees was selected based on company records showing that each member of the cohort had worked for at least one month at one of four jobs in which there was potential exposure to 2,4,5-T or TCP (Ott et al., 1980). Within the cohort, 157 had been involved with one or more of these }obs for less than one year. Production of 2,4,5-T began at the facility in 1950 and was shut down in 1971. An industrial hygiene survey conducted at the plant in 1969 revealed that air concentrations of 2,4,5-T varied from less than 0.1 to 6.2 mg/m . Estimated time weighted average (TWA) concentrations of 2,4,5-T varied from , 0.2 to 0.8 mg/m , depending upon the job. TWA concentrations of 1.6-9.7 mg/m TCP were also reported. The product specifications for 2,4,5-T in 1966 called for a maximum of 1 ppm TCDD. Information on the TCDD content of 2,4,5-T prior to 1966 was not reported. In addition to these exposures, the unit under which the process was organized was also responsible for the production of 2,4,5-trichlorophenoxypropionic acid, 2-methyl-4-chlorophenoxyacetic acid, and styrenebutadiene latex. Thus, many members of the cohort were potentially exposed to a variety of other substances during their employment. 10-5 �None of the individuals within the cohort had ever been known to have had chloracne or porphyria cutanea tarda, which might have indicated excessive exposure to TCDD. The only death due to a malignancy was one case of respiratory cancer in a 63-year-old man who had been exposed to 2,4,5-T for eight years and who had been known to smoke up to two packs of cigarettes per day. Ott et al. (1980) will continue surveillance of persons with known exposure to 2,4,5-T because of the limited scope of this survey. An incident that occurred at this facility in 1964 involved 49 TCP production workers who reported to the industrial medical department with skin conditions subsequently diagnosed as chloraxine. A followup mortality survey of 61 employees known to have been working in the building during 1964 was reported by Cook et al. (1980). No information was supplied concerning exposures to TCDD. The vital status of the cohort through December 1978 was determined. According to Ott et al. (1980), the TCP production plant was separate from the 2,4,5-T production unit, so for this cohort there presumably was no exposure to 2,4,5-T or the other herbicides manufactured. Of the four deaths reported for the cohort, three were due to malignant neoplasms: one adenocarcinoma (site unknown), one fibrosarcoma, and one glioma. None of these three decendents had any record of having developed chloracne while employed at the plant. All three were known smokers. After categorizing iob classification into high and low potential exposure, it was determined that only the worker who died from adenocarcinoma had held a job in an area of high potential exposure. Cook et al. (1980) concluded that the data suggested that TCDD is not a potent human carcinogen, because of the lack of any apparent dose-response relationship, and because the latency period of 14 years was sufficient to allow the identification of a potent human carcinogen. The authors also recommended further research on other cohorts to determine whether TCDD has weak carcinogenic potential in humans. In a letter to Lancet, Honchar and Falperin (1981) reviewed these two studies and the report by Zack and Suskind (1980—see Section 10.1.2). After combining the cohorts from these three studies and some additional unpublished data from Zack, Honchar and Halperin (1981) found that of 105 deaths among workers exposed to TCP or 2,4,5-T, three (2.9 percent) were due to soft-tissue sarcoma. They compared this to an 0.07 percent incidence of deaths in 1975 in American males between the ages of 20 and 84 years. Although realizing that none of the four cohorts showed an excess incidence of soft-tissue sarcoma, the authors concluded that a common pattern was suggested by the combined data. 10.1.6 Summary and Evaluation Studies of the incidence of cancer mortality have been conducted on three major occupational groups for which there has been worker exposure to phenoxyacetic acids and/or trichlorophenol, both of which are known to be contaminated with TCDD. Generally, large excesses in the incidence of cancer mortality, which would suggest a strong carcinogenic effect, have not been demonstrated. From these studies, it is also impossible to implicate any one herbicide or TCDD as being responsible for the small number of cancer deaths documented, because of the confounding factor of mixed exposures. 10-6 �A significant increase in tumor morbidity among a group of Swedish railway workers exposed to pbenoxyacetic acids (Axelson and Sundell, 1977) was demonstrated only after reevaluating previous data (Sundell et al. 1973; Axelson and Sundell, 1974). A later unpublished report (Axelson et al., undated) on the same study group showed that the incidence of death from stomach cancer was significantly higher than national statistics would indicate (two actual cases versus 0.33 expected). The total incidence of deaths from cancer was not significantly high. Workers were known to have been exposed to other herbicides, and exposure levels of 2,4,-D, 2,4,5-T, and TCDD were not available. Case-control studies have pointed to an association between exposure to phenoxy acids and/or chlorophenols used by Swedish forestry and agricultural workers, and the development of malignant mesenchymal soft-tissue sarcomas (Hardell, 1977; Hardell and Sandstrom, 1978; Hardell and Sandstrom, 1^79; Eriksson, 1979), The authors suggested that the risk was not limited to phenoxy acids that contain TCDD (Eriksson et al., 1979). Similar case-control studies have also revealed an increased relative risk of exposure to phenoxyacetic acids or chlorophenols and the development of malignant lymphoma (Hardell, 1979; Hardell et al., 1980). It is impossible to ascertain the contribution by 2,4-D, 2,4,5-T, or TCDD alone, as there was concomitant exposure to other herbicides and chemical agents in these study populations. The third major group of persons studied that have been exposed to 2,4,5-T and TCDD were workers involved in trichlorophenol (TCP) or 2,4,5-T production. Two investigations were conducted on populations with known large exposures following plant accidents. In one study (Theiss and Ftentzel-Beyne, 1977), a significantly higher incidence of death from stomach cancer was found among a group of men exposed to TCP and TCDD following the accident at the BASF facility in Germany in 1953. This finding was not supported in a study of a group of workers exposed to TCP and TCDD following the accident at Nitro, West Virginia (Zack and Suskind, 1980). A larger cohort and a longer observation time was available for the latter study. Two cohort studies of workers exposed to 2,4,5-T and/or TCP, both containing TCDD, have not individually reported an excess incidence of cancer deaths (Ott et al., 1980; Cook et al., 1980). However, when these data were combined with the data of Zack and Suskind (1980) and unpublished data by Zack, a pattern was observed in which exposure to 2,4,5-T and/or TCP led to a greater-than-30-fold increase in the mortality incidence of soft-tissue sarcoma, compared to the background incidence of U.S. males (Honchar and Halperin, 1981). It is worth noting that of the three cases of soft-tissue sarcoma found, two were described in workers involved in TCP production where there was no known exposure to 2,4,5-T reported. The third case was of an employee engaged in 2,4,5-T synthesis. No cases of mortality from soft-tissue sarcoma were described in the only published study of a cohort of 2,4,5-T production workers (Ott et al., 1980). The number of cases of soft-tissue sarcoma described in these studies is too small to make any firm conclusions regarding the risk from exposure to 2,4,5-T or TCDD. Although these findings do not contradict the Swedish case 10-7 �control studies of forestry and agricultural workers, exposure to TCP is a confounding factor in the assessment of the carcinogenic risk of 2,4,5-T and TCDD. More information may become available following analysis of the mortality experience of a larger cohort currently being assembled by the National Institute of Occupational Safety and Health (Honchar and Halperin, 1981). 10.2 ANIMAL STUDIES The following sections describe animal bioassays that have been conducted on 2,4-D, 2,4,5-T, and TCDD. Animal studies on picloram, monuron, and cacodylic acid, compounds for which no human studies are available, are also reviewed in the latter parts of the section. 10.2.1 10.2.1.1 2,4-D Mice Two strains of mice, C57BL/6 x C.3HAnf and C57BL/6 x AKR, 18 of each sex, were fed 46.4 mg 2,4-D per kg by gavage until the mice were 4 weeks of age (Innes, 1969; Bionetics, 1968). Thereafter, the mice were fed 149 mg 2,4-D per kg of diet until they were killed for examination at approximately 18 months of age. Another group of C57BL/6 x AKR mice, 18 of each sex, were fed 2,4-D at higher doses of 100 mg/kg from day 7 to 28, and 323 mg/kg of diet from day 28 to 18 months. No significant increase in tumor incidence within the combined groups or compared to 338 untreated mice was reported at either level. Similar results were obtained in mice fed 2,4-D isopropyl, butyl, or isooctyl esters. IARC (1977) reported that the tumor incidence in individual groups was also comparable to controls. Groups of these same two strains of mice were given 2,4-D by subcutaneous injection on the 28th day of life at doses of 215 or 464 mg/kg. Additional groups of both strains were given subcutaneous injections of 21.5 mg 2,4-D butyl ester per kg, 100 mg 2,4-D isopropyl ester per kg, or 21.5 mg 2,4-D isooctyl ester per kg. When tumor incidence was compared to 613 untreated animals, a significant excess of total tumors .and reticulum cell sarcomas was found for the mice treated with 2,4-D isooctyl ester. No increase in tumor incidence was found in any other treated group. Arkhipov and Kozlova (1974), using groups of 100 OSVA x S57/VL hybrid female mice, found no tumors in mice fed 2,4-D at one-tenth the LD (dose not stated) throughout the animals' lives. The same result was reported for a second group of animals following weekly applications of two drops of a 10 percent 2,4-D solution on the skin. However, a serious lack of detail in reporting the experimental protocol and results makes this study difficult to evaluate. 10-8 �10.2.1.2 Rats Groups of 25 male and 25 female Osborne-Mendel rats were maintained for two years on diets containing from 5 to 1,250 mg 2,4-D per kg diet (Hansen et al., 1971). Analysis of 2,4-D by gas chromatography did not detect TCDD or 2,7-dichlorodibenzo-p-dioxin (limit of detection was 1 ppm). For male rats receiving the highest dose, the number of animals with malignant tumors was found to be significantly greater than in controls (7/25 as compared to 3/25). No specific target organ was apparently affected and the authors noted that the tumors found in treated rats were of the type commonly seen in aging Osborne-Mendel rats. The authors concluded that their study, like that of Innes (1969), supported a lack of carcinogenic effect of 2,4-D in animals. In contrast, Arkhipov and Kozlova (1974) found two rats with tumors among a group of 120 male and 45 female randomly bred rats fed 2,4-D at one-tenth the LD5Q (dose not specified) for 2 years. The tumors found were a fibroadenoma of the mammary gland, which was also seen in one control rat, and a hemangioma of the mesentery. Lack of detail in the report make these results difficult to evaluate, but it is apparent that these results failed to demonstrate significant carcinogenic activity of 2,4-D in rats. 10.2.1.3 Summary and Evaluation 2,4-D has been tested in feeding studies using small numbers of mice and rats and has not been found to exert a strong carcinogenic effect (Innes, 1969; Bionetics, 1968; Hansen et al., 1971; Arkhipov and Kozlova, 1974). A significant increased incidence of tumors was found among mice given 2,4-D isooctyl ester by subcutaneous injection. IARC (1977) concluded that no evaluation of the carcinogenicity of 2,4-D could be made based on these studies due either to inadequate reporting or the small number of animals used in the tests . 10.2.2 10.2.2.1 2,4,5-T Mice Groups of 18 male and 18 female mice of each strain C57BL/6 x C3HAnf and C57BL/6 x AKR received commercial 2,4,5-T at a dose of 21.5 mg/kg daily from age 7 to 28 days (Innes, 1969; Bionetics, 1968). Thereafter each group was fed 60 mg 2,4,5-T per kg of diet until the mice were approximately 18 months of age, when they were killed and examined. When compared to 338 untreated animals, the combined group of treated animals did not show an excessive number of tumors. Similar groups of both strains were given single subcutaneous iniections of 2,4,5-T on day 28 of life at a dose of 215 mg/kg. The tumor incidence in the combined group did not differ from a group of 613 untreated mice. IARC (1977) also reported that in both the feeding and injection study the tumor incidence within individual treated groups did not differ from controls. Although the 2,4,5-T was reported to be 98 percent pure (Innes, 1969; Bionetics, 1968), the TCDD content was not reported. 10-9 �No increase in tumor incidence was reported among a group of 20 male and 19 female XVII/6 mice given 100 mg 2,4,5-T per liter in the drinking water for 2 months, followed by 80 mg 2,4,5-T per kg of diet for the animals' lifespan (Muranyi-Kovacs et al., 1976). However, when a group of 22 male and 35 female C3Hf mice were treated similarly, a significant increased total incidence of tumors was found among female mice. When tumor types were separated into incidental tumors (discovered in an animal which died from some other cause) and nonincidental tumors (diagnosed during life or causing the death of the animal), treated C3Hf males and females showed an increased incidence of nonincidental tumors. No difference was found between treated C3Hf and control mice in the occurrence of incidental tumors. Nonincidental tumor types included leukemias, cutaneous tumors, a variety of sarcomas, and hepatomas diagnosed in living animals. Occasional rare tumor types were found among treated C3Hf mice; these included two cutaneous squamous-cell carcinomas and one osteosarcoma. The authors attributed the increase in tumorigenesis in C3Hf mice to 2,4,5-T, since their sample was reported to contain only between 0,02 and 0.03 ppm TCDD. However, they suggested that the effect produced by 2,4,5-T in C3Hf mice may have been dependent upon the species of animal tested since the carcinogenicity of 2,4,5-T could not be replicated in mice of strain XVII/6. Further testing in other animal strains was recommended. 10.2.2.2 Rats Kociba et al. (1976) examined the carcinogenic potential of 2,4,5-T in Sprague-Dawley rats in one well-designed study. Groups of 100 rats (50 of each sex) were fed 3, 10,.or 30 mg 2,4,5-T per kg mixed in the diet for two years. The 2,4,5-T was reported to contain less than 0.33 ppm TCDD. The body weights and food consumption of randomly selected rats were checked periodically to confirm the dosage. The highest dose level was associated with some degree of tpxicity, including increases in relative kidney weight, urinary excretion of coproporphyrin and uroporphyrin, and slight morphological changes in the kidney, liver, and lungs. Death rate and food consumption were not changed as compared to 86 male and 86 female control rats. At the end of the feeding period, rats were killed and all major organs were examined for gross and histological changes. Female rats fed the lowest dose were found to have a higher incidence than controls of interfollicular C-cell adenoma of the thyroid, but this was attributed to an unusually lower incidence of this lesion in controls than would be expected historically in this strain. No other increased oncogenic response could be demonstrated in male rats or female rats fed the two highest doses. The authors concluded that 2,4,5-T was not carcinogenic in Sprague-Dawley rats in doses high enough to induce toxic changes. 10.2.2.3 Summary and Evaluation 2,4,5-T has been tested in chronic feeding studies using four strains of mice and one strain of rat (Innes, 1969; Bionetics, 1968; Muranyi-Kovacs et al., 1976; Kociba et al., I979b), and by single subcutaneous injection in two strains of mice (Innes, 1969; Bionetics, 1968). An excess number of tumors was found in only one of the strains of mice tested, indicating that a species 10-10 �specific effect was observed. Muranyi-Kovacs , et al. (1976) supported this idea by citing evidence showing that the rate of metabolism of 2,4,5-T differs among animal species. Kociba et al. (I979b) questioned the validity of the finding of a positive carcinogenic effect of 2,4,5-T in one mouse strain because no specific target organ was involved, an observation that they reported is usually seen in animal bioassays of carcinogenic substances. IARC (1977), based on the mouse studies of Innes (1969) and MuranyiKovacs (1976), concluded that no evaluation of the carcinogenicity of 2,4,5-T could be made due to the small number of animals used in the tests. The data presented by Kociba et al. (1979b), which appeared after the IARC review, supports the previous findings of a lack of a strong carcinogenic effect by 2,4,5-T. 10.2.3 10.2.3.1 TCDD Mice The carcinogenic effects of TCDD were examined in Swiss-H/Riop mice. Preliminary data were reported in Toth et al. (1978), and completed data appeared in Toth et al. (1979) and Sugar et al. (1979). In this bioassay, five groups of 100 male and female mice were given various combinations of 2,4,5-trichlorophenoxyethanol (TCPE) and TCDD by gastric intubation once per week for one year. Two groups of 200 mice served as controls. The. dose of TCPE varied from 0.7 to 70 rag/kg while the dose of TCDD varied from 0.00007 to 0.112 mg/kg. The purity of the TCPE or TCDD was not stated. Similarly, three groups of 45 male Swiss-H/Riop mice were given TCDD in doses of 0.007, 0.7, and 7.0 mg/kg (Toth et al., 1979). Forty-five male mice served as controls. After the one-year exposure period, all animals were kept for their lifetimes. At death all maior organs were examined histologically. A relatively high incidence of hepatomas and hepatocellular carcinomas was seen in male control animals, indicating the sensitivity of this strain to developing liver tumors. The incidence of liver tumors was approximately doubled among the two groups of male mice receiving either 67 or 70 mg TCPE per kg, which contained 0.112 and 0.007 mg TCDD per kg, respectively. The difference was statistically significant compared to controls. No increase in tumor incidence was observed for any of the female mice or the male mice receiving lower doses of TCPE. However, in one of these lower TCPE dose groups, the amount of TCDD administered was 0.07 mg/kg, 10 times higher than in one of the two groups with tumors. Toth et al. (1979) and Sugar et al. (1979) concluded that the carcinogenic effect observed was due to TCPE and that TCDD did not have a tumor-enhancing effect at doses of 0.07 mg/kg or less. When TCDD was administered alone to three groups of male mice, a significant increase in liver tumors was found in the group receiving 0.7 mg/ kg, but not in the groups given 0.007 or 7.0 mg/kg (Toth et al., 1979). No excess of deaths occurred in any group. Thus, a dose-response relationship was not observed. At the two highest doses, about one-half of the animals 10-11 �showed skin lesions caused by TCDD. This was the only other toxic effect reported. The authors concluded that TCDD had a liver tumor enhancing effect in Swiss mice at a threshold dose of 0.7 rag/kg, but this does not explain the smaller incidence at 7.0 mg/kg. 10.2.3.2 Rats Nine groups of 10 male Sprague-Dawley rats were placed on a diet containing from .001 to 1,000 ppb TCDD (Van Miller et al., 1977) for 78 weeks. The purity of the TCDD used was not reported. All surviving animals were killed after an additional 17 weeks and their major organs examined histologically. At the three highest doses (50, 500, and 1000 ppb), all rats died between the second and fourth week of the experiment. The food intake for the six remaining groups was comparable to controls. Only one animal in these groups died before the 30th week. All of the animals fed I and 5 ppb TCDD died by the 90th week. By the end of the experiment, 60 percent of the control rats had died, a figure higher than that among groups fed from .001 to 0.5 ppb. The overall incidence of neoplasms in the six treated groups was 38 percent, with no neoplasms seen among control rats or rats fed the lowest TCDD dose ( . 0 ppb). All 10 rats fed the highest dose (5 ppb) 001 had neoplasms, but no other dose-effect relationship was observed among the remaining groups and no statistical analysis of the data was provided. There was a variety of neoplasms reported with no particular target organs affected. Van Miller et al. (1977) concluded that while this study did not prove that TCDD was carcinogenic, the possibility exists that TCDD enhanced the development of neoplastic changes that were induced by an unknown agent. He based this idea on the observations that few tumors were found in the liver, which is the site of TCDD localization, and that the wide variety of neoplasms found is not consistent with the findings of many other known chemical carcinogens. In another study using Sprague-Dawley rats, Kociba et al. (1978, 1979a) maintained rats on diets of 0.1, 0.01, and 0.001 mg TCDD (99 percent pure) per kg daily for two years. Analysis of the diets showed that the animals were ingesting a diet containing an average concentration of 2.193, 0.208, and 0.022 ppb, respectively. Fifty rats of each sex were used per group. At the end of the two-year feeding period, all rats were killed and tissues examined. A variety of non-neoplastic changes were found in the liver, lymphoid tissue, and respiratory organs in rats fed the highest dose of TCDD ( . mg/ 01 kg). The mortality rate was significantly increased in female rats given this dose. At 0.01 mg/kg, the body weights of females were significantly decreased, and non-neoplastic liver and lung changes were observed in both males and females. At the lowest TCDD dose, no general toxic changes were noted, but females had a higher incidence of local hepatocyte swelling. At the highest dose level (0.1 mg/kg), the incidence of hepatocellular carcinomas and squamous cell carcinomas of the lung, hard palate, nasal turbinates, and tongue was significantly increased. The incidence of a variety of 10-12 �other tumor types in treated rats was decreased compared to controls. In rats fed 0.01 mg/kg, the incidence of liver nodules was increased in females only; no other tumor-like lesions were observed in low-dose females or males given the two lowest doses. Kociba et al. (1978, I979a) concluded from these data that TCDD, at doses high enough to induce toxicity and non-neoplastic tissue changes, could alter the incidence of tumors in rats. 10.2.3.3 Studies of TCDD Cocarcinogenesis and Tumor Promotion and Initiation in Mice The general lack of organ specificity reported for TCDD-induced tumorigenesis, and the observation that TCDD has been found to increase tumor incidence in animals only at toxic doses, has led some investigators to speculate that TCDD promotes tumorigenesis, but is not necessarily by itself carcinogenic (Van Miller et al,, 1977; Rappe, 1979). A number of studies have been undertaken in two-stage animal models to investigate the tumor-promoting ability of TCDD. In these studies, a subthreshold dose of a known carcinogen is applied to the skin of an animal (initiation phase). This is followed by repetitive application of a noncarcinogenic tumor promoter (promotion phase). In the studies reviewed in this section, TCDD has been tested as a tumor initiator as well as promoter. The first such study of TCDD as a tumor initiator was reported by DiGiovanni et al. (1977). Using groups of 30 female CD-I mice, 2 ug of TCDD was applied to a shaven area of the back. One week after initiation, 5 ug of the known tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) was applied to the same area twice weekly for 32 weeks. A group of positive control animals received the known tumor initiator 7,12-dimethylbenz(a)anthracene (DMBA) followed by TPA. The incidence of papillomas and carcinomas was observed weekly. The dose of TCDD used was sufficient to kill one-third of the animals by 32 weeks. TCDD showed a weak tumor initiating ability, producing an average of 0.1 papillomas per mouse. The average number of papillomas in positive control animals was not stated; however, in an experiment using TCDD and DMBA simultaneously as initiators, it was reported that the effect was approximately additive and gave rise to an average of 2.2 papillomas per mouse. The authors concluded that TCDD is a weak tumor initiator in this system and does not greatly influence the effect of DMBA, a known tumor initiator. Berry et al. (1978) showed that TCDD did not promote papilloma formation when it was applied twice weekly following DMBA initiation. The dose used was 1 ug per mouse for each application. Positive contol mice given DMBA followed by TPA promotion had an average of 8.1 papillomas per mouse. No papillomas were observed on TCDD promoted mice. A group of mice receiving 1 ug TCDD twice weekly but without DMBA initiation also failed to develop papillomas. The authors also reported that the dose of TCDD used was the maximum tolerated dose; a higher dose could not be used because of excessive mortality. In a continuing investigation using the same system described above, Berry et al. (1979) tested the effect of TCDD on DMBA-induced tumorigenesis. The same results are also reported in DiGiovanni et al. (1979). Groups of 10-13 �30 female CDI mice were treated once with DMBA and twice weekly with the tumor promoter TPA. However, three groups were pretreated once with 1.0 ug TCDD I, 3, or 5 days before DMBA was applied. Tt was found that TCDD did not enhance, and in fact inhibited, DMRA-initiated tumorigenesis. A similar result was achieved when benzo(a)pyrene instead of DMBA was used an an initiator (DiGiovanni et al., 1979). The extent of inhibition of DMBA-induced tumorigenesis by TODD was found to increase with increasing dose and pretreatment time. j Examination of DMBA metabolic products found in TCDD-treated mice revealed that TCDD was a potent inducer of the monooxygenase system which converts DMBA to its noncarcinogenic hydroxylated products. The authors concluded that nontoxic doses of TCDD inhibited skin carcinogenesis induced by polyaromatic hydrocarbons, whereas TCDD itself failed to promote skin tumors at near-toxic doses. A similar finding in a different species was presented by Cohen et al. (1979), who reported that TCDD inhibited skin tumorigenesis of DMBA and benzo(a)pyrene in Sencar mice. A second effect of TCDD on polyaromatic hydrocarbon induced tumorigenesis was described in a report by Kouri et al. (1978), who found that a subcutaneous injection of 100 ug TCDD per kg given simultaneously with a subcutaneous injection of 3-methylcholanthrene (3-MC) markedly increased the incidence of tumors, characterized as injection-site fibrosarcomas, in DBA/2 mice. When TCDD was given intraperitoneally 2 days prior to or simultaneously with 3-MC, the carcinogenic incidence increased only slightly or remained unchanged compared to mice given 3-MC alone. Identical testing of C57BL/6 mice, using only the intraperitoneal dose route for TCDD, did not affect 3-MC-induced carcinogenesis. The subcutaneous route of TCDD application, which was tumor enhancing in DBA/2 mice, was not tested in C57BL/6. TCDD itself, at a dose of 100 ug/kg injected once intraperitoneally, did not induce tumors in either C57BL/6 or DBA/2 mice. This dose was sufficient to kill 30-70 percent of the animals within the 36-week observation period. Kouri et al. (1978) concluded from this study that TCDD, at a dose causing death in 30 to 70 percent of treated mice, did not appear to be carcinogenic in the survivors examined 36 weeks later. However, TCDD at the same dose injected subcutaneously, which was less lethal, did enhance carcinogenicity of 3-MC in DBA/2 mice and is, therefore, a cocarcinogen. They suggested that when TCDD was given together with 3-MC, metabolism of 3-MC to the ultimate carcinogen was made more efficient by TCDD induction of arylhydrocarbon hydroxylase (AHH) or other monooxygenases. The DBA/2 strain, known as an "unresponsive" strain, was shown to be relatively resistant to the carcinogenic effect of 3-MC due to poor induction of AHH by polyaromatic hydrocarbons. Thus, the TCDD was believed to aid in the induction of enzymes required to convert 3-MC to its carcinogenic metabolites. The C57BL/6 strain, on the other hand, is very responsive and sensitive to 3-MC. It was believed by the authors that TCDD induction of AHH had little effect on enhancing 3-MC metabolism since this strain of mouse inherently exhibited the maximal limit for 3-MC-induced tumorigenesis in this system. Berry et al. (1979) disagreed with this suggestion of Kouri et al. (1978), claiming that by using simultaneous administration of TCDD and 3-MC, not enough time was available for TCDD to induce AHH. In the study by Berry et al. (1979), time was a necessary factor in the successful inhibition of 10-14 �DMBA tumorigenesis by TCDD, which presumably was caused by TCDD induction of the monooxygenease system responsible for DMBA detoxification. Other possible mechanisms of cocarcinogenesis were not ruled out in the experiments of Kouri et al. (1978). It appears, then, that TCDD is capable of exerting two different effects on polyaromatic hydrocarbon induced tumorigenesis. Both effects are dependent upon the species and carcinogen tested, the time between TCDD pretreatment and application of the carcinogen, and the dose route used. It is highly unlikely that these types of effects will ever be demonstrated in humans. However, as recommended by Berry et al. (1979), more work is needed to elucidate the mechanisms of TCDD inhibition or enhancement of chemically induced carcinogenesis to permit extrapolation of these effects to humans. 10.2.3.4 Summary and Evaluation TCDD has been shown to enhance the incidence of liver tumors in one strain of mice (Toth et al., 1979), but only at doses high enough to induce skin lesions. A dose-response relationship was not observed. Two studies were published in which TCDD was tested in Sprague-Dawley rats (Van Miller et al., 1977; Kociba et al., 1978, 19793). In both, an excessive incidence of tumors was demonstrated using only toxic doses of TCDD. No specific target organs were affected and in only one of the studies (Kociba et al., 1978, 1979a) could a dose-response relationship be derived. Although the high toxicity of TCDD.has complicated investigations into TCDD-induced carcinogenesis, nontoxic doses have been used in both the mouse and rat studies and have failed to induce carcinogenesis. The observations that TCDD can induce tumors only at toxic doses and without affecting specific target organs have led to the hypothesis that TCDD may be a tumor promoter. This has been studied in a number of systems. TCDD has been found to be a very weak tumor initiator when used in combination with a noncarcinogenic tumor promoter (DiGiovanni et al., 1977). It has not been shown to be a tumor promoter when applied in combination with carcinogenic polyaromatic hydrocarbons such as DMBA or benzo(a)pyrene (Berry et al., 1978, 1979; DiGiovanni et al., 1979), and can, in some systems, inhibit tumorigenesis caused by polyaromatic hydrocarbons. It has been suggested that the latter may be the result of the induction of detoxification pathways by TCDD. TCDD has been shown to act as a cocarcinogen with 3-methylcholanthrene in one mouse system. However, this effect was specific for the species of mouse tested and the route of TCDD administration used. More studies are needed on TCDD enhancement of carcinogenicity induced by other chemicals before any conclusive statements can be made regarding humans. As of April 1981, final reports of two NCI bioassays of TCDD were in review and a decision has been made to begin a third test. 10-15 �10.2.4 Picloram 10.2.4.1 Mice Two groups of B6C3F1 hybrid mice, containing 50 of each sex per group, were fed picloram (90 percent pure) in the diet in time weighted average doses of 5,062 and 2,431 ppm for 80 weeks (NCI, 1978). Mice were observed an additional 33 weeks8, were killed, and all organs were examined for gross and histological changes. The incidence of lesions in the treated mice were compared to matched and pooled control groups. At the end of 17 weeks, one low-dose and five high-dose females had general body tremors. During the first year of feeding, the condition of the mice was reported to be comparable with controls. During the second year of feeding, clinical signs of toxicity became increasingly evident among treated mice. Slight hyperactivity, the presence of rough hair coats, and abdominal distention were noted in treated animals. There was no significant excess mortality among treated mice as compared to controls. At the termination of the study, no significant difference between treated animals and controls was apparent in the incidence of neoplastic and nonneoplastic lesions. 10.2.4.2 Rats Two groups of Osborne-Mendel rats, 50 of each sex per group, were fed time weighted average concentrations of 14,875 and 7,437 ppm picloram in the diet for 80 weeks (NCI, 1978). The rats were observed for an additional 10 weeks, after which they were killed and examined for gross tissue and histological changes. The incidence of lesions was compared to matched and pooled control groups. During the second 6 months of feeding, treated rats were observed to have a moderate incidence of diarrhea, hematuria, and rough hair coats. Clinical signs of toxicity became more apparent in the test animals during the second year of feeding and included dermatitis, tachypnea, dark urine, diarrhea, and vaginal bleeding. Upon examination of tissues, feci of cellular alteration and neoplastic nodules of the liver were seen in treated rats. The increased incidence of neoplastic nodules in the liver was significant only for females receiving the higher dose of picloram. Statistical analysis showed a significant dose-related trend among female rats towards increasing liver nodule incidence with increasing dose. These nodules were interpreted to be benign tumors by the examining pathologist. Heptatocellular carcinoma was observed in one low-dose male and one high-dose female; no hepatocellular carcinoma was observed among matched controls. This finding was reported to be statistically insignificant, A high incidence of follicular and C-cell hyperplasia and neoplasia of the thyroid gland was observed among low-dose male and high-dose female rats, but this finding was not significant. 10-16 �10.2.4.3 Summary and Evaluation In the only x<rell-controlled study found on picloram, female rats fed picloram at doses high enough to induce clinical signs of toxicity developed a significant excess of neop]astic nodules in the liver (NCI, 1978). No other carcinogenic lesions were found in treated rats or mice. The investigating pathologist concluded that picloram is not carcinogenic in these strains of mice and rats, but could induce benign liver tumors in female Osborne-Mendel rats . However, after calculating the upper and lower confidence limits of the relative risk interval, it was found that the upper limit of relative risk was greater than 1. The result of this statistical test was interpreted as indicating that the bioassay design could have failed to reveal a positive carcinogenic effect. The statistical analysis of relative risk suggests that more studies be done before any definite conclusions are made regarding the possible carcinogenicity of picloram. 10.2.5 10.2.5.1 Monuron Mice Male and female mice of two hybrid strains, C57BL/6 x CBH/Anf and C57BL/6 x AKR, were fed 215 mg commercial monuron/kg by gavage daily until the mice were 4 weeks of age (Innes, 1969; Bionetics, 1968). Thereafter, the mice were given 517 mg monuron per kg of diet until they were killed for examination at approximately 18 months of age. The authors reported that a significantly increased incidence of pulmonary adenomas occurred among the treated animals when the combined groups of treated animals were compared with untreated controls (10/66 versus 20/338). The incidence of total tumor types, hepatomas, and reticulum cell carcinomas was not significantly different between control and treated animals. However, IARC (1976) reported that tumor incidence reported in this study was significant only for lung adenomas in males of one strain (6/16 versus 9/90). It is not clear from examining the original report (Innes, 1969; Bionetics, 1968), which did not contain a statistical comparison of test and control groups by sex and strain, how the conclusion made by IARC (1976) was formulated. Rubenchik et al. (1970) fed 50 mixed-breed and 45 C57B1 mice 6 mg monuron in milk once per week for 15 weeks. The mice were held for 27 months. The number and survival rates of the control mice were not specified. The first tumor observed in the mixed-breed mice was found after 16 weeks, and in C57B1 the first tumor was discovered after four weeks. At these times, 23 (46 percent) mixed breed mice and 26 (58 percent) C57B1 mice had survived. A variety of "reactive changes" reportedly was found early in the mice, including lymphocytic infiltrates, catarrhal inflammation, proliferation of epithelium of the bronchi, and focal necrosis of the liver. It was not stated whether these changes occurred in control or treated animals or at which point during the investigation these changes were noted. A total of 13 tumors was reported in the mixed-breed mice and seven tumors occurred in the C57B1 strain. Most of the tumors consisted of benign hepatomas, hepatocellular 10-17 �carinoma, alveolar carcinoma, and kidney cancer. The survival rate of the treated mice at the end of the experiment was not reported and no statistical analysis of the data was provided. Separate groups of both sexes of two hybrid strains of mice were given single subcutaneous injections of 10 mg monuron per kg on the 28th day of life and observed until they were approximately 18 months of age (innes, 1969; Bionetics, 1968). No significant increase in tumors was noted. T.ARC (1976) commented that a single injection may not be an adequate basis for discounting monuron-induced carcinogenesis. 10.2.5.2 Rats Hodge et al. (1958) maintained groups of 30 male and 30 female Rochester albino rats on diets containing 0.0025, 0.025, and 0.25 percent monuron for 2 years. Because of a respiratory infection, 70-90 percent of all groups, including controls, died. Of the remaining animals, gross and microscopic examination of all major tissues reveaed no carcinogenic lesions. IARC (1976) noted that a lack of detail in reporting data made this study difficult to evaluate. Fifty random-bred male rats received 450 mg monuron per kg daily in their diet for 18 months and were observed for an additional 9 months (Rubenchick. et al., 1980). The first tumor was discovered after 18 weeks, at which time 32 rats were still alive. The number of rats surviving for the 27-month experimental and observation period was not stated. Fifteen rats were found to have tumors with no specific target organ excessively affected. No tumors were found in any of the 30 control rats, which IARC (1976) found unusual. 10.2.5.3 Summary and Evaluation Two studies in which mice of different strains were fed monuron were able to demonstrate the induction of tumors, with the lungs and liver being the major target organs (Tnnes, 1969; Rubenchick et al., 1970). One of these studies (Rubenchick et al., 1970) also showed that mixed breed rats fed monuron developed tumors, but no specific target organ was apparent. The report by Innes, 1969 (details of which are provided in Bionetics, 1968) used a small number of animals in test groups. Innes (1969) recommended that monuron be subjected to further study. Serious reporting deficiencies are evident in the publication by Rubenchick (1970), especially with regard to data on control animals, which make evaluation of the study difficult. Based on the information presented for monuron and in the absence of any human data, IARC (1976) concluded that the data suggest that monuron is carcinogenic. In light of the deficiencies of the data presented here, final conclusions should await publication of a chronic feeding study in mice and rats that is currently underway by the National Cancer Institute. 10-18 �10.2.6 Cacodylic Acid Two strains of mice, C57BL/6 x C^H/Anf and C57BL/6 x AKR, 18 of each sex, were fed 464 mg cacodylic acid/kg by gavage daily until the mice were 4 weeks of age (innes, 1969; Bionetics, 1.9681. Thereafter, mice were fed 121 mg cacodylic acid per kg of diet until they were killed for examination at approximately 18 months of age. In a parallel experiment, 18 of each sex of the same two hybrid strains were given 464 mg cacodylic acid per kg by subcutaneous injection on the 28th day of life and observed until they were approximately 18 months of age. No significant increase in tumor incidence, as compared to 338 untreated mice, was reported from use of either dose route. The number of animals used in this test was relatively small, so it is difficult to draw any firm conclusions regarding the lack of carcinogenic potential of cacodylic acid. 10.3 SUMMARY OF CARCINOGENIC POTENTIAL OF HERBICIDES There is no evidence available to suggest the presence of a potent carcinogenic risk to humans exposed to 2,4-D, 2,4,5-T, and TCDD. Some studies have indicated that a causal relationship may exist between exposure to .phenoxyacetic acids and the development of soft-tissue sarcomas. This has come primarily from case-control studies of Swedish forestry and agricultural workers, and cohort studies of workers exposed to trichlorophenol, 2,4,5-T, and TCDD during herbicide production. However, confounding factors exist which make it impossible to implicate 2,4-D, 2,4,5-T, or TCDD specifically. In the case of the Swedish studies, a relatively high proportion of patients with soft-tissue sarcomas were found to have been exposed to phenoxyy acids in forestry and agricultural work. In addition to exposure to 2,4-D and 2,4,5-T, these patients had known exposure to other phenoxy acids and chemical agents. Studies of production workers, which included employees exposed at high levels following accidents as well as employees exposed during normal production operations, are not conclusive because all workers were known to be exposed to trichlorophenol, the precursor of 2,4,5-T. In these cohort studies, a total of only three deaths from soft-tissue sarcoma has been reported, two of which did not involve exposure to 2,4,5-T. Studies of these cohorts is continuing, and the National Institute of Occupational Safety and Health is currently developing a registry of trichlorophenol and 2,4,5-T production workers so additional cohorts may be available in the future. However, from the viewpoint of 2,4,5-T and TCDD carcinogenicity in humans, any future study of production workers will always have the confounding variable of trichlorophenol exposure. Additional study populations, relatively free of trichlorophenol exposure, should be identified for investigation. It is generally suspected that TCDD, a contaminant of 2,4,5-T and trichlorophenol, may have been a contributing factor in the development of the soft-tissue sarcomas. This idea is supported by animal studies, one in which male mice fed combinations of trichlorophenoxyethanol and TCDD showed a significantly higher incidence of liver tumors than control mice. However, tumor incidence was related to the dosage of trichlorophenoxyethanol, and not TCDD. This study and others in which mice and rats have been fed TCDD have shown that TCDD can increase tumor incidence but only at doses sufficient to 10-19 �induce other toxic effects. This observation, along with the wide variety of tumor types found in animal studies, led to the hypothesis that TCDD may be a tumor promoter rather than a chemical carcinogen; that is, a second bio-, chemical event would be required before TCDD could promote carcinogenesis. This hypothesis has been tested in two-stage animal models. TCDD has been shown to be a weak tumor initiator in mice exposed to TCDD and a noncarcinogenic polyaroraatic hydrocarbon. TCDD has not been shown to be a tumor promoter in these systems and, in fact, can inhibit tumorigenesis by polyaromatic hydrocarbons. Evidence has suggested that the mechanism for inhibition involves the induction by TCDD of metabolic pathways that detoxify polyaromatic hydrocarbons. In one instance, TCDD was shown to enhance the tumorigenicity of 3-methylcholanthrene, but this effect was reported to be specific for only one mouse strain and dose route. 2,4-D and 2,4,5-T, when tested in animal bioassays, have consistently failed to induce tumors in mice and rats. In one instance, one strain of mouse fed 2,4,5-T showed a high tumor incidence but no specific target organ was affected. Three other strains similarly tested were not susceptible, indicating that a species-specific effect may have been involved. The small number of animals used in these bioassays prohibits making a definitive conclusion that 2,4-D and 2,4,5-T are not carcinogenic under any circumstances, but it does not appear that these compounds are potent animal carcinogens. A similar conclusion may be made with regards to picloram and cacodylic acid, but only one study for each was available. Two studies have suggested that monuron is carcinogenic in mice and rats; however, one study was of limited design and the other had serious reporting deficiencies. Monuron is currently being tested by NCI, and additional studies on picloram and cacodylic acid are needed. 10-20 �CHAPTER 10. REFERENCES Arkhipov, G. N., and Kozlova, I. N. (1974) Research on the carcinogenic properties of the herbicide amino salt 2,4-D. Vop. Pitan. 5:83-84. Axelson, 0., and Sundell, L. (1974) Herbicide exposure, mortality and tumor incidence: An epidemiological investigation on Swedish railroad workers. Work Environ. Health ll(l):21-28. Axelson, 0., and Sundell, L. 74(35):2887-2888. (1977) Phenoxy acids and cancer. Lakartidningen Axelson, 0., Sundell, L., Anderson, K., Edling, C., Hogstedt, C., and Kling, H. Herbicide exposure and tumor mortality: An updated epidemiological investigation on Swedish railroad workers. Unpublished paper. 10 pp. Cohen, G. M., Bracken, W. M., Lyer, R. P., Berry, D. L., Selkirk, J. K., and Slaga, T. J. (1979) Anticarcinogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dixoin on benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene tumor initiation and its relationship to DNA binding. Cane. Res. 39:4027-4033. Cook, R. R., Townsend, J. C., Ott, M. G. (1980) Mortality experience of employees exposed to tetrachlorodibenzo-p-dioxin (TCDD). J. Occup. Med. 22:47-50. Di Giovanni, J., Juchau, M. R., Berry, D. L., and Slaga, T. J. (1979) 2,3,7,8-tetrachlorodibenzo-p-dioxin: potent anticarcinogenic activity in CD-I mice. Biochem. Biophys. Res. Commun. 86:577-584. DiGiovanni, J., Viaje, A., Berry, D. L., Slaga, T. J., and Juchau, M. R. (1977) Tumor initiating ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 in a two-stage system of mouse skin carcinogenesis. Bull. Environ. Contain. Toxicol. 18(5):552-556. Davring, L., and Hultgren, K. (1977) Cytogenetic effects on invivo bonemarrow cells of Mus musculus induced by a commercial 2,4,-T ester product. Hereditas 85:123-134. 10-21 �DiGiovanni, J. , Viaje, A., Berry, D. L. , Slaga, T. J. , and Juchau, M. R. (1977) Tumor initiating ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254 in a two-stage system of mouse skin carcinogenesis. Bull. Environ. Contain. Toxicol. 18(5):552-556. Eriksson, M., Hardell, L., Berg, N. 0., Holler, T., and Axelson, 0. (1979) Case-control study on malignant mesenchymal tumors of the soft tissue and exposure to chemical substances. Lakartidningen 76:3872-3875. Hansen, W. H., Quaife, M. L., Habermann, R. T., and Fitzhugh, 0. G. (1971) Chronic toxicity of 2,4-dichlorophenoxyacetic acid in rats and dogs. Toxic. Appl. Pharmacol. 20:122-129. Hardell, L. (1979) Malignant lymphoma of histiocytic type and exposure to phenoxyacetic acids or chlorophenols. Lancet 1(8106):55-56. Hardell, L. (1977) Malignant mesenclrymal tumours and exposure to phenoxy acids - a clinical observation. Lakartidningen 74:2753. Hardell, L. , Eriksson, M., and Lenner, P. (1980) Malignant lymphoma and exposure to chemical substances, especially organic solvents, chlorophenols and phenoxy acids. Lakartidningen 77(4):208-210. Hardell, L., and Sandstrom, A. (1979) Case-control study: Soft-tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols. Br. J^ Cancer 39:711-717. Hardell, L., and Sandstrom, A. (1978) Malignant meseuchyraal soft-tissue tumors and exposure to phenoxy acids or chlorophenols. Lakartidningen. 75:3535-3536. Hodge, H. C., Maynard, E. A., Downs, W. L., and Coye, R. D. (1958) Chronic toxicity of 3-(p-chlorophenyl)-l,1-dimethylurea (Monuron). Am. Arch. Ind. Health 17:45-47. Honchar, P. A., and Halperin, W. E. (1981) 2,4,5-T, trichlorophenol, and soft tissue sarcoma. Lancet Jan. 31:268-269. Innes, J. R. M., Ulland, B. M., Valerio, M. G., Petrucelli, L., Fishbein, L., et al. (1969) Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: a preliminary note. J. Natl. Cancer Inst. 10-22 �International Agency for Research on Cancer. (1977) IARC monographs on the evaluation of the carcinogenic risk of chemicals to mart: Some fumigants, the herbicides 2,4-D and 2,4,5-T, chlorinated dibenzodioxins and miscellaneous industrial chemicals. 15:41-299. International Agency for Research on Cancer. (1976) IARC monographs on the evaluation of carcinogenic risk of chemicals to man: Some carbamates, thiocarbamates and carbazides. 12:167-176. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Gehring, P. J. (1979a) Long-term toxicologic studies of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) in laboratory animals. Ann. N.Y. Acad. Sci. 320:397-404. Kociba, R. J., Keyes, D.G., Lisowe, R. W., Kalnins, R. P., Dittenber, D. D., Wade, C. E., Gorzinski, S. J., Mahle, N. H., Schwetz, B. A. (1979b) Results of a two-year chronic toxicity and oncogenic study of rats ingesting diets containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Fd. Cosmet. Toxicol. 17(3) :205-221, 1979. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., et. al. (1978) Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46:279-303. Kouri, R. E., Rude, T. H., Joglekar, R., Dansette, P. M., Jerina, D. M., Atlas, S. A., Owens, I. S., and Nebert, D. W. (1978) 2,3,7,8-Tetrachlorodibenzo-p-dioxin as cocarcinogen causing 3-methylcholanthreneinitiated subcutaneous tumors in mice genetically "nonresponsive" at Ah locus. Can. Res. 38(9):2777-2783. Muranyi-Kovacs, I., Rudali, G., and Imbert, J. (1976) Bioassay of 2,4,5-trichlorophenoxyacetic acid for carcinogenicity in mice. Cancer 33(6):626-633. Br. J. Ott, M. G., Holder, B. B., and Olson, R. D. (1980) A mortality analysis of employees engaged in the manufacture of 2,4,5-trichlorophenoxyacetic acid. J. Occup. Med. 22(l):47-49. Rappe, C. (1979) Dioxiner och dibensofuraner tva substansgrupper i blickpunkten. Lakartidningen. 76:(1-2):21-24. Rubenchik, B. L., Botsman, N. E., and Groban, G. P. (1970) The carcinogenic effect of the herbicide monuron. Vopr. Onkol. 16(10):51-53. 10-23 �Sugar, J., Toth, K., Csuka, 0., Gati, E., and Somfai-Relle, S. (1979) Role of pesticides in hepato-carcinogenesis. J. Toxicol. Environ. Health 5(2/3):183-191. Sundell, L., Rehn, M., Axelson, 0. herbicides. (1973) An epidetniological study concerning Thiess, A. M., and Frentzel-Beyme, R. (1977) Mortality study of persons exposed to dioxin following an accident which occurred in the BASF on 13 November 1953. Presented at the Fifth International Conference of Medichem - Occupational Health in the Chemical Industry, 9 pp. Toth, K., Somfai-Relle, S., Sugar, J., and Bence, J. (1979) Carcinogenicity testing of herbicide 2,4,5-trichlorophenoxyethanol containing dioxin and of pure dioxin in Swiss mice. Nature 278(5704):538-549. Toth, K., Sugar, J., Somfai-Relle, S., and Bence, J. (1978) Carcinogenic bioassay of the herbicide 2,4,5-trichlorophenoxyethanol (TCPE) with different 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) content in Swiss mice. Progress in Biochemical Pharmacology 14:82-93. Tung, T. T. (1973) Primary cancer of the liver in Viet Nam. Chirurgie 99(7):427-436. U.S. Department of Health, Education, and Welfare. (1978) Bioassay of picloram for possible carcinogenicity. National Cancer Institute Carcinogenesis Report Series No. 23. 91 pp. Van Miller, J. P., Lalich, J. J., and Allen, J. R. (1977) Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8-tetrachlorodibenzop-dioxin. Chemosphere (9):537-544. 10-24 �APPENDIX ONGOING EPIDEMIOLOGIC RESEARCH In this appendix, summary information on five epidemiologic investigations of Vietnam exposure to Herbicide Orange are presented. These five studies are planned to begin shortly or are currently underway, and include: • Ranch Hand II: U.S. Air Force • Birth Defects and Military Service in Vietnam: Control, USPHS • Epidemiologic Studies of Agent Orange: • Proportional Mortality of Vietnam Veterans, Other Veterans and Matched Controls in New York State, Exclusive of New York City: State of Center for Disease Veterans Administration New York • Epidemiological Study of Soft-Tissue Sarcoma: State of New York. It is likely that additional epidemiologic research on Vietnam veterans will be initiated at the Federal and State levels. This appendix, therefore, should be considered only an indication of the kinds of studies already planned or underway, rather than an exhaustive listing of all epidemiologic research on Vietnam veterans currently being considered. In the following sections, each of these five studies is addressed. A.I RANCH HAND II: U.S. Air Force RANCH HAND II was developed by the U.S. Air Force to determine whether long-term health effects exist and can be attributed to occupational exposure to Herbicide Orange. Project RANCH HAND II uses a nonconcurrent prospective design entailing mortality, morbidity, and followup studies. The study population to be investigated are Air Force personnel involved in the RANCH HAND Organization, the Air Force unit that flew C-123 spray missions during the Vietnam Conflict. Data will be obtained from Air Force personnel records. The exposed cohort consists of approximately 1,200 individuals. The control population of 12,000 will be selected from a control universe of 25,000 crew members and support personnel from other units assigned to duty in Southeast Asia. Ten statistically equivalent control individuals will be matched to RANCH HAND personnel for the variables of age, type of job, and race. The mortality analysis will utilize a one-to-five ratio design of one RANCH HAND subject to fifty percent of the randomly selected controls. The study population will be followed yearly over a 20-year period. A-i �The morbidity design will utilize a one-to-one ratiOj with the first of the randomized mortality controls entered into the questionnaire and physical examination phase of the study. Questionnaire data will be used to reconstruct occupational, social, and medical histories to quantitate morbidity endpoints and confounding factors. Comprehensive physical examinations will be performed, emphasizing dermatologic, neuropsychiatric, hepatic, immunologic, reproductive, and neoplasic conditions. Questionnaires will be administered using personal and telephone interviews. Blind assessment protocols will be used to avoid bias and limit data variability. Physical examinations and questionnaires will be developed for followup in years 3, 5, 10, 15, and 20 of the study. Inferences about disease state will be developed by identifying symptom complexes on physical findings. Comparison of these symptoms between groups will be utilized to calculate relative risks from baseline data which, if appropriate, will be used in the followup analyses. Statistical methodologies to be used include combinational and correlational analyses to provide statements of probability of disease state, subclinical state, and to correct for over-reporting. Regression techniques will be applied to a normalized exposure index among exposed individuals exhibiting symptoms and/or signs to clarify disease state or syndrome. Mortality data will be analyzed using several different approaches, including age, age-disease specific rates, standardized mortality rates, and modified life table approaches, as well as logistic and multiplicative models. Questionnair'es and physical examinations will be analyzed using log-linear models for dichotomous or polytomous data to verify the appropriateness of the standard statistical methodologies. Continuous variables will undergo covariance analysis to remove non-controlled effects, followed by the use of a paired difference statistic. Group scoring techniques will be used as appropriate. A.2 BIRTH DEFECTS AND MILITARY SERVICE IN VIETNAM: Control, USPHS Center for Disease The Birth Defects Branch of the Center for Disease Control (CDC) is presently conducting a study of Birth Defects and Military Service in Vietnam. The study uses a case-control design. The case population is composed of approximately 7,000 to 8,000 selected babies born with one or more serious birth defects during the years 1968-1980 registered in CDC's Metropolitan Atlanta Congenital Defects Surveillance Program (MSCDSP). The control population will be selected from live births in the metropolitan Atlanta area during the same time-period, between 1968-1980. Criteria for case-matching include race, month and year of birth, and hospital of birth. The ratio of cases to control is two to one. The sample size of 3,000 randomly selected control families was selected on the basis that it would give fairly good sensitivity to a small increase in risk for all malformations identified. Case and control population candidates �identified are traced, contacted, and secured for participation. Participants are interviewed using telephone and mail questionnaires. Family members of case and control groups interviewed are the mother, father, and grandmothers of the infant. Factors identified as possible sources of bias include: sociodemographic characteristics, age at pregnancy, familial history of birth defects, major organic diseases in relatives, and other environmental exposures. Analysis is being performed upon four major groupings: • All defects combined • Individual category of defects • Groups of babies affected with similar patterns of multiple defects • All babies born with defects which may have been caused by a fresh dominant mutation. The analysis proposed is a combination of the search for confounding and using the Mantel-Haenszel approach multidimensional contingency table analysis based upon the log-linear model. For all defects combined and some of the larger specific categories, the log-linear approach will be used. For smaller defect categories only the Mantel-Haenszel approach will be used, although the log-linear analysis done for all defects combined may provide some guidance as to possible higher order interactions. For very small categories of defects, not much searching for confounding will be done. A.3 EPIDEMIOLOGIC STUDIES OF AGENT ORANGE: Veterans Administration The draft protocol for "Epidemiologic Studies of Agent Orange" was developed for the Veterans Administration by the Division of Epidemiology, School of Public Health, University of California at Los Angeles. The purpose of the proposed studies is to determine whether exposure of ground troops to Herbicide Orange resulted in health effects. The draft protocol uses a historical cohort study design, with cohort groups defined using Army and possibly Marine Corps records for the period 1965 through 1971, the period of heaviest herbicide application. Casualties, both immediate and delayed, will be excluded from the cohort. The study will be also limited to draftees and one-term enlisted men. Estimation of exposure for cohorts will be defined on two or more levels to observe dose-response effects. Estimated company exposure levels will be constructed using the HERBS tape and hand abstracted Army Records. These data will be used to generate time-piace-company exposure grids by computer using mapping data and algorithms. AFT �Companies which represent the maximum range of exposure to Herbicide Orange from high to low will be selected. All individuals serving in identified companies wil be initially included in the cohort. These troops will then be checked through personnel records in St. Louis for other Vietnam service time, discharge status, and most recent whereabouts. All members of the cohort will then be traced to determine their vital status. All living cohort participants will be examined using standardized protocol procedures including questionnaire, laboratory testing, and physical examination. The sample size of the study groups have not been given at this time. The sample size estimates to be developed will be based upon the expected disease frequencies, the expected variances of the measurements and the population size available to the study in each exposure group. The details of the analysis will not be specified until the details of the data to be collected are known; however, it is expected that the analysis will begin with simple descriptive statistics and later proceed to testing specific hypotheses. Preliminary to the historical cohort study, UCLA proposed three mortality studies. The first is a proportionate mortality study to attempt to determine if there are unusual causes of death or patterns of death among Vietnam veterans or a specific subgroup of Vietnam veterans. The second study is designated to estimate actual death rates of Vietnam veterans and Vietnam-era veterans who did not serve in Vietnam. The third study is a case-control study utilizing death as an outcome measure for case selection to examine subgroups of Vietnam veterans for evidence of higher risk of death. UCLA also proposed morbidity studies to address the question of whether there is an unusual morbidity experience among Vietnam veterans as compared to nonveterans or among subgroups of Vietnam veterans. The first study proposes using information from the Agent Orange registry to determine the frequency distribution of complaints. The second study proposes to examine veterans claims files for two time periods and to compare the morbidity experience of Vietnam and non-Vietnam veterans, Vietnam veterans within combat and noncombat units, and within areas exposed and unexposed to Herbicide Orange. To determine possible differences in patterns of claims following Vietnam to a similar group of males, UCLA proposes to sample claims from Korean War veterans for comparison with the Vietnam veterans. A.4 PROPORTIONAL MORTALITY OF VIETNAM VETERANS, OTHER VETERANS, AND MATCHED CONTROLS IN NEW YORK STATE, EXCLUSIVE OF NEW YORK CITY: State of New York The protocol, "Proportional Mortality of Vietnam Veterans, Other Veterans, and Matched Controls in New York State, Exclusive of New York City," was developed by the New York State Department of Health, Division of Epidemiology and Office of Biostatistics. The purpose of the study is to determine �whether post-war mortality among Vietnam veterans differs from other veterans or other Upstate New York men, and to identify which disease categories merit further study. The mortalities of Upstate New York men 18 to 29 years old at any time during the period 1962 through 1971 will be sampled and followed through 1979. The deceased men of this cohort will be identified through New York State death certificate files. Veterans status will be obtained from information on death certificates and from the Veterans Administration. The proportional mortality rate (PMR) is the ratio of deaths from a specific cause to all deaths reported in that population. The adjusted proportional mortality rates will be calculated for Vietnam veterans, nonVietnam veterans and nonveterans. Comparison will be of veterans serving in Vietnam to other veterans matched on age, race, educational level, and marital status, or adjusted for these factors. This matching is necessary in that these factors may affect the selection of individuals for military service and, in addition, who is at risk for the various causes of death under study. The expected number of deaths for each cause (age adjusted by the indirect method) and the significance of its deviation from the observed number will be determined by an adaptation of the Mantel-Haenszel procedure using age-specific contingency tables and computation of a continuitycorrected chi-square. In addition to the PMR analysis, standard mortality ratios may be calculated for Vietnam era veterans, if the denominator population of veterans of New York State, exclusive of New York City, can be approximated using census data. A. 5 EPIDEMIC-LOGICAL STUDY OF SOFT-TISSJE SARCOMA: State of New York The State of New York, Department of Health, Office of Public Health, Division of Epidemiology is also conducting an "Epidemiological Study of SoftTissue Sarcoma." The study uses a case-control design. The case group is composed of male residents of New York State, exclusive of New York City, who were in the age group 18 to 29 years during the period 1962-1971, reported to the N.Y. State Cancer Registry as having soft-tissue sarcoma first diagnosed through December 31, 1980. Each case will have two overlapping controls, with each group having a control-to-case ratio of one-to-one. The first control group will be made up of live males selected from drivers license files matched on 5-year age group and zip code. Alternate controls will be selected and stratified for race during analysis. Case group members will be matched against the license files to determine their representativeness on the basis of having a drivers license. �The second control group will include nonliving males deceased for all cases except cancer. For each dead case, two death certificates will be selected for men of the same five-year age group, years of education, race, and health systems area (a ten-county area). In addition to the negative control, a positive control will be attempted to be obtained. Positive controls will be aged-matched individuals who served in Vietnam. Pathology reports and slides will be reviewed using a standard classification system. Reviewers will be blinded as to military history. Distribution of pathology data will compare Vietnam veterans to non-Vietnam veterans and nonveterans. Data on cases and controls will be collected using primarily telephone survey techniques with standardized questionnaires. Personal interviews will also be conducted. Data collected will include smoking history, alcoholism, occupation, and other pertinent exposure factors. Data collected will be checked against records of the Veterans Administration. Data will be analyzed using multivariate statistical techniques, such as the linear logistic model for matched analysis. Special attention will be made to correct for: systematic bias due to nonresponse; preferential recall by case and control subjects; systematic differences of access to medical care between cases and controls; eligibility variance for military service; and overall confounding. The Part II study will analyze data on occupation and industry reported on death certificates. Subjects will include all males of New York State, with the exception of New York City residents, listed on death certificates as dying of soft-tissue sarcomas during the period January 1, 1970 to December 31, 1979. Controls will be selected from death certificate files, matched on dates of birth, years of education, and health systems area. Occupation and industry data from the death certificate will be analyzed to determine if specific occupations or industries are represented in the soft-tissure sarcoma group. Findings of Part II will be used to generate hypotheses for further study. A-O � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5777 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Description An account of the resource <strong>Corporate Author: </strong>JRB Associates, McLean, Virginia Date A point or period of time associated with an event in the lifecycle of the resource 1981-10-01 Title A name given to the resource Review of Literature on Herbicides, Including Phenoxy Herbicides and Associated Dioxins, Volume I: Analysis of Literature ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/405cce8f9a2f562af5193bc437aed08b.pdf 9936f414054ee7b8935a7ff35c636166 PDF Text Text Item D Number °5776 D ^t Scanned Author Corporate Author RBPOrt/ArtlGlB Title Briefing Notes: Briefing to Dr. Custis/Acting General Counsel, 11 Sept 1981 Journal/Book Title Yeer Month/Day Color D Number of Images ° Descripton Notes Thursday, March 28, 2002 Page 5776 of 5780 �BRIEFING NOTES 11 Sept 1981 Briefing to Dr. Custis/Acting General Counsel I. Agent Orange Events That Will Impact Upon VA University of Nebraska Fat Biopsy Report J.R.B. Literature Analysis DOD Information on RH Abort Missions A. Fat Biopsy Report TQDD levels from 20-186 ppt found in adipose tissue from three Vietnam veterans "heavily exposed" to Herbicide Orange. Other tissue samples from "non-exposed" Vietnam veterans and controls <[20 ppt. Significance Dr. Gross et al have probably distributed report. May expect Vietnam veterans and press to respond in very near future. VA Action Options 1. Take no action and write article for New England Journal of Medicine relating Dr. Gross's values to VA's description of exposure and supporting medical data. 2. Call Dr. Gross and explain "Scientific Method" or lack of in selecting individuals, determining exposure index, and no knowledge on "jobs" since Vietnam. Offer to incorporate medical data into "one," more complete manuscript. 3. Submit manuscript to Agent Orange Working Group (AOWG) Science Panel for evaluation and go public with evaluation. Submit medical data at same time. Following evalution, submit manuscript for publication. Advise Dr. Gross of action. May want to submit manuscript to Panel of VA Scientists. AOWG Science Panel meets 15 September 1981. B. JRB LITERATURE ANALYSIS JRB has been given extension of 15 days (to 30 September) to complete manuscript. Extension given to incorporate primary references, fat biopsy information (previously published) and review additional articles on cacodylic acid. �BRIEFING NOTES 11 Sept 1981 Briefing to Dr. Custis/Acting General Counsel (Continued) Significance Time delay may provide signal to vet groups on potential problems. However, VA interested only in a "First-Pate Report" from JRB. VA has not inputed to scientific assessment; rather has been instrumental in obtaining references unavailable to JRB. Preliminary review conducted by Major Young (unofficial and at JRB's request—to check completeness of 1iterature searches); his assessment is that the Literature Report will have significant impact. Report may "alarm" many groups because of volume of scientific data validating extreme toxicity and cellular persistance of TCDD. VA Response Options 1. Upon receipt of report, submit to appropriate Congressional committees and convene VA Advisory Group to receive thorough briefing on report. Prepare press releases. 2. Upon receipt, submit report to AOW3 Science Panel, VA Advisory Committee Membership (via mail) and to appropriate Congressional committees with letter explaining action. Input literature and VA Environmental Physicians attendance at Dioxin Symposiums to show "Continuing Education Process." C. POD Information on Ranch Hand Abort Missions On 10 September 1981, Major Young attended DOD/HHS meeting on new records uncovered by Army record search indicating that Ranch Hand aircraft aborted 92 times during period 1965-1971. During aborts entire contents (21,000 gallons) was jettisoned within 20-40 second. Many of these aborts occurred during take-offs from Ranch Hand bases (Bien H0a and Da Nang) and may have resulted in exposure of thousands of military personnel. HHS/DOD have brought in Dr. Pat Honchar (NIOSH) to review preliminary information and to make report to AOWG Science Panel on 15 Septemer. Significance Report to AOWG will mean report will go public. News media and vet groups will make "hay" since it may mean more veteans exposed then previously thought. Coupled with Dr. Gross biopsy report on TCDD in Vietnam veterans' fat, it may elicit response from Congress. Information will also complicate conduct of VA EPI Study on ground troops. May force EPI study of "Vietnam Experience." —3— �BRIEFING NOTES 11 Sept 1981 Briefing to Dr. Gustis/Acting General Counsel (Continued) RECOVIMENmTION Administrator and VA staff must be fully aware of situation and be prepared to make statements. Advise Administrator to indicate VA following BCD record search carefully and providing assistance as requested (note: Major Young and CHECO data). Also VA working on and with AOW3 Science Panel. � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5776 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Briefing Notes: Briefing to Dr. Custis/Acting General Counsel, 11 Sept 1981 ao_seriesVIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5775 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Letter from Juan A. del Real to Martin Anderson, April 24, 1981 ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/6ea80c1b7bc1340ea788546984841053.pdf fc6ae1d81d7df46e872fe2cad7a946e6 PDF Text Text Item D Number °5774 n Not Scanned Author Corporate Author ROpOrt/ArtlClB TltlB Correspondence Regarding Assignment of Alvin L. Young to the Veterans Administration for a Two-Year Term, February 26, 1981 Journal/Book Title Year Month/Day Color Number of Images u ° Descriptor] Notes Thursday, March 28, 2002 Page 5774 of 5780 �Veterans Administration Memorandum TO: Date: February 26, 1981 Chief Medical Director (10 Assignment of Alvin L. Young, Major, USAF, to Office of Environmental Medicine 1. The purpose of this memorandum is to solicit your approval and support of a request that the Air Force assign Major Alvin L. Young to the Office of Environmental Medicine for a period of approximately two years following completion of his current assignment in July of this year. Major Young has frequently voiced his personal desire to work with the Veterans Administration on the Agent Orange program. The recent reversal of the plan to assign Major Young to the U.S. Air Force Academy has made his detail to this office a very real possibility which in my opinion would be of great benefit to the VA. 2. Major Young holds a PhD in Herbicide Physiology, and has been intimately associated with the Agent Orange program for the past twelve years. He is a recognized expert on dioxins and the phenoxy herbicides and has published two books on the subject. Of particular interest is his involvement in the development of the protocol for the Air Force Ranch Hand Study. 3. It is my belief that Major Young's assignment to this office would materially enhance the performance of our mission in the following areas: a. Provide additional expert guidance in the conduct of the literature analysis mandated by P.L. 96-151. b. Help with managing the contract for the design of the epidemiological study mandated by P.L. 96-151. c. Aid in maintaining close liaison with the Air Force's conduct of the Ranch Hand Study. d. Provide an additional ib-house source of expertise in a number of areas related to the whole agent orange issue. VA FORM 2105 MAR I960 �2. Chief Medical Director (10) 4. Major Young has stated to me his strong conviction that an assignment to the Office of Environmental Medicine would give him great personal and professional satisfaction and that it would have considerable potential for the enhancement of his military career. In addition, he has told me that he has received informal assurances of support from his present commanding officer as well as the Office of the Air Force Surgeon General. 5. In the event that he is assigned to usr it would be most beneficial to Major Young to be assigned for administrative purposes to the Air Force Surgeon General. This would provide eligibility for housing and other benefits at Boiling Air Force Base. 6. Attached is a proposed letter from you to General Myers and a second letter from the Administrator to the Secretary of Defense soliciting their support for this initiative. Major Young's orders to the Air Force Academy have recently been cancelled and since he is a line officer he is potentially available for a wide variety of assignments. Therfore, provided you agree with my proposal, it would be desirable to act expeditiously in this matter. BARCLAY M. SHEPJ^RD, M.D. Special Assistant to the Chief Medical Director for Environmental Medicine (102) Attachments �Department of Medicine and Surgery Washington, D.C.J 20420 Veterans Administration I.t. Gen. Paul W. Myers, USAF, MC Surgeon General United States Air Force Boiling AFB, D.C. 20332 Dear Paul: This will confirm my telephone conversation with General Chesney and our mutual agreement supporting the request to have Major Alvin Young assigned to our Office of Environmental Medicine for a period of approximately two years to help with our Agent Orange program. In addition we have agreed that the Veterans Administration will reimburse the Air Force for his salary. Dr. Barclay M. Shepard who coordinates our Agent Orange activities assures me that Major Young, whose assignment to the Air Force Academy has been deferred, would very much like a 2-year "hitch" with us, since it would keep him in his area of greatest interest and expertise. We in turn would be very pleased to have him join us. Major Young has been very helpful to us on a number of projects in the past, including his work on our recently released film on Agent Orange and his participation in several VA meetings and conferences. For these efforts we are most grateful to him as well as the Air Force for permitting him to take part in these activities. I have asked the Administrator to forward a similar request to the Secretary of Defense, enlisting his support for this assignment. Since you concur, it would be beneficial to Major Young if he could be attached to your office for administrative purposes, so that he might be eligible for base housing at Boiling. Thank you very much for your help in this matter. Sincerely, DONALD L. CUSTIS, M.D. Chief Medical Director In Reply Refer To: 102 �Office of the Administrator of Veterans Affairs Washington, D.C. 20420 Veterans Administration Honorable Caspar W. Weinberger Secretary of Defense Pentagon - Room 3E880 3E88 Washington, D.C. 20301 %i^Tk^t^l \ ftst* / **•- 4DMlt -^ * " Dear Mr. Secretary: The Agent Orange issue remains a major concern to many Vietnam veterans and therefore, to this agency. The Veterans Administration is proceeding to carry out the mandate of P.L. 96-151 which requires us to conduct a long-range epidemiological study of veterans exposed to phenoxy herbicides and a review and analysis of the world's literature on these chemical agents. The successful completion of these efforts will require the specialized knowledge and skills of those individuals most familiar with the many aspects of this complex issue. It has been brought to my attention that one of the most widely recognized experts in the use of herbicides by the Department of Defense is Major Alvin L. Young, USAF. Major Young, who holds a Ph.D. in Herbicide Physiology, has been intimately associated with the Agent Orange program for the past twelve years and has been the principal author of two major publications on the subject. In view of these considerations and the fact that he will soon complete his present assignment at Maxwell Air Force Base, I would very much appreciate your support in requesting that Major Young be assigned for a period of approximately two years to the VA's Office of Environmental Medicine which serves as this agency's focal point for coordination of Agent Orange activities. During the past two years Major Young has worked closely with several members of our staff on a number of Agent Orange related projects and for this we are most grateful to him and to the Air Force. He is familiar with our organizational structure and interagency activities, and he has developed many contacts with the scientific community both inside and outside the federal government. �2. Secretary of Defense Major Young's assignment to the Veterans Administration would, I believe, prove to be mutually beneficial in that he could serve as an additional point of contact in this agency for Air Force personnel involved in the conduct of the Ranch Hand Study with which he has had considerable personal experience. The VA views this study as probably the most significant research effort currently underway in the search for answers to many of the questions related to the possible health effects of exposure to Agent Orange. I am most grateful for all the help which the Air Force and other elements of the Department of Defense have provided to this agency and I look forward to our continued cooperation in our efforts to arrive at a more complete understanding of the many facets of the Agent Orange issue. Sincerely, RUFUS H. WILSON Acting Administrator cc: J. H. Moxley, III, M.D. Assistant Secretary of Defense for Health Affairs Berne Orr Secretary of the Air Force Lt. Gen. Paul W. Myers Surgeon General, USAF � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5774 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Correspondence Regarding Assignment of Alvin L. Young to the Veterans Administration for a Two-Year Term, February 26, 1981 ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/742fb1feee1a8c20cef0b6e448ebec28.pdf 3884496b05eb23656880f4b3169be2f3 PDF Text Text item D Number °5773 Author Young, Alvin L. D Corporate Author Report/Article Title Handwritten Notes on VA Meeting, Tuesday 3 February Journal/Book Title Year Month/Day Color Number of Images u ° Descriptor) Notes Thursday, March 28, 2002 Page 5773 of 5780 �(M >UWLpacts ' A 9-tt$W ,. 7 ��<\ "~ ^ OywUxtrtr\ /vLoSru*^ * ^ � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5773 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Young, Alvin L. Title A name given to the resource Handwritten Notes on VA Meeting, Tuesday 3 February 1981 ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/649b3c1f714174fbc54a34b374ef9fdb.pdf d83ff69d94e7b873e92eb5d39c4ab82e PDF Text Text Item D Number 05772 G ^1 Scanned Author Corporate Author Roport/Artlde TltlO Summary Report of the Public Meeting of the Interagency Work Group to Study the Possible LongTerm Health Effects of Phenoxy Herbicides and Contaminants held September 22,1980, Washington, D. C. Journal/Book Title Year Month/Day Color n Number of Images ° DOSCrlptOn NOtBS Articles and correspondence are also included but were not scanned. Thursday, March 28, 2002 Page 5772 of 5780 �SUMMARY REPORT OF THE PUBLIC MEETING OF THE INTERAGENCY WORK GROUP TO STUDY « THE POSSIBLE LONG-TERM HEALTH EFFECTS OF PHENOXY HERBICIDES AND CONTAMINANTS held September 22, 1980 Washington, D.C. �Panel Members from the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants Joan Z. Bernstein, Department of Health and Human Services, Chair Dr. John A. Moore, Department of Health and Human Services Leslie A. Platt, Department of Health and Human Services Maj. General William Augerson, M.D., Department of Defense Dr'. Donald Barnes, Environmental Protection Agency Dr. Michael Gough, Congressional Office of Technology Assessment Dr. Patricia Honchar, Department of Health and Human Services Dr. Phillip Kearney, U.S. Department of Agriculture Dr. David Logan, Occupational Safety and Health Administration, Department of Labor Dr. Harold Margulies, Department of Health and Human Services Guy McMichael, Veterans Administration Dr. David Rail, Department of Health and Human Services Kathy Schroeher, Department of Health and Human Services Dr. Barclay Shepard, Veterans Administration �INTRODUCTION The Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants was established by the White House in December 1979. It oversees all Federal research efforts regarding the possible health effects of phenoxy herbicides, such as Agent Orange, and is charged with reporting to the public the results and implications of this research. The Work Group is chaired by Joan Z. Bernstein, General Counsel of the Department of Health and Human Services. It includes representatives of the Departments of Defense and Health and Human Services and the Veterans Administration. Representatives of the Environmental Protection Agency, the Departments of Agriculture and Labor, the White House Office of Science and Technology Policy and Congress' Office of Technology Assessment also participate as observers. On August 29, 1980, a notice was published in the Federal Register announcing that a public meeting would be held on September 22 in Washington, D.C. The public was invited to appear before the Work Group to summarize written statements and to submit questions for response by the Work Group during the meeting. SUMMARY OF THE PROCEEDINGS Following opening statements by Ms. Bernstein and Dr. John A. Moore, Chair of the Work Group's Scientific Panel, eleven persons representing organizations or speaking as individuals presented statements to the Work Group. The Work Group then responded to twenty-one questions which had previously been submitted in writing by eight individuals, as well as to several questions from the floor. Mr. Stuart Eizenstat, Assistant to the President for Domestic Affairs and Policy, also made a brief statement. Statements and questions for the record were received from a number of persons who could not attend the meeting. Written responses to such questions have been provided by the Work Group and are included in this report. Approximately 75 persons attended the meeting. �OPENING STATEMENTS Joan 2. Bernstein Chair Interagency Work Group I would like to summarize briefly what the Work Group has done since we came into existence. Our task is extensive. We were directed to oversee, coordinate, and set priorities among relevant Federal Government research activities. We were to design a research agenda and organize the means by which that research agenda would be carried out. I believe we are making significant strides in carrying out that task. So far, we have identified all research activities being conducted by the Federal Government relating to phenoxy herbicides and Agent Orange; identified those areas where additional research is required; and arranged for necessary funding to be made available on a cooperative basis to meet those research needs. I am pleased to note that the Work Group's interim research agenda has been endorsed by the National Research Council of the National Academy of Sciences. In addition, members of our Scientific Panel have been in contact with scientists outside of the Federal Government who are involved in related research activities, including scientists in Europe and elsewhere who are conducting follow-up studies of occupational exposures to phenoxy herbicides. The Work Group takes seriously our pledge to conduct a thorough, objective and scientifically impeccable examination of the possible health effects of exposure to phenoxy herbicides, including Agent Orange. As I have said in testimony before the Congress, we owe the Vietnam veterans and their families nothing less. We also take seriously the health concerns of Vietnam veterans. We do not underestimate the veterans' very real worries about their health or the health of their offspring. While we are making our best efforts to fulfill our commitment to the public and especially to the Vietnam veterans and their families, it is becoming increasingly apparent that science is not likely to be able to answer all of our questions. Nevertheless, the Work Group intends to carry out the work that can be done and must be done in a thorough and timely manner. �Given what we know about phenoxy herbicides and recognizing that additional scientific inquiry will take time, the Work Group asked its Scientific Panel to report on current knowledge about Agent Orange and the time that will be required before gaps in our knowledge will be filled. In preparing its report, the Panel reviewed research already under way as well as research still in the planning stage. The Panel concluded that, with the exception of a few studies whose results will be known in the next few months, it is unlikely that our scientific knowledge about the long-term health effects of Agent Orange will increase significantly in the next six months and that two to three years longer will be required. A major stumbling block to conducting studies of the effects of Agent Orange on the health of Vietnam veterans continues to be an inability to identify a population of ground troops, the nature and extent of whose exposure to Agent Orange can plausibly be reconstructed or documented with any degree of reliability. Unfortunately, records which were kept of Agent Orange spray missions and coincident ground troop locations, along with names of individual troop members, may not be adequate to document the nature and degree of exposure of individual ground personnel to Agent Orange. For that reason, the Work Group examined other ways to obtain data on the health effects of Agent Orange on veterans. The Work Group believes the most promising alternative at this time is the Epidemiologic Study of Ranch Hand Personnel proposed by the Air Force. Therefore, the Scientific Panel and the full Work Group' seriously considered the merits of the Ranch Hand study, which is designed to determine what health effects, if any, have occurred among this heavily exposed population. The Work Group concluded that the study will be useful, although its results will not be able to be used to establish a quantitative risk for specific health decrements among ground troops. This is because the exposure of Ranch Hand personnel to Agent Orange is estimated to have been much greater. However/ if the Ranch Hand study does detect adverse health effects, those results should be useful in providing a focus as to the type of health effects that nay possibly occur in other veterans. �Furthermore, neither the Ranch Hand study nor any future studies of ground troops will tell us whether Agent Orange is the cause of particular health effects among veterans, especially if the studies do not identify any rare or unique diseases associated with Agent Orange exposure. Moreover, many of the health effects about which concerns have been raised by veterans and others are already known to be found in the general population as the result of other factors. What the Ranch Hand study and similar studies will be able to do, however, is define an association between exposure to Agent Orange and an increased risk of particular health effects. Given the limitations on what scientists may be able to tell us in the future about the general long-term health effects of Agent Orange and its health effects on individual veterans, the Scientific Panel recommended and the Work Group agreed that additional studies should be conducted which focus on the health status of Vietnam veterans. Such studies should determine whether service in Vietnam, rather than solely Agent Orange exposure, may have placed Vietnam veterans at a greater risk of suffering certain adverse health effects. The Work Group also agrees that the focus of the Veterans Administration's epidemiologic study of Vietnam veterans exposed to Agent Orange or dioxins, which is required by P.L. 96-151, should be broadened to include an examination of the overall health status of Vietnam veterans as a result of their service in Vietnam. We are pleased that the Senate recently voted to authorize the VA Administrator to expand the scope of the VA study in that manner. We are gratified by the show of confidence in the Work Group's judgment in this regard as indicated by the Senate's support of this modification. I also believe that the overall approach of the Senate proposal for a framework by which decisions can be made about the veterans' claims for benefits based on adverse health effects suffered as a result of exposure to Agent Orange or other aspects of service in Vietnam is a creative response-to a critical aspect of the Agent Orange problem which deserves careful consideration* �The Work Group also recommended to Stuart Sizenstat that the Ranch Hand study be conducted by the Air Force. We conditioned our approval on a commitment by the Executive Branch and the Congress that the evaluation may have to continue for a period of time much longer than five years — and perhaps up to 20 years — in order to improve the chances of detecting and validating latent or subtle effects. The Work Group recommended that the Ranch Hand study be conducted by the Air Force because it is convinced that significant delays in beginning the study -- and thus in obtaining even preliminary results — would otherwise occur. However, the Work Group recommended that the conduct of the study be overseen for at least the first five years by an independent monitoring committee, comprised of representatives of the Work Group, scientists from the private sector and academia, and persons with scientific backgrounds nominated by veterans organizations. Mr. Eizenstat has concurred with the Work Group's recommendations. In addition to the Ranch Hand study, the Work Group has considered and initiated a number of other scientific activities. VA, DOD, and HHS will be signing an interagency agreement in the next several weeks to fund a birth defects study to be conducted by HHS' Center for Disease Control. The purpose of the two-year study is to determine whether Vietnam veterans are at an increased risk of siring children with birth defects, a major concern among veterans. The study is not expected to provide data on infertility among Vietnam veterans or on reproductive problems other than major birth defects. However, it is consistent with the Work Group's view that additional studies should be conducted which focus on service in Vietnam as a possible causal factor. The birth defects study will also build on the results of an important study released last month on whether exposure of male mice to Agent Orange is associated with birth defects among their offspring or infertility. Dr. John Moore, Chair of our Scientific Panel, is one of the authors of the study. The study found no significant increase in birth defects among their offspring. �The mala mouse study, together with ongoing tests of the mutagenicity of the constituents of Agent Orange, should permit our scientists to form a reasoned opinion in the next few months as to whether a scientific basis exists for concerns that Agent Orange exposure may increase the risk of males siring children with birth defects. Vietnam veterans are also concerned that they may be suffering from a higher incidence of cancers than is expected in a population their age. In connection with that expressed concern, the Scientific Panel reviewed one German and four Swedish scientific papers on the carcinogenicity of the chemical constitutents of Agent Orange. The Panel concluded that despite the studies' limitations, they do show a correlation between exposure to phenoxy acid herbicides and an increased risk of developing soft tissue tumors or malignant lymphomas. The Panel also noted that independent verification would further validate these studies. The National Cancer Institute and the National Toxicology Program have completed a cancer bioassay on TCDD, the dioxin contaminant in Agent Orange. The results confirm earlier reports that TCDD is carcinogenic in laboratory animals. The Work Group is convinced that we need to conduct a large-scale study of the Vietnam veterans population. We need to know whether Vietnam veterans are as healthy as a population of their size, with comparable age and other characteristics, who did not serve in Vietnam. If not/ we need to know what specific health problems are occurring with abnormal frequency. We can then further refine our inquiry to try to determine the likely cause or causes of a particular health effect occurring with higher than normal frequency. Again, on behalf of the Work Group, I would like to welcome all of you to this meeting. We look forward to a productive exchange on these important issues of public concern. I will introduce the gentleman who has served a very important function and has already been introduced once, Dr. John Moore, the Chair of our Scientific Panel. �Dr. John A. Moore Chair Scientific Panel For those of you who are' scurrying for the written copy of what I was going to say this morning, I will save you some effort. Since you have that, you can read it any time you like. What I am going to say will not necessarily follow that document. As the title of the Work Group signifies, our responsibilities extend beyond Agent Orange. But in the months we've been in existence, there is no question that a substantial amount of our time has been focused on the Agent Orange issue and, to a lesser extent, on the public health implications of phenoxy acids which might be associated with their use in non-Vietnam circumstances. What is the issue? At the sake of being a little redundant, I think the basic issue as it relates to Agent Orange is that there are veterans and others who are concerned that Agent Orange use in Vietnam, and obviously presumed exposure to that substance in Vietnam, is having, has had or will have adverse health effects, principally in the long-term. By long-term, I think what we are talking about is that months, years, or even decades after exposure to those agents, one might come up with a sequelae of health impairments that is a consequence of that earlier exposure. Let's pursue this a little further. What is the nature of some of these health concerns? I think it is fair to say that the health concerns have no single focus. Indeed, the variety of health concerns alleged or feared ranges from cancer to infertility to birth defects in offspring to nervous disorders and to skin lesions, to mention but a few. Why Agent Orange? Certainly I think it is reasonable to assume that somebody was exposed any time roughly 100 million pounds of a chemical are used in a country over a period of several years. Events that led to its curtailed use and final suspension in the Vietnam war were a consequence of concerns that exposure to 2/4,5-T, or specifically the TCDD contaminant in 2/4,5-T, might be associated with birth defects. �Certainly a variety of incidents occurred in this country that also build on the concern of some people that health consequences may be associated with it. To mention but a few, there are allegations or concerns of an increased incidence of birth defects or miscarriages among women exposed in places such as Alsea, Oregon. The Long Island Railroad has workers who are concerned that they may be at risk of cancer. The EPA. action of a year and a half ago to suspend the use of 2,4,5-T in a majority of its applications in this country also, I think, raised suspicions that something is going on. 2,4,5-T, or more particularly its TCDD contaminant, has been a subject of scientific fascination for the last decade, simply because of its exquisite toxicity as well as the way in which it asserts its toxic effect. A number of scientists have said that TCDD is the most toxic man-made chemical known to man. To hear a statement of that sort certainly catches the attention of even the casual listener. Certainly the Vietnam concerns, as well as the domestic issues I have mentioned, have received their play in the media, I think there is a general awareness that there is something that relates to 2,4,5-T or TCDD and, of course, Agent Orange. 2,4,5-T represented 50 percent of what was Agent Orange. What do we know? 2,4,5-T has been in use in this country and throughout the world for several decades in a variety of applications. A number of industrial accidents occurred associated with its production and manufacture. The first accident occurred in this country in 1949 — some 31 years ago. Most accidents involved a relatively small number of workers who were heavily exposed. In a number of instances, a long time has elapsed since their exposure. Therefore, we can learn something from those people as to what has happened to their health. Some of the first evidence that 2,4,5-T — more specifically TCDD — can cause chloracne came out of studies of those people. Those studies have shown that chloracne can persist for decades after exposure. In other instances, the chloracne disappeared. The evidence out of Seveso, Italy, where three years ago there was an accidental exposure of the general population to TCDD, rather than the worker population, was that chloracne appeared. �In that case/ as is typical in most of the known exposures, the chloracne was somewhat mild and transient and disappeared over a period of time. The instances of chloracne which have persisted for a number of years or decades are clearly sequelae of some of the massive exposures in occupational accidents. There has also been evidence of liver effects, decreases in the ability of the nerves to conduct impulses and alterations in lipid metabolism. Without exception, I believe all of the occupational exposures have involved males. No females were involved in these occupational accidents. However, as near as I know, there is no information dealing with fertility, birth defects or birth outcome among the offspring of these exposed workers. The data simply has not been collected. There is no unequivocal data from a follow-up of these people with regard to long-term effects. Some of the known exposed populations have been poorly followed. We also have the problem of small population — you won't see something in that population. Indeed, I think one of the major frustrations, which also applies to the Agent Orange issue, is that no specific disease has occurred ~ no unique constellation of findings — with the possible exception of chloracne. As to the liver effects, the lipid effects or any of the other type of effects, one can find a variety of possible causes. These effects are not unique, like mesothelioma, a rather unique tumor such that when one sees it, you almost have to say it is asbestos-related because it is so rare. Another example is vinyl chloride and angiosarcoma. This just does not occur as far as we know as a consequence of Agent Orange or phenoxy acid exposure. If it did, it would be much easier. A number of ongoing activities are following occupational exposures. One is the NIOSH dioxin registry. It is trying to enroll and follow-up on in a systematic fashion all workers exposed to 2,4,5-T or dioxin in a worker setting, with the hope being that if you combine them together, you might have a big enough population to look at from which you can draw some conclusions. �Taking that one step further, we are actively trying to establish an international registry which will allow us to pick up a number of the accidents that have occurred outside of this country, most notably some in Germany, Czechoslovakia and Britain, Again, the intent is that we would have a large population from which to glean some results. Follow-ups are already ongoing of some of these past exposures. We have maintained contact particularly with the British and Czechoslovakians to find out what is the current health status of their exposed worker populations. Not much has been reported on them in the literature in recent years. We were told in both instances that further reports are due out between now and the end of the year. Seveso, Italy, while not a long exposure from the standpoint of the time that has elapsed since exposure, does represent a rather significant exposure'given the number of people involved. This is also being followed. There are also a number of studies going on in this country some of which you will hear about today from the witnesses who will appear later. A West Virginia accident which occurred in 1949 is currently in the process of being looked at simultaneously by two studies. One study is being conducted by the Mt. Sinai School of Medicine and the other by the University of Cincinnati. This is certainly information we hope will give us some insight as to the nature of the effects associated with long-term consequences of exposure. We certainly want to follow all of these studies. The reason we have an interest in following these as far as they are related to Agent Orange is very simple. They involve known exposures/ so there is no issue of who was exposed and who was not exposed. They also involve very heavy exposures and one of the basic foundations of toxicology is that there is typically a dose response relationship between exposure and the likelihood of health effects. Indeed, the chances of finding health effects among the most heavily exposed population are much greater than among those who were maybe casually exposed. Also, as I mentioned, the time lapse that has occurred in some of these exposures will give us some hint as to the long-term sequelae. Any effects associated with these studies will give us, I would say, concrete ideas as to the nature of health effects that should be looked for in all exposed populations. �11 A number of toxicity studies of TCDD have been done and some are ongoing. TCDD is known to be a teratogen in mice and to be fetotoxic in low dose levels in almost all animal species that have been looked at. It is known to persist in the body for a long period of time. It is not the type of chemical that, if you get exposed at 10 o'clock this morning, is gone by 12 o'clock today or tomorrow. It is known to persist for up to two months in the tissues of animals exposed. However, one of the problems with this type of information is that there is marked species variability as to how long it stays in the body. In some species, it is removed rather rapidly and in others, it may persist for up to a couple of months. When you try to extrapolate this data to a human population, you don't know which animal species to use. TCDD has been found to be a carcinogen in animal studies. It was associated with increased carcinogenic risk in three rat studies and one mouse study. TCDD is also known to be associated with iramunosuppression. However, there is also evidence that low doses of exposure might enhance one's immune capabilities. There are no clearcut black and white answers yet. The immuno enhancement is clearly a finding that the Italian scientists reported as a consequence of their follow-up of some of the children exposed in Seveso, Italy. It tends to suggest that the effects on the immune systems -— or one's ability to resist infectious disease -- may be related to dose. Very low doses might stimulate immune capabilities and a higher dose level might result in a more profound type of suppression. There is certainly species variability as to the nature of the type of toxicities observed. In general, I would say the animal toxicity studies give a good qualitative suggestion as .to the nature of effects or the organ systems attacked. What they don't do is give any quantitative estimate as to the likelihood of effects in humans from a given exposure. The inability to give a quantitative estimate, say with cancer, certainly is not unique to TCDD. That is one of the problems with our current state-of-the-art with regard to all cancer studies. We can say a risk is associated with it with some degree of assurance. But science has difficulty trying �12 to establish the degree of that risk — in a billion, one in a hundred. one in a million, one But again, the animal toxicity studies, as do the occupational studies, suggest the type of health effects to look for in any study in humans. A number of ongoing studies are trying to determine if TCDD, 2,4-D, 2,4,5-T or those chemicals in combination have the ability to cause a mutagenic insult or to cause genetic damage. A variety of efforts are under way to develop better analytical methods for detecting TCDD. A lot of studies under way are trying to understand various facets of how TCDD does what it does. I would characterize these studies as more in the nature of basic research thrusts. To summarize, I think the studies that are available certainly have told us a lot. Unfortunately, however, I don't think they have told us enough. There is still much to learn. Often many of the studies raise more questions that need to be pursued than the number of questions they answer. But certainly we need to continue these types of studies and someday, hopefully, we will get some definitive answers. However, I don't think we can sit back and wait for this magical data to appear. We don't know if it will appear next week, next month, next year or ten years from now. Indeed, it probably does not need to be stated to this audience that the veterans' concern is an issue now, not a decade from now or two decades from now. The concern is right now. There are a number of veterans who feel they are suffering health impairments as a consequence of their exposure in Vietnam, and I don't think we can wait for the classic epideraiological studies in occupational or laboratory settings to give us those answers. Obviously, the simple thing to do is the straightforward approach — identify veterans who were exposed and design and conduct a study on those veterans. Quite likely, you will have more than one study on veterans because of the wide variety of disease parameters that need to be looked at. However/ as has been pointed out, we seem to have trouble getting to first base on this approach because we are having great difficulty identifying a population to study whom we can say with some degree of certainty was exposed. �13 Obviously, a classic exception to that is the Ranch Hand population of roughly 1100-odd Air Force personnel involved in the distribution of roughly 100 million pounds of Agent Orange. We will come back to this group a little further along. One of the frustrations everyone in general as well as the Science Panel has is why can't ground troop personnel be identified. Simply stated, no systematic records were kept for this purpose to enable us to go back and benefit from the existence of those records a number of years later. Another fact that certainly confounds the issue is that our leaving of Vietnam was not done in the most orderly fashion. Indeed, there is very good evidence that some of the records we do have are not neatly filed away/ as a result of the rapid pullout that occurred. That is especially true of the army records, as I understand it. Efforts are under way to see if we can identify ground troop personnel. We on the Scientific Panel are not interested in identifying who among all of the better than 2 million veterans in the Vietnam theatre were or were not exposed. We can certainly get on with the types of studies that we feel need to be done with a more modest population of several thousand. So our search is maybe more limited than the records needs of others. An effort has been under way for several months to identify a battalion of marines and battalion of army personnel in Vietnam to see if we can find any evidence that would correlate their position on any one day or days with the HERBS tapes the Air Force maintained. The tapes identify generally where they sprayed Agent Orange, on what days they sprayed it and on what days they came back and sprayed a second or even a third time. That effort has recently been completed and it is ray understanding that a report was transmitted to the Scientific Panel last Friday for our review. We will review it thoroughly. We are also aware of the fact that the General Accounting Office was consulted on the design and conduct of this search effort. We have talked to GAO and intend to talk to them some more to get the benefit of their findings as well as what insights they may have from their previous effort which led to the GAO report suggesting that marine records might serve us in identifying gound troop personnel. I might mention in this regard that early on in the search Dr. Honchar and I did spend a number of hours with Department of Defense personnel during which they outlined to us the nature of their approach and what they hoped to accomplish. �Without prejudicing what we will find when we examine the report in detail, I think I would be less than candid if I did not share with you that, at least from the verbal briefings I have received on the content of this report, one need not be overly optimistic that DOD has succeeded in its efforts to identify with any degree of certainty ground troops who were exposed to Herbicide Orange. That's not to say that it didn't happen, but only that they cannot verify it. I am sure there will be further attempts after we review the DOD report and talk to GAO and others. I am sure other ideas may pop up that would suggest other avenues of approach. I think it is quite likely or at least possible that, after all of these efforts, one could end up in a circumstance in which you have only odds that a population was exposed. The odds may be such that we cannot say with any degree of certainty that we have a population of sufficient size who were expo'sed for scientific studies. But you might get an odds ratio better than simply putting all the names of Vietnam veterans in a hat and pulling out some to use for an epideraiological study. The certainty of their exposure may be 20 percent, 30 percent or 80 percent, but it will nevertheless be only a probability and not necessarily a fact. One has to be aware that if this is the best that can be done, there will be problems in trying to interpret study results. • Coming back to Ranch Hand, there certainly has been some concern over the design and conduct of the study. I might point out that it has received five reviews, the last of which was by the Scientific Panel and full Interagency Work Group. As a result of our review of the previous four peer reviews, as well as our own evaluation, we did recommend that the Ranch Hand population be studied, and be studied under the aegis of the U.S. Air Force. The reasons we came to this conclusion were several-fold. First, there were 1100-odd people who were heavily exposed, in my opinion, and I think they deserve to have the benefit of knowing what is happening or what might happen to their health. Probably more important for the larger issue of all Vietnam veterans, the study may identify some health effects. To the extent that it identifies some health effects, and as we draw on the studies of occupational exposures, the health effects identified should be incorporated into a larger study, such as a VA epidemiclogical study. �The Work Group and the Scientific Panel have been asked to perform an oversight function as to the Ranch Hand study while it is performed by the Air Force. In doing this, we intend to augment the Scientific Panel with nongovernment scientists who are experts in the area as well as to freely call upon scientists anywhere on an ad hoc basis when a particular issue comes up that requires a great depth of specific expertise. We certainly hope that veterans and veterans organizations will also identify for us scientists whom they feel are qualified to participate in this effort. One issue has come up several times — and I think it still exists in the minds of some — and that regards credibility. I might say that, in closely reviewing all of the peer reviews that the Air Force Ranch Hand protocol had been subjected to, there was never any concern on the part of the reviewers that the Air Force could not conduct the study in a credible manner. I think the issue really is concern that the public might perceive the Air Force conducting its study as less than objective. I personally do not hold that view and I don't think the previous peer reviewers held that view. I think the attempt to get an outside group of scientists as part of the oversight group should also go far in allaying the concerns on the part of veterans or the public that the study just won't be done in a proper manner and whatever results come out will not be made public. Indeed, they will be made public. Time was another factor that was important to our recommending that the Air Force conduct'the study. Certainly it was one that was considered, although it was not the prime factor. There is no doubt that the Air Force will be able to mount that study in a shorter time than could most any other group I can think of. They have been involved? they have some of the machinery and some of the early processes in place. I think that's important. To the degree we can have the Ranch Hand study far enough along so that we start to get results emerging from that study before a VA epidemology study begins, we will have the benefit of whatever findings may be emerging to incorporate into the design of the general veterans study. Finally, the last matter I would like to mention is the issue of widening the scope of the VA study. Our recommendation that any epidemiclogical study of veterans should go beyond a singular focus on Herbicide Orange does not imply that we feel Agent Orange should be dismissed or relegated to a minor role in such an effort. �16 In fact, as I understand it, the VA is mandated toy law to conduct a study of Vietnam veterans that relates to their Herbicide Orange exposure. I don't think anything has been suggested that would allow them to do otherwise. What we are suggesting is that, in the design and conduct of such a study, other causal factors be considered. Why other causal factors? One, obviously, because there is some difficulty in identifying an Agent Orange-exposed population. However, and primarily, we failed to perceive the logic behind Agent Orange being the sole possible agent considered, because there are other causal possibilities involved in some or part of the health effects claimed, if, indeed, they are related to Vietnam experience. For example, the animal bioassays are one of the pieces of information that causes concern that TCDD, and therefore Agent Orange, may be associated with cancer risks. You cannot lose sight of the fact that, using similar type methodologies, other chemicals used in Vietnam also were found to be associated with increased carcinogenic risk. Dapsone, one of the anti-malarials used in Vietnam, was found to be positive in cancer bioassay studies. Picloram, one of the ingredients in another herbicide, has similarly been found to be associated with possible cancer. Also, I think one intuitively questions whether a single agent, Agent Orange in this case, can be associated with the entire spectrum of health complaints claimed to result from Herbicide Orange exposure. As I just mentioned, other possible agents should also be considered. I feel it would be a disservice to design and conduct just an Agent Orange study. ' If the study comes out negative, suggesting there is no association, I think a disservice will have been done if indeed there were other chemical factors or a constellation of factors in Vietnam which could be associated with those health impairments. Indeed, you might have gotten the specific answer on Agent Orange to be "no", but you might have missed the broader issue of whether there was something in Vietnam associated with health effects in veterans. �Stuart Eizenstat Assistant to the President for Domestic Affairs and Policy I appreciate the work the Work Group has been and will be doing. This for me is a very important meeting. Last December when I asked Secretary Harris to establish the Interagency Work Group, I indicated that one of its main responsibilities would be to assure that all relevant research findings would promptly be made available to the public and to the Congress in a comprehensive and comprehensible manner. That communication goes both ways. Those of you who have submitted data and testimony today provide a very valuable service to the government's efforts to determine the long-term health effects of*exposure to phenoxy herbicides, particularly Agent Orange, and to establish a sound compensation policy. Let me assure you that all of the testimony presented here today will be analyzed by the Interagency Work Group and the relevant agencies and will be responded to promptly and appropriately. We need to work together on this difficult and important issue. This kind of interchange provides a forum in which to do so. We painfully recognize that fully conclusive scientific answers will not be avaiable for a long period of time, indeed if ever. But basic information from a wide variety of sources about the possible adverse health effects from exposure to Agent Orange in Vietnam is coming in. The preliminary results of the Ranch Hand study and the planned VA study should be available in a few years. These, combined with the results of various studies of industrial accidents, the Center for Disease Control's birth defects study and other human and animal studies, should produce the development of an informed government policy on compensation long before the definitive scientific data is available. While initial decisions may need to be modified or expanded as additional data comes in, it is our firm intention to address policy issues as soon as basic data permits. In addition to seeking answers to Agent Orange exposure, the Interagency Work Group recommended to me in their August 1 report that it will also be important to determine whether service in Vietnam itself may have placed Vietnam veterans at a higher risk of suffering certain adverse health consequences. �I have endorsed this important recommendation. Veterans in service to their country in Vietnam were exposed to other herbicides, chemicals, drugs, environmental hazards and stressful conditions. We owe it to•each and every one of those Vietnam veterans to determine, if possible, whether service in Vietnam has predisposed him or her to particular health problems. The medical community would be in a far better position to offer useful preventive health suggestions and to provide earlier medical treatment if they know what to look for. This approach is consistent with the VA's current statutory scheme of benefits which is less concerned with the cause of given disability than relating it to a particular period of military service. Let me assure those of you who are rightfully concerned/ as we are, about Agent Orange that we remain committed to finding firm answers to whether Agent Orange exposure produced adverse health effects. Any inquiries we may undertake into the more general area of service in Vietnam will not/ under any circumstances, replace the Agent Orange studies that various agencies will be conducting. They will be additive — in addition to — not in place of the investigation concerning Agent Orange. Of that, I can assure you. In closing, I appreciate how frustrating it is for veterans to be told to wait until data comes in. Many veterans have illnesses or have produced deformed children and believe deeply that exposure to Agent Orange was the causative factor. Many have questions about their future health and future well-being. We owe them answers so that that cloud of doubt and uncertainty will be removed as swiftly and promptly as possible. Until the evidence is in, we can and we must offer them the compassionate and competent medical care and counselling that each so richly deserves. The Veterans Administration is committed to providing that care and we at the White House are committed to assuring that they provide it. The VA's outreach efforts are being expanded and its educational programs for health professionals intensified, partly because of the firm and resolute actions taken by many of you on behalf of Vietnam veterans. All of your suggestions regarding how we can appropriately respond to the concerned or sick Vietnam veteran will be carefully and sensitively reviewed. I greatly appreciate your willingness to be here today to offer criticism and constructive suggestions and make recommendations. I hope we can continue to keep the channels of communication open because we share the same goals. To that, Jodie, I again want to compliment you on the work you have done. I think you have made �the Intaragency Work Group widely respected for its objectivity and I ara committed to working with you and the panel to assure that these critically important issues are addrsssed honestly and objectively. The facts will fall where they may. _. �SUMMARIES OF STATEMENTS FROM PUBLIC PARTICIPANTS Dr. Robert Tardiff, Executive Director of the Board on Toxicology and Environmental Health Hazards of the National Academy of Sciences (STAS) : o TGDD is an extremely potent toxicant to the female reproductive system of laboratory animals and has also been found to be carcinogenic. o NAS will soon complete a brief report on an evaluation of data relating to herbicide spray missions and the development of congenital malformations in South Vietnamese children. The data is equivocal at best; if Agent Orange caused any birth defects in South Vietnam, the absolute number must have been quite small. o NAS would welcome the opportunity to review the Air Force protocol for the Ranch Hand study as revised by the Air Force in response to the Academy's recommendations. o NAS has reviewed the German and Swedish studies on occupational exposures to phenoxy herbicides and TCDD. NAS concluded that at best the studies point to an association between occupational exposure and cancer/ but that it is unlikely that attribution can be established with respect to exposure to any particular chemical. o Additional studies should be conducted which focus on evaluation of the health status of individuals accidentally exposed to high levels of TCDD and on intensive and controlled studies of human surrogates, e.g., laboratory animals. Mr. Lewis Milford, National Veterans Law Center'(NVLC): o The Work Group should independently analyze data provided by DoD on the results of its records search for populations of ground troops exposed to Agent Orange, o The Air Force will face a conflict of interest in conducting the Ranch Hand study because the Air Force conducted the spraying and Air Force personnel are engaged in the research and promotion of chemical warfare efforts. �o The Work Group's recommendation that the Veterans Administration should expand the focus of its epidemiology study to include service in Vietnam is premature and should not be implemented until the Work Group independently evaluates DoD efforts to identify populations of ground troops. o NVLC is concerned that the Work Group's recommendation as to the VA epidemiology study represents an abandonment of scientific investigations focused on Agent Orange. o The presumed high exposure of Ranch Hand personnel to Agent Orange should be documented. Mr. John Sommer, Assistant Director of the National Veterans Affairs and Rehabilitation Commission of the American Legion: o The study of the Ranch Hand population should not minimize efforts to identify a population of ground troops exposed to Agent Orange. o The American Legion supports studies that focus on the health status of Vietnam veterans to determine whether service in Vietnam may have placed veterans at a higher risk of suffering certain health decrements, but only with the assurance that scientific studies on the long range health effects of Agent Orange not consequently be de-emphasized. o The American Legion believes that it is important that a study be conducted to determine whether or not increased cancer incidence is directly related to Agent Orange exposure. o The American Legion strongly believes that the scientific study presently being conducted by the Veterans Administration should be completed by an independent agency. o The American Legion is monitoring Agent Orange screening programs at VA medical facilities for appropriate examinations; its report will be made available to the Work Group. Mr. Frank McCarthy, President of Agent Orange Victims International: o A full-blown retrospective and prospective epideraiological study of all 2.4 million veterans is the true answer in terms of a study. �The Veterans Administration is failing to provide veterans with proper medical examinations, tests, treatment and compensation, despite its own directives. Physical examinations conducted by the VA should include.basic cancer tests; veterans with skin rashes should be examined by dermatologists knowledgeable about chloracna; veterans and their families should receive genetic counselling. Dr. James Dwyer, a statistician in the Psychology and Sociology Department of the State University of New York at Stony Brook who is working with Citizen Soldier to analyze data collected from a health questionnaire distributed to Vietnam veterans: o While the Citizen Soldier survey falls short of a true experiment (e.g., its measures of exposure are indirect, symptomatology evidence is basad on retrospective self-reporting), the data may be of considerable importance because they are probably the only source of information on patterns of symtomatology among over 4,000 Vietnam veterans that includes a sufficient number of cases to achieve a reasonable level of statistical power. o 60 variables — or pieces of information — from the survey have been coded. Observed associations will be subject to numerous causal and spurious explanations; thus, analysis must rely on identifying patterns of findings that speak to the merits of various competing explanations. o Evidence from the data are only moderately suggestive of a causal relationship between Agent Orange and birth defects. o The evidence is rather highly suggestive of a causal relationship between exposure to dioxin and liver disease. Mr. HaroId Co11ins, Assistant Executive Director of the National Agricultural Aviation Association: o NAAA is conducting a health study of its members. NAAA represents about one-third of the agricultural aviation businesses in the U.S. o The study also includes information on the health of the pilots' siblings and the siblings' families. �23 o Results of the study will be made available to the public on December 3, 1980. Mr. George E. Gandenberger administered ground security for a defoliation mission of an area in Vinh Hu Village, Ho a Dong District, Go Cong Province, Republic of Vietnam: o Informed reading of the HERBS tapes indicates that very few areas in Vietnam were not exposed to combinations of chemicals through spray, drainage and consummables. o Not all herbicides used during the Vietnam conflict met product standards for use in the United States. Herbicides should be the object of treatment, not study. o Third country nationals from Australia, New Zealand, the Phillipines and Korea should also be advised that they may have suffered environmental damage. o The Federal government should develop a data processing program for correlation of data from the various public and private epidemiclogic studies now under way. Mr. Robert Muller, Executive Director of Vietnam Veterans of America: o The Veterans Administration should begin a national outreach program advising veterans of the availability of the VA medical system. o The Veterans Administration should grant service connection for chloracne. o The VA should provide access to its medical system for Agent Orange-related medical examinations on a service-connected priority basis. o There can be no responsible governmental policy on the Agent Orange issue without a credible scientific review process. Compensation policy must rest on science. Public confidence in Agent Orange policy will depend on public confidence in the process for reviewing and developing scientific evidence. o The government must face in a manner open to public scrutiny and involvement the policy questions at stake in.establishing compensation and a full range of health care for Agent Orange-related disabilities. �Mr. Alfred Baxter/ Chairman of the National Forest Products Association Committee on Forest Industry Chemicals and President of the American Forest Institute: o The forest products industry supports careful use of substances which could be harmful to human health, active concern about the potentially harmful effects of such subs-tances, careful and rigorous scientific inquiry, and necessary governmental controls which properly weigh related social and economic concerns. o NFPA will provide the Work Group with all studies sponsored by it relating to health effects and phenoxy herbicides, o The forest products industry is willing to join in cooperative support of independent scientific studies of the relationship between phenoxy herbicides and neural tube birth defects and cancer. Mr. Jon R. Furst, Chairman of the National Veterans Task on Agent Orange: o The Work Group should verify data provided by DoD and VA before making recommendations based on that data. o The Air Force role in the development of a chemical warfare capacity creates a conflict of interest for the Air Force in its conduct of the Ranch Hand study. o Conclusions as to whether sufficient numbers of exposed ground troops can be identified for study are premature. o A Vietnam service study in addition to a study.of exposed ground troops is much more acceptable than deleting one for the other. The Task Force sees the two studies as addressing different concerns. o A study of Ranch Hand personnel should be conducted, but it should not be conducted by the Air Force, even if an independent monitoring committee is established. The Work Group should reconsider the merits of having independent researchers conduct the Ranch Hand study. Dr. Tschirley, Department of Botany and Plant Pathology, Michigan State University: o A scientific dispute resolution conference held in June 1979 concluded that 2,4,5-T is not the sole �That conference also concluded that levels of TCDD greater than 100 parts per million have not been detected in any environmental sample associated with normal use of 2/4,. S-T; 2,4,5-T itself is of minor ecologic concern. The conference concluded that 2,4,5-T is not a carcinogen nor a mutagen; however the Swedish studies published in 1979 and 1980 were not available at the time the conference was held. Copies of the written statements of each witness are available from the Office of General Counsel, Department of Health and Human Services, Room 716E, Hubert H. Humphrey Building, 200 Independence Ave., S.W., Washington, D.C. 20201. RESPONSES BY THE WORK GROUP TO QUESTIONS PROM THE PUBLIC Following the presentations, Work Group members responded to questions submitted in writing by* the public. Each question was read to the Work Group by Mr. Leslie Platt. Each question is set out in full below, with the name of the individual and, where appropriate, the organization which submitted the question. Robert Muller, Vietnam Veterans of America; "If the Veterans Administration epidemiological study conducted pursuant to Public Law 96-151 is broadened to include examination of the overall health status of Vietnam veterans as a result of their service in Vietnam pursuant to the Work Group's recommendation, does that entail that the P.L. 96-151 study will not address the impact of Agent Orange?" DR. MOORE: As my introductory remarks this morning pointed out, it is certainly not the intent of the Science Panel that the study's focus on Agent Orange should dim at all. I think the statement of Mr. Eizenstat certainly underscored that. DR. GOUGH: May I add something? I spoke with a member of Senator Cranston's staff today. Senator Cranston does not intend that an expanded study would mean that we would do away with the Agent Orange study. In Senator Cranston's mind, the Agent Orange study was mandated by Congress and will be carried out. Robert Muller: "If an attempt would be made to study the impact of Agent Orange, does that mean that the Work Group envisions at this time that, subject to change in the protocol development process, two studies should be done under Public Law �96-151, one to look at a cohort which represents a best guess at an exposed population and one to consider service in South Vietnam, or does it mean that one study will be undertaken which will contain a subgroup or cohort which is the best guess at an exposed population?" DR MOORE: All of the above. I think at this stage in the evolution of the design of that protocol, one clearly can't definitively state that it will be one study with two, three, or four subsets or whether it will be two studies or what the case will be. I don't think it would be prudent to speculate because it would be pure speculation. Robert Muller; "Will the Work Group submit the report expected from the Department of Defense on their efforts to determine if a ground troop cohort could be found to the General Accounting Office for comment?" MS. BERNSTEIN: We always have the right to submit something to GAO, and the answer is yes. GEN. AUGERSON: • As I mentioned this morning, we found it best to invite the GAO in to tag along with us, as we cut our teeth on the details of this record search. I have an impression of what they think, but I think it would be better to comment at some future time after we have formally submitted it to GAO. Dr. Moore will eventually get his copies of the report and I can visualize that he might invite other members of the Scientific Panel to take a look, not just at the report, but at how the records review was conducted. To reiterate what I said this morning, we have not given up on trying to identify personnel' on the ground who were probably exposed. We have just found it extraordinarily difficult to go into the records on the basis of spray flight missions from Ranch Hand and come to grips with who was where on the ground. We have gotten some useful suggestions to that end today and we will explore those. Mr. James F. Lee, Jr., Laurel, Maryland: "With all the talk about 2,4,5-T exposure and TCDD exposure, I would like to know whether or not the Food and Drug Administration, U.S. Department of Agriculture, EPA or any other Federal agency is conducting Federal testing programs of any kind to analyze random samples of food for TCDD and for 2,4,5-T residues? "If it is being done, I would like to know what residues, if any, they have found, how extensive their sampling is, and what the limit of detection is on their analytic method. If �27 such sampling is not being done, I would like to know if it is being planned or studied by any Federal agency. "It would appear that all the current concern and evaluation of whether there continues to be exposure of people despite the present ban is important." DR. KEARNEY: I am aware of six kinds of studies being conducted on various food items that might be of interest. One is m'other's milk, one is beef fat, one is beef liver, one is cow's milk, one is fish, and one is wildlife. As of today, I am aware of four studies on mother's milk. No TCDD has been detected in mother's milk at a level of sensitivity of one part per trillion. In the beef study, the number of animals involved varies, but I believe that about 35 animals were investigated. One animal had a level of 68 parts per trillion in the fat; two had lower levels and none was detected at a level of 10 parts per trillion. No TCDD was detected in 43 beef liver samples? no TCDD was detected in cow's milk. No TCDD was detected in fish caught near a pond in a rice growing region in the United Sates where we have used 2,4,5-T extensively for weed control. There is a report from Vietnam, from Messelson and Bachman, of a fish caught there that contained measurable residues of TCDD. In the area of wildlife, until we supported a study ourselves in the Department of Agriculture, I was unaware of any TCDD residues in wildlife. There is a study now that found nine positive samples in deer in a forest treated with 2,4,5-T. The highest sample was about 5 parts per trillion. These are the studies I am aware of. There are probably other studies going on that are unknown to us that may not necessarily be in the Federal sector. I think we do have a fairly good overview of what is being done, however, and these are the levels we are seeing. DR. BARNES: I might be repeating on some of these. The beef fat-beef liver study that was mentioned is in the process of being reanalyzed. The way it is done is to have the samples analyzed by two different laboratories. One laboratory analyzes the samples, and the samples are then reanalyzed at a second laboratory. 'There are two different studies nearing completion on deer and elk in different forested areas of the western part of the country. Dating back sometime ago, small roadside animals were analyzed. �23 The agency is also looking into analyzing materials from the rice growing sectors of this country, which would include catfish and crawfish. Those samples have been collected and extracted and are now undergoing analysis. The agency plans to continue to look into areas which we think need to be looked into for TCDD contamination. The purpose of this as far as our agency is concerned is not to look at the food supply directly, since that is the purview of the Food and Drug Administration. For those who are interested, many of these samples have been summarized in the exposure assessment which SPA will introduce into evidence in its hearings in the next ten days. This will become public information at that time. Maurice Loir, Military Order of the Public Heart; "We have heard a variety of statements concerning persistence of TCDD. We expect that some statement will be made by the Scientific Panel to help clarify this point." DR. MOORE: As we read this question, we were unclear as to what Mr. Loir meant by "persistence." Two contexts came to mind: one is persistence in the human body or animal body and the other is persistence in the environment. We will try to give the answer to both. I will try to give you the one with regard to persistence in animal tissue or the body and Phil Kearney will try the environmental definition. First of all, no human data is available that addresses the issue with regard to persistence. There is some human data that might suggest that if indeed the analytics were correct, TCDD can be detected in human tissue. But with regard to persistence, e.g., how long it had been there and how long it would stay there, we have no data. That is one of the major data gaps we have in trying to extrapolate a lot of the laboratory data. With regard to a number of animal studies done in a variety of species, there is significant variability with respect to the length of time TCDD will stay in the body. But if you will allow me to generalize, one can say that the half-life can vary anywhere from 28 days to possibly 60 days. �29 What is meant by half-life value is that during period of 60 days, one half of what was in your body previously would be gone. In an additional 60 days, of what was left would be gone. In another 60 days, one-half of that, etc. the 60 days one-half etc., DR. KEARNEY: Let me talk to the more difficult of the two and state at the beginning that the answer is complex, which means that, scientifically, we are not sure. But looking at the phases of the environment, we know that on plant surfaces and in water the molecule is subject to photodecomposition. The force of sunlight can alter the molecule and alter its toxicity. On the soil and in the soil, the molecule does appear to be rather persistent. We ran a study, using that term "half-life" again, at Beltsville and we found the half-life was about one year. Other people have studied it and it ran anywhere up to 500 days. It depends on the soil it's in. A warm, moist soil high in organic matter is different from a sandy soil in a colder climate. We have no absolute numbers but we think the half-life is about one year. James F. Lee, Jr.; "I would like some information on research being done by the National Institute of Environmental Health Sciences (NIEHS) to examine the effect of TCDD on the immune system of animals. The effect of transplacental and neonatal exposure is also being checked. "What is the status of these experiments, when will they be completed, and are there any reports available to the public?" DR. MOORE: Most of those experiments have been completed and printed and have been available from anywhere from six months to two, three or four years in the open scientific literature. Recent testimony by Dr. Michael Luster of NIEHS at the EPA suspension hearings might be a good source because in that testimony he tried to summarize what all of the studies meant. His testimony would also give the references to the majority of the individual studies that are in the open literature. So the studies are completed and reports are available. Mr. Lee is correct in his assumption that the effects were associated with transplacental and neonatal exposure. �Maurice Loir: "Regarding the teratogenicity of TCDD, it has been noted that little real information exists regarding exposed males and unexposed females. "A recent study with rodents has been reported. We ask if further studies are planned, particularly with primates rather than rodents? It is our feeling that the use of primates could be regarded as far more definitive than any test with rodents." DR. MOORE: I know of no further studies being planned. Further, I don't agree a priori that the use of primates might be more definitive with~~respect to experimental data, particularly if you are talking about birth defects. The reasons for that statement are severalfold. First, the number of primates one would have to use to end up with a meaningful result I think far exceeds our resources as far as being able to get primates for research in this country. Most of our primates are imported and come from South America, India or Southeast Asia and most of those countries have banned the export of primates to this country and to the world. Second, quantitative extrapolation of results using the rhesus monkey or some of the other primates is questionable. A number of scientists are of the opinion that the dose effects seen in some of the studies done in primates logically don't suggest they should extrapolate directly to man. The monkey in this case gives the appearance of being super-sensitive and that's an opinion and not fact. Also, given the fact that the rhesus monky and some of the other primates are so sensitive to certain toxic effects, I think if you studied reproductive effects on exposed males, you would end up with overt toxicity before you could get a chance to study male fertility. Finally, if indeed the thrust of the question is to endocrine effects, maybe the rehesus monkey would be good. Constance See, Alexandria, Virginia; "The National Center for Toxicological Research is developing pharmacokinetic models for 2,4,5-T and dioxin to assess whether teratogenic responses between laboratory animals differ because of enzyme activity. �Tl "I would like to know what the researchers, Drs, Young and Holson, have discovered so far. I would like to know if they have made a final report of any kind, whether such report is available to the public and, if not completed, when they expect to complete their experiment?" DR. MOORE: I called the Center for Toxicological Research. They are not doing any work with TCDD and I don't believe they are planning any in the near future. They have done work on pharmacokinetic studies with 2,4,5-T, principally in one species The results of that study are in internal review. I believe there is an abstract available from the Society of Toxicology Meeting held last year or this year. The senior author has recently left that organization and I am sure that his leaving will slow down development of the final report, as is typically the case. I might mention that outside of NCTR but germane to the thrust of the question, there have been a number of studies that have show that there are different effects among strains of species, principally rodents. Marianne T. Anderson, Alexandria, Virginia; "The first project I am interested in is being done by Dr. Borzelleca at the School of Pharmacy, Virginia Commonwealth University on the effects of TCDD on the male reproductive system and I would like the following information, if possible. "One, preliminary or final results; two, has a final report been issued; and three, how can I obtain a copy of the final report?" DR. MOORE: I was afraid when found something we had not found. he said they never did the studies studies for the simple reason that safety facilities to be able to do I read this one that they I called Dr. Borzelleca and and don't plan to do -the they don't have adequte such studies. Marianne T. Anderson: "In the same area, Dr. Lee, with the National Institute of Environmental Health Sciences, is also studying the reproductive toxicity of TCDD and I would like to know the following: "One, the current status of the research; two, whether potential human health effects of TCDD will be assessed in the study; and three, whether a final report of the research is available at this time?" �32 DR. MOORE: This is one we did not catch. There are such studies being done by Dr. Lee and they basically concern the . • effect on male offspring of TCDD administered to pregnant animals. Dr. Lee is interested in testicular function of one sort or another. I did not have the chance to talk to Dr. Lee before this meeting, but we will get an activity sheet filled out which will give the status and time needed for completion. Constance See; "The National Institute of Environmental Health Sciences is sponsoring research by a Dr. Peterson at the University of Wisconsin at Madison on effects of TCDD on the lever and pancreas of animals. He was also developing a blood clearance test to detect liver problems caused by TCDD. "I would like to find out whether such research is actually going on, whether such a blood clearance test has been developed and whether there is any published reference on his research which I could obtain?" DR. MOORE: We did do a search of published literature and there was no citation of a Dr. Peterson, which suggests it is not published if he has done something. Marianne T. Anderson: "Dr. W. Piper of the University of Nebraska Medical School is conducting research on toxic effects caused by TCDD through the impairment of endocrine function and interference with the heme biosynthetic pathway in testes. I would like to know the following: "One, what his research has found to date; two, when it will be completed; and three, whether-or not he has written any reports on his research which are available to the public?" DR. MOORE: Again, on incomplete information, this is a brand new grant, less than one year old, and I would suspect that his research has very few findings to date. Maurice Loir: "Since other herbicides and pesticides were used in Vietnam, what is being done to discover the extent of synergetic effects? " DR. MOORE:' Nothing. �33 Maurice Loir; "We are concerned with the type of exposure test animals face. Since the Vietduc Medical Center in Hanoi, Vietnam has reported finding TCDD residue in fish, it would appear that a major form of exposure comes from the use of water. Is this factor being fully considered in planning the' various studies?" DR. MOORE: We are certainly aware of the fact that this is a possibility, based on the Baughman/Meselson data that came out of Vietnam showing levels of TCDD in butterfish and information from this country showing levels of TCDD in some fish downstream from the industrial plants' involved in production of these types of products. I think the more distinct possibility is that there is a likelihood that such residues may be found in the biota rather than the water per se. It is a very water insoluble compound. Perhaps Dr. Barnes would like to add to that. DR. BARNES: I would like to just mention again that we have a study ongoing of catfish and crawfish in the rice growing areas of the country with that idea in mind. In addition, EPA has a water analysis network in which we look for 2,4,5-T in waterways. This is a continuing activity of the agency. DR. KEARNEY: I would note TCDD is a very insoluble molecule. One would suspect that it would be liquid filling. DR. BARNES: Along that line, in natural waters there is suspended sediment and organic material which could act as a carrier and there might be reason to go back and look. Lewis Milford; "The Scientific Panel concluded that the exposures of the Ranch Hand personnel are known and documented and that attempts to identify the exposures of other non-Ranch Hand subpopulations of Vietnam veterans have, proved impossible. Please provide the documentation relied upon to support this conclusion and the method of investigation to arrive at this conclusion. In particular, please explain whether and in what manner any independent evaluation of exposure data was conducted or is contemplated by the Work Group." DR. MOORE: Part of the association between exposure of personnel is logic. If you are involved with 80 million pounds of material, you must have been exposed to it at some time. �34 Also, various statements of Ranch Hand personnel describe the lack o£ precautions they took with regard to trying to reduce' their exposure. They often flew in T-shirts and they did not by any stretch of the imagination use routine protection measures. We also heard a report from the Air Force, which was trying to identify the degree of exposure that may have been a consequence of being Ranch Hand personnel. There was, for example, the function of being the pilot or the console operator in the back, etc. That report is not yet publicly available, principally because the person who did it is using it for a graduate thesis. According to academic rules, it's not available for publication until he defends his thesis. We intend to look at that report in detail once it is available, which I understand is just a matter of a few weeks. Lewis Milfordt "The Work Group recommended the conduct of the Ranch Hand study by the Air Force on the express condition that an independent peer review committee monitor the conduct of the study. With- regard to this recommendation, please explain, a) whether and which veterans orgnizations were consulted before this recommendation was prepared; b) how the establishment of such a committee would alleviate problems of credibility where the Air Force is perceived to have a clear conflict of interest in promotion of chemical warfare weaponry? c) whether organizations other than the Air Force were considered as possible candidates for the conduct of the study of the Ranch Hand, and if so, the reasons for rejecting such alternatives; d) the significance of possible delays in the decision to approve an Air Force controlled study and whether the need for credibility that might be restored by the conduct of the study by a group other than the Air Force may offset the negative impact further delays may cause." MS. BERNSTEIN: I will try to respond to that first. As to part (a), whether and which organizations were consulted before this recommendation was prepared, no formal consultations were held with any group as we went about preparing these or other recommendations. That was not for reasons of cutting out outside people, but simply because as the Work Group established itself and went about trying to do its work, we found early on that the best way for us to get the best product and best judgment from people was to proceed informally, without formal votes, without formal procedures. However, as we went about forming our recommendations, we did talk to a 'lot of people, including congressional committees �35 and congressional staff and various veterans organizations. I would not say we talked to every single one, but we had informal consultations as we moved along. I would not try to document that in any sense of a-listing of groups consulted or not consulted for any purpose. But I felt strongly and I think others as well that we worked together best by achieving consensus on a whole variety of recommendations as we moved along. If anyone would like to add to that, feel free to interrupt. Part (b) concerns the review committee. On the issue of credibility, I will express my own personal view on that but will try to characterize the views of the Work Group as well. After working on the Ranch Hand study, thinking about it and reviewing the work of others, I came to believe that the credibility issue was less an issue of a formal conflict of interest than it was an issue of general perception of lack of credibility. In straightforward lay language, people said nobody will believe the study if the Air Force does. it. To me, that means it is a judgment call. That is to say, there are some people who, I suppose, would say we don't ever believe Group A just because we've had experience with Group A over the years and we will never trust them no matter what they tell us. 3y the same token, others would say we don't see any reason not to, unless they do something wrong or don't have the competence in the first place. What I was doing was trying to determine how to assess a perception. There is no scientific way or any scientific inquiry that can help you very much in that. The best you can do is try to get a feel for it by talking to a lot of people and getting many expressions of views. If then a perception problem remains, the best way of reassurance is to have a continuing mechanism to evaluate what it is that that group is doing. It's as simple as that for me. It's similar to an oversight committee of the Congress continuing to look at an agency as it goes about its work. The agency or department can't be doing too much wrong if there is an external group watching over its shoulder, keeping it open and making sure there are no biases consciously or unconsciously being expressed. �For me it was just one way in which to add some reassurance to those who continued to have a perception of lack of credibility I did not feel that lack of credibility. But I thought it was a wise judgment to take that last step to insure continuing credibility. That is the way I would explain our judgment on that. GEN. AUGERSON: May I add a footnote to that? MS. BERNSTEIN: Absolutely. GEN. AUGERSON: I must say I was surprised by this idea of linking the way in which medical people in the Air Force might somehow affect the outcome of the Ranch Hand study because of some perception of the interest, or the lack of it, of the Air Force in chemical warfare. I guess it is a form of left-handed compliment that assumes that somehow we are well enough coordinated in one of our large military departments that medical people thumping chests and examining people would somehow be susceptible to influence from some of the more weapons-oriented people. I assure you that is not the case. 1 think whatever happens in terms of national policy about chemical warfare will be driven more by present and future determinations of a threat and the United States' posture vis-a-vis that threat than any regrettable or unintended consequences of what has happened in the past. DR. GOUGH: One issue we have heard a lot of today, and I guess everyone in this town hears all the time, is the question of government credibility. I don't think any agency of the government or the government as a whole will establish credibility if, every time a delicate and sensitive subject comes along, the government farms it out to an independent agency. MS. BERNSTEIN: I was hoping you would add that because I know most people in our group and others we talked to were concerned with that aspect. Increasingly there seems to be a sort of almost adjudicatory — as we lawyers think about it — movement that suggests that anybody who has had anything to do with anything can never make another statement on it because it will be somehow suspect I think we were concerned about it. I don't think we overreacted, but I think we did feel that we did not want to get to a state of affairs in which a par~t of the government could never examine itself and its operations. �37 DR. LOGAN: It may be important, too, to mention that many of us on the task fores were impressed with the Air Force presentation of the Ranch Hand study and the sophistication and obvious level of expertise put into the development and design of the protocol. I think that the scientific expertise that went into that protocol was apparent to members of the committee here and further led to a feeling that the Air Force was going to be very responsible in the conduct of the Ranch Hand study. MS. BERNSTEIN: In response to the last two parts of the question, the first being that if organizations other than the Air Force were considered and so forth, I think the answer to that is yes, other organizations were considered. Again, in our customary way of working, they were considered by the Scientific Panel and the Work Group generally. They were not considered in any formalized way. We did not ask people to propose and reject other organizations and so forth. However, we did consider whether it would be sensible to pursue other organizations, particularly those within the government. Again, I could not give you a formal statement of reasons for rejecting each of them because we very soon reached the conclusion — I don't mean without deliberation but quickly — that there was a consensus that the Air Force met the standards which we were setting and therefore we did not have to seriously pursue other organizations. On the question of the significance of delay, we all felt that there had already been considerable delay in conducting this study and other studies. That was one of our primary concerns. There have been a couple .of years of not very productive activity in the sense of simply not getting on with it. I think we were all very sensitive to delay. Surely there would have to be delays if, at this very late stage of the game, after the extensive development of protocols and the extensive reviews by not only our little organization, but the National Academy of Sciences and others as well, we recommended that yet another organization go back and start over again. I for one — and I think the rest of the group as well — was convinced that delays would be automatic and that is what we were concerned about. Would anyone like to add anything? �33 Lewis Milford! "With regard to tha issue of exposure, the Scientific Panel suggested that 'actual' exposure may be the standard needed to conduct a valid epidemiological study. Please explain the standard of exposure the Scientific Panel believes is necessary for such a study, with particular attention to how such a standard may or may not be met, given the existing quality of exposure data." DR. MOORE: I think that question gets to the crux of the issue with respect to the quandry caused by the lack of data in some archives somewhere that describe the companies — "exposed on such-and-such a date." I doubt that we'll get data, if you want to compare it to Ranch Hand, that will say this person was on duty on these dates in Vietnam at this period of time and he flew this many missions, so you can roughly calculate the exposure. I don't think you will find that with regard to ground troops. I think what one might best come up with is some way down the road being able to get the odds that some particular unit might have been exposed at some particular period in time. It's something like a weather forecast and I think it can be wrong. I do hope in the efforts we are making we will be able to identify a group and that if it is the best one can get without having unequivocal data saying they were exposed, there would be general agreement saying that it's probably one of the better groups you could look at in terms of ground troops that might have been exposed. But you will never get the unequivocal data, I don't think, unless something magic appears that I am now unaware of. Lewis Milfordt "The Scientific Panel recommended that a Vietnam service study, rather than an Agent Orange study, be the focus of future scientific investigations. With regard to this recommendation, a) explain whether it would be necessary to identify a subpopulation of veterans exposed to Agent Orange at some time during the conduct of such a study and, if so, the approximate time within the conduct of such a study that this information should be developed, e.g., before proceeding or sometime after possible ill health effects are identified. b) If the answer to Part (a) is that it is not necessary to identify a population exposed to Agent Orange, please explain why not. �39 c) Explain whether the difficulty with developing exposure data has played a role in this recommendation. d) Explain the basis for the expressed belief of Work Group members that veterans are concerned only with whether Vietnam service may have caused them ill health effects/ and not whether Agent Orange may be the cause of their ill health problems. e) Explain whether Vietnam service focus could or is contemplated to produce data on the possible correlation between Agent Orange and ill health effects. DR, MOORE: With regard to the first question, the study is not yet designed so any answer would be speculative at this time. I think one needs to use some logic in looking at Vietnam veterans and gi*ve some consideration to what they did while they were in Vietnam. Certainly, there is a large contrast between a fighter pilot sitting in an air base versus someone who was out on search and destroy missions most of the time he was there. I would expect and hope that when Vietnam veterans who are going to be studied are identified, these factors will be considered in selecting which people should be part of such a study. Consistent with that logic, assuming we can come up with some units where there is evidence that suggests that if any units on the ground were exposed to Herbicide Orange these stand the likelihood of being the most likely exposed or the more repetitively exposed and more intensely exposed, whether it be engineer units such as postulated this morning or whatever, these units should be a subset. I would hope in doing this, the timing would be such that we could identify those subsets of populations before the study began. That might be subject to revision if other data becomes available somewhere down the line. But I think the thrust would be to identify populations or subsets of populations before you begin. I can skip part (b) because the answer was "yes" to part (a). Part (c) asks whether difficulty with developing exposure data has played a role in this recommendation. Certainly, the people who considered and made the recommendation were aware of the fact that we had difficultities identifying ground troops. There is no question that it was factored into the decision. I don't think it was the major reason for the decision. �Part (d) asks for the basis for the expressed belief of Work Group members that veterans are concerned only that Vietnam service may have caused ill health effects and not whether Agent Orange is the cause of their ill health problem.. The word that jumped out at me was the word "only". As a general point of clarification, I don't think we have used the word "only. Speaking for myself, the basis for my belief that veterans are concerned about Vietnam service is based, pure and simple, on some of the conversations I've had with veterans. Part (e) asks whether the Vietnam service focus could or is contemplated to produce data on the possible correlation betweeen Agent Orange and ill health effects. We hope it will but I don't know if it could. We will certainly try to end up with something that will produce data on whether a correlation exists between Herbicide Orange exposure and health. Terry Jemison, U.S. Medicine: "In testimony prepared for a House hearing on Sepember 16, 1980, Dr. Moore said studies conducted by Dr. Lennart Hardell in Umea, Sweden and others show a correlation between TCDD exposure and an increased risk of cancer, but he said these data do not lend themselves to establishing a quantitative risk for veterans exposed to Agent Orange. He said a study specifically of veterans is needed, suggesting the VA study could detect any excess cancer appearing only ten years after exposure and a positive finding would establish service connection. "One, would a relative risk in the range of the Hardell studies constitute such a positive finding? "Two, how large a study population would be needed to place the study beyond criticism for size — what numbers?" DR. MOORE: I think my statement was that the VA study might pick up excess cancer and the reason I say might is the fact that a lot of the cancers are not developed ten years post-exposure. That may be a small point. DR. HONCHAR: With regard to the first question, the relative risk in the studies conducted by the Swedes clustered around six. That is epidemiologically considered to be a positive relative risk, i.e., one worth noting, important and so on. What would then be done with the relative risk relative to other possible relative risks would be a social policy decision. �41 The second question is rather difficult because it depends on what kind of study is being done and on what particular cancers nay be looked at as end points. In the case of an epidemiclogical control study, the number of cases of soft tissue sarcomas, for example, that you would need to investigate would be one consideration, into which you would have to factor an estimation of the number of people who might have been exposed — some estimation from the population your numbers are drawn from. With regard to a mortality study, which is an entirely different kind of epidemiological study, different considerations have to be taken into account as to the appropriate size of the cohort required to-detect cancers down to a certain point so that the study has a degree of power. It's a broad question to answer specifically. Lewis Milford; "In order to understand the reasons for the recommendations discussed here, we request access to any documentation, records or other materials of the Work Group that would explain in greater detail the scientific and policy bases for the Work Group and Scientific Panel." MS. BERNSTEIN: I believe we have already made everything public. We have no intention not to. I would like to ask Les, because my voice is getting tired, to detail the written information we have already made public and will continue to make public. MR. PLATT: The written material of the Work Group and its Scientific Panel is comprised of the monthly reports of the full panel as well as reports by the Scientific Panel. In addition, we have been preparing updates of scientific activity timetables and detailed funding charts for each of the activities covered by the timetables to supplement those supplied in the spring in the initial round of reporting by the involved agencies. Those reports, as well as any writings of members on their activities and the testimony of Work Group members, together with the report of this and similar proceedings in the future, constitute the totality of the Work Group's writings, except for correspondence with individual persons or organizations who write in to us. �42 We have been making it all public and intend to continue to do so. Anyone who would like a copy of the summary report of today's proceedings, please be sure to complete the form and we will put you on our mailing list for this report and for the write-up of our monthly meetings. In addition, anyone who would like to submit additional questions may do so until the end of this month and we will endeavor to incorporate responses to those questions into the summary report of today's proceedings. If anyone has additional material, please get it in to me before the end of the month. Roger Runningham, reporter; "Is there any government research going on"or anticipated at the Mayo Clinic on Agent Orange." DR. MOORE: I know of none. Barbara Saunders; "What is the relationship of the ingredients in Agent Orange to sevin, malathion, diazinon and insecticides in general? Do not these insecticides include derivatives of Orange?" DR. MOORE: Sevin is a carbamate whereas malathion is an organo-phosphate. Diazinon is not related to phenoxy acids, I know that for a fact. I guess by process of elimination, we would say these insecticides do not include derivatives of Orange. Is that correct? DR. KEARNEY: That's correct. QUESTIONS FROM THE FLOOR MS. BERNSTEIN: We will now open the floor for questions. If you will go to the microphone/ state your name and if you are affiliated with an organization, let us know what that is. You may direct your questions to individual members of the panel, if you like. Mr. Furst? MR. FURST: Dr. Moore, I am told that the means by which an animal would be exposed to TCDD can greatly change the manner in which it is found to have a toxic effect. For instance, I am told that there is a great difference in the reaction laboratory animals have to small exposures over a long period of tine as opposed to a single exposure of the same general dosage. Can you tell me how that works? �43 Specifically, I am told that in order to cause the same kind of poisoning in laboratory animals who have been exposed over a long period of time, they have to increase the single dose a great deal. DR. MOORE: TCDD is a lipophilic compound, which means it tends to migrate to the fat. There has been some speculation that there could be some differences depending on the route of exposure, that is, whether you got it on the skin, whether you ingested it, etc. With most lipophilic compounds, there isn't much difference as to whether it will get in. This is known to be true for TGDD. I would not say it's exactly the same, but they are generally the same. With regard to differences in the type of response one will get ~ whether you have one whopping big dose or little doses over a long period of time — there is a difference and there are a variety of reasons for it. Let me give one generality. With these types of compounds, what happens is that you have somewhat of an absorption phenomenon. If you get one huge dose all at once, it passes through the body before you fully absorb it, whereas if you get small incremental doses, you will absorb most of it and continue to build body burden. One of the best examples I can think of is the PBB problem in Michigan where the acute LD-50 was 25,000 rag. But if given in incremental doses in periods of 30 to 45 days, the LD-50 dropped by almost two orders of magnitude. MR. FURST: Thank you. MR. GANDENBERGER: My name is George Gandenberger. I'm not affiliated with an organization, but I was directly involved in spraying. I have a study of approximately 150 different studies, really a bibliography, related to TCDD and 2,4,5-T which was published earlier 'last year. It seems to establish that there is some causation for illness related to both substances. I also have a copy of my separation physical from Vietnam which explained what I went through in terms of treatment and then had a rubber-stamp entry, "Items 20 to 38 reviewed and answer found to be no medical significance." �Now, I had no medical records, they were in Saigon and I was not. Many people in the service have the same situation. I know a fellow in the 101st Airborne who has four Purple Hearts and zero medical records. If the VA insists on having direct causation in terras of medical treatment before they will provide service connection, then there is no point to this group's study in the first place. As long as they hold out for that, for many people who were directly exposed there is no way to substantiate that putative exposure. How can you accommodate that unless you go to area-wide eligibility? MR. McMICHAEL: While medical records are very valuable in establishing claims for service connection on disabilities, the absence or presence of those medical records are not necessarily conclusive. What we attempt to do in establishing service connection is ascertain whether or not there is a disability of which the veteran is currently complaining and then try to relate it to a period of service. Obviously, we have medical records that aid, but that's not the only way service connection can be established. Other testimony, your own testimony and the testimony of those you served with can serve to establish service connection in a given case. MR. GANDENBERGER: Sir, I don't want to dispute you, but I have several responses from the VA office in Newark saying there was no service connection because there are no medical records, I'm sure many other people can come up with the same type of response. MR. and have Board of evidence claim. McMICHAEL: If you have filed a claim for compensation been turned down, I would urge you to appeal it to the Veterans. Appeals and urge you to submit whatever you believe you have that relates directly to that MR. GANDENBERGER: Thank you. �ADDITIONAL QUESTIONS FROM THE PUBLIC RECEIVED AFTER THE MEETING The following questions were received from Rayma D. Whited Plummer, the Brotherhood of Vietnam Veterans, Inc., Austin, Texas: Interagency Work Group 1. "What were your results on the male mice study?" Attached is a copy of the report's abstract. 2. "Why wasn't the study of male mice by Dr. Lars Davring in 1977 considered? (Hereditas, 80:255-262 (1975) (Inst. Genestics - University of Lund Sweden))." The study by Dr. Lars Daving in Heriditas 80:255-262 (1975) is entitled "Effects of a 2,4,5-T Ester On Early Oogenesis, Fertility and Development in Drosophila Melanogaster." The study utilized the female fruit fly, not male mice. Dr. Moore scanned the article and found it does not talk about or refer to mice. 3. "Why did Dr. Moore tell me that he wasn't aware of any other male mice study?" Dr. Moore is not aware of any other male mice studies. 4. "Why was the VA study of the fatty tissues of veterans and controls rejected and what made it unable to comprehend? (as stated by Dr. Moore)" Attached is a copy of the Science Panel's report on these studies which provides the information you request. 5. "Have you received a copy of the Dow Chemical Co. reproductive study on exposure to 2,4,5-T (and/or Agent Orange)? If so, what are its findings?" Dr. Moore is aware of a study performed by Dow Chemical scientists entitled "Three Generation Reproductive Study of Rats Given 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in the Diet" which appeared in Toxicology and Applied Pharmacology 50: 241-252 (1979). A copy of the abstract which summarized the study findings is attached. 6. ."As per questioned (sic) in my previous letter, what are the effects of contamination when a person receives a wound such as a punji stick with artery involvement? �Since the punji stick and the animal exrement it was dipped in would both be contaminated by dioxin, the dioxin would therefore get directly into the main blood stream thru artery involvement in the wound. Once it is in the blood stream, where does the dioxin go? To the fatty tissues or where? And what happens once in the blood stream?" Dr. Moore cannot factually respond to your query since there is no data relevant to your question. Is your assumption that a punji stick would be contaminated with dioxin verifiable? One can speculate that dioxin entering the blood stream of a nan would rapidly clear and partition into the fat and/or liver. 7. "Have you attempted to produce a duplicate batch of Agent Orange and study it? If so, what were your findings? If not, why haven't you?" The male mouse study by Lamb, et al, was performed using a mixture of 2,4-D, 2,4,5-T and TCDD that simulated Agent Orange. 8. "What are your statistics on deaths suspected to be related to Agent Orange or exposure to 2,4,5-T and 2,4-D? What are the statistics on birth defects from exposure to Agent Orange or 2,4,5-T and 2,4-D? What are the statistics on the symptoms of exposure (for example: cancer - how many), etc." There are no reliable statistics available for the type of end points you list. It is anticipated that the CDC Birth Defects Study, Air Force Ranch Hand Study and proposed VA study will provide such data. 9. "What are the effects of the antimalarials and Agent Orange when combined?" There is no information available to answer this question. 10. "Why was I asked if ray husband (and many others asked) was given antimalarials? What is it you suspect about the antimalarials?" Dr. Moore does not know why you or your husband were asked if he took antimalarials. There is data that indicated that one of the antimalarial drugs was associated with an increased incidence of cancer in animal studies. � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5772 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Summary Report of the Public Meeting of the Interagency Work Group to Study the Possible Long-Term Health Effects of Phenoxy Herbicides and Contaminants held September 22, 1980, Washington, D. C. ao_seriesVIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5771 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Letter to Alvin L. Young from F. A. Zacharewicz, June 26, 1980 ao_seriesVIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5770 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Letter to Alvin L. Young from F. A. Zacharewicz, April 19, 1980 ao_seriesVIII https://www.nal.usda.gov/exhibits/speccoll/files/original/02cc894d5079e208581e69bb965a965e.pdf bbc8390039a5c0ce003f7ae775dd899f PDF Text Text Item D Number 05769 G NotScannBd Author Corporate Author Report/Article Title Herbicide Orange Questionalre, Rough Draft Journal/Book Title Year Month/Day Color n Number of Images ° DeSGrlPton NOtBS Accompanied by cover letter from F. A. Zacharewicz to Alvin L. Young, March 21,1980. Thursday, March 28, 2002 Page 5769 of 5780 �Medical Center St. Louis, MO 63125 Veterans Administration March 21, 1980 • Major Alvin L. Young, Ph.D. Herbicide Orange Project Epidemiology Division USAF School of Aerospace Medicine/ESS Brooks AFB, Texas 78235 Dear Major Young: This is the rough draft of the Herbicide Orange questionnaire compiled by our staff with input from Mr. Ronald DeYoung and Mr. Michael Skyer. Please review this and let us have your comments at your earliest convenience. We also intend to go in greater detail at our next content meeting. Sincerely, F. A. Direct Medical :al Regional lucation Center Enclosure In Reply Refer To: 657 /14A-JB �HERBICIDE ORANGE QUESTIONAIRE to: Month: from: Month: Year: 1. Dates of Military Service Year: to: from: 2. Dates of Service 1n Vietnam: Month: Year: Month: from: 3. Dates of other foreign or overseas service: Month; Year: to: Month: Year: Year: 4 Branch of Service: (Circle appropriate letter) a. Army b. Navy c. Air Force d. Marines e. f. Coast Guard Others: Specify 5. Unit served in Vietnam: (Columns II and III are to be used for multiple assignments) COLUMN I COLUMN II COLUMN III a. Division b. Brigade c. Regiment d. Battalion e. Company f. Others: Specify:_ 6. Names of base camps while stationed in Vietnam: Approximate duration of ,stay from: MO: YR: _MO: MO: MO: to: MO: YR: YR MO: YR �7. Field operations while in Vietnam: (List code names Veteran can remember such as Dewey, Canyon, Sherwood Forest, lA-Drang) a. b. c. 8. Identify other oversea operations involving contact with toxic chemicals a. b. c. 9. Which mi-litary corp in Vietnam did veteran serve? (Circle applicable letters) a. b. c. d. e. I Corp II Corp Ill Corp IV Corp Unknown 10. Does veteran recall military operations in defoliated areas? Yes No 11. Was Veteran attached to: (Circle applicable letters) a. b. c. d. e. f. g. h. 1. Air Force Operation Ranch Hand _ Armor Aviation Transporation Infantry Engineers Artillery Chemical Corp Others: Specify 12. Identify Landmarks, firebases or towns near defoliation: (such as Black Virgin Mountain, LZ English, Rock Pile and Firebase Moore) a. b. c. Identify defoliated areas Veteran recalls on or near base camp: (Such as Clean Field of Fire, Dead defoliated areas) a. b. �14. What was the Veteran's military occupation code number: Primary Code _____ (MOS) Secondary Code If Code is unknown give duties or job title. Was Veteran's actual duties different from those identified above. YES NO If YES is checked please specify 15. Did Veteran handle or use chemical weapons while in Vietnam or elsewhere? YES NO If YES*is checked please identify_types of chemical a. Gases: CS CN BZ b. Irrantants c. Insecticides d. Herbicides e. Unknown Identify method of contact with chemicals a. Bulk chemicals b Spray operations c. Operations in recent contaminated areas 16. Does veteran recall any exposure to toxic chemicals? YES: NO: If YES is checked please identify types of chemical a. Gases: CS CN~~~ BZ b. Irrantants c. Insecticides d. Herbicides e. Unknown �17. "Identify methods of exposure to chemicals a. Direct contact with bulk chemicals b. Direct contact with spray chemicals. c. Contact with recently sprayed plants. d. Contaminated foods e. Contaminated water f. Digging in contaminated soil. 18. A. Identify the type of spraying operations used in Vietnam a.. Fixed wing aircraft b. Helicopters c. Truck mounted sprayers d. Hand held spraying equipment e. Others: Specify B. If fixed wing aircraft was used, was it a. Silver in color b. Camoflouge painted c. Unknown (does not remember) C. Identify approximate time of day of fixed wing spraying operation a. Early morning b. Mid-day c. Late afternoon d. After dark �19. Identify Veteran's frequency of exposure (Total number of days exposed, such as guard duties every third night on perimeter and duration of stay in defoliated areas) a. One to ten days b. Eleven to twenty days c. Twenty-one to thirty days d. Thirty-one to forty days e. Forty-one or more days f.. Unknown 20. Does veteran describe exposure as a. Severe b. Moderate c. Mild d. Uncertain 21. Did Veteran eat locally grown fruits or crops YES NO If YES is checked please circle applicable letters a. Mangos, papayas, bananas, plantain b. Sweet potatoes c. Rice 22. Did Veteran locally obtained meats or fish YES NO If YES is checked identify type of a. Meats b. Fish c. Shell fish d. Wild game �23 ''HOW often did Veteran eat foods obtained locally a. Frequently b. Occasionally c. Not at all „ Does Veteran recall tne sources of his drinking water . a. Base camp 1. River 2. Stream 3.' Unknown source b. Field 1. Rivers 2. Stream __ 3. Paddies 4. Lakes _ 5. Ponds - 6. Shelled craters 7. Collected rain water 8. Others: Specify ___ �25. Did the veteran experience Before going to Vietnam While in Vietnam a. Skin rash Blisters,boil or running sore hives* others b. • Frequent headaches c. Stomach ulcers t e. Severe diarrhea f. Jaundice g. Nervous system Psychological problems (see VA Form 10-20681 for additional symptoms) 26. Did the veteran seek medical care while in Vietnam YES NO If YES was check identify medical personnel a. Corpsmen/aid station b. nurse/physician at field hospital c. 27. Others: Specify Identify "reasons" for seeking medical care a. wounds b. other injuries c. Illness Within two years after return from Vietnam More than two years after return from Vietnam �28. Provide (complete) the following: Approx.dates of tr: Symptoms of illness: Where treated: (such as 24th Evac. Hos.,lst Infantry, Division Hospital, Battlion aid Station) 29 Was Treatment effecti ve: Identify the type of anti-malarial medication the veteran received a. Big orange pill/Chlorogainc/Primaquine b. Big pink pill/Chloroquine c. Little white pill/Dapsone d. Others/unknown 30. Did Veteran take other prescribed drugs while in Vietnam? YES NO If YES was checked list name of medications 31. Identify other substances such as non-tobacco cigarettes or other non-prescribed drugs. (This is an attempt to identify illegal or controlled substances used by veterans while in Vietnam) �32. Identify alcohol usage (Responses are optional) Before going to Vietnam While in Vietnam After leaving Vietnam a. Heavy b. Moderate c. Light d. None 33. Does the veteran recall ever working in an occupation where chemicals were used? Before going to Vietnam While in Vietnam After leaving Vietnam Type of chemical if known 34. Does Veteran recall being exposed to chemicals around the house and/or farm? (Circle appropriate letters) a. Lawn weed killers b. Pesticides c. Insecticides d. Agriculture chemicals 35. Identify Veterans's use (Circle appropriate letter) a. Heavy b. Moderate c. Light d. None e. Ex-smoker � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5769 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Herbicide Orange Questionaire, Rough Draft ao_seriesVIII Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 205 Folder The folder containing the original item. 5768 Series The series number of the original item. Series VIII Subseries III Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Letter from F. A. Zacharewicz to Alvin L. Young, March 7, 1980 ao_seriesVIII