1 15 3 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. 157 Folder The folder containing the original item. 4758 Series The series number of the original item. Series VII 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 Waters, Michael D. Shahbeg S. Sandhu Vincent F. Simmon Kristien E. Mortelmans Ann D. Mitchell Ted A. Jorgenson David C. L. Jones Ruby Valencia Neil E. Garrett Title A name given to the resource Study of Pesticide Genotoxicity https://www.nal.usda.gov/exhibits/speccoll/files/original/7aa48f39dd3e63ec3e115d276f12253e.pdf 3693e7c0ebde2fac53f6a00684077d6c PDF Text Text Item D Number °3063 Author Simmon, Vincent F. Corporate Author Stanford Research Institute ROUOrt/ArtlGlB Titto Evaluation of Selected Pesticides as Chemical Mutagens: In Vitro and In Vivo Studies n NotScannad Journal/Book Title Year 1977 Month/Day May Color D Number of Images 253 Descrtoton Notes Contract No. 68-01-2458 Tuesday, November 13, 2001 Page 3063 of 3112 �U.S. HEPARTMEHT OF COMMERCE National Technical Information Service PB-268 647 Evaluation of Selected Pesticides as Chemical Mutagens 'In vitro' and 'In vivo' Studies Stanford Research Institute, Menlo Park, Calif Prepared for Health Effects Research Lab, Research Triangle Park, N C May 77 �EPA-600/1-77-Q28 May 1977 •Environmental Health Effects Research Series PB 2BB 647 EVALUATION OF SELECTED PESTICIDES AS ^ CHEMICAL MUTAGENS In Vitro and In Vivo Studies 5 4 REPRODUCED By NATIONAL TECHNICAL „ ,..„,'.' " " "u i • * INFORMATION SERVICE Health Effects Research Laboratory /•,>• •n i I n I . SPRINGFIELD. VA. 22161 Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, North Carolina 27711 U. S DEPARTMENT OF COMMERCE n �RESEARCH REPORTING SERIES Research reports of the Office of Research and Development, U S. Environmental , Protection Agency, have been grouped into nine series These nine broad categories were established to facilitate further development and application of environmental technology Elimination of traditional grouping was consciously planned to foster technology transfer and a maximum interface in related fields. The nine series are: 1 Environmental Health Effects Research 2. Environmental Protection Technology 3. Ecological Research 4. Environmental Monitoring 5. Socioeconomic Environmental Studies 6. Scientific and Technical Assessment Reports (STAR) 7. In'eragency Energy-Environment Research and Development 8. 'Special" Reports 9. Miscellaneous Reports This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RESEARCH series This series describes projects and studies relating to the tolerances of man for unhealthful substances or conditions. This work is generally assessed from a medical viewpoint, including physiological or psychological studies in addition to toxicology and other medical specialities, study areas include biomedical instrumentation and health research techniques utilizing animals — but always with intended application to human health measures. This document is available to the public through the National Technical Information Service. Springfield. Virginia 22161 �TECHNICAL REPORT DATA (Pt.'asc read Instructions oil the rftrrse before completing) 1 REPORT NO. ORT NO. J2. EPA -600/1-77-028 3. RECIPIENT S ACCESSION-NO. I 4. T I T L E AND S U B T I T L E 5. REPORT DATE EVALUATION OF SELECTED PESTICIDES AS CHEMICAL MUTAGENS In Vitro and In Vivo Studies 7. AUTHOR(S) May 1977 6. PERFORMING O R G A N I Z A T I O N CODE 8. PERFORMING ORGANIZATION REPORT NO LSU-3493 Vincent F. Simmon, Ann D. Mitchell and Ted A. Jorgenson 9. PERFORMING ORGANIZATION NAME AND ADDRESS 1C. PROGRAM ELEMENT NO. Stanford Research Ins'itute Menlo Park, California 94025 1EA615 11. CONTRACT/GRANT NO. 68-01-2458 12. SFONSORING AGENCY NAME AND ADDRESS 13. TYPE Of REPORT AND PERIOD COVERED Health Effects Research Laboratory - RTP, NC Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, N.C. 27711 14. SPONSORING AGENCY CODE 600/11 15. SUPPLEMENTARY NOTES 16. ABSTRACT Twenty pesticides being reviewed as part of the EPA Substitute Chemical Program were studied for mutagenic activity by several in vitro and in vivo test procedures. The pesticides reviewed were: monocrotophos, bromacil, cacodylic acid, captan, chlorpyrifos, dinoseb, DSMA, r'enthion, folpet, azinphos-methyl, tnalathion, methomyl, monuror., MSMA, parathion, patathion-methyl, quintozene (PCNB), phorate, simazine, and trifluralin. Ten of the twenty compounds were evaluated in vivo by the mouse dominant lethal test. All twenty compounds were tested in vitro. None of the ten compounds tested in the mouse produced a dominant lethal response. Ten of the twenty compounds were mutagenic i.i one or more in vitro assays. Two were mutagenic in all of the in vitro assays: capcan and folpet. KEY WORDS AND DOCUMENT ANALYSIS DESCRIPTORS b.lDENTIFIERS/OPEN ENDED TERMS RELEASE TO PUBLIC COSATI |-'icld/ClOUp 06 F 06 T pesticides In vivo analysis in vitro analysis mutagens 3. [ J I S T R I 8 U T I O N S T A T E M E N T C. 19. SECURITY CLASS <This Report) UNCLASSIFIED 2O. SECURITY CLASS IThiSpagt) EPA Form 2220-1 (9-73) I . 21. NO. OF PAGES �EPA-600/1-77-028 May 1977 EVALUATION OF SELECTED PESTICIDES AS CHEMICAL MUTAGENS In Vitro and In Vivo Studies By Vincent F. Simmon, Ann D. Mitchell, and Ted. A. Jorgenson Stanford Research Institute Menlo Park, California 94025 Contract No. 68-01-2458 Project Officer Michael D. Waters Environmental Toxicology Division Health Effects Research Laboratory Research Triangle Park, N.C. 27711 U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF RESEARCH AND DEVELOPMENT HEALTH EFFECTS RESEARCH LABORATORY RESEARCH TRIANGLE PARK, N.C. 27711 10. �DISCLAIMER This report has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. ii �FOREWORD The many benefits of our modern, developing, industrial society are accompanied by certain hazards. Careful assessment of the relative risk of existing and new man-made environmental hazards is necessary for the establishment of sound regulatory policy. These regulations serve to enhance the quality of our environment in order to promote the public health and welfare and the productive capacity of our Nation's population. The Health Effects Research Laboratory, Research Triangle Park, conducts a coordinated environmental health research program in toxicology, epidemiology, and clinical studies using human volunteer subjects. These studies address problems in air pollution, non-ionizing radiation, environmental carcinogenesis and the toxicology of pesticides as well as other chemical pollutants. The Laboratory develops and revises air quality criteria documents on pollutants for which national ambient air quality standards exist or are proposed, provides the data for registration of new pesticides or proposed suspension of those already in .»se, conducts research on hazardous and toxic materials, and is preparing the health basis for non-iouizing radiation standards. Direct support to the regulatory function of the Agency is provided in the form of expert testimony and preparation of affidavits as well as expert advice to the Administrator to assure the adequacy of health care anj surveillance of persons having suffered imminent and substantial endangeraent of their health. This report describes the testing of a series of twenty technical grade pesticide chemicals for genotoxic properties by use of a battery of in vitro and in vivo methods. The battery includes tests for gene and chromosomal mutations and primary damage to DNA as measured by effects on DNA repair recombination. Since DNA is chemically similar in all species, test results from a variety of ceils and organisms are relevant in assessing the potential genetic hazard of pesticide chemicals in humans. John H. Knelson, M.D. . Director, Health Effects Research Laboratory iii �ABSTRACT Twenty pesticides being reviewed as a part of the EPA Substitute Chemical Program were studied for mutagenic activity by several in vivo and in vitro test procedures. Ten of the twenty compounds were evaluated in vivo by the mouse dominant lethal test. All twenty compounds were tested by the following in vitro procedures: Unscheduled DNA synthesis (UDS) in human fibroblasts (WI-38 cells); reverse mutation in Salmonella typhimuriuis strains TA1535, TA1537, TA1538, and TA100 and in Escherichia coli WP2; mitotic recombination in the yeast Saccharomyces cerevisiae D3; and preferential toxicity assays in DNA repairproficient and -deficient strains of E^. coli (strains W3110 and p3478, respectively) and Bacilluj subtilis (strains H17 and MAS, respectively). None of the ten compounds tested in the mouse produced a dominant lethal response. Ten of the twenty compounds were mutagenic in one or more ill vitro assays. Two were mutagenic in all of the in vitro assays; captan and folpet. In a heritable translocation study in mice, under the experimental procedures employed, captan at 5000 ppm in the diet of male mice for 8 consecutive weeks produced a heritable mutagenic event in F^ generation male mice. iv �CONTENTS LIST OF TABLES ill SUMMARY 1 TWENTY PESTICIDES EVALUATED BY SRI FOR MUTAGENIC ACTIVITY 3 INTRODUCTION 4 DOMINANT LETHAL TEST IN THE MOUSE 6 General Experimental Animals and Chemicals Determination of Acute Toxicity Maximum Tolerated Dcse Study Treatment Levels Administration of the Compounds Test Groups Necropsy and Evaluation Results and Discussion , MAMMALIAN HI VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS General Experimental Cell Culture Dilution of Compounds Controls UDS Assays Interpretation of Results MICROBIOLOGICAL ASSAYS 6 6 6 7 7 7 8 8 9 9 12 12 13 13 13 13 14 15 17 General 17 Experimental 18 Salmonella typhimurium Sfains TA1535, TA1537, TA1538, and TAiOO 18 Escherichia coli WP2 19 Escherichia coli W3100/p3478 and Bacillus subtilis H17/M45 . 20 Saccharomyces cerevlsiae P^ 21 Aroclor 1254-Stimulated Mai.abolic Activation System ... 22 Results and Discussion 22 DISCUSSION 25 REFERENCES 26 APPENDIX A: Mutagenasis Studies of Pesticide Compounds - Mouse Heritable Translocation Test - Captan 171 �TABLES DOMINANT LETHAL TEST 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Chl-Square Test of the Fertility Index - Monocrotophos .... Average Implants per Pregnant Female - Monocrotophos . . . . . Average Dead Implants per Pregnant Female - Monocrotophos. . . Chi-Square Test of the Dsath Index - Monocrotophos Number cf Dead Implants per Total Implants - Monccrotophos . . Chi-Square Test of the Fertility Index - Bromacil Average Implants per Pregnant Female - Broraacil . Average Dead Implants per Pregnant Female - Bromacil Chi-Square Test of the Death Index - Brcmacil .... Number of Dead Implants per Total Implants - Bromacil Chi-Square Test of the Fertility Index - Captan Average Implants per Pregnant Female - Captan Average Dead Implants per Pregnant Female - Captan Chi-Square Test of the Death Index - Captan Number of Dead Implants per Total Implants - Captan Chi-Square Test of the Fertility Index - Folpet . Average Implants per Pregnant Female - Folpet Average Dead Implants per Pregnant Female - Folpet Chi-Square Test of the Death Index - Folpeto Number of Dead Implants per Total Implants - Folpet Chi-Square Test of the Fertility Index - Azinphos-Methyl ... Average Implants per Pregnant Female - Azinphos-Methyl . . . . Average Dead Implants per Pregnant Female - Azinphos-Methyl. . Chi-Square Test of the Death Index - Azinphos-Methyl Number of Dead Implants per Total Impxants - Azinphos-Methyl . Chi-Square Test of the Fertilitv Index - K_lathion . . . . . . Average Implants per Pregnant Temale - Malathion Average Dead Implants per Pr<gnant Female - Malathion Chi-Square Test of the Death Index - Malathion Number of Dead Implants per Total Implants - Malathion .... Chi-Square Test of the Fc.vility Index - Parathion Average Implants per Pregnant Female - Parathion Average Dead Implants per Pregnant Female Chi-Square Test of the Death Index - Parathiou Number of Dead Implants per Total Implants - Parathion . . . . Chi-Square Test of the Fertility Index - Parathion-Methyl. . . Average Implants per Pregnant Female - Parathion-Methyl. ... Average Dead Implants per Pregnant Female - Parathion-Methyl . Chi-Square Test of the Death Index - Parathion-Methyl Number of Dead Implants per Total Implants - Parathion-Methyl. Chi-Square Test of the Fertility Index - Quintozene (PCNB) . . Average Implants per Pregnant Female - Quintozene (PCNB) ... vi 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 �43 44 45 46 47 48 49 50 Average Dead Implants per Pregnant Female Quintoaene (PCNB) Chi-Square Test of the Death Index - Quintozene (PCNB).... Number of Dead Implants per Total Implants Quintozene (PCNB) Chi-Square Test of the Fertility Index - Phorate Average Implants per Pregnant FemaL. - Phorate Average Dead Implants per Pregnant Female - Phorate Chi-Square Test of the Death Index - Phorate Number of Dead Iw lants por Total Implants - Phovate 70 71 72 73 74 75 76 77 UDS ASSAYS 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 DNA Repair Synthesis Assay of Monocrotopbos 78 DNA Repair Synthesis Assay of Monocrotophos with Metabolic Activation 79 DMA Repair Synthesis Assay of Bromacil 80 DNA Repair Synthesis Assay of Bromacil with Metabolic Activation 81 DNA Repair Synthesis Assay of Cacodylic Acid. . . . . . . . . 82 DNA Repair Synthesis Assay of Cacodylic Acid with Metabolic Activation 83 DNA Repair Synthesis Assay of Captan 84 DNA Repair Synthesis Assay of Captan with Metabolic Activation 85 DNA Repair Synthesis Assay of Chloropyrifos 86 DNA Repair Synthesis Assay of Chloropyrifos with Metabolic Activation 87 DNA Repair Synthesis Assay of Dinoseb 88 DNA Repair Synthesis Assay of Dinoseb with Metabolic Activation 89 DNA Repair Synthesis Assay of DSMA 90 DNA Repair Synthesis Assay of DSMA with Metabolic Activation 91 DNA Repair S>nthesis Assay of Fenthion 92 DNA Repair Synthesis Assay of Fenthion wi th Metabolic Activation 93 DNA Repair Synthesis Assay of Folpet 94 DNA Repair Synthesis Assay of Folpet wich Metabolic Activation 95 DNA Repair Synthesis Assay of Aziuphos-Methyl 96 DNA Repair Synthesis Assay of Azinphos-Methyl with Metabolic Activation 97 DNA Repair Synthesis Assay of Malathion 98 DNA Repair Synthesis Assay of Malathion with Metabolic Activation. . . . . 99 DNA Repair Synthesis Assay of Methomyl. 100 PNA Repair Synthesis Assay of Meuhomyl with Metabolic Activation 101 DNA Repair Synthesis Assay of Monuron 102 vii �76 77 78 79 80 81 62 83 84 85 86 67 88 89 90 DNA Repair Synthesis Assay of Monuron with Metabolic Activation DNA Repair Synthesis Assay of MSMA DNA Repair Synthesis Assay of MSMA with Metabolic Activation. •,..'...'. DNA Repair Synthesis Assay of Parathion DNA Repair Synthesis Assay of Paraf^-.jn with Metabolic Activation DNA RepcJ.r Synthesis Assay of Paiathicn-Msthyl DNA Repair Synthesis Assay of Parathion-Methyl with Metabolic Activation DNA Repair Synthesis Assay of Quintozene (PCNB) DNA Repair Synthesis Assay of Quintozene (PCN3) with Metabolic Activation DNA Repair Synthesis Assay of Phorate DNA Repair Synthesis Assay of Phorate with Metabolic Activation DNA Repair Synthesis Assay of Simagine DNA Repair Synthesis Assay of Sitna/ine with MetaboT'.c Activation DNA Repair Synthesis Assay of Trifluralin DNA Repair Synthesis Assay of Trifluralin with Metabolic Activation 103 104 105 106 107 2; '3 109 110 Ill 112 113 114 115 116 ' 117 MICROBIOLOGICAL ASSAYS 91 92 93 94 95 96 97 98 99 100 101 102 lf>3 104 In Vitro Assays with Salmonella typhimurium Results of Assays with Escerichia coli WP2 Microbial Inhibition in Escerichia colj. and Bacnius sub til is In Vitro Assays with Saccharomyces cercvisiae Monocrotophos In Vitro Assays with Saccharomyces cerevisiae L^omacil Ir. Vitro Assays with Saccharomyces cerevisiae Cacodylic Acid '. In Vitro Assays with Saccharomyes carevisiae Captan In Vitro Assays with Saccharomyces cerevisiae Chloropyrifos In Vitro Assays with Saccharomyces cerevisiae Dinoseb . In Vitro Assays with Saccharomyces cerevisiae In Vitro Assays with Saccharomyces crreviaiae Fenthion In Vitio Ar-scyR with Saccharomyces cerevisiae Folp^-t ~ In Vitro Assays with Sacrharomyces cerevisiae Azinphos-Methyl In Vitro Assays w<th Sacch?romyces cereviaiae Malathion vi.i 118 . 138 148 D3 150 D3 151 D3 352 D3 153 D3 154 D3 155 D3 - DMSA . . . 156 D3 157 D3 158 D3 159 D3 160 �105 106 107 108 109 110 111 112 113 114 In Vitro Assays with Methomyl _In Vitro Assays with Monuron In Vitro Assays with In Vitro "assays with Parathion In Vitro Assays with Parathion-Methyl In Vitro Assays with Quintozene (PCNB) In Vitro Assays with Phorate In Vitro Assays with Simazint In Vitro Assays with Trifluralin In Vitro Mutagenesis Saccharomyces cerevisiae D3 161 Saccharomyces cerevisiae D3 162 Saccharomyces cerevisiae D3 - MSMA . . . 163 Saccharomyces cerevisiae D3 164 Saccharomyces cerevisiae D3 165 Saccharoayces cerevisiae D3 166 Saccharomyces cerevisiae D3 167 Saccharomyces cerevisiae D3 168 Saccharomyces cerevisiae D3 169 with Salmonella typhimurium 170 ix �ACKNOWLEDGMENTS Stanford Research Institute wishes to thank the EPA project officers for their guidance and assistance during the course of this project. Dr. Robert E. KcGaughy, Office of Research and Development, Washington, D.C., was Project Officer from the beginning of trie project in June 1974 until July 1975. Project responsibility then was transferred to Dr. Ronald L. Baron, Health Effects Research Laboratory, Research Triangle Park, North Carolina, until January 1976. At that time, Dr. Michaal D. Waters, now Chief of the Biochemistry Branch in the Environmental Toxicology Division, Health Effects Research Laboratory, Research Triangle Park, North Carolina, became Project Officer. Dr. Waters has continued in this project responsibility to the present. �INTRODUCTION The Federal Insecticide, Fungicide, and Rodenticide Act designates the Environmental Protection Agency as the governmental body responsible for the safety of all pesticides used in the United States. More recently, the Federal Environmental Pesticide Control Act (PL 92-516) strengthened EPA's regulatory responsibilities in the area of pesticides to include intra- as well as inter-state commerce. To be federally registered, a pesticide must have been determined not to be hazardous to health or to the environment when used according to its labeling restrictions. Thus, relative to new law as well as to specific directives included in Public Law 93-135, 1973, EPA now is conducting a thorough review of the implications of using alternate chemicals, including older registered pesticides, for pest control. In the pesticide review process, EPA emphasizes development of scientific criteria for evaluating the safety of compounds substituted for those pesticides found to be hazardous. In addition to reviewing and evaluating the literature on pesticides and maintaining liaison with industry and academia, the strategy program includes laboratory studies to obtain additional data. One of these laboratory programs is directed toward gathering mutagenesis data on a selected number of compounds. EPA's program is timely and responsive to one of the recommendations included in the President's Scientific Advisory Committee Report of September 1973, Chemicals and Health. In that document, the Committee recommended that "Regulatory agencies should take steps to insure that new scientific data raising the possibility of new or extended hazards from chemicals in use are subject to careful process of scientific review for merit interpretation." Development of methods for evaluating the mutagenic hazard of chemical compounds has advanced markedly in the last few years. In contrast to the undefined empirical tests used a short time ago, procedures now �available can detect chromosome breaks and other genetic changes caused by chemical stress. Mutant strains of microorganisms in cell culture and mammalian fibroblast cells in tissue culture are effective in vitro systems for reliable detection of presumptive gene mutations, whereas the mammalian dominant lethal test is a recognized test for the assessment of chromosome damage to germinal cells.' Today many pesticide chemicals in commercial use have not been / invi. tigated adequately for their mutagenic hazard. With the public's increasing concern about possible pollution of our environment by chemicals, the widely used pesticides must be evaluated. In this project, SRI used test methods that are appropriate for these evaluations and that are in use by the scientific community. Under contract to EPA, SRI examined 20 pesticides for mutagenic activity using a combintation of in vivo and in vitro mutagenicity assay systems. The 20 pesticides tested and their sources are listed in the' following two tables. The assays used were the dominant lethal test in mice (only ten compounds); unscheduled DNA synthesis (UDS) in human fibroblasts (WI-38 cells); reverse mutation in Salmonella typhimurium strains TA1535, TA1537, TA1538, and TA100 and in Escherichia coli WP2; mitotic recombination in the yeast Saccharomyces cerevisiae D3; and preferential toxicity assays in DNA repair-proficient and -deficient strains of IJ. coli (strains W3110 and p3478, respectively) and Bacillus subtilis (strains H17 and M45, respectively. Based on positive responses in both Tier I (in vitro test) and Tier II (Drosophila) mutagenic studies, it was recommended that a heritable translocation test (Tier TII) in the mouse be conducted �I to further assess therniitagt..ii;potential of Captan. The results of these further studies are reported as Appendix A. The experimental procedures and results for the mammalian dominant lethal test, the 'JDS assay, and the microbiological assays are described in the separate suctions that follow. �IN VIVO AND IN VITRO MUTACENESIS: SUMMARY DATA FOR EPA PESTICIDES Positive Response, +; Negative Response, - Mouse Salmonella Dominant Pesticide Lethal* typhlmurlunt Escherlchla coll WP2 Saccharomyces cerevistae (His* Reversion) (Try* Reversion) (Mltotic Recombination) -MA Monocrotophos Bromacll Cacodyllc AcH Captan Chlorpyrllos Dinoseb DSMA +MA -MA +«A -MA - - - + + + + - 4- 4 - - . 4 - - . •(• - Fenthlon - + _ _ . - UDS - _ - 4> + 4. . + 4 4 . (DNA Repair) - - - - - - . . . . . _ - - + 4 . _ . - 4 - . _ + _ _ - . . - . - - - - - - - 4 Parathlon-methyl Qutntozene (PCNB) - - - - - 4 > * + t - - . - Phorate - . - ' Trlfluralln . _ . _ - - - _ • - * Only ten pesticides were tested by the dominant lethal procedure. t See page 170. t Marginally positive. - - - - - - . - . - - 4 . , - - - . + . - - - - - 4. - - . + + - - + - +KA 4 . 4 . - Parathlon Slmazlne . + -MA + _ . + - - - - + - - ' _ + - - Methoaiyl Monuron MSMA - - Folpet Azinphos-methyl Malathlon Bacillus subtil is (Relative Toxlctty) 4MA - - E«chertehla coll (Relative ToxlcUy) _ - . _ _ . �TWENTY PESTICIDES EVALUATED BY SRI FOR MUTAGENIC ACTIVITY Common Name* Monocrotophos Bromacil Cacodylic Acid Captan Chlorpyrifos Dinoseb DSMA Fenthion Folpet Azinphos-methyl Malathion Methorny1 Monuron MSMA Parathion Parathion-methyl Quintozene (PCNB) Phorate Simaztne Trifluralin Trade Name of Compound Tested Azodrin-5 Manufacturer Batch or Lot Number Purity </0 Supplier 55.0 Manufacturer Shell Chemical Company Batch H, 9-SCL-77 Hyvar E.I. DuPont de Nemours T80619/40 Phytar Ansul Chemical Company Phyton 138 Orthoside 406 Chevron Chemical Company 5X640 Dursban Dow Chemical Company MM-1114-1 (603-D1) Dow Chemical Company Premerge MM 200554 Ansar Ansul Chemical Company 8100 Baytex Chemogro 4-15-2026 Phaltan Chevron Chemical Company SX579 Chemogro Guthion 411-0229 Malathion American Cyanamid Company 40216006.300 Lannate E.I. DuPont de ilemours 6602-82 Telvar E.I. PuPont de Nemours T-40817-20 Ansul Chemical Company Ansar 170 H.C. Monsanti Chemical Company AD 1236 Niran Methyl Parathion Monsanto Chemical Company AD 0659 Olin Mathieson Chemical Terrachlor Technical Corporation American Cyanamid Company MC85 Thimet Primatol Ciba-Geigy Chemical Co. FL-740846 Eli Lillv & Company Treflan X-26290 95.9 65.6 Technical 98.8 Battelle Battelle Battelle Battelle 97.7 80.1 9o.O Technical iyc'nr.lc.al Technical 99.0 97.0 58.4 99.0 80.0 99.0 Battelle Battelle Battelle Battelle Battelle Eattelle Battelle Battelle Battelle Battelle Battelle Battelle 97.7 97.7 Battelle Battelle. Battelle * Common name as approved by the International Organization for Standardization. �DOMINANT LETHAL TEST IN THE MOUSE General In Che dominant lethal test, the ten compounds under investigation were fed in the diet to proven male breeder mice for 7 weeks. After this period, each male was mated with two adult virgin females for 7 days; these females were then replaced by two others for another breeding. The sequence was continued for 8 weeks. This procedure emphasizes possible mutagenic effects on the male sperm, the normal female acting as a carrier to reveal in her offspring abnormalities that may have occurred in the male. We evaluated effects by examining the condition and state of fetal development during the middle to latter stages of gestation. Experimental Animals and Chemicals Adult ICR/SIM mice from a closed, random-bred colony were used for the acute toxicity and maximum tolerated dose determinations as well as for the dominant lethal assay. These male and female mice were supplied by Simonsen Laboratories, Gilroy, California. The males were 3- to 4-month-old proven breeders, and the females were 10- to 12-week-old virgin stock. At the direction of EPA, the Battelle Columbus Laboratories obtained the pesricides from the manufacturers and subsequently provided SRT. with aliquots for the studies reported here. Each pesticide was a "technical" grade product (or equivalent) and was provided in sufficient quantity for us to complete all aspects of the experimental program. Excess supplies were refrigerated or frozen, should they be needed for future reference. �We investigated the solubility of each compound using water, propylene glycol, polyethylene glycol, corn oil, or carboxymethylcellulose to determine the most appropriate vehicle for administration. Compounds were administered orally, by gavage for the acute toxicity (LDcQ) detPtminations, and via the diet for the maximum tolerated dose and dominant lethal studies. Determination of Acute Toxicity Although acute toxicity information on some of the compounds was available in the literature, we conducted confirmatory tests on all Lo obtain an LD__ under our laboratory conditions and for the ICR/SIM strain of mouse. If no data were available, we conducted a preliminary range-finding test, followed by a determination of the oral LD^Q. Maximum Tolerated Dose Study Based on the acute toxicity data and available information from the literature on dose levels known to cause adverse responses when administered in the diet, several dose levels were selected and administered in the diet to adult male mice for 2 weeks. Treated males then were caged with two adult virgin females each for 7 days; these females were replaced by two others weekly for 2 weeks. The females were examined daily for the presence of vaginal (mating) plugs. At midterm of pregnancy, the females were sacrificed and examined for total implants, as well as for early and late fetal deaths. For this work, we defined a maximum tolerated dose as that dietary level which may produce u^ to a 20% weight loss, mild but transient clinical signs, no inhibition of breeding performance, and no mortality. Thus, these initial studies provided information on changes in body weight, acceptability of the diet, clinical signs, mortality, and breeding performance. Treatment Levels For the dominant-lethal study, three dose levels were administered. The highest was the maximum tolerated dose or 5 g/kg (a maximum level �agreed on by EPA and SRI), whichever was lower. The intermediate and lower dosages were one-half and one-quarter of the highest dose, respectively. Administration of thu Compounds Each pesticide was fed in the diet to adult male mice for 7 weeks. An appropriate amount of compound initially was dissolved or suspended \ in corn oil; then the compound-oil^concentrate was added at a level of 3% \ to a finely ground commercial diet of .known composition. The use of corn oil assured even distribution of the compound and prevented stratification of the test material in an otherwise dry diet. Diets were prepared at 2-week intervals and were refrigerated at 4°C until fed to the animals. Fresh diet was placed in the feed containers every ot~h'er~"day- ...._ to minimize the loss of compound through instability or volatility. Test Groups Two reference control groups were included in this project. One was run at the beginning of each of the two dominant lethal series, five pesticides being ran concurrently. In this manner, reference breeding and implant data were obtained at two time periods, as was information on each shipment of research animals. Males in these groups were fed a finely ground commercial diet supplemented with corn oil at 3%. Control groups were treated in the same manner as the compound test groups. Two positive control groups were run concurrently with each of the two series of five pesticide tests. For these groups, the known mutagen triethylenemelamine (TEM) was administered as a single intraperitoneal injection of 0.2 mg/kg approximately 2 hours before the first mating. A commercial pelleted diet was available at all times. Each control and experimental test group contained 20 adult male mice. At the end of the 7-week compound treatment period, each male was allowed to breed with two virgin females over a period of 7 days. Females were replaced weekly for 8 weeks. 8 �Necropsy and Evaluation Females were sacrificed at midterm of pregnancy. A complete necropsy was performed to determine if an intercurrent infection was present; such a condition can induce preimplantation loss and early fetal deaths. At sacrifice, each female was scored for early fetal deaths, late fetal deaths, and living fetuses (all of which provide a total implant score). The following parameters indicate effects in dominant lethal studies: Total implants (live fetuses plus early and late fetal deaths), total dead (early and late fetal deaths), and dead implants per total implants. Total implants and dead implants were analyzed for significance by the t-test. The index of dead implants per total implants was analyzed statistically by the t-test on arcsine- (or angular) transformed data, as described in Experimental Design (Theory and Application). index was computed for each female. fertility and death indices. Results and Discussion Single-dose oral acute toxicity data are as follows: Compound LD50 Monocrotophos 17 mg/kg Bromacil 3.04 g/kg Captai. > 15 g/kg Folpet > 10 g/kg Azinphos-methyl 15 mg/kg Halathion 1196 mg/kg Parathion This Other parameters analyzed were the ' 17 mg/kg Parathion-methyl 39 mg/kg Quintozene (PCNB) > 10 g/kg Phorate 6.59 mg/kg �After evaluating the acute toxicity data and those from subsequent maximum tolerated, dose studies, we selected the following dosage levels for the dominant lethal studies: Treatment Levels (mg/kg of Diet) Compound M^nocrotophos Bromacil Captan Folpet Azinphos-methyl Malathion Parathion Parathion-methyl Qulntozene (PCNB) Phorate 15, 30, 60 1250, 2500, 5000 1250, 2500, 5000 1250, 2500, 5000 20, 40, 80 1250, 2500, 5000 62.5, 125, 250 20, 40, 80 1250, 2500, 5000 5, 10, 20 Throughout the experiment, the biological criteria used to evaluate mutagenic effects in the mouse showed no consistent responses that could be attributed to treatment. Although we found occasional statistical differences between control and compound treated groups, they were random and did not suggest a time or dose-response effect. Summary data on the fertility index, implantations per pregnant female, dead implants per pregnant female, death index, and number of dead implants per total implants are presented by compound ao follows: Tables 1 through 5, Monocrotophos; Tables 6 through 10, Broiaacil; Tables 11 through 15, Captan; Tables 16 through 20, Fclpet; Tables 21 through 25, Azinphos-methyl; Tables 26 through 30, Malathion; Tables 31 through 35, Parathion; Tables 36 through 40, Parathion-Methyl; Tables 41 through 45, Quintozene (PCNB); and Tables 46 through 50, Phorate. Two copies of a description of the statistical analysis procedures used for dominant lethal tpsts and computer printouts of the raw data and the statistical analyses are on file with the current Project Officer, Dr. Michael P. Waters, Environmental Toxicology Division, Health Effects Research Laboratory, EPA Environmental Research Center, Research Triangle Park, North Carolina 27711. 10 �The following statistical procedures were used: Chi-square test of the fertility index; Armitage test for ? linear trend in proportion for the fertility index based on dose levels, based on logarithms of the dose levels, and based on dose levels including the control group; t-test of the number of imp1antacions in pregnant females; Regression fits of implantations on dose and log dose and with and without control group included; t-test of the (Freeman-Tukey transformed) preimplantation losses in pregnant females; t-test of the number of dead implants; Chi-square test of the death index; Armitage test for a linear trend in proportion for the death index, based on dose levels with and without control group included and based on logarithm's of the dose levels; Probit analysis of the proportion of pregnant females with one or more dead implants; t-test of the (Freeman-Tukey transformed) number of dead implants (dead implants/total implants); Control group analyses of variances for number of pregnant females, number of implantations per pregnant female, preimplantation loss per pregnant female, number of dead implants per pregnant female, ratio of dead implants to total implants per pregnant female; and t-test of the number oc corpora lutea in pregnant females. Careful review and statistical evaluation of the data show that folpet, captan, parathion-methy1, parathion, phorate, malathion, bromacil, monocrotophos, quintozene (PCNB), and azinphos-methyl are ~f , *y not mutagenic in the mouse by the dominant lethal test. 11 �MAMMALIAN IN VITRO JNSCHEDULED DNA SYNTHESIS ASSAYS General Many mutagenic and carcinogenic agents have been shown to induce unscheduled DNA synthesis (UDS) in an in vitro tissue culture system of mammalian cells. UDS is a form of mammalian repair synthesis that involves at least two processes. The first is interaction of the agent with DNA, resulting in damage of the DNA. The second, which follows, Is incopor- »;tion of nucleotides to repair the DNA. I!DS may be considered a fairly universal system because it occurs in wide variety of mammalian cell types and because it has been observd in all stages of the cell cycle (GQ, -'i, 1?, and M) other than S_, the normal DNA synthetic phase.2'3 (UDS it. not. observed during S_-phase be- cause the high Jevel of incorporation o£ mcleotides dicing the scheduled DNA synthesis obscures the relatively low level of incorporation of nucleotides during unscheduled DNA synthesis.) An additional feature of UDS is that it may detect a level ' f DNA damage higher than that revealed by examination of chromosomeal aberrations'* because some DNA repair results in ll.ttle or no de> ••ctn'ole char f. in chror.jsome morphology. For each compound tested, a:i in vitro metabolic activation system should be incorporated for a parallel series of UDS assays since some compounds may be ineffective in producing DNA damage unless they are first activated by a microsomal preparation from a mammalian liver homogenate. The UDS system we have developed is unique in thai., at the end of each assay, DNA is extracted from human diploid fibroblasts (WI-38 cells) so that the exteii. of repair may be expressed per unit of DNA. Ke have found that this UDS assay system affords sensitivity and precision without sacrificing efficiency or economy. Under separate contact, NCI approved our use and validation of this system for the prescreeniivj; r>f che:..ical carcinogens. With the approval of the EPA project offi, used this system for testing the 20 substitute pesticides, with without metabolic activation. 12 �Experimental Cell Culture WI-38 cells grown in T-25 tissue culture flasks were used for the UDS assays. Replicate cultures of these cells were initiated in Eagle's Basal Medium (BME) containing 10% (v/v) fetal calf serum and anreomycin, an antibiotic specific for PPLO*. For 1 to 2 weeks preceding the UDS assays, the cells were grown in medium containing 0.5% serum. This pro- duced contact-inhibited cells in synchronous cultures in the Gj phase of the mitotic. cyclo. To reduce further the possibility of incorporation 3 of H-TdR by an occasional S-phase cell that might escape the contactiiihibition synchrony and thus obscure measurements of UDS, the cultures were preincubated for 1 hour with 10~2 M hydroxyurea (HU) before each assay, and 10~2 M HU was added during each subsequent step of the assays. Dilution of Compounds Chemicals to be tested were made up immediately before use and were diluted in appropriate solvents (water, ethanol, or DMSO), the final concentration of solvent being one that did not produce a cytotoxic effect after repeated testing. Sonification and pH adjustments were used to ensure maximum solubility or even suspension of the stock solutions of the compounds. The highest concentration was diluted further in solvent and then in culture medium to give several log dilutions of each compound. All compounds were in apparent solution and within the physiological pH range when tested, except as otherwise noted in the tables. Controls The positive controls were 4-nitroquinoline-N-oxide (4NQO), a com- pound that induces UDS in the absence of a metabolic activation system, and dimethylnitrosamine (DMN), a compound that induces UDS only with metabolic activation. The negative controls were the solvents diluted in culture medium. * As an additional check against the presence of PPLO, which could incorporate tritiated thymidine (3H-TdR) and thus ob re measurementr of UDS, stock cultures were analyzed monthly for the presence of PPLO. The results of these analyses wore consistently negative. 13 \ �UPS Assays The contact-inhibited WI-38 cells were incubated at 37°C with log dilutions of the substitute pesticides and with 1 pCi/ml of 3H-TdR (sp act, 6.7 Ci/tnmole). For testing in the absence of metabolic activation, the cells wer.T exposed simultaneously to the substitute pesticide and to 3H-TdR for 3 hours. For testing with metabolic activation, the cells were exposed to the substitute pesticide, to 3H-TdR, and to 500 rag/ml of the 9000 x £ supernatant fraction of a liver homogenate from adult male Swiss-Webster mice, with appropriate cofactors,* for 1 hour; then the cells were incubated with only 3H-TdR for an additional 4 hours. The shorter exposure time for metabolic activation testing was used to preclude cytotoxic effects of the liver homogenate preparation. Both approaches included a postincorp- oration incubation with unlabeled thymidine. DMA was extracted from the cells by a modification of the PCA-hydrolysis procedure;5 one aliquot of the DNA solution was used to measure the DNA content, after reaction with diphenylamine,6 and a second aliquot was used for scintillation counting measurements of the extent of incorporation of 3H-TdR. Results were expres- sed as incorporated per unit of DNA and were compared with the background rate of incorporation. We have defined as an acceptable assay one in which the response of the positive control compound is predicted, within the 95% confidence limits, by regressions of average dpm/yg DNA versus average dpm/pg for background. The regressions that follow are based on data that we have acquired in previous testing: Type of Testing Regression"*" Sample Size (n) Correlation Coefficient (r) Without metabolic activation YI = 696 + 17.45 ( ) X* 48 0.7668 With Metabolic activation Y2 = 263 + 13 0.9639 1.83 ( ) X* *Nicotinamide, 3.05 mg/ml; glucose-6-phosphate, 16.1 mg/ml: MgCl2'6H20, 5.08 mg/ml; NADP, 0.765 mg/ml. ''"Regressions over a range of background dpm/yg DNA of 0 to 450. *Yj = Average dpm/ug DNA for 10~5 M 4NQO (positive control). Y2 = Average dpm/yg DNA for 5 x 10~? M DMh (positive control). X = Average dpm/ g DNA for background (negative control). 14 �If the observed average level of incorporation for the positive control compound is outside the 95% confidence limits of the regression, we assume that sorie variation has occured in the experimental procedures and repeat the test. Interpretation of Pesults In a report to the National Cancer Institute,7 we presented the results of tests performed without metabolic activation on 40 compounds of known carcinogenicity. We have analyzed these results using either the parametric One-Way Classifiction Analysis of Variance or the nonparametric Kruskal-Wallis One-Way Analysis of Variance, depending on which was more appropriate.* At the 99% confidence limits, all the ultimate carcinogens significantly elevate the incorporation of 3H-TdR into the DNA. The noncarcinogenic compounds, with one exception, fail to elevate significantly the incorporation of 3H-TdR at this level of confidence. Thus, the 99% confidence limits of these statistical analyses apparently can be used with reasonable accuracy to predict the biological significance of the response to a chemical. The number of compounds we have tested with metabolic activation is insufficient to establish a correlation between statistical significance and biological significance. Therefore, v;e assumed that the 99% confid- ence levels of the analyses of variance used without metabolic activation also apply for testing with metabolic activation. Results and Discussion Tables 51 through 90 present the results of the UDS testing, with and without metabolic activation, of the 20 substitute pesticides. Tables 51 and 52, the DNA repair synthesis assays of monocrotophos, include detailed summaries of the cell culture and experimental conditions for these assays. The assays presented in the following taoxes (53 through 90) were conducted under similar conditions. In routine testing in the *If there is reason to believe that the variances of each of the treatments in a test are equal (i.e., Bartlett's test of the variance is negative), the parametric analysis is the appropriate one. If the variances are not equal, the nor.parametric analysis is the appropriate one. 15 �absence of metabolic activation, six samples each are used for five log concentrations of each test compound and for the negative and positive controls. However, because of the expense of the metabolic activation preparations, for all compounds except bromacil we tested three replicate samples in the presence of metabolic activation and used three concentrations of the test compound (selected on the basis of the testing without metabolic activation). Based on the criteria for positive responses, we observed significant increases in unscheduled DNA synthesis in the absence of metabolic activation after exposure of the cells to only two substitute pesticides, monocrotophos and parathion. In the presence of metabolic activation enzymes, significantly increased UDS was detected for five substitute pesticides: monocrotophos, captan, folpet, azinphos-methyl, and monuron. Compared with those of negative controls, the levels of 3H-TdR incorporation were greatly reduced in the absence of metabolic activation at the highest concentrations tested for captan, folpet, azinphos-methyl, and monuron, the same four compounds that induced UDS only in the presence of metabolic activation. The reduced levels of incorporation may be inter- preted as cytotoxic effects or as inhibition of repair caused by the highest concentration of the test compounds. A similar effect was observed in the presence of metabolic activation for only one compound, captan, and this was observed at a higher concentration than had been tested without metabolic activation. Stich et al.8 have discussed the problem of cyto- toxicity and possible inhibition of DNA repair systems by some chemicals and have stressed that, whereas such factors may obscure measurements of UDS, often a close relationship exists between concentrations that induce UDS and concentrations that are cytotoxic or that inhibit repair. because of the cytotoxic or inhibitory effects of the substitute pesticides, it should not be assuued rithout further testing that monocrotophos and parathion would be carcinogenic without metabolic activation or that the other four substitute pesticides that induced UDS in the presence of metabolic activation are procarcinogens. The positive UDS results indicate that these six substitute pesticides should be tested more extensively, with the testing to include evaluations of the effects of these chemicals in in vivc bioassays. 16 �MICROBIOLOGICAL ASSAYS General SRI examined twenty pesticides for mutagenicity by in vitro microbiological assays with Salmonella typhimurium (TA1535, TA1537, TA1538, TA100), Escherichia coli WP2, repair-deficient and -proficient strains of Bacillus subtilis and E. coli, and with the yeast Saccharomyc e s cerevisiae D3. An Aroclor 1254-stimulated, rat-liver-homogenate metabolic activation system was included in each procedure, except the relative toxicity assays, to provide metabolic steps that the bacteria are either incapable of conducting or that they do not carry out under the assay conditions. The purpose of this study was to determine whether the compounds elicited a mutagenic response in microorganisms. The assay procedure with S_. typhimurium has been proven to be 85 to 90% accurate in detecting carcinogens as mutagens, and it has about the o same accuracy in identifying chemicals that are not carcinogenic. The assay procedure with J3. cerevisiae is about 50% accurate in detecting carcinogens as agents that increase mitotic recombination. E. coli WP2 and the microbial sensitivity assay are two additional methods of detecting mutagens. The combination of these four assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. 17 �Experimental Salmonella typhimurium Strains TA1535, TA1537, TA1538, and TA100 The J^. typhimurium strains used at SRI were obtained from Dr. Bruce Ames of the University of California at Berkeley.10"12 All are histidine auxotrophs (his~) by virtue of mutations in the histidine operon. In addition to the mutations in the histidine operon, the indicator strains have mutations in the lipopolysaccharide coat (rfa ) and deletions that cover a gene involved in the repair of uv damage (uvrB~). The rfa~ mutation makes the strains more permeable to large molecules, thereby increasing their sensitivity to these molecules. The uvrB~ mutation decreases repair of some types of chemically damaged DNA and thereby enhances sensitivity to some mutagenic chemicals. Strain TA1535 is + reverted to histidine prototrophy (his ) by many mutagens that cause basepair substitutions. Strains TA1537 and TA1538 are reverted by many frameshift mutagens. TA1537 is more sensitive than TA1538 to mutation by some acridine and benzanthracenes, but the difference is quantitative rather than qualitative. TA100 is derived from TA1535 by the intro- duction of the R factor plasmid pKMlOl.13 The introduction of this plasmid, which confers ampicillin resistance to the strain, greatly enhances the sensitivity of the strain to some base-pair substitution mutagens. We have shown that mutagens such as benzyl chloride and 2-(2-furyl)-3-(5-nitro-2-furyl) acrylasiide (known as AF2) can be detected in plate assays by TA100 but not by TA.1535. The presence of this plasmid also makes strain TA100 sensitive to some frameshift mutagens—e.g., ICR-191, benzo(a)pyrene, aflatoxin B,, and 7,12-dimethylbenz(a) anthracene. All the indicator strains are stored at -80°C. For each experiment, an inoculum from frozen stock cultures is grown overnight at 37°C in a nutrient broth consisting of 1% tryptone and 0.5% yeast extract. After stationary overnight growth, the cultures are shaken for 3 to 4 hours to ensure optimal growth. crystal violet. Each culture is checked for sensitivity to The presence of the rfa mutation makes the indicator strains sensitive to this dye, whereas the parent strain, rfa*, is not sensitive to the dye. However, the mutation is reversible, ^leading to 18 �the accumulation of rfa cells in the culture. Therefore, the cells must be tested routinely to ensure their sensitivity to crystal violet. Each culture also is tested by specific mutagens known to revert each test strain (positive controls). To a sterile 13 x 100 mm test tube placed in a A3°C heating block, we add in the following order: Assays in agar (1) 2 ml of 0.6% agar* (2) 0.1 ml of indicator organisms (3) 0.5 ml of metabolic activation mixture (optional) (4) Up to 100 pi of a solution of the test chemical.** For negative controls, we use steps (1), ( ) and (3) (optional) 2, ard 100 vil of the solvent used for the test chemical. "This mixture is stirred gently and then poured onto minimal agar plates.t After the soft agar has set, the plates are incubated at 37°C for 2 days. The number of his revertanr.s (colonies that grow on plates lacking a sufficient amount of histidine to support colony formation) are counted and recorded. Some of the revertants are routinely tested to confirm that they are his]*", require biotin, and are sensicive to crystal violet (rfa~). Escherichia coli WP2 The 1Z. coli WP2 (uvrA~) used in this project was given to us by Dr. D. McCalla. ' A procedure similar to the one used with Salmonella is used to measure the reversion of WP2 to tryptophan independence. However, instead of containing a trace of tryptophan in the top agar, the minimal agar plates contain 1.25 g of oxoid broth per liter to provide * 0.6% agar contains 0.05 mM histidine and 0.05 mM biotin. t Minimal agar plates consist of 15 g of agar, 20 g of glucose, 0.2 g of MgSO,.7 H20, 2 g of citric acid monohydrate, 10 g of K2HP04, and 3.5 g of NaHNH4P04.H20 per liter. **Solvents used as appropriate include: water, dimethyl sulfoxlde, ethanol, and benzene. 19 �the trace of tryptophan required for enhancement of any mutagenic effect of the test chemical. Alternatively, reversion of the mutated tryptophan gene, WP2 may undergo a forward mutation in a tryptophan tRNA gene to obtain tryptophan independence. We do not distinguish experimentally between the true revertants and the phenotypic revertants (although the latter tend to form smaller colonies). Escherichia coli W3110/p3478 and Bacillus subttlis H17/M45 The JE. coli strains W311C and p3478 were obtained from Dr. H. Rosenkranz.16 Strain p3478 is a polA~ derivative of strain W3110. It carries a single, revertable mutation in a gene for a DNA polymerase; Gross and Gross showed that this mutation is involved in DNA repair synthesis. This mutation increases the sensitivity of strain p3478 to chemicals that lead to alterations (damage) of the DNA. Therefore, we can assay for chemicals that damage DNA by comparing the relative sensitivity of the two strains (p3478 and W3110) to the test chemical. The IJ. subtilus strains H17 and M45 were obtained from Dr. Kada.18 Strain HI7 (rec'*') is derived from H17 but is deficient in the genetic recombination mechanism necessary to repair DNA damage. Cells deficient in this repair mechanism are killed more easily by chemical mutagens than are wild-type cells (rec*). If the chemical is toxic to rec~ cells, Lut at the same concentration is not toxic to rec"*" cells, the chemical probably is a mutagen. Inoculums from frozen stocks are grown overnight in nutrient broth at 37°C with shaking. To 2 ml of nutrient broth containing 0.6* agar is added 0.1 ml of the test culture. The suspension is mixed and poured onto plates containing nutrient broth and 2% agar. After the soft agar has solidified, a sterile filter disc impregnated with the test chemical is placed in the center of the plate. The plates are incubated at 37°C for 16 hours, and the width of the zone of * Tryptone, '%, and O.j% yeast extract, supplemented with 5 pg of thymine/nl to prevent selection of thy"1" revertants. 20 �toxiclty or inhibition of growth is then measured. We usually must test several concentrations of chemical to detect accurately differences in the zones of growth inhibition because higher initial concentrations lead to steep concentration gradients that may reduce the differences in growth inhibition of the two strains. The positive control for this asray is 1 ml of l-phenyl-3,3-diraethyltriazene placed on the disc. A zone of approximately 40-mra width is observed (52 and 61 mm, respectively). An additional control is 30 vig of chloramphenicol placed on a disc. Equal zones of inhibition are expected in all four strains (approximately 30 mm) since the toxicity of this chemical does not depend on a mechanism that leads to DNA damage. All assays are performed at least three times. Saccharomyces cercvisiae D3 The yeast S^. cerevisiae D3 is a diploid heterozygous for a mutation in an adenine-metabolizing enzymes.*' Cells homozygous for this mutation produce a red dye when grown on medium containing adenlrie. Adenine- requiring homozygotes can be generated from ths heterozygotes by mitotic recombination. Many mutagens increase the frequency of mitotic recombi- nation. Mitotic recombination is indicated by the development, of colonies with red pigmentation, and the degree of conversion to this pigmented colony indicates the tnutagenicity of a compound or its metabolite.20 The Saccharomyces test strain from the liquid nitrogen is grown overnight at 30°C with aeration in 1.0% tryptcne and 0.5'* yeast extract. The cells are washed twice in 0.067M P04 buffer (pH 7.4) and resuspended in the same buffer at a concentration of 10° cells/ml. The iH vitro yeast mitotic recombination assay in suspension consists of 5 x 10 washed, stationary-phase yeast cells in 1 ml of 0.067M P(>4 buffer (pH 7.4) and 50 rag/ml of the test chemical (or a fraction of the concentration required to give 50% killing). The. suspension is incubated at 30° for 4 hours. After incubation, the sample is diluted serially in sterile saline and plated on tryptone-yeast-agar plates. 21 �Platas of a 10"^ dilution are incubated for 2 days at 30°C, followed by 2 days at 4°C to enhance the development of the red pigment indicative of adenine-regative homozygosity. To detect red colonies or red sectors, we scan the plates with a disrecting mi:roscc?e at 10 x magnification. Plates of a 10 dilution are incubated for 2 days at 30°C for determination of the total number of colony-forming units. The in vitro yeast itotic recombination assay in suspension with metabolic activation is conducted as above with the addition of the metabolic activation system to the incubation mixture. Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic mutagens (e.g., dimethvlnitrosamine) are inactive •inless they are converted to their active form by being metabolized. Ames et ai. 1 have described the metabolic activation systems we use. Adult male mice are given a single 500-mg/kg intraperitoneal injection of a polychlorinated biphenyl (Aroclor 1254). 2 Four days after the injection, the animals' food is removed. On the fifth day, the mice are killed. The liver are removed aseptically and placed in preweighed, sterile glass beakers. The organ weight is determined, and all subsequent operations to the metabolic activation step are conducted in an ice bath. The orgaa is washed in an equal volume of cold, sterile 0.15 M KC1 (1 ml/g of wet organ), minced with sterile surgical scissors in three volume of 0.15 KC1, and homogenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x j>, and the supernatant is removed and stored in liquid nitrogen. To the postmitochondrlal supernate are added MgCl2, KC1, glucose-6-phosphate, TPN, and sodium phosphate (pH 7.A). Results and Discussion All the pesticides submitted to SRI for examination were tested at least three times in the microbiological assays. sented here are an average of those experiments. 22 The results pre- �Table 91 presents the results of the microbiological assays in agar with Snlmoiyl la typhimurium. In this histitline reverse-i.-utation assay system, two pest icidcs--captar» and folpct—were riutagonic. For each chemical, we observed an increase in the number of histidineindcpendcrt revertants on strains TA1535 and TA100 but not on strains TA98, TA1537, or TA1538. These results suggest that these pesticides can alkylste DNA, rousing mutations of the base-pair substitution type. This conclusion is consistent with the r.mtagenic activity of these compounds in assays with E. coli WP2 (Toble 92), which is sensitive to base-pair substitution mutagens. Although liver hoirogcnate activation was not required for mutagenic activity, the mut.igenic activity was enhanced somwhat with activation at some doses. A toxic effect (reduction of the number of mutants) was observed at doses of 100 pg of each compound. Table 92 presents the results of assays with _E. coli WP2. Essentially, the results were identical to those obtained with j. [ typhimuriun! TA1535 and TA100; captan and folpet were ir.utagenic, but none of the other pesticides was mutagenic. Table 93 presents the results of the assays for microbial inhibition in repair-deficient and-proficient strains of B. Aul>Jtili§. and ¥,. .celt.. Folpet, captan, ch]oropyrifos, and dinoseb all gave toxic zones that were larger on t:io repair-deficient strains than on the repair-proficient strains, indicating a mutagenic response. Toxic chemicals that do not act by damaging DNA (e.g., chloramphenicol) should give equivalent zones of toxicity. However, many ii not all nutagens damage DNA and, if the damage is not repaired, can result in cell death. Thus, a given concentration of mutagen may be toxic for a repair deficient strain but not for a strain the effectively repairs its DNA. Tables 94 through 1J3 present the results of the assays for mitotic roconib invit ion in Saccharomyccs _t-erovi siae D3. A positive response in t h i s assay is indirated by an increase of more than threefold in the absolute number of m i t o t i c reron.M riaiivs por m i l l i l i t o r as well as ir the relative number of mi I otic rocomhinants per 10" survivors. 23 Folpet, �captan, tnonocrotophos, cacodylic acid, and azinphos-methyl increased mitotio recombination significantly and are considered positive by these procedures. Methyl parathion gave a marginally positive response. Our results indicate that 7 of the 20 pesticides examined give positive responses in one or more of the four microbiological assay procedures. Although a mutagenic response in a microorganisms does not mean that a chemical is a mutagen in humans, the combination of four separate assay system greatly enhances the probability of detecting potentially hazardous chemicals. Folpet and captan are mutagenic in all four assay procedures. Chloropyrifos and dinoseb are positive in the microbial sensitivity. Monocrotophos, cacodylic acid, and azinphosmethyl are positive in the yeast assays. �DISCUSSION Of the 20 pesticides tested for mutagenic activity, 9 were clearly mutagenic in one or more in vitro assays. Of these 9, 2 w=:re mutagei ic in all the in vitro assays, but none of them produced a dorainaii-; I.-tlal response in the mouse. In the Salmonella assays, these chemicalR c.-,i r.c.-i base-pair substitution mutations but not frameshift mutations. The absence of activity in the dominant lethal assay may be dui to a lack of sensitivity of the mouse to these types of compounds; for example, N-methyl-N'-nicro-N-nitrosoguanidine and other alkylating agents that cause base-pair substitution mutations do not all cause dominrnt lethality. Another explanation for the absence of activity may be that those pesticides did not reach the gonadal tissues in sufficient amounts >_o cause a mutagenic event. None of the other 6 pesticides was mutagenic in all the in vitro assays. The combination of assays used in this program is one means of identifying those pesticides that may present a mutagenic health hazard. Those that show positive responses in several experimental systems should be evaluated more thoroughly before they are substituted for other pesticides already considered as a risk to the environment. 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A study 01" the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxgribonucleic acid. Biochem. J. 6£, 315-323 ( 9 6 . 15) 7. A. D. Mitchell. Potential Prescreens for Chemical Carcinogens: Unscheduled DNA Synthesis, Task 2. Final report prepared for National Cancer Institute under Contract N01/CP-33394, SRI Project LSU-2735 (April 1976). 8. H. F. Stich, D. Kleser, R.H.C. San, and B. A. Laishes. The Use of DNA repair in the luentification of carcinogens, precarcinogens, and target tissue. In Canadian Cancer Conference: Proceedings of the Tenth Canadian Cancer Conference, Vol. 10. P. E. Scholefield (ed.), University jf Toronto Press, Toronto, Canada, 1973, pp. 83-110. 9. J. McCann, E. Choi, E. Yamasaki, fiid B. P. Ames. Detection of carcinogens as mutagcn? in the Salmonella microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA 72_, 5135-5139. 10. B. N. A.nes, r. C. Gurney, J. A. Miller, and H. Bartsch. 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Andersor., E. E. Slater, and H. S. Rosenkranz. Rapid detection of mutagans and carcinogens. Cancer Res. 31, 970-973 (1971). 17. J. Gross and M. Gross. Genetic analysis of an IJ. coli strain with a mutation affecting DNA polymerase. Nature 224. 1166-1168 ( 9 9 . 16) 18. T. Kada. Mutagenicir.y testing of chemicals in microbial systems. New Methods in Environ. Chem. Toxicology 127-133 (November 1973). 19. F. K. Zimmermann and K. Schwaier. Induction of mitotic gene inversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alkylating agents in S a cc haroayce s cerevisiae. Mol. Gen. Genet. 100, 63-69 (19C7). 20. D. J. Brusick anJ V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives 6^, 83-96 (1973). 21. L. D. Kier, E. Yamasaki, and B. N. toes. Detection of mutagenic activity Jn cigarette smoke rondensates. Proc. Nat. Acad. Sci. USA 2 , 4159-4163 (19/4). 1 22. B. N. Amec, J. McCann, and E. Yamasaki. Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagi-ntcity Test. Mut. Res. 3J_, 347-364 (1975). 27 On mutagenicity of nitrofurans. �Table I CHI-SQUARE TEST OF THE FERTILITY INDEX - MOHOCROTOPHOS 1 DECREE OF FREEDOM V E M I U E CONTROL HEEK N N FERT. PRO MTD INDEX CMISO 74-10 N N PRQ MTD IS MG/KO FERT. INDEX <M I SO 74-10 N N PRO MTD 30 MO/KO FERT. INDEX CM I so 74-10 N N PR8 MTD 60 MG/KO FERT. INOE.« CM I SO TFM MG/KO N n PRO MTO FERT. INDEX CHISQ 0.00 MULTIPLE rHEATMENT .07 18 40 .5 4 4.14 .7 7 .6 9 20 40 .0 5 .75 2.01 19 40 .7 4 .05 22 40 .5 5 4.79*1 25 40 .3 6 0.00 21 40 .2 5 .20 23 40 .57 .8 0 25 38 .6 6 0.00 1 28 40 .70 0.00 23 40 .7 5 2 26 40 .5 6 0.00 31 40 3 23 40 .57 0.00 30 40 4 27 40 .7 6 0.00 25 40 .3 6 39 .5 8 40 .7 6 .03 16 40 .0 4 6.11* 29 40 .72 1.28 16 40 .0 4 4.06* 27 39 .9 6 .03 .5 4 22 40 .5 5 0.00 32 40 .0 8 3.72 .4 8 IS 40 .8 3 6.07» 27 40 .7 6 .6 0 30 40 .75 1.42 27 38 .71 .4 2 CO 5 24 40 .0 6 0.00 33 6 24 38 .63 0.00 27 7 30 39 .9 7 0.00 25 40 .3 6 1.81 21 40 .2 5 4.9l» 28 38 ,T4 .07 27 36 .S 7 .02 8 27 38 .71 0.00 28 40 .0 7 .02 20 40 .0 5 2.78 24 38 .3 6 .24 26 36 .72 .02 * I SIGNIFICANT AT P LT 0.05 INCREASED ABOVE CONTROL �Table 2 AVERAGE IMPLANTS PER PRECXANT FEMALE - MONOCROTOPHOS MEEK CONTROL 7»-10 15 MG/K3 7»-18 30 MO/KG 7«-10 60 NO/KG TE* .2 MG/KG MULTIPLE TREATMENT 16-11.25 316/ 29.10.90 20.12.10 leo/ iao/ 16*11.25 293/ 27.10.85 19.12. 58 "I 267/ 22.12. 1* *I 3*8/ 32.10.87 2 . 2 *5 21. 166/ 15*11.07 266/ 27. 9.85* 25.10.80 2*8/ 21*11.81 356/ 30.11.87 23*11.96 255/ 25»10.20 ** 273/ 27.10.11" 21-12.1* 316/ 26.11.29 306/ 27.11.33 20«11. »0 291/ 2*. 12. 12 322/ 26.12. 3d "I 319/ 28«11.39 2*9/ 23-10.83 let/ 18-10.50 2 303/ 26*11.65 332/ 31.10.71 2*2/ 2»5/ 23.10.65 356/ 30.11.87 239/ * 309/ 27.11.** 31*/ 25.12.56 2T*/ 5 27*/ 2*>11.*2 372/ 33.11. 27 270/ 6 302/ 2*>:2.58 32V 27.12.11 275/ T 3»6/ 30«11.53 285/ 25.11. »0 2S5/ 8 292/ 27.10.81 3I3/ Z8.ll.18 228/ 1 M * • •• I S I G N I F I C A N T AT f» LT 0.05 S I G N I F I C A N T AT P LT 0.01 INCREASED ABOVE CONTROL �Table 3 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - MONOCROTOPHOS KEEK 15 CONTROL NO/KG 74-10 30 MO/KG 74-10 60 MG/KG TEM .2 MG/KG MULTIPLE TREATMENT 1 13/ 2 e/ 2 . .6 8 4 2. 6 28/ 23" 1.22 10X 18. .56 10X 16* .3 6 62X 29. 2.14" lot 20. .0 5 2X 16. .13 77X 2 . 2.85** 7 19. .31 3/ 31. .10*0 3 9/ 23. .9 3 16/ 30. .3 5 .7 4 9X 22> .*» 87X 32. 2.72** 4 2/ 27. .0? 7/ 2. 5 .28 * 26X 2 . 1.18* 2 9/ 15- .0* 6* 11X 2. 7 .41* 5 ll/ 4 2 . .6 4 22/ 33> .7 6 12X 2. 5 .8 4 9/ .43 22X 30* .73 6 21/ 24« 16/ 27. .9 5 14X 2. 3 .1 6 6X ,24*«D 17X 27. .63 7 30/ 30. 1.00 ll/ 25* .4 4 4X .19 ax 28> .9 2 lix 27« .41 9X 24. .38 11X 2. 6 .2 4 8 • •• D 19/ 27. .8 8 .70 Z4/ S1SNIFKANT AT P LT 0.05 S I G N I F I C A N T AT P LT 0.01 DECREASED BELOW CONTROL 2 . .6 8 8 9X 7X 212. 0 .35 2125- �Table t> CHI-SQUARE TEST OF THE DEATH INDEX - MOHOCROTOPHOS 1 DECREE OF FREEDOM 74-10 VEHICLE CONTROL N N DEATH DDI PAG INDEX N CHISO N 15 74-10 MO/KG N DEATH »DI PHO INDEX CHISQ N 30 KG/KG DEATH tot PRG INDEX CMlsO 74-10 N N «OI PRG «0 M6/KG .? MG/KG TE* N N MDI PH<5 DEATH INDEX DEATH INCCX CM I SO .38 .4 0 25 29 .H6 13.27 ** 26 27 .6 9 24.26 ** 25 32 .* 7 7.05 ** 8 2? .30 3.07 30 .33 .00 27- .37 2.36 9 27 .33 .07 26 .38 .03 CM ISO MULTIPLE TREATMENT 1 10 2 7 24 3 9 23 28 0.00 11 .27 0.00 3 .9 3 0.09 11 .36 4 2 27 • 0» 0.00 5 9 .38 0.00 24 6 15 24 .3 6 0.00 7 8 30 .7 2 0.00 8 12 27 .4 4 0.00 , ** SIGNIFICANT AT P IT 0.01 .8 4 .35 31 .10 1.84 30 .7 3 .01 23 7 25 7 6 18 .9 3 .01 7 20 .35 .07 2 16 .13 .2 5 7 .37 .03 6 22 .27 .8 2 19 9 16 .8 2 2.54 11 22 .50 9.20** 15 .60 11.21 «* .33 .00 12 25 .8 4 .21 8 21 .38 .07 27 .48 .6 5 11 23 .8 4 .52 S 25 .20 8 25 .32 .02 4 21 .19 .9 0 7 28 .5 2 .02 .4 5 .17 7 20 .5 3 .12 8 24 .33 .27 11 13 IS 33 28 7.48 19 10 10 �Table 5 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - MONOCROTOPHOS MEEK 74-10 CONTROL 15 H6/KG T»-10 30 HG/KO 74-10 60 Mb/KG MULTIPLE TREATMENT 28/ Z*9« .11 10/ 189- • Ob 8/ 303' .03 3/ 33Z' .01 10/ Z«Z» .* 0 .0* 16/ 356> .* 0 9/ Z39> Z/ 309' .01 7/ 31*« .02 Z6/ 27*. 5 ll/ 27*' .0* ZZ/ 37Z» .6 0 1Z/ 270« 6 Zl/ 30Z« .07 16/ 3Z7' .05 7 30/ 3»6« .9 0 ll/ Z85* .0* 8 19/ Z9Z' .07 Z*/ 313> .08 1 Z u> 13/ 319' . 4 0 3 9/ 2*5» » • SIGNIFICANT AT f UT 0.05 •• SIGNIFICANT AT P LT 0.01 D DECREASED BELOW CONTROL . .6 0 62/ 316» Z/ 180' .01 «/ Z67» .03 77/ 293« .6 2" .* 0 B7/ 348' .25" .09" 9/ 166' .05" ll/ 266< .«»•- .* 0 9/ Z»8« .0* Z2/ 356« .06 I*/ 275» .05 6/ 255' .02»D 17/ 273* .6 0 */ 25S» .02 8/ 316' .03 ll/ 30A' .* 0 7/ ZZ8> .43 9/ 291' .03 ll/ 3?2« .03 10/ 1 0 8' ,ZO»* �CHI-SQl'ARE TEST OF THE FERTILITY INDEX - BROHACIL 1 DEGREE OF FREEDOM *EEK V E H I C L E CONTROL N N PBG HTD FERT. INDEX CHISO 74-06 N N 1250 HG/KG FERT. PRG MTO INDEX CHISO 74-06 N N PRG MTD 2500 HO/KG FERT. INDEX 74-06 CHISO N PRG .87 29 5000 MS/KG TEM N FERT. MTO INDEX CHISO 40 .72 0.00 39 .9 6 .03 32 40 .0 " 3.72 MTO INDEX CHisa N PRG 0.00 29 0.00 27 N FERT. . MQ/KG ? MULTIPLE, TREATMENT U) 1 28 40 .70 0.00 21 40 .52 1.90 23 40 .7 5 40 .2 7 2 26 40 .65 0.00 24 40 .0 6 .05 26 38 .8 6 .01 25 40 .63 3 23 40 .7 5 0.00 29 40 .72 1.37 19 38 .0 5 .»» 25 40 .3 6 4 27 40 .7 6 0.00 25 40 .63 .05 22 38 .8 5 .41 28 40 .0 7 27 40 .7 6 .6 0 5 24 40 .0 6 0.00 31 40 .77 2.09 22 38 .8 5 .00 27 40 .7 6 .22 30 40 .75 1.42 6 24 38 .3 6 0.00 31 40 .77 1.30 25 38 .6 6 0.00 27 40 .7 6 .03 27 38 .71 .24 40 .72 .16 22 38 .8 5 2.98 29 40 .72 .16 27 36 .75 .02 39 .2 7 .03 24 38 .3 6 27 40 .7 6 .01 26 36 .72 .2 0 7 30 38 .9 7 0.00 29 8 27 38 .71 o.oo 20 .4 2 .05 0.00 �T.fcle 7 AVERAGE IMPLANTS PER PREGNANT FEMALE - BROHACIL KEEK CONTROL 74-06 1250 MG/KO 74-06 3500 MG/KG 74-06 5000 MG/K3 TE« .2 HO/KG MULTIPLE TREATMENT 1 319/ 28*11.39 23S/ 21.11 .19 27s/ 23*12.09 32g/ 29*11.31 316/ 29*10 .90 26.10.92 304/ 25*12.16 293/ 8 27*10 . 5 2 303/ 26.11.65 2S3/ 2-.. 10.54 2B4/ 3 245/ 23.10.65 33S/ 29.11 . 5 5 225/ 19.11.84 319/ 25.12.76 "I 34B/ 32*10 .87 4 309/ 27-11.4* Z90/ 25*11 ,6fl 268/ 22*12.18 346/ 28-12.36 26»/ 37. 9 .85 « 5 2T4/ 24.11.42 361' 31*11 .65 261/ 22.ll.B6 323/ 27*11.96 35ft/ 30*11.87 6 302/ 24*12.58 368/ 8 31*11 . 7 294/ 25*11.76 278/ 27*10.30" 273/ 27*10 .11 •* 29.12 .24 2S3/ 22.11.50 357/ 29*12.31 306/ 27.11 .33 28.11 .36 277/ 24.11.54 307/ £7*11.37 322/ 3" 26*12 . 8 ! 7 346/ 30.11.53 3SS/ 8 292/ 27*10.81 318/ • SIGNIFICANT AT P LT 0.05 •• SIGNIFICANT AT P LT 0.01 I INCREASED ABOVE CONTROL �Table 8 AVERAGE DEAD IMPLANTS PER PREGNANT FEU ALE - BROMACU. KEEK 7«-06 CONTROL 1250 HO/KG 74-06 2500 KG/KG 74-06 5000 MS/KG .2 MG/KG TEM MULTIPLE TREATMENT 1 13/ 4 28 • . 6 8/ 21* .8 3 10/ 23* .43 17/ 2' 9 .9 5 62/ .*. 29* 2 1 . .0 5 8/ 26* .Jl 5/ 25* .0 2 77/ 27. 2 8 * .5. .21 12/ 19* .63 10/ 25* .40 87/ 32* 2.T2« IS/ 2 . .5** 8 Jl' 27. .1 4. 8/ 27* .30 «/ 30« .73 2 8/ 26* .31 12/ 24* 3 9/ .39 6/ 29* 23> 4 2/ 27* .07 7/ 25* .8 2 16/ 2. 2 .73** 5 ll/ 24* .6 4 12/ 31* .39 IS/ 22* .8 6 6 21/ 24* .8 8 7/ 31* .23**D 16/ 2' 5 • 64 21/ 27* .78 17/ 27* .63 7 30/ 30* 1.00 ll/ 29* .8 3 14/ 22* .4 6 14/ 4 2. .8 9 ll/ 27* .41 8 I")/ IS/ 28. .4 5 a/ 2. 4 .33 9/ 27. ll/ 2. 6 .«2 27* .TO • S I G N I F I C A N T AT P LT 0.05 •• S I G N I F I C A N T AT P LT 0.01 D DECREASED BELOW CONTROL .33 �Table 9 CHI-SQUARE TEST OF THE DEATH INDEX - BROKACIL 1 DECREE OF FREEDOM •EEK VEHICLE CONTROL N N ttDI PRO DEATH INDEX CHISO 7»-06 N N HOI PRG 1250 MO/KB DEATH INDEX CHISO 74-06 N N MDI PRG 2500 MO/KO DEATH INDEX CHISO 74-06 N N «DI PRG 5000 M6/KO TEN .2 MG/KO DEATH INDEX CHISQ N MDI PKG DEATH INDEX N CHISO MULTIPLE TREATMENT 1 10 28 .36 0.00 2 7 26 .27 0.00 7 21 .33 .02 7 23 .30 24 .42 .4 6 a 26 .31 9 19 .7 4 .05 11 22 .0 1 9 2 " . 0 12 22 .55 .74 10.65**D 13 25 • 52 • 21 22 .5 4 7 24 .9 2 10 3 9 23 .9 3 0.00 6 29 .21 1.32 4 2 27 .07 0.00 6 25 .24 1.62 5 9 .'48 0.00 12 24 31 .9 3 .6 1 .24 .3 6 C'OO 5 31 8 30 .27 0.00 7 29 8 12 27 .4 4 11 28 »* D SIGNIFICANT AT F LT 0.01 DECREASED BELOW CONTROL 6 7 15 2* 0.00 .9 3 .4 0 .01 .01 10 .01 0.00 1.24 .70 1* 29 .8 4 .8 4 25 29 .6 8 13.27" 5 25 .20 .6 0 26 27 .6 9 2">.26*« 6 25 .4 2 .7 6 25 32 .8 7 70" .5 28 .50 8 27 .30 3.0? 14 10.11" 8 27 .30 10 27 .7 3 12 29 . M .4 8 T 27 .6 2 1.30 .09 2.36 10 30 .33 10 2 ' .37 9 27 .33 .07 26 .8 3 .03 10 .00 2.36 �Table 10 NUMBFR OF DEAD IMPLANTS PER TOTAL IMPLANTS - BROMACIL »EEK 74-06 CONTROL 5COO M6/KG .2 MS/KG MULTIPLE TREATMENT 1 .04 */ 235* .03 2 8/ 303* .03 12/ 253* .05 3 I 13/ 319* 9/ 245* .04 6/ 335* 4 2/ 309* .01 7/ 2 0 9. 17/ 328* .05 62/ 316* .0. 2. 284* .03 5/ 304* .02 77/ 293* .6. 2* .02*0 .05 IO/ 319* .03 87/ 348* .25** .02 16/ 268* .6* 0* 15/ 346* .04** ll/ 266* .04* .03 15/ 261* .) 16 8/ 323* .02 22/ 356* .6 0 .02 **D 16/ 294* .5 0 21/ 278* .08 17/ 273* .6 0 I*/ 253* . 06 I4/ 357* .4 0 ll/ 306* .4 0 9/ 307* .03 ll/ 322* .3 0 ll/ 274. .0* 12/ 361* 6 21/ 302* .07 7/ 368* 7 30/ 346* .09 ll/ 355* .03 8 19/ 292* .7 0 IS/ 318* .5 0 S I G N I F I C A N T AT P LT 0.05 S I G N I F I C A N T AT P LT 0.01 DECREASED BELOW CONTROL e/ .0* 12/ 225* 5 • •• D IO/ 278* a/ 277* .03 �Table li CHI-SQUARE TEST OF THE FERTILITY INDEX - CAPTAN 1 DECREE OF FREEIOM • EE.K VEHICLE CONTROL N N PRO NTD FERT. INDEX CHISQ 74-02 N N PRG MTO 1250 MO/KG FERT. INDEX CHISO 74-02 N PRS 74-02 «(ooo NG/KG 2500 N FtRT. MTO INDEX CHISO N N PRG MTO FEBT. INDEX CHISO .? MG/KG TEN N N PRG MTO FERT. INOEJ CHISO 1.37 MULTIPLE TREATMENT LO 00 40 .2 7 1.37 29 40 .? 7 4.07.t 30 40 .75 .10 27 39 .9 6 .6 0 .5 7 .4 5 31 40 .7T .8 9 32 40 .0 8 1.5T .7 7 1 23 40 .7 5 0.00 34 40 • 8! 31 40 2 27 39 .9 6 0.00 30 40 .75 .10 36 40 .0 9 . 3 26 40 .5 6 0.00 23 40 .7 5 .21 30 40 4 27 29 29 40 31 3» •82 1.35 33 40 .2 8 32 40 .0 8 1*03 27 41) .72 40 6 0.00 .7 6 0.00 26 38 .8 6 .02 30 40 •75 0.00 26 38 .8 6 .02 30 40 .* 7 .72 0.00 27 38 .71 .01 34 40 .5 8 1.20 30 38 .9 7 .16 27 38 .71 3S •O .01 30 40 •75 o*oa 26 38 .8 6 .02 27 36 .5 7 .00 38 .7 8 .6 2 34 40 .5 8 .9 0 27 38 .1 7 .3 4 26 36 .? 7 .8 2 .7 6 40 5 1.67 \ 7 29 40 .72 0.00 27 8 32 40 .0 8 0.00 33 * I SIGNIFICANT .AT K T 0 0 . .5 INCREASED ABOVK CONTKOL .0» 0.00 .01 �Table i : AVERAGE IMPLASTS PER PREGNANT FEHALE - CAPTAN III <K CCNTHOL 7*-0<! MS/KG 74-02 2500 7«-OZ 5000 MG/KG .2 KG/KG MULTIPLE TREATMENT 1 265/ 23*11.S.. 3f)7/ 3*«11 .38 339/ 31*10^* 310/ 29* 10. 69 31f</ 29*10. 90 27*10. 85 2 s 305/ 27.11.30 328/ 30=10 .93 375/ 36=10. »2 3*2/ 30*11. *0 293/ 3 268/ 26.10. 11 252/ 23.10 .96 313/ 30x10. *3 3*5/ 31, U. 13 3»n/ 32*10.87 « ?88/ ?7«10.67 32»/ 31*10. »5 359/ 33*10. »" 333/ 32= 10. *! 26iS/ 27* 9. 85 5 33*/ 29=11. ">2 2HH/ 2b*10 .85 3*8/ 30*11. bO 298/ 26- 1!. *6 35*/ 30*11. 87 6 3J3/ 29.11.1* 312/ 27.11 .56 361/ 3*»10.*2 3»0/ 30* 27*10. 11 30x10.97 309/ 26. ;i. 33 11. «» 273/ 329/ 30»>/ 27.11.33 383/ 34'll.^S 301/ 27«11. JS 322/ 26*12. 38 7 323/ 29.11.1* 30V/ 27.11 .** fl 3 8,/ 3J.11.91 3»2/ 33*11 .58 • SIGNIFICANT AT . LT 0.05 •• SIGNIFICANT AT P LT 0.01 �Title 13 AVERJWS : KftK 7»-02 CONTROL 1250 NG/KO IMPLANTS PER PKKbNAKT TltHlX 74-02 2500 HO/K6 T»-0.» MG/KG 5000 K8/K8 WULTIPLE TREATMENT 1 R/ ?3* .35 17/ 34. .50 19/ 3. .* .61 IS/ 29> .52 «>2/ 24* 2.M** 2 ll/ 27* .41 lb/ 30. .50 J7/ 3 ' .» 6 . 14/ 30* ,»7 77/ 27* 2.«5»* 3 16/ ?•>•* ,»>2 lb/ 23* 16, 30* IS/ 31* .8 4 87/ 3?« <.72** 2. 7 .) 4 30. .73 .70 .S3 4 16/ 27« .59 I2/ 31* .39 !'•>/ 33* .3 • ll/ 32< .3* ll/ 5 15/ 2. 9 .32 ?•>/ 26* .92 9' . 30* .JO ll/ 26. .42 22/ 6 9/ z** ,-jl 13/ 27* .48 21' 34* .2 6 IS/ 30' .60 17/ 27* .63 7 21/ 25* .72 15/ 27. .56 I*/ 30* .47 «/ 26* .31 ll/ 27* •*! •1 13/ 32* .41 12/ 33* ,36 ll/ 34* .32 7/ 27* .26 ll/ 26r .»2 • SIGNIFICANT AT P LT O.O1) •• SIGNIFICANT f.T P UT 0.01 �Tible 14 CHI-SQUAKE TEST OF THE DEATH INDEX - CAPTAN 1 DECREE OF FREEDOM VEHICLE CONTROL KEfcK N N WDI PRG DEATH INDEX CHISO 74-02 N N •01 PRS MO/K6 DEATH INDEX CHISO 74-02 N N «DI PRO 2500 KG/KG DEATH INDEX CHISO 74-02 N N HOI PRG 5000 MS/KG DEATH INDEX CHISO .04 .? MS/KG TEN N N WDI PRG DEATH INDEX CHISO MULTIPLE TREATMENT 1 8 23 .35 0.00 2 9 27 .33 0.00 10 25 29 .6 . 12.49" .08 26 27 .6 9 20.79" .00 25 32 .78 7.B5" 8 27 .30 .32 .02 12 31 .39 .00 12 29 .<! .7 2 .07 12 36 .33 ,'J7 10 30 .33 30 .37 .02 13 31 .42 7 33 .21 1.85 8 12 .25 1.02 34 .9 2 a 30 3 10 26 .38 0.00 12 23 .2 5 .6 4 4 11 27 .41 0.00 8 31 .6 2 .6 8 5 11 29 .38 o.oo o.oo o.oo o.oo 10 26 .8 3 .6 0 7 30 .23 .87 11 £6 .2 4 .00 10 30 .33 .01 9 27 • 33 .03 15 34 .44 1.20 10 30 .33 0* 10 27 .37 .22 12 27 .4 4 .0 0 11 30 .37 .01 7 26 .27 .71 9 27 .33 .12 8 33 • 24 .39 9 34 .26 .19 5 .J9 1.15 26 .38 .00 6 8 29 ." 2 7 12 29 .41 32 • 34 8 «« 11 SIGNIFICANT AT PLT 0.01 11 27 10 �Table IS NUMBER OF DEAD IKPLANTS PER TOTAL IKPLAKTS - CAPTAK 74-0? CONTHOl 1250 MG/KG 74-02 2500 MG/KG 74-02 5000 MO/KO TE» .? MULTIPLE TREATMENT 1 B/ 265> .03 IT/ 3e7» .4 0 19/ 3 9 3' .06 IS/ 3>0* .05 6?/ 3,t. .0' 2* T7/ 293> .6 ?" VI f 348. .5 2" 2 ll/ 305* . 4 0 IS/ 328* .5 0 17/ 375* .US 14/ 342* .04 3 16/ 268* .6 0 16/ 252* .6 0 16/ 313* .uS IS/ 345* .34 4 J6/ 2«B« .06 12/ 324« .04 15/ 359* '4 0 ll/ 333' .03 ll/ 266. .4 0 5 IS/ 334* .4 0 2*/ 282* .9 0 9/ 348* .03 9' ll/ 2 8 .4 0 22/ 356. .6 0 6 9/ 3?3* .3 0 13/ 312* .04 21/ 361* .06* IB/ 340* .05 17/ 273* .6 0 7 21/ 323* .07 15/ 309* .05 14/ 329. .11-. 8/ 309. .03 ll/ 306* . 4 0 8 13/ 3*1' .03 Ii/ 332» .03 ll/ 3B3« .03 V 30;- .02 ll/ 322* .03 • SIGNIFICANT AT P LT 0.05 •• S I G N I F I C A N T AT P LT 0.01 �CHI-SQUARE TEST OF THE FERTILITY INDEX - FOLPET 1 DECREE OF FREEDOM 74-03 VEHICLE CONTROL • EEK N N PNG MTD FERT. INOtX N CHISJ N 1250 MG/KG CERT. PHU MTU INDEX CHlaa 74-03 N N PRG MTU 2500 MC/Kli FERT. INDtX CHlSa 74-OJ 5000 Mto/KG TEH N N HR« MTu FEHT. INDEX CMISS 30 40 .5 7 2.91 29 .2MG/KG N N "KG KTO fERT. INDEX CHISU 40 .2 7 1.37 MULTIPLE TREATMENT 29 40 .72 .0 7 .03 23 40 ,!>7 .12 3b 40 .8 8 2.90 27 39 .9 6 .6 0 31 40 .77 .V8 32 40 .80 1.57 32 40 .80 1.57 18 40 .45 3*25 30 40 .75 .24 27 40 .7 6 .6 0 .£3 35 40 .8 8 1.9b 30 40 .75 0*00 21* 4U .70 U.OO 27 38 • 71 • 01 40 .77 .07 27 J6 .5 7 .00 40 .75 .07 2» J6 • 72 .28 23 40 .7 5 0.00 «. 40 .7 6 2 27 39 . .9 6 0.00 2« 40 3 26 40 .5 6 0.00 2b 40 .3 6 o.uo 4 27 40 .7 6 0.00 30 ;40 .75 .4 2 5 29 40 .72 U.OO JO 40 .75 O.UO 26 40 .65 6 29 40 .72 0.00 30 40 .75 0.00 28 4o .7o 40 .70 o.uo 27 40 .67 40 .7 6 1.03 29 .72 7 8 29 32 40 .72 40 .80 0.00 28 0.00 27 1.J7 .8 4 1 40 (U6 .28 31 JO 0.00 �Table 17 AVERAGE IMPLANTS PER PREGNANT FEMALE - FOI-PET •ELK COSTAOL 7*-Oi IZSl) Mtf/KD M-03 £*>00 M6/K6 7»-03 9000 Mo/Mi .ZMG/iVb IE" MULTIPLE TKtATMtNT 30«/ 27*11.*! J2*/ 29*11.17 32B/ 30*10.93 316/ 29*10.90 27*11.30 30*/ ZB.10.b6 257/ 23*11.17 390/ 33*11.1* 293/ 27*10.85 26*10.31 271/ 2b*10.8* 33«/ 31. 10. 1 1 327/ 32*10.22 3*8/ 12*10.87 299/ 3o* 9 9 .7 18J/ 18*lu>l7 JZO/ 30*10.67 266/ 27* 9 8 .5 26*12.^3 1 Z6S/ 23*11.5? 2 JOS/ 3 Z68/ * Z88/ j7.lu.67 5 33»/ Z9*11.SZ 333/ 30*11.10 31tt/ 399/ 35*1 l.*0 3S6/ 30*11.87 6 3Z3/ 29*11. 1» 33«/ 30*11.13 29«/ ZB.10.sO 312/ 28*11.1* Z73/ 27*10.11 7 3Z3/ 29*11.1* 307/ 28*10. >6 311/ 27*11.32 3!>6/ 31«ll.»t> 306/ 27*11.33 8 3«l/ 32*11.91 301/ Z'«1U1' 3*3' Z9-U. VO 3*6/ 30*11.53 322/ 26*12.38 • SIGNIFICANT AT f LT 0.05 •• SIGNIFICANT AT P LT U.01 �Table 18 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - FOLPET • EtK CONTROL 74-03 2SOO M G / R & 1250 HO/KG mJLUPLt 1 7»-03 5000 IE* T«! «fMENT 23< .35 1U/ .37 25/ 29» .6 8 13/ 30« .43 2 a/ ll/ 2. 7 .41 la/ Z8» .64 20/ 2' 3 .7 0. 21/ 35» 3 16/ 26> .62 U/ 2b. .52 19/ 31. .61 ll/ 32« * 16/ 27. .59 16/ 30. a/ la* .4 4 7/ 26« .J5 16/ 28» .57 27* .3 5 V/ 62/ 2 ' «.1»" 9 .60 TV 2 . 2.e5** 7 .34 87/ 32' 2.72** 30. ,23»D ll/ 27. .41 16/ 35s .46 22/ 30. .73 13/ Ha* .46 \v 27. .63 5 15/ 29. .52 12/ 30« .40 6 9/ 29« .31 17/ 30« .57 7 21/ 29. .72 U/ 2B« .43 10/ 27' .37 la/ 31- .8 5 ll/ 27« .41 a 13/ 32' .41 ll/ 27. 20/ Z9« .9 6 13/ 30' .43 ll/ 26" .42 • •• D S I G N I F I C A N T AT P LT 0.05 S I G N I F I C A N T AT P LT 0.01 DECREASED BELOW CONTROL .41 �CHI-SQUARE TEST OF THE DEATH INDEX - FOtPET 1 DECREE OF FREEDOM VEnICLt CONTROL N N HOI PRl> DEATH INUtX CHISU /»-03 USO MG/Kti N N DEATH • UI HKW JNDEx CfllSU 7»-03 N N •01 PRI> 2500 DEATH iNUtX 7--03 CHI SO N N »Ul PRb 5000 Mb/KU OtATH JNBEX CHISU TEH N N »L)I PhO DEATH INDEX CHISU MULTIPLE TNLATKENT 1 8 23 .35 0.00 a 27 .JO .VI 13 24 .5 4 .<0 10 30 .33 .03 25 <:v .6 8 12.49** 2 9 27 .3 3 00 .0 13 *8 ,6 » .I S 12 23 .52 1.12 IS 3b .3 4 .b 2 in il .6 9 2.9* 07. «!b .36 .01 V 31 .29 .22 « 32 .2b 2S J2 .78 7.85** .37 .00 7 la .39 . UJ 1.27 8 tJ .30 .32 8 26 .31 .07 • 34 .00 10 JO • 33 .01 •32 • 01 10 *1 .7 3 .2 2 3 10 26 .38 0.00 V 4 11 27 .41 0.00 11 30 5 11 29 •38 0.00 10 30 .33 .01 11 30 .7 3 ,ti 11 6 7 8 7 30 12 35 6 29 •8 2 00 .0 2* • 39 29 .41 0-CO rt «!B .9 2 .b4 * 2T .33 .12 13 31 • 42 • 05 » <f .33 • 12 0.00 4 .33 «0» 16 29 .55 1.89 11 30 .37 • 01 10 <6 .38 .04 12 11 32 •34 ** SIGNIFICANT AT PIT 0.01 2' .43 V .23 •-.«-. 28 �Tible 20 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLAXTS - FOLPET WttK 7»-OJ CONTROL 12bO MO/KG 7»-OJ 5000 Mt>/f\G Tt* .?MG/Kb HULTIHLE TKtATMtNT 1 8 265* ' .03 10/ JOB* .03 2b/ 32** .8 0 13/ 328* .0* «2/ 316* .0 2" 2 ll/ 305* .* 0 lb/ 30*. .6 0 20/ 257* .UB* 21/ 390* .05 77/ 293* .6 2" 3 16/ 268* .6 0 U/ 271. .05 I*/ 33*. .6 0 ll/ 327. * lb/ 288. .6 0 If/ 29V = .05 8/ 1«3« 5 15/ 33*. .0* 12/ 333. .0* •V 318* 6 9/ 323* .03 I// J3»- .05 7 21/ 323* .' 0 \i/ 307. 8 13/ 381. .03 ll/ 301* '•' • SIGNIFICANT AT P LT 0 0 .5 •• SIGNIFICANT AT V LT 0.01 .03 87/ 3*8. .5 2" 7/ 320* .02 ll/ 266* • 0* .03 16/ 391' .0* 22/ 356' .6 0 16/ *9»« .Ob 13/ 312* .0* 17/ 273. .06 .«* 10/ 311* .03 18/ 356. .05 ll/ 306. .0* .* 0 20/ 3*5' .6 0 13/ 3*6* .0* ll/ 322. .03 .0 �Table 21 CHI-SQUARE TEST OF THE FERTILITY INDEX - AZINPHOS-METHYl, 1 DECREE OF FREEDOM VEHICLE CONTROL MEEK N N PRO MTO FERT. INDEX CHISQ 74-09 N N PUG MTD 20 74-09 MG/K6 FERT. INDEX CHISQ N N PR6 MTD MULTIPLE «. 05 *0 MG/KO FERT. INDEX CHISQ 74-09 •» N PRU MTD DO MO/KO FEPT. INDEX TEM n Chi SO PRO 0.00 29 MG/KO N FERT. MTD INDEX CHISQ 40 .2 7 0.00 TREATMENT 1 28 40 .70 0.00 34 40 .5 8 1.79 28 40 .70 .6 0 27 40 2 26 40 .65 0.00 29 40 .2 7 .3 2 27 40 .67 0.00 27 40 .7 6 0.00 27 39 .9 6 .03 24 38 .3 6 .08 32 40 .80 3.72 .8 5 .35 27 40 .7 6 .6 0 1.42 3 23 40 .57 0.00 33 40 .2 8 4.82*1 29 40 .72 1.37 4 27 40 .67 0.00 30 40 .5 7 .4 2 2» 40 .2 7 .6 0 21 36 5 24 .60 0.00 29 40 .2 7 .9 8 26 40 .5 6 .05 20 34 .9 5 .9 7 27 40 .7 6 • 03 28 34 .2 8 .8 7 25 40 .3 6 .71 .11 26 40 .5 6 40 6 24 38 • 63 0.00 30 «0 .5 7 7 30 .9 7 0.00 27 40 .7 6 8 • I 27 38 38 .1\ 0.00 SIGNIFICANT AT P LT 0.05 INCREASED ABOVE CONTROL 26 40 .5 6 1.81 24 34 .11 22 34 .5 6 30 40 .75 27 38 • 71 .24 .30 f 36 .5 7 .02 .10 26 i6 .2 7 .02 .12 2.41 �T.;ble II AVERAGE IMPLANTS PER PRECNANT FEMALE - AZINPHOS-METHVL *EEK CONTROL 74-09 20 MG/KG 74-09 40 MG/KG 74-09 80 MG/KB .2 KG/16 MULTIPLE TREATMENT 1 28*11.39 3T5/ 34*11.03 300/ 28*10 .71 306X 27*11 .33 31*/ 29*10.90 2 303/ 26*11.65 335/ ;«*u.ss 312/ 27*11 .56 310/ 27*11 .*8 293/ 27*10.85 3 vO 319/ ?45/ 23*10.65 379/ 33*11.48 33B/ 6 29*11 . 6 272X 24.11 .33 34«/ 32*10.87 4 309/ 27*11.44 367/ 30*12.23 369/ 29.12.72**! 247/ 21*11 .76 266/ 27, 9.85 * * 274X 24*11.42 337/ 29*11.62 310/ 26.11.92 228/ 20*11 3S6/ 30*11.87 6 302/ 24*12.58 34S/ 30*11.50 * 276/ 27*10 .22** 323X 28*11 .5** 273/ 27*10.11 ** 7 346/ 30*11.53 288X 27*10.67 278/ 25*11 .12 262/ 24*10.92 30A/ 27*11.33 8 292X 27.10.fll 306/ 26*11.77 293/ 26.11 .?T 250/ 22*11 .36 322/ 2 . 12. 39 **I 6 • S I G N I F I C A N T AT P LT 0.05 •• SIGNIFICANT AT P LT 0.01 I INCREASED ABOVE CONTROL .0 4 �Table 23 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - AZINPHOS-METHYL MEEK 7*-09 CONTROL 20 MG/KG V4-09 40 MS/KB 74-09 80 H8/KO TEM .? XG/K6 MULTIPLE TREATMENT 1 13/ .6 * 13/ 34. . 8 3 3*/ 28> 1.21 . 19/ 2 8/ 26. .31 16/ 2. .5 9 5 15/ 27> .56 19/ 2727« 3 9/ 23. .39 31/ 33. .4 9 2. .8 9 2 ll/ 2. 4 28> 4 2/ 27. .07 21/ 30- .0. 7* 5 ll/ 24. . 6 4 IS/ 29» .2 5 6 21/ 8 2. .8 4 10/ 30. ,33«*D 7 30/ 30. 1.00 8/ 8 • •• D 19/ 27. .70 13/ S I G N I F I C A N T AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL 27. .30 26. .50 a/ 31/ 2 . I.OT** 9 7/ .4' 29> 2 1 . .0 7 62/ .70** 77, 27. 2.85** .6 4 87/ 32. 2.72** .33* ll/ 27. .41* .5 5 22/ 30. .73 .32**D 17/ 2. .3 7 6 .23 ll/ 2120. 17/ 2 . .3 7 6 9/ 28> a/ 25. . 2 3 19/ 2 . .T9 4 ll/ 2 . .41 7 10/ 3 2 . .8 6 a/ 2 . .36 2 ll/ 2 . .42 6 6/ 2ft. �Table 24 CHI-SQUARE TEST OF THE DEATH INDEX - AZINPHOS-METHYL 1 DECREE OF FRF.EEOM VEHICLE CONTROL N PRG N DEATH INDEX CHISQ 74-09 N N MOI PRO 20 MO/KG DEATH INDEX CHiSQ 74-09 N N MOI PRS 40 H6/KO DEATH INDEX CHISQ 74-09 N N MOI PRO 80 MG/KO .? KG/KG TE" N N MOI PHG DEATH INDEX CHI SO .1 4 .1 0 25 29 DEATH INDEX CH1SO MULTIPLE TREATMENT 1 ! 10 28 .6 3 0.00 12 34 .5 3 .5 0 14 28 .0 5 .6 6 11 27 .Hft 13.27** 2 7 26 .27 0.00 11 29 .38 .34 8 2* .30 .01 12 27 .4 4 1.09 26 27 .6 9 24.26** 3 9 23 .39 0.00 16 33 .48 .18 5 29 .IT 2.11 a 24 .33 .01 25 32 .78 T.OS** 4 2 27 .7 0 0.00 13 30 .3 4 7,70** 14 7 21 .33 3.65 5 9 24 .38 0.00 13 29 .45 .07 7 20 .35 .02 24 .3 6 0.03 a 30 .27 8 28 .9 2 8 30 .7 2 0.00 7 27 .6 2 24 .2 * .T6 27 .*» 0.00 .38 8 22 .6 3 .8 0 6 7 8 * ** D 15 12 SIGNIFICANT AT t LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL 10 26 29 .8 4 9 53" . 6 26 .23 .4 6 9 27 .33 3.25 .6 0 7 25 .8 2 .4 0 .03 8 26 .31 .55 5.61**D 10 4.73 *a 8 27 .30 3.07 10 30 .33 .00 10 27 .37 2.36 9 27 .33 .07 26 .31 .03 10 �Table 25 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - AZINPHOS-METHYL *EEK T»-09 CONTROL 20 Mt/KO 74-09 40 M6XKO 74-09 80 M6XKO TEM .2 MOXK3 MULTIPLE TREATMENT 1 13X 319. .04 13X 375* .03 34X 300* .11 19X 306* .6 0 62X 316* .20" ISX 312* .05 19X 310* .6 0 77X 293* .26" 38 3 . .02 MX 272« .* 0 BTX 3 8 4. .5 2" 7X 247* .03* MX 266. .04* .5 0 22X 356. .6 0 ,3D 0" 17X 273* .6 0 .07 MX 306. .04 .0* MX 322* 2 •Ji M «X 303« .03 16X 335* .05 3 9X 245. .04 31X 379. .08 2X 309. .01 21X 367. .06 " 4 5 11X »* .04 15X 337. .4 0 6 21/ .07 10X 345. .os «»o 7 30X .09 8 19X i'/2« .07 • •• D ax 2 8 8 . .03 ]3X 3o>* S I O N i r t C A N T AT P LT 0.05 S I G N I F I C A N T AT P LT 0.01 DECREASED BELOW CONTROL .04 ax 31X 369. 6X 310. .08" .02 17/ .•6 . 6 0 .7. ax 278. 10X 2»3* .03 • 03 llx 228. 9X 323* 19X 262* ex ?5,» • 03 �•Vable 26 CHI-SQUARE TEST OF THE FERTILITY INDEX - KM./.THION 1 DEGt_E OF FREEDOM KEEK VEHICLE CONTROL N N PRG MTD FERT. INDEX CHISO 7»-07 N N PRG MTO 1253 MO/KG FERT. 1NOEX CHISO 74-07 N N PRG MTO 2500 MG/KG F£RT. INDEX 7»-07 5000 MG/KG N N .6 0 29 40 .2 7 CHISQ FERT. PRG MTD INDEX CHI sa TFH .? MG/KO N N FV3 HTO FERT. INOE'. CHISQ 0.00 MULTIPLE TREATMENT 1 28 40 .70 O.OC 31 40 .7 7 .26 26 40 .5 6 2 26 40 .5 6 0.00 30 40 .5 7 .4 5 24 40 .0 6 .05 31 39 3 23 40 .57 O.bO 23 38 .61 .00 24 4? .0 6 0.00 33 40 4 27 40 .7 6 0.00 25 38 .6 6 .01 22 38 .8 5 .1 4 .01 22 36 ^^ • 58 .61 .8 7 1.26 19 34 .6 5 5 6 2* 40 .0 6 0.00 21 36 2-, 38 .63 0.00 28 36 40 .72 .9 7 1.40 27 39 .9 6 .03 .82 4.82*1 32 40 .to 3.72 27 40 .67 .6 0 1.42 40 .67 .06 .02 *0 .5 8 5.08*1 30 40 .75 .15 35 40 .8 8 5.02*1 27 38 .71 .4 2 32 40 .0 8 .03 27 36 .5 7 .02 37 40 .2 9 4 7 * . 2 : 26 36 .? 7 .02 7 10 38 .79 0.00 30 36 .3 8 .03 19 34 .6 5 8 27 38 .! 7 0.00 30 36 .3 8 .6 9 16 34 .7 4 3.36 SIGNIFICANT AT P IT 0,05 INCREASED ABOVE CONTROL 29 34 3.39 ** I 0.00 �Table 27 AVERAGE IMPLANTS PER PREGNANT FEMALE - MALATHION KEEK CONTROL 74-07 1250 MO/K3 74-07 2500 MG/KG 74-07 5000 MG/KG TEM .2 "G/KG HM.TIPLE TREATMENT 319/ 28«11.39 346/ 3L11.16 4 298 / 26.11 . 6 321/ 29*11.07 316/ 29.10.90 2 303/ 2.16 61.5 363/ 3.21 01.0 267/ 24.11 .12 3S6/ 31.11.48 293/ 2.08 71.5 3 24S/ 23.10.65 281/ 2 . 2 2 *I 31.2 30S/ 24.12 .71*1 402/ 33.12.18*1 348/ 32.10.87 " » 309/ 2 . . 4 7 U 4 304/ 25.12.16 256/ 6 22.11 . 4 300/ 27.11.11 266X 27. 9.6-3* 5 274/ 24.11,42 238/ 21.11.33 243/ 22.11 . 5 0 386/ 34.11.35 356/ 3 .' 1 8 0 ..7 6 30?/ 24.12.58 318/ 28.11.36 * 226/ 8 19.1* . 9 391/ 35-11.17" 273/ 27.10.11** ' 3*6/ 30.11.53 337/ 3.12 01.3 235/ 1 . 2 .37*1 91 3SZ/ 3 . 1 00 21. 306/ 2.13 71.3 8 323/ 3 . 0 7 0 1 . 7 187/ 6 16.11 . 9 397/ 37>10.73 322/ 2.23*! 61.8* 1 VI 4 • •• I 292/ 27.10.81 SIGNIFICANT AT f LT 0.05 SIGNIFICANT AT P LT 0.01 INCREASED ABOVE CONTROL �Table 28 AVERAGE DEAD IMPHXTS PER PREGNANT FFVALE - MALATHION MEEK 7»-07 CONTROL 1250 MG/KG 74-07 2500 MG/KG 74-07 5000 MG/KG .2 MG/KG TEH MULTIPLE TREATMENT 1 13/ 28. 2 i 12/ .46 8/ 26. .31 3 9/ 23. .39 30. .40 a/ u/ .46 ll/ 31* .35 77/ 27. 2.85" 14/ 23. .61 14/ 2. 4 .58 14/ 33. .42 87/ 32. 2.72" .56** 22. .36* 18/ <, 31« .8 5 1 4 2/ 27. .07 14/ 25. 5 U/ 24. .46 4/ 21. .19 e/ a/ o 21/ 24. .88 21/ 28, .5 7 20/ 7 30/ 30. 1.00 10/ 30. .33 6/ 8 19/ 27. .TO 19/ 30. .63 7/ • SIGNIFICANT »T P LT 0.05 •• SIGNIFICANT AT P LT 0.01 2. 6 .31 13/ 29. .5 4 62/ 29. 2.14" 24. 7/ 27. .26 ll/ 27. .41* 2 . .36 2 a/ 34. .24 22/ 30. .73 19. 1.05 23/ 35. .6 6 17/ 27. .63 19. .32 IS/ 32> .7 4 ll/ 27. .41 16. .4 4 32/ 3 . 7 .6 8 ll/ 2 . .42 6 �Tlt>l* 29 CHI-SQUARE TEST Of THE DEATH IPDEX - MALATHION 1 DECREE OF FREEDOM •EEK VEHICLE CONTROL N N N DEATH •01 PRG INDEX 74-07 CHI SO N 1250 NO/KG C^ATH •DI PRO INDEX CHISQ 74-07 N N •01 PRG 2500 MG/KG DEATH INDEX CHISO 74-07 N N •01 PR3 5000 MG/KO TEN .2 MB/KG N DEATH INDEX CHISO N DEATH •oi PHG INDEX CHISO MULTIPLE TREATMENT 1 10 28 .6 3 0.00 13 31 .2 4 .5 0 7 26 .7 2 .6 1 10 29 .4 3 .03 25 29 .6 • 13.27" .8 0 9 24 .0 3 .25 8 31 .6 2 .4 0 26 27 .»6 24.26 " .10 24 .«» .03 9 33 .27 .! * ZS 3Z 5 27 .19 .6 6 7 34 .21 1.26 2 7 <I6 .27 O.OP 8 30 .7 2 3 9 23 .9 3 0.00 7 23 .30 11 1.24 4 2 27 .07 0.00 10 25 .40 6.04* S 22 .23 5 9 24 .38 0.00 3 21 .1* 2.01 6 22 .7 2 .18 15 Z4 .3 6 0.00 11 6 7 8 30 .27 0.00 8 12 27 .4 4 0.00 * SIGNIFICANT AT P LT 0.05 »• SIGNIFICANT AT P LT 0.01 28 .9 3 1.93 6 1* .2 3 2.91 16 35 .6 4 1.01 7 30 .3 2 0.00 4 19 .21 .01 12 32 .8 3 .41 30 .7 4 .1 0 7 16 .* * .7 0 16 37 343 .03 14 .0 7 7.05 ** 8 27 .30 3.07 10 30 .33 .00 10 27 .7 3 23 .6 9 27 .33 .07 26 .8 ' .3 0 10 �Table 30 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - MALATHIOS »EEK CONTROL 74-07 1250 MG/KG 7*.or 2500 MG/KG 74-07 SOOO MG/KG .2 HO/KG TE" MULTIPLE TREATMENT .5 0 8/ 2 8 9. .03 13/ 321« .4 0 2" 6?/ 316' . 0 8/ 303. .03 12/ 363. .'03 ll/ 267. .4 0 ll/ 356« .03 77/ 3 9/ 245. .04 14/ 281. .05 14/ 305. .05 14/ 402* .03 87/ 348. .25" 4 2/ 309. .01 14/ 304. .5 0" B/ 2S6. .03 T/ 300' .02 ll/ 266* .04» .20 0* a/ .03 8/ 386. .2 0 0 22/ 3 6 5 . .6 0 2 . .9 20 / 2 6 23/ 391« .6 0 17/ 273' .6 0 1 13/ 319. .04 2 18/ 346. 293* .6 2" 5 ll/ 274. .04 4/ 238. 6 21/ 302. .07 21/ 318. .7 0 7 0 30/ 346. . 9 10/ 337. .03 6/ 235. .03 15/ 352. .0* ll/ 306. .* 0 8 19/ 292. .07 19/ 323. .06 7/ 187. .4 0 32/ 397. .8 0 ll/ 322* .03 • S I G N I F I C A N T AT P LT 0.05 •• SIGNIFICANT AT P LT 0.01 D DECREASED BELOW CONTROL 23 4. �CHI-SQUARE TEST OF THE FERTILITY IXDEX - PARATHION 1 DEGREE OF FREEDOM VEHICLE COM»OL MEEK N N FERT. PRG MTO INDEX 74.01 N CHISO N 62.5 MG/K6 FERT. PRG MTO INDEX CMlSB 74-01 N N PRG MTO 125. NQ/K6 FERT. INDEX CHISQ T*-01 0. M6/KG N N PRG MTO FEBT. INDEX CKISJ TFM MR/KG s PHG MTO 29 N FEHT. INOFX CHISO . 0 .? 7 1.37 J9 .• •9 .6 0 1.57 5 MULTIPLE TREATMENT 1 23 40 .57 0.00 25 40 .63 .05 27 40 .7 6 .8 4 22 40 ., '5 .72 23 40 .57 .72 27 .Ob 32 40 .0 0 0.00 2 27 39 .9 6 0.00 32 40 .80 .71 23 40 .57 3 26 40 .5 6 0.00 23 40 .7 5 .21 20 40 .50 1.28 26 40 .5 6 4 27 40 .7 6 0.00 25 40 .3 6 .05 23 40 .57 .48 2B 40 .70 0.00 27 40 .*7 5 29 40 .72 0.00 25 40 .3 6 .51 25 40 .3 6 .51 30 40 .75 0.00 30 40 .75 6 29 40 .72 (.00 26 40 .70 23 40 .57 1.37 J2 40 .80 .8 2 27 J1 .71 .01 40 2.61 2-» 40 .72 .6 0 27 36 .75 40 30 40 .5 7 .07 «6 36 .72 .no .?« 40 .72 (0 .1 29 • 32 41 .0 8 (0 .( 33 7 29 O.(0 .' .2 .6 0 21 40 .2 5 .2 * (( .0 29 40 .2 7 .8 2 .06 0.00 �Table 32 AVERAGE IMPLANTS I E R PREGNANT FEHAJ.E - PARATHION WEEK CONTROL 74-01 62.5 HG/K3 74-01 125. 7*-fll 250. Mb/«r, re- "G/KU MULTIPLE TREATMENT 1 2 Wi VO 3 2t5/ 23*11 .52 305/ Z68/ 26b/ 25*10 .6* 31*/ 27*11.63 22*/ 22*10 .!«• 31*/ 29*10.90 291/ 27*10. «* 3»fl/ 32*10. «7 27*11 .30 337/ 32=10 .53 250/ 23=10. H7 23<S/ 23*10 .35 26*10 .31 257/ 23*11 .17 246/ ^0*12.30 >*I 277/ 2«>«10 .65 27*10.67 277/ 25*11 .08 Z53/ 2i«ll.uO 311/ 2**11 .11 26*/ ?.7t 9.85 5 334/ 29*11 .52 29Y/ 2b*ll ,V6 28S/ 2S«11.»0 339/ 30*11 .30 35«i/ 3«»11.R7 29*11 .14 322/ 2t>=ll .50 259/ 23*11.^6 331. / 32*10 .3* 271/ 27*10.11 21*11. X* 339/ 29*11 .69 30»>/ 27*11.33 29*11. b9 355/ 30*11 .83 322/ 26*12. 3B * 6 2B8/ 323/ 7 323/ 29*11 .1* 322/ 29*11.10 236.' 8 38l/ 33*11 .91 393/ 33*11 .91 336/ • S I G N I F I C A N T AT f LT 0.05 *• SIGNIFICANT »T P LT 0.01 I INCREASED ABOVE CONTROL �Table 33 AVERAGE DEAD IMPLAXTS PER PREGNANT FEMALE - PARATHIOK NECK 74-01 CONTROL 62.5 MG/KG 74-01 125. MG/KG T4~01 250. HG/KG MULTIPLE TREATMENT 1 8/ 22* .6 3 6?/ 2 . 2.14** 9 .3 4 17/ 23* .74 77/ 2 . 2.85** 7 8/ 26* 8/ 2 . 3 .35 18/ 2. 5 .2 7 ll/ 2 . .41 7 27. .41 14/ 32. .4 4 10/ 23. 2 ll/ 3 6 6 16/ 2 . . 2 7/ 23. .30 10/ 20* .0 5 .35 B7/ 32. 2.72** 4 5 16/ 27. . 9 9/ 2. 5 .36 5/ 23. .20 2* »2' 26* 1.50 ll/ 27. .41 S IS/ 2. 9 .52 13/ 2. 5 .52 14/ 2. 5 .6 5 ll/ 30. .37 22/ 30. .7* 6 9 9/ 2 . .31 10/ 2. 8 .36 9/ 23. .39 14/ 3. ? .4 4 17/ 27. .63 7 9 21/ 2 . .72 15/ 2. 9 .2 5 23/ 21. 1.10 22/ 2 . .6 9 7 ll/ 27. .41 33* .9 3 9/ 2V. .31 6/ 30* I)/ 2A> .2 * 8 13/ 32* .1 4 13/ • SIGNIFICANT M P LT O.OS •• S I G N I F I C A N T AT P LT 0.01 D DECREASED BELOW CONTROL .0 2 �Table 34 CHI-SV'ARE TEST OF THE DEATH INDEX - PARATHION 1 DEGREE OF FREEDOM •EEK VEHICLE CONTROL N N «DI PRG DEATH INDEX CMISO 74-01 N N •01 PRS 62.5 MG/KG DEATH INDEX CHISU 74-01 N N HOI PRG 125. "G/KO DEATH INDEX 7«-01 CM I SO N •U. N .? MG/KG 250. MO/KG OEATH PRG INDEX ChlSU N K »CI P«G CEATH INOFX CHISU MULTIPLE TREATMENT 1 8 23 .35 o.oo 2 9 27 .33 0.00 .4 1 .25 9 27 .33 .04 5 22 .23 .32 25 <>9 .MA 12.49** 32 .34 .04 a 23 .35 .04 7 23 .30 .01 26 27 .If, 20.7<*.» 7S* .5. 6 25 11 3 10 26 .8 3 o.oc 7 23 .0 3 .8 0 8 20 .0 4 .4 0 7 26 .27 .* 3 25 32 ,7« 4 11 27 .41 0.00 a 25 .32 .13 4 23 .17 2.11 10 2a .6 3 .01 H *7 .30 .32 5 11 29 .38 0.00 7 25 .8 2 .23 9 25 .6 3 .02 8 30 • 27 .42 10 30 .11 .01 8 29 .28 0.00 9 28 .32 .01 7 23 • 31 .00 10 27 .37 .22 0.00 12 29 .41 .07 11 33 .33 .03 6 7 12 29 • 41 8 ' 11 32 .4 3 ** SIGNIFICANT AT PIT 0.00 0.01 • 30 • VI 10 32 21 .57 .6 6 11 29 • 38 o.oo ? ^r •n .12 9 29 .31 .00 5 30 .17 1.70 in />>> .1* .10 12 �Table 35 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - PARVTHION *EEK 7*-01 CONTROL 6Z.5 KG/KG 74-01 125. MG/nO 74-0) 350. M6/KG IEM MO/KO MULTIPLE TREATMENT .03 IB/ 26t>« .07 ll/ 314. .4 0 2 ll/ 305. .04 14/ 337. .04 10/ 250" .04 3 16/ 26Bz 7/ 257. .03 10/ 24f>. c .4 0 1 \ ' 4 5 6 8/ 265> .06 16/ 2BB. .06 9/ 277. .03 IS/ 334. .04 13/ 299. .4 0 10/ 322> .03 9/ 323" .03 7 21/ 323. .07 0 15/ 3 2 2 ' .5 8 13/ 381. . 3 0 9 . .03 13/ 3 3 • SIGNIFICANT AT P LT 0.05 •• SIGNIFICANT AT P LT 0.01 0 DECREASED BELOW CONTROL e/ 224. .4 0 62/ 316. .20** 17/ 230* .07 77/ 293. .?!•>** 277* .03 «7/ 34R. .25** .02*0 42/ 311. .14 ll/ 266. .4 0 14/ 28b. .AS ll/ 339. .03 2?/ 356. .6 0 9/ 25V. .03 14/ 331« .04 17/ 273« .06 23/ 2 6 3 . .10 22/ 339» .6 0 ll/ 30*. .4 0 .20 0* ll/ 322* .03 s/ a -3. 9/ 336. .3 0 e/ 6/ 355* �CHI-SQUARE TEST OF THE FERTILITY INDEX - PARATHTON-KETHYL. • DEGREE OF FREEDOM 74-05 VEHICLE CONTROL DECK N N FERT. PUG MTU INDEX cms'u N N PNCi MTU 20 74-OS M6/K6 FtRT. INOEX H CMIbU N PHG MTU 40 74-05 FtRT. 1NOLX CHliU N N PHO MTO TEM 80 FERT. INCH CMlSQ MS/KG N N PXI> MTU INUL« CM I Si. MULTIHLE TREATMENT 1 W 23 40 .57 0.00 ' 9 40 .72 1.47 24 40 .60 0.00 29 40 .72 1.37 29 40 .72 1.37 27 39 .9 6 0.00 33 40 ' .82 l.<:5 23 40 .57 .72 29 40 .72 .01 27 39 .9 6 .06 3 26 40 ,65 30 40 .75 .34 32 40 .80 1.57 2M »o .70 .6 0 32 40 .40 1.57 4 27 40 .7 6 o.oo v.oo 38 40 .5 9 8.«1"I 26 37 .70 .00 C6 39 .7 6 .03 2 40 ' .7 6 .6 0 5 29 40 .72 0.00 J2 40 »0 .1 7 0-00 6 29 40 .72 0.00 33 40 7 29 40 .72 0.00 36 8 32 40 .80 0.00 32 2 *• I SIGNIFICANT AT P LT 0.01 INCREASED ABOVE CONTROL .80 .<rB 24 *0 .0 6 .9 6 30 40 .75 0.00 30 .«2 .65 30 40 .75 0.00 24 40 .0 6 .9 8 27 38 .71 .01 40 .0 9 2.95 32 40 .BO ,tS 33 40 .? 8 .5 6 27 36 .7* .00 40 .80 .08 35 40 .88 .37 34 40 .85 .9 0 26 36 .72 .28 �T«ble 37 AVERAGE IMPLANTS PER PREGNANT FEMALE - PAXATHION-METHYL •EEK CONTROL 74-05 20 MG/KO 74-05 40 MG/KG 80 Hto/KG .2 TEM MULTIPLE TREATMENT 1 23*11.52 301/ 29*10.38* 268/ 24*11 .17 303/ 29*10.45* 316/ 29*10.90 293/ 27*10.85 305/ 27*11.30 34 7/ 33*10.52 241/ 23*10 . 8 4 323/ 29*11.14 3 268/ 26*10.31 306/ 30*10.20 331/ 32*10 .J4 297/ 28*10.61 34H/ 32.10.87 28 8/ 27*10.67 417/ 38*10. «7 275/ 3 26*lo . 8 281/ 2*08 61.1 26 6/ 27* 9 8 .5 300/ 24*12 .SO 368/ 30*12.27 35a/ 30*11.87 i. , 26S/ 4 5 334/ 29*11.52 38V/ 32*12.16 6 323/ 29*11.14 J66/ 33*11.09 334/ 30*11 .13 279/ 24*11.62 273/ 27*10.11 32*11 . 6 1 399/ 33*12.09 306/ 27.11.33 3S.11 .63 402/ 34*11.82 322/ 26. tit. 38 7 323/ 29.11.14 401/ 36.11.14 3*7/ 381/ 32*11.91 347/ 32.10. 84» 407/ 8 • SIGNIFICANT AT P IT 0.05 •• SIGNIFICANT AT P LT 0.01 �Table 38 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - I-ARATHION-KETHYL •UK 7»-Oi CONTHOL 20 «G/Kt» 74-05 *0 Mb/Kb 7«-Ob 80 It/US Tt« .2 HULTIHLE TREATMENT 1 23* .35 9/ 29* .31 2 s 8/ ll/ 27m .»! 10/ 33. .JO .62 B/ 30. .27 *D .9 5 !«/ 3(t* 29m .52 23/ 9/ 29m .31 20/ .72 .! * 3 16/ 26m 2b/ 2»m l.u* * 16/ 29* .55 2<<m a/ 23* .Jb 9/ 22/ 32. .6S 3«/ .«7 b/ £6» .jV «D 32. .72 17/ 2«« 33" .61 10/ 30* 7/ 36. .19*0 ll/ n/ 32m .3* 18/ 62/ .». 29« . ! . <!.es,. .31 77/ 2/m 2tf« 1.21 87/ 32> 2.72*. 9/ 26* .35 ll/ 27« •*1 ./I 12/ 30« .*0 22/ 30. .73 .J3 6/ 2»* .25 17/ 27« .63 32m .J« lb/ 33m .»S ll/ 27m .»! 35* .bl 12/ 3»« .35 ll/ 26* .42 t 5 6 IS/ 7 21/ 8 13/ 32m 29m • SIONIFICANT AT P UT 0.05 •• S I G N I F I C A N T AT P LT 0.01 D DECREASED BELOW CONTROL �Table 39 CHI-SQUARE TEST OF THE DEATH INDEX - PARATHION-METHYI. VEHICLE CONTROL KEEK N N DEATH *DI PRG INDEX CHI SO 74-05 N N • 01 PRG 20 MO/KG DEATH INDEX CHlbU 74-Ob N N •DI PRG 40 74-OS DEATH INOtX Cut SO N N •01 PRO 80 m>/*« DEATH INDEX CHISU .00 HG/KG TEH ft N • DI PNG UIAT>: INDEX CHlSg MULTIPLE TREATMENT 1 8 23 2 9 .35 0.00 a 29 .8 2 .07 12 24 .5k .be V 29 .31 2V 25 29 .6 0 12.49** 27 .33 0.00 9 33 .7 2 .05 6 23 .6 2 .6 0 a .8 2 .03 26 27 .6 9 20. 7*** 10 26 .8 3 0.00 8 30 .7 2 .»3 14 31 .* » .2 0 9 28 .2 3 .4 0 25 J2 .78 7.85** 11 27 .1 4 o.oo 14 38 .37 .00 5 26 .19 1.98 7 26 .27 .0 6 a 27 .30 .32 11 29 .38 0.00 11 32 .34 .00 24 .42 .00 10 3D .33 .01 10 30 .33 .01 8 29 .8 2 0.00 11 33 • 33 .05 8 30 .7 2 .5 0 6 24 • 25 •01 10 27 .37 .2 2 7 12 29 • 41 0.00 6 36 .17 3. ' 4 8 32 .5 2 i.ia 13 33 • 39 • 01 •» 27 .33 • 12 a 11 .4 3 0.00 8 32 .5 2 .JO 7 35 .0 2 1.10 a 34 • 24 .9 4 10 26 .38 .00 . 3 4 5 6 ** 32 SIGNIFICANT AT F LT 0.01 10 �Table 40 NUMBER OF DEAD IMPLANT? PER TOTAL IMPLANTS - PAHATHION-METHYL WEtK CONTROL 7*.Ob 20 MG/KG >*-05 «0 M6/KO 7»-OS TEM 60 .2 *G/M> MULTIPLE TREATMENT 1 „ j 8/ 265* .03 9/ 301* .03 2S/ 268* 16/ 303* .OS 62/ 316* .0* 2. 2 ll/ 305. .0* 10/ 3*7> .03 8/ 2*1» .1)3 •>/ 323* .03 77/ 293* .6* 2. 3 16/ 268* .6 0 a/ 306. .03 22/ 331. .07 3»/ 297* .11 87/ 3*8* .5. 2* 18/ M7 = .C* S/ 27S* 9/ 281* 16/ 288* .06 5 15/ 33** .0* 6 «/ 323. 7 r • •• D • 91* .03 ll/ 266* .0* 23/ 389* .6 0 17/ 30u« .6 0 12/ 368* .03 22/ 356* .6 0 .03 20/ 366* .OS 10/ 53*^ .) 13 6/ 279* .02 17/ 273* . 0* 21/ 323* .07 7/ *01* .02*0 ll/ 357* .03 16/ 399* .0* ll/ 306* .0* 13/ 381* .03 ll/ 3*7* .03 18/ *07* • u* 12/ *02* .03 ll/ 322* .03 <' S I G N I F I C A N T AT P LT 0.05 S I G N I F I C A N T AT t» LT 0.01 DECREASED BELOW CONTROL • U2*D �Table 41 CHI-SQUARE TEST OF THE FERTILITY IND.'X - qUINT07.ENE (PCNB) 1 DECREE OF FREEH- .1 VtHJCLE CONTROL N PPG N FERT. MTU INOE» CHisa T4-oe 1250 MO/KS N N PRO HTO FERT. I NOES CHISO 74-OB N N PR6 HTD 2500 «G/K6 FERT. INDEX CHISO *.-OB 5000 KG/KG N N PR3 HTO FERT. INOE« CM I SO TEH .? MR/KG N N PR 6 HTO FEBT. INOF< CHISO HULTIPLE TREATMENT 40 00 2 .70 o.co 29 40 .72 0.00 .10 40 .5 7 .6 0 31 'V . .77 .6 2 29 40 .* 7 0.00 26 40 .5 6 0.00 21 34 .2 6 .00 22 40 .5 5 .7 4 26 40 .5 6 .5 0 27 39 .0 6 .3 0 0.90 25 38 .6 6 .27 26 38 .8 6 .8 5 29 40 .72 1.37 32 40 .00 3.72 30 40 40 3 23 4= .57 * 27 40 .7 6 0.00 33 38 .7 8 3.09 .5 7 .2* 28 .70 0.00 27 *0 ,t7 .06 5 24 40 .60 0.00 19 38 .50 .4 4 ;:a 4U .0 7 .9 4 33 40 .82 3.»>*I 30 40 .75 1.42 6 24 38 .63 0.00 23 38 .61 0.00 26 40 .5 6 .00 36 40 .0 9 6.47*1 27 38 .71 .24 7 30 38 .79 0.00 27 38 .71 .8 2 27 40 .7 6 .8 7 28 40 .0 7 .»2 27 36 .75 .02 8 27 38 .71 0.00 30 3D .9 7 .8 2 31 40 ,77 .15 30 40 .75 .02 26 36 .? 7 .02 * I SIGNIFICANT AT P IT 0.05 INCREASED ABOVS CONTROL �Table 42 AVERAGE IMPLANTS PER PREGNANT FEMALE - QUINTOZENE (PCNB) »t£K 7*-OH CONTROL 1250 WG/K& 7*-08 2SCO H6/K6 74-08 5000 HO/KG IE* MULTIPLE TREATMENT 1 3l9/ 28*11.19 349/ £9* 12 .03 338/ 30*11.^7 361/ 31«11.6S 3I6/ 29*10 .90 2 303/ 26*11.65 222/ 5 ?U 10 . 7 23s/ 22*10.68 259/ 26» 9.96* 293/ 27.10 .85 11 .84 32S/ 26*12. S4 **I 353/ 29*12. 17*1 34«/ 32*10 .«7 11 .88 IV* 11 . 6 2 23* 11 .61 3»9/ 30=11.63 317/ 28*11.32 26h/ 27* <t .ft5* 342/ 28*12.2! 376/ 33»11.39 35(S/ 30*11 .87 291/ 26*11.19 ** 400/ 36-11.11* 273/ 27*10 .11 «* 287/ 27*10.63* 292/ 27*10. nl 302/ 28*10.79 306/ 27*U .33 354/ 30* 11 336/ 31*10. H» 333/ 30*11.10 322/ 26*12 .38**! 3 ?*5/ 23*10.65 296/ 25* 4 309/ 27*11.44 392/ 33* 5 274/ 24*11.42 214/ 6 302/ 24il2.Se 2h7/ 7 346/ 30*11. 5J 27*10.81 8 • •• I 292/ .; S I G N I F I C A N T AT l> LT 0.05 S I G N I F I C A N T »T f LT 0.01 INCREASED ABOVE CONTROL .80 �Table 43 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - QUINTOZENE (PC.SB) •EfK CONTROL T4-OH 1250 "G/KO 74-08 2500 MG/K6 T'.-Ob 1000 HG/KG TFM MULTIPLE TREATMENT 1 13/ 28* .46 ll/ 29* .8 3 12/ 30* .*0 ll/ 31* .35 62/ 2 . 2.14 ** 9 27. 2.85«» 2 8/ 26. .31 12/ 21. .57 IS/ 22* .68 6/ 26* .23 77/ 3 9/ 23* .39 13/ 25* .52 7/ 2*« »*7 IS/ 29* .52 87/ 32" 2. 72 ** 4 2/ 27« .07 lb/ 33" ,»5»* 7/ 30* .<-3 10/ 2«* .36* ll/ 27* .41 * 5 ll/ 24m .46 10/ . .3 5 21/ 2£« .75 16/ 33* .*« 2?/ 3. 0 .73 6 4 21/ 2 . ,8H B/ 23* .35*0 «/ Z6« .35 **D 16/ 36. .44*D IT/ 27, .63 7 30/ 30* 1.00 ll/ 27* .41 ; ' 27* 6 ,b9 20/ Z8« •M ll/ 2T« .41 a >9/ 27« 1Y/ 30* .7 5 10/ .32 13/ 30* .3 4 ll/ 26- .2 4 • •• D .TO SIGNIFICANT AT C LT 0.05 S I G N I F I C A N T AT P LT 0.01 DECREASED BELOW CONTROL 31* �Table 44 CHI-SQUARE TEST OF THE DEATH INDEX - QL1 IST07.F.NE (FCNB) 1 DECREE OF KREKUOM VEHICLF CONTROL N N uni PRO DEATH INDEX CMISQ 74-08 N N •01 PhG U50 MG/FG DEATH INDEX CHISO 7*-<18 N HOI N 74-00 DEATH pRu INDEX SOOO MS/KB N MR/KG N PnO DFATH INPF* 25 49 .8f. 13. ?7 ** N CHISO uOl c«e INCEx CHISO « 31 .2s .07 2 26 CHISO MULTIPLE TREATMENT .31 .! 0 1.35 9 22 .»t .51 .00 6 26 .23 .82 5.44 * 6 30 .20 .12 13 20 .6 4 6 26 .Jl 14 .03 <i 2, .24 .4* .27 0.00 10 21 ,4fl .9 3 0.00 11 25 .4 4 2 27 .07 0.00 12 33 .36 9 .3fl 0.00 9 19 .47 15 24 , .63 0.00 6 23 .6 2 8 30 .27 0.00 10 27 .37 .44 0.00 13 30 .3 4 2» .3fi 2 7 26 3 9 23 * 5 7 30 7 10 6 10 0.00 1 24 8 12 • *• D SICNIFICANT AT T LT 0 0 .5 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL 27 4.91 D .6 0 2.15 ?ft 27 .o>, 2 . 26" * 11 29 .30 .4 0 25 32 ,7B 7.05** .M7 7 28 • 2S A 27 ,?n 3.07 .14 13 33 .31 .02 10 30 .31 .00 .31 3.06*D 12 36 .33 3.84*D 10 27 .17 2.36 27 .52 2.01 10 28 .36 .21 31 .9 2 .9 0 12 30 .40 .00 1.96 9 27 10 2* .11 .rt? .3* ,«3 �Table 45 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - QUINTOZENE (PCNB) XEEK 74-08 CONTHOL 1250 KG/KG 74-08 2500 M6/K6 74-08 5000 MG/KO .2 MO/KG MULTIPLE TREATMENT 1 13/ 319. .4 0 ll/ 349. .03 12/ 338* .* 0 ll/ 361* .03 6?/ 316* .20** 2 8/ 303. .03 12/ 222. .OS IS/ 235. .06 6/ 259. .02 77/ 293* .Ztf* 3 9/ 245* . 4 0 13/ 296. • 04 7/ 326. • 1)2 *0 IS/ 353* .04 B'7/ 34B* .5 2" 4 2/ 309* .01 IS/ 392* .4* 0* 7/ 349* .02 10/ 317* .3 0* ll/ ?6f>* .04* 5 ll/ 274* .4 0 10/ 214* .5 0 21/ 342* .6 0 16/ 376* .4 0 22/ 3«>6« .* 0. 9/ 291. 6 21/ 302* .07 8/ 267* . ( *D 13 16/ 400* .4 0 I7/ ?73= .6 0 7 30/ 346* .09 ll/ 287. .04 16/ 292. .05 20/ 302. .07 ll/ 306* .4 0 8 ]9/ 292* .0? I?/ 354* • OS 10/ 336* .03 13/ 333« .4 0 ll/ 322* .03 • SIGNIFICANT AT f LT O.OS •• SIGNIFICANT AT P LT 0.01 D DECREASED BELOW CONTROL .03*0 �Ttble 46 TEST OF THE FERTILITY INDEX - PHOROTE 1 DECREE OF FREEDOM VEHICLE. COMRUL N N PKli MTU FERT. INDEX ChlSU 74-04 N N HHO MTU 74-0* FtHT. INDEX CMtbU N N PHO MTU 10 Mb/KU FEHT. INUEX CnliO 7«t-04 TtM 20 N N PKO MTU FERT. INDEX Cnisu .? MG/KG N N PHti M f U INDEX CHISU 40 .7? 1.37 MULTIPLE TKEATMtHT 1 23 40 .7 5 0.00 2J 40 .57 .Ub 21 40 .2 5 .5 0 «4 ! 40 .0 6 il.OO 24 2 27 39 .9 6 0.00 30 4U .75 .10 2J 40 ,57 .72 29 40 .72 .01 27 3* .*« .06 .05 24 4j ' .60 ,U5 31 40 .77 .»« 32 40 .80 1.57 40 .65 O.UO 3i 40 .0 8 1.03 27 40 .7 6 .6 0 3 26 40 .5 6 0.00 26 40 .65 4 27 , 0 .7 * 0.00 26 40 .65 O.UO 26 b £0 40 .72 0.00 lib 4u .63 .51 27 40 .7 6 .6 0 31 40 .77 .07 3D *0 .75 0.00 6 29 40 .72 (1.0(1 25 40 .63 .SI 26 4U .5 6 .3 ^ 33 40 .02 .S 6 2' 3» .71 .01 7 29 40 .72 0.00 30 40 .75 O.UO 29 4U .72 .06 3S 40 .80 1.95 27 Jb .75 ,OU 32 40 .9 6 0.00 26 40 .65 1.97 2* 40 .72 «<* 30 4U .75 .07 J6 JO .7? .2« * �T«ble 47 AVERAGE IMPLANTS PER PREGNANT FEMALE - PHORATC • EtK 7«-0« CONTROL. ?0 Nti/KO IKK .2 MG/KO MULTIPLE TRfcATMtNT 1 26S/ 23*11.52 241/ 23*10.48* 22U 21*10 .52 * 269/ 24.11 .21 316/ 29*10.90 2 305/ 27*11.30 326/ 30*10 .87 25S/ 23.11 .09 3U7/ 29*10 .59 293/ 27*10.85 26.11.50*1 *S7/ 24.10 .71 34 1/ 31.11 .00 348/ 32.10.87 266/ 27* 9.85 3 268/ 26.10.31 299/ 4 288/ 27.10.67 291/ 26.11.19 277/ 26.10 .65 353/ 32.11 .03 5 334/ 29.11.52 2T8/ 312/ 27.11 .s6 36V/ 31.11 .90 3S6/ 30.11.87 6 323/ 29.11.14 305/ 25.12 .20*1 *9S/ 26.11 .35 389/ 33.11 273/ 27.10.11 7 323/ 29.11.14 3J6/ 30.11.20 317/ 29.10 .V3 • 07/ 35.11.63 30A/ 27.11.33 381/ 32-11.91 31b/ 26*12 • 12 33b/ 29*11 05 J24/ 30*10 .do* 32<!/ 26*12.38 o • SIGNIFICANT AT P LT 0.05 •• S I G N I F I C A N T AT P LT 0.01 I INCREASED ABOVE CONTROL 2bxll .12 .'* �Table 48 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - PHORATE •tEK 7»-U» COMTHOL b MG/KS 1U MG/Kli ft* ZU MULTIPLE TKtATMtNT !«/ 21. .„ , 3/ 2*» .13 .53 «/ 23* .17 17/ 29* 2. 4 .SO 15/ 31* 26i 1.00 12/ 32* 1 B/ 23* .35 9/ 23. .39 2 ll/ 27* ,»1 16/ 30* 6?/ 29* 2.1* " .59 77/ 27* 2.85** .»B 87/ 32. 2.72** .38 ll/ 27* 3 16/ 26. .62 U/ 26* .50 U/ » U/ 27. .59 IS/ 26. .S8 26/ 5 IS/ 29* ,52 13/ 25* .bZ ll/ 27. .»! 13/ 31* ,*2 22/ 30* .73 6 9/ 29. .31 13/ 25* .52 7/ 26* ,i7 7/ 33* .21 17/ 27. .63 7 21/ 29. ,li U/ 30* ,»3 22/ 29. . /o 8 13/ 32* .«1 10/ 26* .8 3 26/ 2. 9 • •• D /-• S I G N I F I C A N T »T f LT 0.05 S I G N I F I C A N T AT f LT U . 0 1 DECREASED BELOW CONTROL .40 .! * 7/ 35* .20*0 ll/ 27. .! * ll/ 30* .37 ll/ 2. 6 .*2 �ruble 49 CHI-SQUARE TEST OF THE DEATH INDEX - PHORATE 1 DECREE OF FREEDOM «EtK VEHICLE CONTMOL N N MDI PRb DEATH INDEX CM1SO 74-04 ft N »UI PKli 5 MG/Ktt DEATH INDEX CHl>« 74-04 H N . 1 PRS 0 10 DEATH lNUt» MG/Ko CttlSU 74-04 20 N N DEATH • Ul HRli INUEr. Mb/nt! .? Mfi/KG N CnlSU N •01 PHG UEATH INDFI rwrca MULTIPLE TREATMENT 1 8 23 .35 0.00 t) 23 .35 .10 7 21 .33 .US 2 2 24 9 27 .08 3.45 .33 0.00 13 30 .43 .<b 2 23 .09 3.07 13 29 .5 4 .37 .27 . « U 24 .SO ,«!9 10 31 .32 . 4 0 .03 15 26 .5tt .v2 11 31 .34 .05 8 3 4 5 6 7 8 * ** 0 10 11 11 26 27 29 8 29 12 11 29 3? .38 .41 .38 .8 2 .«! .34 o.oo 0.00 o.oo o.oc 0.00 O.Ov SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL 7 11 12 10 9 26 26 25 25 30 9 26 .2 4 .8 * .40 .30 .35 2S £i • H6 12.44 • • 26 tl .6 9 20.7V** 25 J2 .T8 7.b5** *7 ..in .32 .01 ,<LZ 7 27 .6 2 .6 4 11 31 .35 .01 10 JO .33 .6 * 7 26 .7 2 .6 0 7 33 .21 .08 10 <7 .37 .2 2 .2 5 .<« S 3b .14 4.66*D 9 iJ .3* .12 • 30 .27 .1* 10 eb .38 .00 .»! .*>7 Ib 14 29 29 .8 * .71 �Table 50 NUMBER 0( DEAD IMPLANTS PER TOTAL IMPLANTS - PHORATE •UK 7»-0» COMHOL 5 /»-0* 7*-0» 10 20 Mb/Kb MO/KG PULTlfLt TKtATMtNT .01*D 62/ 316* . 0 ** 2 17/ 307* .6 0 77/ 293* .26 ** lb/ 3*1* .0* 87/ 3*8* .25 ** 266* .0* 1 «/ Z6S* .03 V/ 2*1* .04 10/ 221* «ub 3/ 264*. 2 ll/ 305* .o« 16/ J?6« .05 »/ 2bb* .UZ 3 16/ 266* .06 13/ ZCV* .U* 1Z/ Z57* .0. » 16/ 2B8* .6 0 lb/ ^l» .05 26/ .J77. tu 9 12/ 3b3* .03 U/ 5 15/ 33»* .04 13/ 270* .05 ll/ 312* .0* 13/ 364* .0* 22/ S56' .06 6 9/ 323* .03 13/ 30b* .0* 7/ 29b» ,u2 7/ 3«9* .02 17/ 273* .6 0 .* 0 ZZ/ 317* ,«7 7/ »07« ,OZ*D ll/ 306* .0* .03 26/ 33b* .US ll/ 3Zt* .03 ll/ 322* .03 7 21/ 323* .07 13/ 336* 8 13/ 381* .03 10/ Jib* • SIGNIFICANT AT P LT 0.05 •• S I G N I F I C A N T AT P LT 0.01 D DECREASED BELOW CONTROL �Table 51 DNA REPAIR SYNTHESIS ASSAY OF Monocrotophos DNA) 4NQO (M) Monocrotophos (M) _£, IO"7 io-6 31 io-8 HT1 10-" io-3 54 70 64 3354 46 86 1273 65* 46 2 35 42 9 46 3 39 39 25 40 36 69 1308 4 34 34 25 40 40 51 975 5 30 26 46 53 64 64 972 37 1135 Sample 1 f 4fc f 20 32 ~ 38 ~ 32 —* 47 50 92 71 SD 7 7 10 7 14 15 168 SE 6 3 5 3 6 6 69 / Mean 6 \169 * Sample deleted from calculations because of low DNA value. Only five samples used. Cell culture and experimental condition'-. T-25 flask cultures of passage 24 WI-38 cells were initiated in medium containing 10% serum. The medium was replaced with medium containing 0.5% serum on day 5 following initiation and subsequently on days 11 and 15. The assay was conducted on day 22. Hydroxyurea (10~2M) preincubaticn = 1 hour. Compound exposure time = 3.hours. 3 H-tdR added with compound. 3 H-TdR incorporation = 1 p,Ci/ml (S.A. = 13.7 Ci/mmole), 3 hours Postinoorporation incubation = medium containing TdR, 3/4 hour. Cells were removed with IK NaOH, 1 minute, 70°C. DNA was extracted by the PCA-hydrolysis procedure and measured fallowing reaction with diphenylamine. Negative control and compound solvent = 0.556 EtOH. 78 �Table 52 DMA REPAIR SYNTHESIS ASSAY OF Monocrotophos WITH METABOLIC ACTIVATION (dpm/|Ag DNA) Monocrotophcs (M) DMN (M) 0_ lO-4^ 10-3 IP"2 Sample 1 55 67 43 87 206 2 54 66 48 84 220 3 52 43 52 82 223 54 59 48 84 216 2 14 4 2 9 1 8 2 1 5 Mean SO S E 5 X 10"a Cell culture and experimental conditions T-25 flask cultures of passage 24 WI-38 cells were initiated in medium containing 10^ serum. The medium was replaced with medium containing 0.5% serum on dny 4 following initiation and subsequently on day 10. The assay was conducted on day 16. Hydroxyurea (10~2M) preincubation = 1 hour. Co.noounci exposure time = 1 hour, with the 9,000 £ fraction of a mouse liver homogena^e. *H-TdR added with compound. 3 H-TdR incorporation = I ^Ci/ral ( S . A . = 6.7 Ci/mmole), 4 hours. Postincorporation incubation = medium containing TdR, J hour. Cells were reiroved with IN NaOH, 10 minutes, 22°C. DNA was extracted by tho> PCA-hydrolysis procedure and measured fol 1 -JNing reaction w i t h diphenylamine. Negative control and compound solvent = 0.55fe EtOH. 79 �Table 53 DNA REPAIR SYNTHESIS TESTING OF BROMACIL (dpm/yg Bromacil (M) 0* Sample 1 2 3 4 5 6 Mean SD SE 195 153 212 230 217 298 218 48 19 4NQO (M) 3t I' D7 6 10" I' D5 10-*f 10- 10-5 207 201 248 129 2670 212 215 121 218 184 220 193 38 15 178 148 144 152 204 144 200 165 28 12 137 2850 105 2688 107 2702 80 2438 74 2662 87 2668 50 134 20 55 156 187 182 251 199 32 13 179 231 240 178 209 34 14 Negative control and compound solvent = 0.5% DMSO. "I" Slight precipitate observed at 10"3 M and lO"1* M 80 �Table 54 DNA REPAIR SYNTHESIS ASSAY OF BROMACIL WITH METALOLIC ACTIVATVON (dpm/ug DNA) DMN (M) Bromacil (M) 0 Sample 1 2 113 102 5 1*1 158 218 6 136' 3 4 Mean 145 SD 41 SE 17 iir7 10-6 10-5 10-" 10~3 191 112 174 189 152 170 165 30 12 91 117 110 82 104 96 100 13 5 102 161 178 131 185 141 133 155 400 397 119 120 110 149 135 167 165 141 37 23 24 15 9 10 105 50 * Sample lost. 81 102 102 91 126 190 5 X ID"2 * 529 645 448 484 �Table 55 DNA REPAIR SYNTHESIS ASSAY OF CACODYi.IC ACID DNA) 4NQO (M) Cucodylic Acid (M) 0* 1C-7 ic-6 io-5 10-* 10- »t Sample i 61 47 __* 31 36 1891 2 31 32 41 48 27 1681 59 25 30 19 45 32 38 __* __* 29 22 28 28 3 1 19 38 36 32 11 4 3 4 5 6 Mean SD SE 18 25 35 34 15 6 3S» 22 27 40 13 34 10 5 5 —* 63 68 23 29 46 23 11 * Negative control and compound solvent = O.S^b DMSO. t Slight lowering of pH at 10"3 M. t Sample lost. 82 10- * 2418 2245 1430 2275 1990 387 158 �Table 56 DNA REPAIR SYNTHESIS ASSAY OF CACODYLIC ACID WITH METABOLIC ACTIVATION (dpm/pg DNA) Cacodylic Acid (M) DMN (M) 0* Sample 1 io-s 10-" io-3 44 25 21 29 381 5 X IO-2 2 30 33 22 25 384 3 23 39 28 21 339 Mean 33 32 24 25 368 SD 11 7 4 4 25 SE 6 4 2 2 15 Negative control and compound solvent = 0.5^> EtOH. 83 �Table 57 DNA REPAIR SYNTHESIS ASSAY OF CAPTAN DNA) Oaptai? (M) _°T 4NQO(M) io-« io-7 io-6 10~5 10- 10-5 •Sample 1 37 43 41 74 81 8 924 2 64 58 S3 60 81 6 947 3 76 so 68 52 57 7 1106 4 76 60 73 37 51 5 801 5 63 61 56 50 72 5 760 6 66 44 66 82 G5 6 884 Mean 64 51 59 59 68 6 904 SD 14 8 12 16 12 1 122 SE 6 3 5 7 5 Negative control and compound solvent = 0.5% DMSO. 84 . 0.4 50 �Table 58 DNA REPAIR SYNTHESIS ASSAY OF CAPTAN WITH METABOLIC ACTIVATION (dpm/|j,g DNA) Captan (M) 0* 10~3 5 x 10~* 89 7 _-t 50 73 5 48 71 5 35 50 77 6 353 SD 7 2 9 1 43 SE 5 1 5 0.6 31 Sample 1 2 / 3 Moan IP"5 10- * 30 53 4O t DMN (M) Negative control and compouud solvent = 0.5^. DMSO. Sample lost. 85 323 i 384 �Table 59 DNA REPAIR SYNTHESIS ASSAY OF CHLOROPYRIFOS (dpm/p£/DNA) Chloropyrlfos _l_ Sample 1 115 f 6 4NQO ( I I) 5 io- io- 10-" 28$ 282* __* 61 10-7 3 io109 __* 10-s 1337 _>± 2 143 129 93 110 67 3 96 102 84 4 72 95 64 110 __* 60 52 5 97 89 6 • 98 85 64 79 Mean 78 92 98 86 103 85 101 64 49 66 SO 17 15 13 11 9 31 220 SE 7 7 6 5 4 15 98 ' 99 __* 1721 68 37 1220 1208 1209 Negative control and compound solvent = 0.5^> DMSO. Sample delete-! from calculations because of low DNA value. Sample lost. 86 1339 �Table 60 DNA REPAIR SYNTHKSIS ASSAY OF CHLOROPYRIFOS WITH METABOLIC ACTIVATION (dpm/jig DNA) DMN (M) Chloropyrifos 0* io-s Sample 1 72 79 70 5; 75 65 3 55 Mean 10- io-3 5 X 10~a 355 75 52 71 67 63 79 384 67 70 69 67 363 SO iO 8 6 14 18 SE 5 5 4 8 11 Negative control and compound solvent = 0.5% DMSO. 87 349 �Table 61 DEN REPAIR SYNTHESIS ASSAY OF D1NOSEB (dpm/u« DNA) Dinoseb (H) 0* Sample 1 2 3 / 4 5 6 Mean SD SE 10-7 10-6 115 101 67 143* 101 68 96 72 97 112 54 61 57 78 92 17 58 82 7 26 11 4NQO (H) io-4t io-» 10~5 103 100 116 106 ICO 79 1721 58 6C 62 66 1220 60 67 1208 73 64 7 60 84 76 1209 82 1339 25 17 220 3 10 7 98 -> Negative control and compound solvent =0.5^ DMSO. Suggestion of precipitate at 10~"* M. Sample deleted from calculations because of low DNA value. S Sample lost. 88 1337 __§ �Table 62 DNA REPAIR SYNTHESIS ASSAY OF DINOSED WITH METABOLIC ACTIVATION DNA) Dinoseb (M) 0* DMN (M) 10~5 10- « 10-3 5 x 10"* Sample 1 72 93 76 71 355 2 75 81 80 64 349 3 55 51 58 89 384 Mean 67 75 71 74 363 3D 10 22 i2 13 18 SE 6 12 7 8 11 Negative control and compound solvent = 0.5% DKSO. 89 �Table 63 DNA REPAIR SYNTHESIS ASSAY OF DSMA DNA) (M) DSMA 7 5 0* io- 10-« io- Sample 1 67 51 79 86 2 5b 54 67 3 44 36 99 4NQO (M) 3 10- * 100 62 902 64 155 49 1241 64 f io- 35 58 1380 990 10-« 4 55 36 400 45 44 59 5 79 62 74 53 55 68 1087 6 105 32 75 107 89 69 971 Mean 68 45 79 70 80 61 1095 SD 22 12 12 23 45 7 182 SE 9 5 5 9 18 3 74 Negative control and compound solvent = H20. Sample deleted from calculations because of low DNA value. 90 �Table 64 DNA REPAIR SYNTHESIS ASSAY OF DSMA WITH METABOLIC ACTIVATION DNA) DSMA DMN (M) (M) 0* 10~5 io-« io-3 Sample 1 44 28 25 38 381 2 30 36 29 36 384 3 23 21 32 28 339 Mean 33 29 29 34 368 SO 11 8 4 5 25 SE 6 4 2 3 li. Negative control and compound solvent = 0.5^ EtOH. 91 5 X 10~s �Table 65 DNA REPAIR SYNTHESIS A3SAY OF FENXHION DNA) 4NQO (M) Fenthion (M) 0* 10-6 10~5 10-* 10-3t ID"5 65 105 154 37 64 2983 337* 34 67 2272 3 89 43 107 83 85 40 36 2552 4 62 46 54 63 4059 5 61 94 34 102 31 63 44 51 64 44 —§ 33 189J 88 41 2583 26 44 13 51 15 11 18 5 6 350 Sample 1 2 6 SD 76 24 SE 10 Mean 1728 857 Negative control and compound solvent = 0.5% EtOH. * Precipitate observed at 10~3 M. * Sample deleted from calculations because of low DNA value. § Sample lost. 92 �Table 66 DNA REPAIR SYNTHESIS ASSAY OF PENSION WITH METABOLIC ACTIVATION DNA) renthion (M) DMN (M) 0 * 10-s 10- « io-3 Sample 1 55 54 60 51 206 2 54 50 46 64 220 3 52 42 64 63 2*3 54 48 57 60 216 SD 2 6 10 7 9 SE 1 4 6 4 5 Mean Negative control and compound solvent = O.S^t EtOH. 93 5 X 10"a �Table 67 DNA REPAIR SYNTHESIS ASSAY OF FOLPET DNA) Folpet (M) 7 4NQO(M) s 0* 10-" io- . IP" io- io-« 10-s Sample 1 37 43 63 45 82 29 924 2 64 58 62 54 58 25 947 3 76 91 108 52 92 31 1106 92 65 26 801 31 760 6 4 76 83 91 5 63 107 72 70 85 6 66 60 84 104 107 29 884 Hear. 64 73 80 71 82 28 904 SD 14 24 18 25 18 2 122 10 7 10 7 1 50 SB Negative control and compound solvent = 0.5% DMSO. 94 �Table 68 DNA REPAIR SYNTHESIS ASSAY OF FOLPET WITH METABOLIC ACTIVATION (dpm/ug DNA) • Folpet (M) DMN (M) 0* 10~5 10" *« 10"3 5 X 10~2 30 49 63 49 —t 40 —t 54 54 82 98 40 58 323 384 35 52 81 49 353 SD 7 3 18 9 43 SE 5 2 10 5 31 Sample 1 2 3 Mean Negative control and compound solvent = 0.5% DMSO t Sample lost. 95 �Table 69 DNA REPAIR SYNTHESIS ASSAY OF AZINPHOS-METHYL . (dpm/iig DNA) 4NQO (M) Azinophos- methyl (M) 0* io-7 io-a 10-s io-« io-'+ io-s Sample I 102 145 99 264* 51 55 804 2 99 115 103 99 105* 39 924 3 77 96 71 82 55 33 629 4 97 125 80 85 34 856 5 85 93 100 —5 79 57 97 761 6 111 77 72 56 68 35 897 Mean 95 108 89 79 63 49 822 SD 12 25 16 15 14 25 87 SE 5 10 7 7 6 10 36 / Negative control and compound solvent = 0.5% DMSO. t Precipitate observed at 10~3 M. * Sample deleted from calculations because of low DNA value. § Sample lost. 96 �Table 70 DNA REPAIR SYNTHESIS ASSAY OF AZINPHOS-METHYL WITH METABOLIC ACTIVATION DNA) DMN (M) 0* 10-* 10-* io-3 Sample 1 30 70 75 42 2 40 66 65 55 323 78 41 54 384 35 71 60 50 353 SD 7 6 18 8 43 SE 5 3 10 4 31 3 Mean —* * Negative control and compound solvent = 0-5% DMSO. Sample lost. 97 5 X 10~a -t �Table 71 DNA REPAIR SYNTHESIS ASSAY OF MALATHION (dpm/pg DNA) Malathion (M) 0* 7 io- 10~6 5 10- 4NQO (M) 10-" 2 3 4 5 6 Mean SD SE ID'5 33 34 23 44 39 40 35 7 3 1943 • Sample 1 10-3 123 125 106 100 114 138 118 14 6 110 111 86 133 116 110 111 15 6 128 124 130 116 127 143 128 9 4 144 78 74 119 132 127 112 29 12 90 126 67 113 91 156 107 31 13 * Negative control and compound solvent » 0.5% EtOH. 98 3626 1538 1264 1737 1651 1626 225 92 �Table 72 DMA REPAIR SYNTHESIS ASSAY OF MALATHION WITH METABOLIC ACTIVATION DNA) DMN (M) Malathion (M) 10-' 0* Sample 1 10-5. 10-" 55 48 46 38 206 49 52 48 220 223 5 X 10-* 2 54 3 52 62 55 37 54 53 51 41 216 SO 2 8 5 6 9 SE 1 5 3 4 5 / Mean * Negative control and compound solvent = 0.5^> EtOH. 99 �Table 73 DMA REPAIR SYNTHESIS ASSAY OF METHOMYL (dpm/vig DNA) Methomyl (M) 4NQO (M) __ ^• * 0 in-7 lu Sample 1 2 135 95 __t 3 165 4 • * 5 io-e XV 10~s 10-4 IO-3 10- • 116 11V 126 88 1442 133 108 116 69 1544 100 129 97 122 69 1518 98 97 115 109 69 1385 72 104 85 103 116 109 70 1423 __t 103 95 93 139 130 61 94 112 115 118 71 1462 118 6 17 9 9 67 32 14 SD 3 7 4 4 30 13 6 SB Mean 133 Negative control and compound solvent =0.5* DMSO. Sample lost. 100 �Table 74 DNA REPAIR SYNTHESIS ASSAY OF METHOMYL WITH METABOLIC ACTIVATION (dpm/p>g DNA) Methoayl (M) DMN(M) 0* 10~s io-4 io-3 Sample 1 44 26 22 24 381 2 30 25 20 25 384 3 23 22 26 30 339 Mean 33 25 23 26 368 SO 11 2 3 3 25 SB 6 1 2 2 15 * Negative control and compound solvent =0.5^. EtOH. 101 5 X 10-a �Table 75 ONA REPAIR SYNTHESIS ASSAY OF MONURON (dpm/p« DMA) 4NQO (M) 0 Sample 1 135 2 133 113 __* 86 97 83 48 1442 93 88 72 47 1544 89 77 81 46 1518 83 75 3 165 4 72 82 49 5 1385 104 118 92 77 84 6 45 1O3 131 110 109 1423 __t 85 38 118 123 92 87 81 SO 46 1462 32 8 9 14 5 SE 4 67 13 4 4 6 2 1 30 / Mean 129 __* Negative control and compound solvent = 0.5% DMSO. Precipitate observed at. 10"3 M. t Sample lest. 102 . �Table 76 DNA REPAIR SYNTHESIS ASSAY OF MONURON WITH METABOLIC ACTIVATION (dpm/jig DNA) Monuron (M) DMN (M) 0* io-s 10-" io-3 Sample 1 30 78 81 74 2 40 88 68 74 323 3 -- 92 63 88 384 35 86 71 79 353 SD 7 7 9 8 43 SE 5 4 5 5 31 Mean Negative control and compound solvent =0.5^ OMSO. Sample lost. 103 5 x 10~* -t �Table 77 DNA REPAIR SYNTHESIS ASSAY OF MSMA (dpm/jig DNA) MSMA (M.) JL IO-7 io-6 67 38 108 2 58 84 3 114 4 44 55 93 68 50 5 79 75 6 105 53 68 73 77 22 29 9 13 23 9 Mean SO SE 235 66 __* 10-s 10-* io- io-» 60 53 61 93 52 68 39 902 61 67 1380 63 66 66 75 48 60 1087 50 Sample 1 t 4NQO (M) 3 59 75 971 60 9 64 62 12 1095 5 74 4 16 7 Negative control and compound solvent = HaO. Sample deleted from calculations because of low DNA value. Sample lost. 104 1241 990 182 �Table 78 DNA REPAIR SYNTHESIS ASSAY OF MSMA WITH METABOLIC ACTIVATION (dpra/ng DNA) MSMA (M) DMN (M) o* IO-8 io-4 io-3 Sample 1 44 27 21 32 381 2 30 25 35 fco 384 3 23 21 34 24 339 Mean 33 24 30 27 368 SD 11 3 8 4 25 SE 6 2 5 3 15 fr Negative control and compound solvent = O.S^t KtOH. 105 & X 10~» �Table 79 DNA REPAIR SYNTHESIS ASSAY OF PARATHION (dpm/Mg DNA) Parathlon (M) 7 6 3 0* Sample 1 2 3 4 5 6 Mean SD SE 10- io- 10~ 10-" 10- 123 125 106 100 114 138 118 14 6 127 129 135 124 137 145 133 7 3 151 155 135 200 160 210 169 29 221 129 157 137 155 126 154 35 14 102 90 104 93 116 102 93 100 10 4 12 5 94 83 72 93 71 86 13 5 * Negative control and compound solvent ~ 0.5% EtOH. 106 4NQO (M) 10-5 1943 1626 1538 1264 1737 1651 1626 225 92 �Table 80 DNA REPAIR SYNTHESIS ASSAY OF PAKATHION WITH METABOLIC ACTIVATION DNA) OMN (M) Parathion (M) 5 3 0* io- io-« 10- Sample 1 44 33 42 20 381 2 30 33 32 23 384 3 2? 35 30 30 339 Mean 33 34 35 24 368 SD 11 1 6 5 25 SE 6 1 4 3 15 * Negative control and compound solvent = 0.5% EtOH. 107 5 x 10-* �Table 81 DNA REPAIR SYNTHESIS ASSAY OF PARATH ION -METHYL DNA) Parathion-Methyl (M) 4>IQO (M) 0* 10-' io-« 10-» 10- * 10~3 10-' Sample 1 36 36 33 34 44 40 411* 2 34 38 32 55 44 23 781 3 86 49 28 31 53 28 782 4 94 56 27 52 41 29 858 5 53 49 35 43 40 29 1296 6 112* 46 41 85 44 27 1103 Mean 61 46 33 50 44 28 964 SD 28 8 5 20 5 6 227 SE 13 3 2 8 2 3 102 / I • w Negative control and compound solvent =0.5% EtOH. Precipitate observed at 10"3 M. $ Sample deleted from calculations because of low DNA value. 108 �Table 82 DNA REPAIR SYNTHESIS ASSAY OF PARATHION-METHYL WITH METABOLIC ACTIVATION DNA) Parathion-Methyl (M) DMN (M) 0* 10-s io-4 io-3 Sample 1 55 44 65 51 206 2 54 54 52 48 220 3 52 57 37 45 223 54 52 51 48 216 SD 2 6 14 3 9 SE 1 4 8 2 5 Mean * Negative control and compound solvent = O.S^EtOH. 109 5 X 10~a �Table 83 DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB) DNA) PCNB (M) o* io-7 Sample 1 61 2 3 4NQO (M) 10-* io-4 35 loss 21 27 31 43 22 21 18 32 44 31 23 18 18 30 25 35 38 30 27 16 32 20 Mean 34 35 20 27 SO 15 6 9 4 5 2 4 / 5 6 SE 110 23 37 10"8 1891 1681 2418 18 22 19 21 27 39 25 20 27 2275 20 26 1990 8 4 2 6 387 3 158 3 Negative control and compound solvent = 0.5% DMSO. Precipitate observed at 10"3 M. . 10~3+ 2245 1430 �Table 84 DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB) WITH METABOLIC ACTIVATION DNA) I PCNB (M) DMN (M) * 0 10-8 i<r« Sample 1 72 75 95 103 355 2 75 79 71 72 349 3 84 73 49 384 Mean 55 67 79 80 76 363 SO 10 5 13 29 18 SE 6 3 8 17 11 IP-3 * Negative control and compound solvent = 0.5^> DMSO. Ill 5 X 10"a �Table 85 DNA REPAIR SYNTHESIS ASSAY OF PRORATE DMA) Phorate e 4NQO (M) (M) 5 3 0* 10-7 io- io- io-« io- t 10~5 1 36 58 38 25 27 56 411* 2 34 45 44 17 50 45 781 3 86 31 107 __§ 22 42 55 782 4 94 26 43 43 SO 858 5 53 40 44 52 37 61 1295 6 112* 56 26 77 39 59 1103 Mean 61 43 52 39 40 55 964 SO 28 13 32 23 8 6 227 SS 13 5 14 9 3 2 102 Sample Negative control and compound solvent = 0.5% EtOH. Precipitate observed at 10~3 M. * Sample deleted from calculations because of low DNA value. § Sample lost. 112 �Table 86 DNA REPAIR SYNTHESIS ASSAK OF PHORATE WITH METABOLIC ACTIVATION DNA) DMN (M) Phorate. (M) 0* io-s io-4 io-» Sample 1 55 45 43 37 206 2 54 59 41 35 220 3 52 63 39 38 223 54 55 41 37 216 SO 2 10 2 1.4 9 SE 1 6 1 08 . 5 Mean * Negative control and compound solvent =0.5% EtOH. 113 5 X 10~* �Table 87 DNA REPAIR SYNTHESIS ASSAY OF SIMAZINE (dpm/Hg DNA) 4NQO (M) Simazine (M) 0* Sample 1 2 3 4 5 6 Mean SD SE io-7 io-6 10-5 10-" 10-3 10-5 195 153 212 230 217 298 218 43 19 165 91 131 138 152 113 132 27 11 151 171 152 146 179 213 169 25 10 195 190 312 290 237 305 255 55 22 177 193 253 281 166 161 205 50 20 369 208 233 165 205 t 2670 2850 2688 2702 2435 2662 2668 134 55 * Media control and compound solvent *• 0.5Z DMSO. t Sample lost. 114- 236 78 32 �Table 88 DNA REPAIR SYNTHESIS ASSAY OF SIMAZINE WITB METABOLIC ACTIVATION DNA) Simazine (M) DMN (It) * 0 io-» io-« io-3 Sample 1 i2 57 64 59 355 2 75 58 60 58 349 3 55 64 61 76 384 Moan 67 60 62 64 363 SO 10 4 2 10 18 SB 6 2 1 6 11 Negative control and compound solvent = O.SJt DMSO. 115 5 X 10~* �Table 89 DNA REPAIR SYNTHESIS ASSAY OF TRTFLURAWN DKA) 4NQO (M) Trifluralln (M) 7 0 * io- Sample 1 56 42 2 68 3 10- 10- 1 0 1 0 10-8 51 71 639 152* 97 123 1125 SI 78 48 68 158* 112 570 4 45 51 76 89 78 83 894 5 50 47 7 9 97 56 8 0 663 6 / 53 29 26 _.t 29 41 59 57 43 S3 986 50 48 58 73 62 87 812 13 17 22 19 28 26 222 5 7 10 9 12 11 91 Mean SO SE * Negative control and compound solvent =0.5% EtCH. Sample lost. $ Sample deleted from calculations because of low DNA value. 116 �Table 90 DNA REPAIR SYNTHESIS ASSAY OF TRIFLURALIN WITH METABOLIC ACTIVATION DNA) Trifluralln (M) 0* 10-" 10- 72 67 79 2 75 58 3 55 Mean DMN (M) 10- 5 X 10~* 51 355 64 61 39 4 74 79 --1" 384 67 66 74 56 363 SO 10 8 9 7 18 SE 6 5 5 5 11 Sample 1 Negative control and compound solvent = 0.5% DMSO. Sanmle lost. 117 �Table 91 IN VI.TRO ASSAYS WITH SALMONELLA TYPHIMURIUM Compound Metabolic Activation ug Compound Added/Plate 95 113 Negative control + • Positive control, 4-o-tolylazo-l-toluidine + £ 1000 8 10 1 5 10 50 100 500 1000 87 101 93 107 97 95 126 101 89 75 79 71 78 113 9 22 14 23 13 17 14 ' ' 16 20 16 14 16 19 18 10 9 9 7 11 10 10 14 13 12 1C 15 9 10 7 10 6 183 11 10 13 10 9 7 6 12 12 11 10 11 15 10 Monoci + + + + + + + 13 13 25 25 1 5 10 50 100 500 Monocrotophos 00 Average Number of Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA1538 •i o 0 •o o 01 �Table 91 (continued) Compound Metabolic Activation \j.g Compound Added/Plate Bromacil ( 0 50 ^1 0.05 50 50 TA100 TA1535 TA1537 TA1538 145 154 Negative control Positive controls B -Proplolactone AF-2 2-Anthramine Average Number of Hlstldlne-Positlve Revertants/Plate 22 25 25 24 16 30 756 372 63 338 1 5 10 50 100 500 1000 120 129 123 117 136 140 101 23 17 31 29 40 30 11 24 13 22 16 18 15 6 26 14 13 15 19 15 11 1 5 10 50 100 500 1000 118 131 157 136 138 145 162 35 28 33 26 30 21 8 21 16 21 14 12 20 5 16 20 19 13 15 20 16 I 0 a �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate 56 72 Negative control Positive control, 4-o-tolylazo-o-toluidine Cacodylic Acid N> o' Average Number of Histidine-Positive Revertants/Plate TA1537 TA100 TA1538 TA1535 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 15 14 12 9 48 42 42 39 43 44 44 69 53 64 DO 64 54 59 17 15 12 18 22 15 IS 17 15 16 15 18 21 14 15 15 15 10 11 11 8 14 15 12 11 15 14 13 7 15 10 ' 150 5 il 8 8 9 9 8 18 8 18 12 19 15 ' '£» n 13 8. �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Platfc Negative control Positive control, 4-o-tolylazo-o-toluidine Captan Average Number of Histidine-Positive Revertants/Plate TA100 TA1537 TA1535 TA1538 72 98 2 2 1 5 10 15 25 50 1 5 10 15 25 50 211 532 822 820 720 Killing 141 210 285 340 330 704 18 14 7 3 8 25 3 100 29 80 76 104 80 Killing 20 60 113 55 71 143 2 5 0 0 0 0 2 2 2 0 0 1 "1 14 16 26 6 22 19 22 26 21 46 44 s? •o ft 0) a �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Average Number of Histidine-Positive Revertants/Plate TA1QQ TA1537 TA1535 TA1538 92 80 18 14 12 16 16 16 Positive control, 4-o-tolylazo-o-toluidine Chloropyrifos NJ H ' 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 66 92 65 88 67 87 79 67 71 87 77 77 I'll 20 22 14 26 • 20 17 13 11 9 11 16 11 13 14 12 15 21 15 17 18 20 15 15 15 18 14 14 11 . )5 168 22 28 24 25 22 17 22 14 16 20 13 16 30 22 g. •» »-• 0 H O •o X fl �Tabie 91 (continued) Compound Metabolic Acti^ar.ion Vg Compound Added/Plate Negative control Average Number of Hisfidine-Positive Revertants/Plate TA1537 TA100 TA1535 TA1538 97 80 Positive control, 4-o-tolylazo-o-toluidine Dinoseb •f + 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 15 15 12 16 12 69 21 12 59 16 57 17 18 67 17 17 82 12 16 91 7 17 Killing Killing Killing 79 13 19 82 14 18 94 12 17 14 83 15 87 13 17 104 12 10 Killing Killing Killing 21 17 • 15 168 15 14 20 17 15 19 Killing 15 14 15 17 14 7 Killing •3 O CD (B er �Table 91 (continued) Compound Metabolic Activation Ug Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine DSMA Average Number of Histidine-Positive Revertants/Plate TAIOQ TA1535 TA1537 TA1538 56 72 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 15 14 12 9 7 13 . 50 56 51 66 71 53 43 54 85 50 -55 50 60 53 12 10 20 15 16 13 12 22 7 16 17 13 15 14 7 10 10 11 7 12 7 8 _ 8 11 9 5 2 10 250 4 5 3 8 8 7 9 8 15 3 5 7 10 8 o CO �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Average Number of Histidine-Posftive Revertants/Plate TA1537 T/UOO TA1538 TA1535 101 102 Positive control, 4-o-tolylazo-o-toluidir.a Azlnphos-methyl + + + + •f 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 26 26 6 3 66 74 74 73 75 107 104 76 69 68 81 65 84 119 39 22 23 30 49 30 31 23 23 24 22 24 30 24 4 3 2 3 4 2 3 1 2 3 3 2 0 0 13 24 13 78 10 11 9 11 10 10 13 20 25 28 21 19 24 23 N P•g VJ �Table 91 (continued) Compound Metabolic Activation Ug Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Fenthion Average Number of Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA1535 m • • i^ • • • 94 80 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 36 20, 10 9 12 12 . 64 97 105 112 100 107 90 114 97 81 90 98 86 89 31 34 32 36 38 42 32 15 17 f/ 16 14 22 2(j 8 11 15 15 16 10 6 10 12 9 12 7 8 10 15 168 13 17 12 14 14 14 12 12 10 17 21 13 10 15 f f^ sr Ho a �Table 91 (continued) Metabolic Activation Negative control vg Compound Added/Plate Average Number of .UO/ * •*.»»•»Hist id imi-Positiye Keveti.au TA1538 "TA1537 TA1535 TA10Q 72 93 + _ 19 15 6 183 25 25 1 5 10 25 50 — Folpet — w . + + + + 4- 127 150 244 300 550 20 35 39 48 111 100 500 1000 Positive control, 4-o-tolylazo-o-toluldine 286 Killing Killing 110 Killing Killing 1 5 10 25 50 100 500 1000 8 20 3 7 11?. 173 241 20 51 79 70 420 218 720 216 532 Killing Killing Killing Killing 7 0 0 ' 2 0 0 0 1 1 10 3 0 0 5 11 14 7 Killing Killing 30 26 35 36 45 48 Killing Killing �91 (continued) Metabolic Compound Added/Plate pg Negative control Positive Histidine-Pcsitive Revertants/Pl TAJ™ -•• — TA1538 89 92 control, 4-o-tolvlazo-o-toluidine Malathion NJ 00 + • f + + 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 54 48 85 99 81 82 61 65 61 99 92 75 90 66 10 10 8 7 7 8 7 12 10 5 6 9 8 9 8 7 8 10 11 12 10 . 6 7 5 7 6 8 7 7 6 12 10 7 7 6 183 3 3 5 7 5 7 4 9 5 4 6 5 4 4 o �Table 91 (continued) Compound Metabolic Activation ug Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Methomyl Average Number of Histidine-Positive Revertants/Plate TA1537 TA100 TA1535 TA1538 128 149 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 18 14 33 20 • 123 112 98 109 110 119 105 145 115 126 129 132 133 122 19 20 16 18 21 17 13 12 10 12 10 13 10 14 28 35 28 27 34 23 24 18 18 21 19 20 20 24 17 22 17 206 14 10 18 14 24 26 21 15 15 i: 20 19 18 14 �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Average Number of Histidine-Positive Revertants/Plate TA1537 TA100 TA1538 TA1535 128 149 Positive control, 4-o-tolylazo-o-toluidine 25 25 1 5 10 50 100 500 1000 Monuron U) o •f + •f + •f 1 5 10 50 100 500 1000 17 12 17 15 126 - 98 108 122 114 122 125 125 142 119 116 108 104 123 15 24 17 19 19 20 22 la 12 12 11 15 18 14 15 15 13 19 16 15 15 10 13 17 15 15 15 11 17 22 6 177 19 18 22 21 29 29 19 21 15 18 21 19 12 17 I I �Table 91 (continued) Compound Metabolic Activation Ug Compound Added/Plate MSMA TA100 TA1535 TA1537 56 72 Negative control Positive control, 4-o-tolylazo-o-toluidine Average Number of Histidine-Positive Revertants/Plate 15 14 12 9 79 69 62 52 41 53 48 79 64 65 67 53 66 63 15 15 14 17 15 17 13 11 15 7 7 12 14 10 7 13 11 11 12 8 12 11 8 10 14 8 7 10 TA1538 7 15 10 250 6 4 25 rs 1 5 10 50 100 500 1000 1 5 10 50 100 500 loon ' 6 6 7 5 5 10 10 9 7 8 8 10 �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluiHine Parathior Average Number of Histidine-Posittve Revertants/Platt. TA10Q TA1535 TA1537 TA1538 95 114 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 19 21 6 13 94 138 85 98 . 87 110 107 56 75 69 76 88 105 103 12 12 13 13 15 13 14 11 16 14 14 17 15 12 7 7 4 4 4 4 3 12 7 5 6 7 8 6 6 7 6 177 8 7 7 8 6 8 13 \5 15 19 8 5 9 12 s? to It O �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine u> U) Parathion-methyl Average Number of Histidine-Positive Revertants/Plate TA100 TA1537 TA1535 TA1538 84 77 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 5CO 1000 64 75 86 75 72 78 46 61 53 60 69 66 67 56 10 10 12 9 8 10 9 7 3 4 11 8 10 10 6 4 10 6 7 7 6 7 8 8 9 7 5 8 9 9 12 4 6 7 6 177 8 8 9 13 10 20 18 14 10 8 11 13 14 16 to 2 o I �Table 91 (continued) Compound Metabolic Activation V> g Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Quintozene (PCNB) Average Number of Histidine-Positive Revertants/Plate TA1537 TA100 TA1538 TA1535 96 101 33 24 9 6 25 25 6 177 99 85 90 87 94 100 107 32 29 34 30 37 27 35 6 7 6 6 5 5 6 15 12 13 7 11 14 11 90 95 91 100 87 119 106 20 25 18 18 23 22 30 9 7 6 3 4 1 1 25 27 29 25 20 26 27 ' 1? 3 tozene i 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 13 25. /— \ o z w �Table 91 (continued) Average Number of Compound Metabolic Activation Negative control + Phorate " i^ T + : + + + . 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 TA10Q TA1535 TAI537 TA1538 96 118 + Positive control, 4-o-tolylar.o-o-toluidine vg Compound Added /Plate 15 17 8 11 8 11 6 177 16 11 11 8 70 65 85 65 72 70 58 101 103 79 89 79 59 70 17 15 17 17 11 14 14 14 11 13 15 15 16 19 8 8 7 5 7 o 7 11 8 10 9 6 6 8 15 10 12 11 5 r p> PI (D �Table 91 (continued) Compound Metabolic Activation V> g Compound AdctJ/Piate Negative control Positive tontrol, 4-o-tolylazo-o-toluidine Simazine Average Number of Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA1535 98 106 25 25 1 5 10 SO 100 500 1000 1 5 10 50 100 500 1000 10 7 8 8 83 72 73 87 85 71 69 84 90 82 83 87 89 120 7 5 7 7 8 3 4 9 4 8 9 8 4 2 15 10 20 10 12 7 11 11 11 16 9 11 io id 25 26 22 266 ?2 20 20 22 17 25 22 22 2i 15 14 15 15 18 ' 9 n �Table 91 (concluded) Compound Metabolic Activation yg Compound Added/Plate Trifluralin 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 TA100 TA1535 TA1537 TA1538 96 80 Negative control Positive control, 4-o-tolylazo-o-toluidine Average Number of Histidine-Positive Revertants/Plate 15 15 17 20 14 8 73 81 74 93 86 76 95 72 80 78 90 81 79 81 11 12 16 19 18 18 lj 10 13 14 15 8 12 12 18 24 29 23 25 22 18 13 16 18 14 16 13 15 15 168 15 14 14 16 15 11 15 9 9 14 13 15 . 11 10 1 H. Ml h-" �Table 92 RESULTS OF ASSAYS WITH ESCHERICHIA COLI WP2 Compound Metabolic Activation Average Number of Tryptophanug of Compound Added per Plate Positlve Revertants per Plate Negative control Positive control, AF-2 Moncrotophos .00 68 73 0.05 0.05 1 10 50 100 SCO 1000 1 10 50 100 500 1000 Bromacil 10 50 100 500 1000 1 10 50 100 500 1000 204 220 89 83 76 77 61 70 90 88 76 73 75 95 65 74 71 70 70 67 71 73 66 70 70 71 �Table 92 (continued) / w 10 Captan ug of Compound Added per Plate - 1 10 m 102 89 82 500 1000 Cacodylic Acid Metabolic Activation 50 100 Compound 91 85 1 10 50 100 500 1000 1 5 10 15 95 76 79 89 81 85 124 381 733 1358 25 50 1755 2600 1 5 10 15 25 50 89 182 + + + + + + - + + + + + + • Average Number of TryptophanPositive Revertants per Plate 423 699 955 1712 �Table 92 (continued) Compound Chloropyrifos Metabolic Activation pg of Compound Added per Plate Average Number of TryptophanPosltive Revertants per Plate 57 57 49 71 52 42 1 10 50 100 500 1000 Dlnoseb 1 10 50 100 500 1000 60 73 53 49 61 49 1 10 50 100 500 1000 64 73 58 55 44 Toxic 1 10 50 73 69 63 65 49 Toxic 100 500 1000 �Table 92 (continued) Compound DSMA Metabolic Activation yg of Compound Added per Plate Average Number of TryptophanPositive Severtants per Plate 1 86 81 67 68 68 10 50 100 500 1000 1 10 50 100 500 1000 Fenthion 1 10 50 100 500 1000 1 10 50 100 500 1000 . 71 69 62 67 73 81 79 59 63 62 56 70 71 64 50 67 64 64 81 �Table 92 (continued) Compound Folpet Azlnphos-methyl Metabolic Activation Vig of Compound Added per Plate 1 5 10 25 50 100 1 5 10 25 50 100 1 10 30 100 500 1000 1 10 50 100 500 1000 Average Number of TryptophanPositlve Revertants per Plate 65 162 170 424 720 1260 74 167 202 900 1680 1880 92 87 83 89 68 88 83 74 87 86 /J 79 �Table 92 (continued) Compound Metabolic Activation Average Number of TryptophanPositive Revertants per Plate 1 10 50 100 500 1000 + + + + + + Methomyl + + •• *• + '•-, 62 54 60 60 54 48 1 10 50 100 500 1000 58 50 55 59 75 64 1 10 50 100 500 1000 Malathion £ yg of Compound Added per Plate 61 76 83 57 63 71 1 10 50 100 500 1000 70 81 78 83 63 74 �Table 92 (continued) ~~ Metabolic Activation pg of Compound Added per Plate Monuron - MSMA + + + + + + - 1 10 50 100 500 1000 1 10 50 100 500 1000 1 10 50 100 500 1000 72 68 57 63 65 63 60 59 47 71 50 61 55 64 57 76 60 63 1 10 50 100 500 1000 55 71 73 71 61 72 Compound + * + + + + . Average Number of TryptophanPositive Revertants per Plate �Table 92 (continued) Compound Metabolic Activation Parathion •f + + + + + Parathion-methyl Ug of Coa; ound Added pe.- Plate 1 10 50 100 500 1000 1 10 50 100 500 1000 1 10 50 100 500 1000 1 10 50 100 500 1000 Average Ntnnber of TryptophanPosltive Revertants per Plate 71 64 66 70 64 64 69 53 76 57 72 66 53 56 60 68 63 52 64 83 60 65 53 71 �Table 92 (continued) Compound Metabolic Activation Ug of Compound Added per Plate Average Number of TryptophanPosltlve Revertants per Plate 1 10 50 100 500 1000 + + + 78 67 54 57 59 62 1 10 50 100 500 1000 Phorate 70 60 57 62 1 10 50 100 500 1000 Qulntozene (PCNB) 63 64 65 49 71 60 1 10 50 100 500 1000 78 86 83 73 90 70 54 :s �Table 92 (concluded) Coropound Metabolic Activation Simazine - Trifluralin + + + + + + - pg of Compound Added per Plate + + + + + + . Average Number of TryptophanPositive Revertants per Plate 1 10 50 100 500 1000 1 10 50 100 500 1000 1 10 50 100 500 1000 55 51 73 54 54 53 64 66 72 56 71 83 75 73 81 86 69 60 1 10 50 100 500 1000 58 63 63 65 70 70 �Table 93 MICROBIAL INHIBITION IN ESCHERICHIA COLI AND BACILLUS SUBTILIS Diameter of Zone of Inhibition (mm) Compound mg of Compound Added to Disc W3110 E. coll p3478 B. subtilis HI 7 m45 Positive control, l-phenyl-3,dimethyltriazene 37 52 40 61 Negative control, chloramphenacol oo 1.0 0.03 34.5 34 32 31 Monocrotophos 1 6 . 6 6 6 Bromacil 1.0 6.5 6.5 6.5 6.5 Cacodylic acid 1 6 6 6 6 Cap tan 0.1 6.5 11 9 19 Chloropyrifos 2.5 6 10 6 11 Dinoseb 1 10 17 8.5 11 DMSA 1 6 6 6 6 Fsnthion 1 6 6 6 6 Folpet 0.1 6.5 6.5 7.5 Azinphos-methyl 1 6 6 6 10 6 �Table 93 (concluded) Compound mg of Compound Added to Disc Diameter of Zone of Inhibition (tma) B. subtilis E. coli n45 p3478 H17 W3110 Malathion 1 6 6 6 6 Me thorny 1 1 6 6 6 6 Monuron 1 6 6 6 6 MSMA 1 6 6 6 6 Parathion 1 6 6 6 6 Parathiow-methyl 1 6 6 6 6 Qulntozene (PCNB) 1 6 6 6 6 Phorate ' 1 6 6 6 6 Simazine 1 6 6 6 6 Trifluralin 1 6 6 5 6 �Table 94 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MONOCROTOPHOS Metabolic Compound Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x ID'7) Control Mitotic Recombinants per ml per 10s 3 (x 10- ) Survivors EXPERIMENT 1 Negative control Ul 4.5 7.9 100 4.5 7.8 0.1 5.8 102 1,650 285 ,4 0.1 4.6 79 1,435 3,120 5 5.7 100 44 77.2 5 4.7 81 30 63.8 9.1 100 8 8.8 8.6 Monocrotophos o 100 5.8 Positive control 1,2,3,4-Diepoxybutane 5.7 100 9.5 5 6.3 69 26 41.2 5 48 . 56 40 83.3 EXPERIMENT 2 Negative control Vonocro tophos 11.0 �Table 95 IN VITRO ASSAYS WITH SACCHAROMVCES CEHEVISIAE D3 - BROMACIL Compound Metabolic Activation Percent Survivors Concentration Cells/ml Percent ol (w/v or v/v) (x 107) Control Mltotlc Recomblnants per 105 per ml (x 1 ' Survivors 0 ) EXPERIMENT 1 4.8 4.7 100 100 3 3 00 .4 0.04 3.4 3.5 71 74 745 683 0.005 0.01 0.05 0.10 05 .0 5.0 4.S 5.0 4.4 2.0 104 94 104 92 42 3 2 3 1 1 6.0 4.4 6.0 2.3 1. 00 005 .0 0.01 0.05 01 .0 0.50 4.4 3.9 4.3 3.8 2.4 94 83 91 81 51 5 3 3 1 3 11.4 7.7 7.0 2.6 12.5 4.5 4.2 100 100 5 3 11.1 7.1 00 .4 00 .4 4.5 4.2 100 100 870 653 0.05 01 .0 0.25 05 .0 5.7 5.4 5.5 49 . 126 120 122 108 7 5 5 1 12.3 9.3 9.V 2.0 0.05 0.10 0.25 O.SO 4 .9 4.7 4.8 49 . 117 112 114 117 4 3 6 1 8. 2 10.8 12.5 2.0 Negative control Positive control 1,2,3.4-Dlepoxybutane Broraacll 6.3 G.4 2191 1951 EXPERIMENT 2 Negative control Positive control 1,2,3,4-Diepoxybutrne Bromacll •f 4-i- 193J 1555 �Table 97 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CAPTAN Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml (x 10-7) Percent of Control Mitotic Recombinants per ml per 10s (x 10-3) Survivors EXPERIMENT 1 Negative control - 7.1 100 3.5 4.9 + 6.5 100 3.0 4.6 003 .0 6.0 84 205 342 0.003 9.1 140 145 159 7.5 100 60 . 100 Captan + in u> EXPERIMENT 2 _ Negative control + Captan 0 .0 03 + 0.0 03 .7 7 5.1 1.5 4 2.0 6.7 10 37 481 85 58 114 �Table 98 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CHLOROPYRIFOS Percent Compound Metabolic Activation Concentration (w/v or v/v) Survivors Percent of Control Cells/ml (x iO~7 ) Mitettc Recombinants per ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 7.5 100 6.0 100 5 7.7 5 8.0 103 133 + Chloropyrif os + 1.5 4 7 10 2.0 6.7 9.1 12.5 EXPERIMENT 2 Negative control 7.4 100 100 01 . 38 . 60 1,045 0.1 5.2 70 903 5 7.9 5 7.4 125 100 3 10 6.3 + Positive control, 1,2,3, 4-diepoxybutane + Chloropyrifos + 1.5 3.5 2.4 4.7 2,750 1,737 3.8 13.5 �Table 99 IN VITRO ASSAYS VITA SACCHAROMYCES CEREVISIAE D3 - DINOSEB Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per ml r,er 10s (x 10-') ourvivors EXPERIMENT 1 Negative control 100 1.5 2.4 7.4 100 3.5 4.7 0.1 3.8 60 0.1 5.2 70 903 0.2 6.2 98 9 14.5 0.2 40 . 54 1 2.5 0.3 Positive control, 1,2,3, 4-diepoxybutane 6.3 .3 5 5 0.3 2.4 32 5 5.5 100 2.5 4.5 5.2 100 2.0 3,8 0.1 4.4 80 3 6.8 0.1 40 . 77 4 10.0 0.2 4.1 75 4.3 83 8 8 19.5 0.2 + + Dinoseb + + 1,045 2,750 1,737 167 20.8 EXPERIMENT 2 _ Negative control + Dinoseb + + 18.6 �Table 100 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DSMA Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10~7J Mitotic Recombinants per ml per 10s (x 10-s) Survivors EXPERIMENT 1 Negative control 7.4 100 7.5 7.7 100 5 4.5 1.1 15 0 4.5 5.2 68 0 7.1 100 3.5 49 . 6.5 100 3 4.6 5 4.7 66 3 64 . 5 3.4 52 7 20.6 + DSMA + 10.1 6.5 EXPERIMENT 2 Negative control + DSMA + �Table 101 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FENTHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10~7) Control Mitotic Recombinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 7.4 100 100 0.1 3.8 60 0.1 5.2 70 5 6.6 105 5 7.5 6.3 + Positive control, 1 , 2 , 3 , A-d iepoxybutane Ul + Fenthion 1.5 2.4 3.5 4.7 1,045 903 2,750 1,737 101 13.6 6.7 7.5 + 9; 5 100 1.5 2.0 6.0 100 4 6.7 7.8 7.1 104 4 5.1 118 6 8.5 EXPERIMENT 2 Negative control + Fenthion + 5 5 �Table 102 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FOLPET Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x ID'7) Control Mitotic Recombinants per ml per 10s 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 7.5 + Folpet 6.0 100 100 1.5 2.0 4 67 . 4.0 3.8 63 6.3 100 1.5 2.3 7.4 100 3.5 4.7 0 .0 03 9.5 151 89 94 0 .0 03 9.1 123 82 90 0 .0 03 + 0. 0 03 53 119 65 298 171 Ul 00 EXPi-TttMEOT 2 _ Negative control + Folpet + �Table 103 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 -AZINPHOS-METHYL Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10~7 ) Control Mitotic Recombinants per ml per 10s (x 10-3) Survivors EXPERIMENT 1 5.7 100 4.5 7.9 5.8 100 4.5 7.8 0.1 5.8 102 1,650 2,845 0.1 4.6 79 1,435 3,120 4.5 5.3 93 15 28.3 4.5 5.8 100 15 25.9 9.1 100 8 8.8 8.6 100 9.5 5 5.7 63 68 119.3 5 6.2 72 80 129 Negative control + POL itive control 1,2,3,4-Diepoxybutane VO + Azlnphos-methyl + EXPERIMENT 2 Negative control + AzinphoE-methyl + 11.0 �Table 104 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MALATHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Recombinants per 105 per ml 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 100 4.5 7.9 5.8 100 4.5 7.8 0.1 5.8 102 1,650 2,845 0.1 4.6 79 1,435 3,120 5 7.8 137 5 6.3 109 11 7 11.1 9.1 100 8 8.8 8.6 100 9.5 5 + Positive control, 1,2,3, 4-diepoxybutane 5.7 81 . 5 76 . 89 88 M + Malathion + '-x 14.1 EXPERIMENT 2 _ Negative control + Malathion + 11.0 13 16.0 8 10.5 �Table 105 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - METHOMYL Comoound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x ID'7) Control Mitotic Recomb< nants per ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 0.1 0.1 1.8 27 266 1,478 1.5 29 184 1,227 2.0 5.0 76 4 2.0 2.7 50 0 3.0 Positive control, 1,2,3, 4-diepoxybutane 5.8 100 100 3.7 56 8 21.6 3.0 4.2 76 6 14.6 5.5 5.2 100 100 3 4.7 85 15 31.9 3 4.4 85 10 22." 6.6 + + Methomyl + + 4.5 6.8 2.5 4.6 80 . EXPERIMENT 2 _ Negative control + Methomyl + 2.5 4.5 2.0 3.8 �Table 106 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MONURON Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml (x ID'7) Percent of Control Mitotic Recombinants per ml per 10s 3 (x 10- ) Survivors EXPERIMENT 1 Negative control Positive control, 1,2,3,4-diepoxybutane 6.6 100 4.5 6.8 5.4 100 2.5 4.6 0.1 1.8 27 266 1,478 0.1 1.5 29 184 1,227 5 3.5 53 3 8.6 5 3.8 70 1 2.6 5.5 2.5 4.5 5.2 100 100 2.0 3.3 5 6.9 125 2 2.9 5 6.2 119 9 14.5 + + Monuron + EXPERIMENT 2 _ Negative control + Monuron + �Table 107 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MSMA Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Recombinants per ml per 10s 3 (x ur ) Survivors EXPERIMENT 1 Negative control - 7.4 7.7 100 7.5 100 5 6.5 5 4.3 58 1 2.3 5 5.4 70 3 5.6 7.1 100 3.5 4.9 6.5 100 3 4.6 5 4.9 69 10.2 20.8 5 5.8 89 • 10.4 17.9 •f MSMA + 10.1 EXPERIMENT 3 _ Negative control + MSMA ' + �Table 10F IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIA^ D3 - PARATHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Rrcombinants per ml per 10s 3 (x 10- ) Survivors EXPERIMENT 1 _ Negative control 5.7 Positiva control, 1,2,3, 4-diepoxybutane 4.5 7.9 5.3 + 100 100 4.5 7.8 Pfi.rathi.on + 5.8 102 1,650 2,845 46 . 79 1,435 3,120 5 + 0.1 0.1 6.5 9 j 4.6 5 5.8 114 1JO 5 8.8 8.8 EXPERIMENT 2 _ Negative control 9.1 8.6 100 8 100 ft.s 5 8.8 96 4 4.5 5 8.2 95 5 6.1 + Par ;thion + 11.0 �Table 109 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PARATHION-METHYL Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10"7) Control Mitotic Rec^mbinants per ml per 10s (x 10-3) Survivors EXPERIMENT 1 Negative control 5.7 + Positive control, 1,2,3, 4-d iepoxybu cane - 102 1,650 285 ,4 46 . 79 1,435 3,120 7.7 135 16 20.8 5 5.4 93 15 27.8 100 8 8.6 100 9.5 5 + 58 . 91 . Parathion-methyl 0.1 0.1 5 + 4 5.8 100 300 74 . 81 19 25.7 5 7.2 84 25 34.7 7.9 J5 4.5 7.8 EXPERIMENT 2 Negative control + Parathion-methyl + 88 . 11.0 �Table 110 IE VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - QUINTOZQIE (PCNB) Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-') Mitotic Recombinants per ml per 10s (x 10-3) Survivors EXPERIMENT 1 Negative control 5.7 + Positive control, 1 , 2 , 3 , 4-d iepoxybutane * . 5.8 100 100 4.5 4.5 7.9 7.8 1,650 2,845 4.6 79 1,435 3,120 3.7 65 3 8.1 2 4.2 72 4 9.5 9.1 100 8 8.8 86 . 100 9.5 5.8 64 4 1 7.0 81 7 2 + 102 1 Quint ozene (PCNB) 5.8 2 + 0.1 0.1 6.8 75 3 2 7.5 87 10 EXPERIMENT 2 Negative control + Qulntozene (PCNB) + + 11.0 6.9 10.0 4,4 13.3 �Table 111 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PHORATE Percent Compound Metabolic Activation Concentration (w/v or v/v) Survivors Cells/ml (x 10~7) Percent of Control Mitotic Recombinants per ml per 105 3 (x ID' ) Survivors EXPERIMENT 1 9.1 100 8 8.6 100 9.5 11.0 5 8.7 96 9 10.3 5 7.5 87 3 4.0 5.7 100 4.5 7.9 5.8 100 4.5 7.8 5 7.5 4 5 7.2 132 124 5.3 9.7 Negativs control + Phorate + 8.8 EXPERIMENT 2 Negative control + Phorate + 7 �Table 112 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - SIMAZINE Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10-7 ) Control Mitotic Recombinants per ml per 10s 3 (x 10- ) Survivors EXPERIMENT 1 _ Negative control 6.6 Positive control, 1 ,2,3,4-diepoxybutane CO 4.5 6.8 5.4 + 100 100 2.5 4.6 266 1,478 1.5 29 184 1,227 3.8 58 3 7.9 5 2.0 37 1 5.0 100 2.5 4.5 5.2 100 2 3.8 5 + 27 5.5 Simazine 1.8 - 5 + 0.1 0.1 7.0 127 7 5 7.0 135 4 EXPERIMENT 2 _ Negative control -t- Simazine + 10.0 5.7 �Table 113 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE DS - TRIFLURALIN Compound Metabolic Activation Percent Concentration fw/v or v/v) Survivors Cells/ml Percent of Control (x 10~7 ) Mitotic Recombinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 7.5 100 1.5 6.0 100 4 5 8.4 112 5 6.0 100 5 2 3.3 6.3 1.5 2.4 7.4 100 100 3.5 4.7 0.1 3.8 60 0.1 5.2 70 903 5 8.7 138 7 80 . 5 8.4 114 3 3.6 Negative control + Trifl'jralin + 2.0 6.7 5.9 EXPERIMENT 2 vO -^ Negative control + Positive control, 1,2,3, 4-diepoxybutane + Trifluralin + 1,045 2,750 1,737 �Table 114 IN VITBO MOTAGENESIS WITH SALMONELLA TYPHIUURIU1I SUMMARY DATA FOR EPA PESTICIDES Po*ltlve Re*pon*e, +; Negative Re*pon*e, - TA100 Pesticide Monocrotophoa Bromacll Cacodyllc Acid Captan Chlorpyrlfoa Dlnoseb DSHA Fenthlon Folpet Azlnphos-metbyl Malathlon Methorny1 Uonuron MSMA Parathlon Parathion-nethy1 Qulntozene (POJB) Phorate Slnazlne Trifluralln - Metabolic + Metabolic Activation Activation TA153S TA1537 TA1838 - Metabolic 4 Metabolic ~ Metabolic + Metabolic - Metabolic + Metabolic • Activation Activation Activation Activation Activation Activation �APPENDIX A MUTAGENESIS STUDIES OF PESTICIDE COMPOUNDS MOUSE HERITABLE TRANSLOCATION TEST CAPTAN 171 �SUMMARY SRI conducted a heritable translocation study of Captan in mice to Investigate whether heritable mutagenic events occur when the compound is ingested repeatedly over an extended period. For 8 weeks, adult male mice were administered Captan in their diet; 60 mice received 2500 ppm, and 61 received 5000 ppm. A control group of 60 adult male mice received an untreated diet during this time. A positive control group containing 66 adult male mice was treated as a control group for 4 weeks and then received the known mutagen triethylenemelamine (TEM) in the drinking water for A weeks. After treatment, all males were bred with two virgin females each to produce an Fj generation, the males of which were raised to maturity. (0 20 Selected per group) F. males were bred to three virgin females each, and presumptive translocates were rebred to three additional females each. A third breeding was conducted with selected nonbrteder and/or presumptive males. Evaluation of the data on fertility, breeding, and litter size distribution for F_ and F. generations does not suggest the presence of translocation heterozygotes in control or Captan-trea?ed male mice. Data on dead implants and rebrecding did, however, suggest the presence of translocation heterozygotes in the group treated with 5000 ppm Captan. Meiotic cell preparations of the testes of t'le presumptive males were evaluated cytogenetically. in the following numbers of F Normal meiotic chromosomes were found males derived from the group specified: 8 of 8 controls, 8 of 8 from the 2500 ppm Captan group, 8 from the 5000 ppm Captan group, and 2 of 2 from the 5000 ppm Captan-treated group derived from traumatized F females. Five of 5 TEM-treated F males and 1 of 8 from the 5000 ppm Captan-treated males showed reciprocal translocations. 172 �The results of this study show that under the experimental procedures employed, Captan at 5000 ppra in the diet of male mice for 8 consecutive weeks can produce a heritable mutagenic event in F. generation male mice. 173 �INTRODUCTION The EPA is reviewing and evaluating the health hazard of pesticides and of substitute candidate pesticides according to available data. Additionally, the Agency is obtaining supplemental laboratory data. The objective is to enable the EPA to select those chemicals that are minimally hazardous when used according to labeling restrictions. SRI is participating in this Suostitute Chemical Program by investigating the mutagenic potential of selected materials by in vitro and in vivo procedures. Captan has been shown to respond in a positive manner in Salmonella typhimurium, Escherichia coli WP2, Saccharonyces cerevisiae, JS. coli (relative toxicity), Bacillus subtilis, WI-38 unscheduled DNA synthesis (UDS) with metabolic activation, and Drosophila melanogaster experiments. It vas not positive in a mouse dominant-lethal test. Based on positive responses in both Tier I ( n vitro test) and Tier II (Drosophila) i mutagenic studies, it was recommended that a heritable translocation test (Tier III) in the mouse be conducted to further assess the mutagenic potential of Captan. In this study, young adult male ICR/SIM mice from a closed, randombred colony were administered Captan in the diet for 8 weeks. After treatment, each male was mated to two virgin females to produce an Fgeneration, the males of which were raised to maturity and bred to three virgin females eacr.. Pregnant females were evaluated against pre- determined selection criteria for identification of suspect F. males, which were rebred and evaluated again. Presumptive F. males were examined cytogenetically. Through this procedure, a heritable mutagenic response can be detected. Potential muLa^enic effects were identified by examination of fetuses during the middle to later stages of gestation. 174 Cytogenetic �examinations were made of meiotic cell preparations of the testes from suspect males for conf iiiurtion of findings obtained from the breeding studies. Reported here, are the results of the heritable study of Captan. 175 translocatirn �MOUSE HERITABLE TRANSLOCAT10N TEST Background Human populations frequently are exposed to man-made chemicals, often at barely detectable levels, for extended periods. To evaluate the genetic hazards of such chemicals, a prudent approach is to study them in mammalian systems so as to maximize detection of a mutagenic response. The study reported here was such an investigation of Captan for its potential to produce heritable genetic defects. Chemical induction of chromosomal aberrations in the me use is a valuable and important experimental aid in understanding the many genetic defects due to chromosomal anomalies in humans. To date, mammalian evaluations of cnemically induced chromosomal aberrations have been attempted with the dominant-lethal of somatic and germinal cells. test and cytogenetic studies Although these procedures can provide useful information, they do not measure heritable genetic effects, the most important mutagenic occurrences that are permanent and transmissible. A need exists for a method to reliably identify compounds that cause heritable chromosomal aberrations in mammalian systems. The mouse translocation procedure appears to be such a system. A well-defined translocation test will demonstrate the fertility of an F male population derived from F males treated with a test agent. Confirmation of a nonbreeder, sterile, or partially sterile response can be obtained by cytological examination of the germ cells from suspected males. Sterility and partial sterility are closely correlated with the induction of translocation heterozygotes. The procedure used in conducting this translocation test was based on experimental techniques described by Leonard and DeKnudt,1 Cattanach et al.,2 Falconer et al ,3 and Generoso.1* We modified this approach, in consultation with government and industry scientists actively engaged in mutagenesis research. 176 �Materials and Methods Animals Male and female ICR/SIM mice were purchased from Simonsen Laboratories. Gilroy, California. The F males were 8 to 10 weeks old. The females " used in the breeding phases were 10- to 12-week-old virgin stock. Chemical Supply A supply of Captan sufficient for all aspects t the experimental program was received from Battelle Columbus Laboratory and EPA-RTP. Lot number SX-640, Chevron Chemical Company, was used for all treatment periods. The excess material has been placed in storage in case it is needed for future reference. Dosage Selection and Compound Administration SRI and EPA staff selected the two dosage levels of Captan to be used in this experimental program. ti For 8 weeks, Captan was fed in diet at 2500 t.id 5000 ppra. An appropriate amount of Captan was dissolved and/or susperded in corn oil. Then the compound-oil concentrate was added at a level of 3% to a finely ground commercial diet (Purina) of known composition. The use of corn oil assured even distribution of Captan and prevented its stratification in an otherwise dry diet. Diets prepared at 2-week intervals were refrigerated at 4°C until fed to the animals. The diet was replaced in the feed containers twice weekly to minimize the possibility of compound loss. Body weights and food consumption were recorded weekly during the 8-week exposure period. Reference Control Males in the reference control group were fed the Purina diet with only corn oil added at a level of 3%. These mice were treated in the same manner as those in the compound test groups. recorded weekly, as was food consumption. 177 Body weights were �Positive Control For the positive control group, the known mutagen triethyl-onemelaraine (TEM) was administered in the drinking water at 0.32 mg/liter fjr 2 weeks and then at 0.124 mg/liter for ' weeks. TEM treatment was initiated after the males had been on the control diet for 4 weeks. 3ody weights and food consumption were recorded weekly. TEM is one of the chemical mutagens that have the demonstrated effect Oi inducing translocations in the F.. progeny of F_ treated males. Genetic Tests After 8 weeks of treatment, the mai.<= in each treatment group were ; mated to two adult virgin females each. A£ -er 1 we.k, each femalo was housed inH<vtdually tnd allowed to deliver its litter. The F_ males were discarded. All litters were raised to weaning age, at wnich time the females were discarded. The F.. males were raised to maturity. At maturity (10 to 12 "eeks of age), 200 F males from ecch experimental group, were selected randomly and housed individually. Three adult virgin females were housed with each F.. male for the first breeding. They were examined daily for the presence of vaginal plugs. These females were sacrificed 14 days after mating, and a uterine analysis was performed for determination of the number of total, live, and dead implants. Males bred to females that produced litters fitting our criteria for presumptive classification as steri]z, partially sterile, or nonbreeder were rebred to three new virgin females each. The same evaluation was made for the second breeding. Our criteria for presumptive classification of a male as "partially sterile," "sterile," or "nonbreeder" are: * "Partially Sterile" Male - If all 3 females are pregnant, each muct have 9 or fewer live implants, with at least 1 having 6 or fewer live implants. - If only 2 of 3 females are pregnant, both must have 9 or fewer live implants, v th 1 having 6 or fewer live implants. 178 �~ If only 1 of 3 female* is pregnant, this female must have ,6 or fewer live implants. • "Sterile" Male - None of 3 females pregnant—previously identified by presence of vaginal plug. • "Nonbreeder" Male - None of 3 females pregnant—not previously identified by presence of vaginal plug. Any F male that did not fit one of these descriptions was considered "normal" and was discarded. For each FI male in the control and compound-treated groups suspected of being a translocate or nonbreeder after 2 or 3 breedings, a cytogenetic evaluation was made of meiotic cell preparations of its tastes. Five males from the positive control group were also subjected to cytogenetic evaluation. Evaluation of Breeding Data F. males w^re identified as sterile, partially sterile, or nonbreeders by the methods outlined above. Individual data were totaled to give the number of observed (presumptive) translocations per treatment group, using a data base of 600 to 800 females per group. Also, for an accurate review of such findings, the F. breeding and litter data were thoroughly evaluated. The various measured evaluated included percentage of pregnancies, average litter size, average number of males and Temalcs, average number of males with f .-males having zero to five or more dead implants, average number of females having zero to five or more dead implants, percentage of females with plugs, and percentage of pregnancies with and without plugs. Meiotic Cell Cytogenetic Studies Cytogenetic examinations were made of the testes of 31 F the two testes from each mouse being examined separately. 179 mice, with The procedures �used for the cytogenetic preparat Ji.s are as follows. to sacrifice the mice. CO- was used The testes were removed, weighed, and placed in an isotonic solution of 2.2% sodium citrate. The tunica of each testis was punctured to release the tubules, which were then rolled on a glass plate to release the cell contents into the isotonic solution. The resulting cell suspension was centrifuged at 800 rpm for 5 minutes; the supernatant was removed, and each pellet of cells was resuspended.in 5 ml of IX sodium citrate hypotonic and held at room temperature for 15 minutes. The cells were centrifuged again at 800 rpm for 5 minutes and the supernatant was discarded. The cells were then treated with Carney's fixative (3 parts methyl alcohol and 1 part glacial acetic acid) to give a total volume of 5 ml, and immediately centrifuged again at 800 rpm for 5 minutes. This procedure was performed twice. Then the cells, suspended in an appropriate amount of fixative, were dropped onto clean, wet microscope slides and allowed to air-dry. were stained with 2% buffered Giemsa for 5 minutes. attached with Permount. The slides Covcrslips were The slides were coded to preclude bias on the part of the scorers. 180 �RESULTS AND DISCUSSION General Table 1 presents the average body weights for mice in the various groups. The body weights of the control, TEM, and 2500 ppm Captan group were within normal limits and comparable throughout the experiment. The 5000 ppm Captan-treatcd group showed a depressed body weight for 5 weeks before demonstrating a recovery trend during Weeks 6 to 8. This body weight depression appeared to be c'ue to the inability of the male mice to acclimate to such a high level of compound in their diet. Table 2 summarizes the average food consumption by treatment group. Both Captan-treated groups (2500 and 5000 ppm) showed a lower average weekly food intake than did the control and TEM-treated groups. During the week immediately following the 1-week F generation mating, one animal rack holding some females that had been mated with the mice given 5000 ppm Captan was accidentally tipped and some cages were spilled onto the fllor, resulting in our inability to identify which males had been mated with these females. sufficient numbers of F There were, however, generation males from those females that were not traumatized to allow us to randomly select 200 F subsequent F generation breedings. males for use in In addition, we held all females that had been traumatized by the tipping of the rack and maintained them throughout the remainder of the study as a separate group. From this separate group we selected 50 FI males (at least one per feriale retained) for use in the F generation breedings and evaluations. FO Generation Information on the breeding performance, litter size, sex distribution, and clinical effects of the F. generation should be included in the evaluation of translocation data, because it may provide valuable reference data. 181 �Table 3 summarizes the breeding and litter performance of the F generation. No adverse effects were observed in the control and 2590 ppm Captan groups. The two 5000 ppm Captan group:; showed a reduced pregnancy rate; the rate for the traumatized females was 29% below that of the controls. Litter sizes for the 5000 ppm groups were slightly below control values. As expected, the TEM group had a reduced pregnancy rate and a litter size 47% below the control level. Table 4 presents litter-size distributions of live young from the F . generation mating. . Although the distribution patterns for the control and Captan-treated animals were within normal ranges for our strain of mouse, there was evident a definite pattern of decreasing litter size and increasing variance and standard -Jeviations between the different experimental groups. As expected, the TEM-treated animals showed the classic shift toward smaller litters. Figure 1 graphically presents the data on the F- generation litter-size distribution. F, Generation j_ Table 5 summarizes breeding data from the first mating of the F generation male mice. In the females mated with TEM males and with males from the 5000 ppra Captan group of traumatized F- mothers, there were 10% fewer females with mating plu^s and an increased percentage of nonpregnant females in comparison with control values; also, these two male groups had an increased percentage of males with no pregnant females. Results from males in the 2500 and 5000 ppm Captan groups were within normal limits for this strain of mouse and comparable with '-.f rrom control males. Litter-size distributions of live implants-- derived from the first mating of F. generation males are pre.-it-nted in Table 6. Responses of control and Captan groups were within normal limits and readily comparable. litter size. The TEM group showed approximately a 13% reduction in Mean litter sizes were 11.72 for the control group, 11.73 for the 2500 ppm Captan group, 11.56 for the 5000 ppm Captan group, 11.88 for the 5000 ppm (traumatized F 182 female) group, and �10.24 for the TEM-treated group. The data on the F generation litter- size distribution are presented graphically in Figure 2. Tables 7 and 8 summarize the data on dead implants per FI male and dead implants per female, respectively. The 2500 and 5000 ppm Captan groups showed a slight increase in total dead implants for both males and fena'es at the 4, 5, and >5 levels when compared with controls. TEM aniuals showed significant increases in dead implants for both males and females. Table 9 summarizes the breeding results by treatment of those F. males classified as presumptive sterile, partially sterile, or nonbreeders after three breedings. Table 10 identifies these F males individually by number and treatment. Details of the breeding and rebreeding data for presumptive F males are presented in Table 11. In the reference control group, 24 of the 200 males were considered as presumptive translocates. rebred, 12 males remained in this classification. When A third mating of selected questionable and/or nonbreeder males reduced the number of presumptive males to eight: 1 nonbreeder, 1 presumptive sterile, and 6 partially sterile (3 of which were questionable partially sterile). For the TEM group, 83 of 200 F mutants after the first breeding. criteria. males were identified as presumptive When rebred, 53 still met the original A third breeding reduced this number to 49; 4 continued to be nonbreeders, 14 were presumptive sterile, and 31 were partially sterile (6 of which were questionable partially sterile). In the 2500 ppm Captan group. 30 of 200 FI -nales were identified as presumptive mutants after the first breeding. still met the criteria: When rebred, 11 5 were nonbreeders, 1 was a presumptive sterile, and 2 were partially sterile (1 of which was questionable partially sterile). The 5000 pr - • Captan group alsc had 30 of 200 F as presumptive mutants after the first breeding. reduced this number to 9. . males identified The second mating After a third breeding, 8 males still met the- 183 �original criteria: 2 were nonbreeders, 1 was a presumptive sterile, and 5 were partially sterile. For the group of F.. males derived from traumatized F and males treated with 5000 ppm Captan, 12 of 50 F as presumptive mutants after the first mating. females males were identified A second breeding reduced this number to 4 and a third breeding further reduced to 2 the number of F males that still met the original criteria; both were partially sterile, with one of them being questionable partially sterile. t' The data on the F and F generations' fertility, breeding, and litter-size distribution as well as the data on the F generation's dead implants and rebreeding show that Captan tends to induce doserelated effects on the reproductive performance of male mice. The data also suggest the presence of translocation heterozygotes tn the 5000 ppm Captan group. Review of the data on dead implants, breeding, and rebreeding for the F.. generation of the TEM-treated group showed, as expected, the potential for the presence of translocation heterozygotes in 24.5% of the F males. Cytogenetic Studies Table 12 presents the findings from the cytogenetic evaluation of meiotic cell preparations from F. males in the Captan groups characterized as nonbreedf.r, presumptive sterile, or partially'sterile. Also, eight control males and 5 of 49 TEM males were evaluated. Whenever possible, 25 spermatocytes per testis were scored. slides were decoded only after all scoring was completed. The The results are summarized as follows: • All eight uiales examined in the control group were cytogenetically normal. • The five TEM males all showed positive reciprocal translocations. • All eight males in the 2500 ppm Captan group were cytogenetically normal. • Seven males in the 5000 ppm Captan group were cytogenetically normal; however, the eighth male (No. 657) showed as a positive reciprocal translocation. 184 �• The two males examined in the 5000 ppm Captan group derived from traumatized F temales vere cytogenet'cally normal. Discussion Increased use of Lhe translocation procedure has revealed that a meaningful relationship exists between the incidence of dead implants in F.. ma tings and the occurrence of a heritable translocation event. Previous experiments at SRI and at Oak Ridge National Laboratories have demonstrated this correlation. The following paragraphs discuss occurrences of dead implants in this study. When females had a total implant count of less than six or when all their implants were identified as dead, we generally considered this to be a result of first breeding or of some factor other than compound treatment (such as background incidence) and excluded those females from this evaluation. Tables 13 through 17 present the total, dead, and live implantation data for suspect translocates of the control, the TEM group, and the three Captan groups. The control group (Table 13) showed a normal implant distribution, with the exception of one F. mala (No. 122) for whom the number of 1 i implants was high. In the TEM group (Table 14), the expected increase in dead implants and the n 3ultant decrease in live implants occurred, although the numbers of total implants were generally normal. This pattern occurred during all breeding periods. i The 2500 ppm Captan group (Table 15) contained seven males in the first breeding with females having high dead implant counts but normal live litter size, according to the criteria. Also, 3 males showed high dead implant counts during the first breeding, with live implant counts fitting the criteria for partially sterile males. This increase in dead implant occurrence was not repeated in subsequent breeding, and all FI males were classified as normal after the breeding phases. 185 �Five F malea in the 5000 ppm Captan group (Table 16) showed an increase in dead implants during the first breeding. In the rebreeding of these males, only male No. 657 continued to show the increase in dead iuplarits and the resultant decrease in live implants. All other P.. males in this group had a normal distribution of dead and live implants for all breedings. Table 17 presents the implant data for f. males derived from traumatized females in the 5000 ppm Captan group. With the exception of an occasional female showing an increase in dead implants, the distribution of total, dead, and live implants was normal. The numbers of total implantations were generally within normal limits for all experimental groups in all breedings. 186 �REFERENCES 1. A. Leonard and G. H. DeKnudt. Mutation Res. 9_, 127 ( 9 0 . 17) 2. B. M. Caltanach, C. E. Pollard, and J. H. Isaacson. Mutation Res. 6, 297 ( 9 8 . 16) 3. D. S. Falconer, D. M. Slizynski, and C. Auerbach. 81 (1952). J. Genet. 51, / A. W. M. Generoso. Proc. Second Annual Environmental Mutagen Soc., p. 9 (March 1971). 187 �Table 1 AVERAGE BODY WEIGHTS IN GRAMS FOR MICE RECEIVING VARIOUS LEVELS OF CAPTAN IN THEIR DIETS Week of Test TEM Control Dietary Levels of Captan (ppm diet) 2500 5000 Initial 33.8 33.4 33.8 33.6 1 2 32.5 32.9 33.3 31.8 33,7 34.6 33.7 31.4 3 34.8 35.4 34.4 32.1 4 5 6 7 8 34.6 35.8 34.4 31.4 34.8 35.9 36.0 33.0 37.3 38.1 37.5 35.3 37.7 38.9 39.3 39.2 39.8 38.9 35.8 36.9 188 �Table 2 AVERAGE FOOD CONSUMPTION FOR MICE RECEIVING VARIOUS LEVELS OF CAPTAN IN THEIR DIETS (Grams of Food Consumed/Mouse/Day) Week of Test TEM Control Dietary Levels of Captan (ppra diet) 2500 5000 1 4.01 4.05 3.74 3.25 2 4.82 5.00 4.72 4.38 3 4.91 5.08 4.90 4.40 4 5.28 5.33 4.93 4.45 5 5.08 5.55 5.44 4.95 6 5.36 5.56 4.95 4.75 7 5.81 6.14 5.63 5.27 5.55 5.74 5.13 4.83 8 189 �Table 3 TRANSTXXATION STUDY OF CAPTAN F0 GENERATION MICE SUMMARY OF BREEDING AND LITTER DATA Parameter Number of FQ males Number of F.. females Number pregnant Percent pregnant Number of nonbreeder males Percent nonbreeders Average live litter size Average number of males weaned/Utter a Control TEM 2500 ppm 60 120 93 77.5 8 13.3 12.30 5.84 66 60 119 91 76.5 8 13.3 12.16 5.68 132 77 58.3 19 28.8 6.55 3.26 5000 PI 31 61 38 62.3 5 16.1 11.68 6.29 5000 ppma 30 59 33 55.9 11.33 5.25 Gr~up of females accidentally tipped off rack folioving 1 week of mating with iOOO ppm-treated mates. These traumatized females, and their offspring, nort of which could not re identified back to a particular FQ male, vere considered as a separate group for the remainder OJL tne study. �Table 4 TRANSLOCATION STUDY OF CAPTAN MOUSE LITTER-SIZE DISTKIBUTION OF LIVE YOUNG DERIVED FROM FQ GENERATION ADULTS Captan (ppm diet) 5000 Litter Size Control 1 0 1 0 0 0 2 0 2 0 1 0 3 0 3 0 0 0 4 0 8 0 0 1 5 0 10 1 0 0 6 0 15 2 1 1 7 1 12 0 0 1 8 2 8 3 0 0 9 9 8 3 1 3 10 6 2 8 6 3 11 8 3 9 5 7 12 18 1 24 8 5 13 22 0 20 9 8 18 1 10 6 1 3 14 TEM 2500 5000^ 15 6 0 6 1 16 3 0 3 0 0 17 0 0 2 0 0 18 0 0 0 0 0 Mean ( ) u 12.30 6.55 12.16 11.68 11.33 Variance ( 3 o) 3.89 5.84 4.97 5.73 6.09 Standard Deviation 1.97 2.42 2.23 2.39 2.47 (o) Traumatized FQ females. 191 �24 1 I I I I I I T i 1 I I I I I ————— Control 22 • ' —— TEM — "— — — — 2500 ppm Capun — • — • — 5000 ppm Captan ........... 5000 ppm Captan 20 (Traumatized Females) 18 16 £14 u U. 10 2 0 ' C ui a 1 10 O mn / X &*$&**• FIGURE 1 8 10 LITTER SIZE 12 14 LITTER SIZE DISTRIBUTION FO GENERATION 192 16 18 �Table 5 TRANSLOCATION STUDY OF CAPTAN F L GENERATION MICE SUMMARY OF BREEDING DATA--FIRST BREEDING Parameter Number of Fl males Number of females Number of mating plugs Percent mating plugs Number pregnant Percent pregnant Number pregnant with plug Percent pregnant with plug Number pregnant without plug Percent pragnar.t without plug Number not pregnant Percent not pregnant Number not pregnant with plug Percent not pregnant with plug Males with no pregnant females Percent males with no pregnant females Traumatized Fo females. Control 200 600 450 75 492 82 434 88 58 12 108 18 16 15 13 6.5 TEM 2500 200 600 376 63 383 64 326 85 57 15 217 36 50 23 39 19.5 200 600 442 74 472 79 419 89 53 11 128 21 23 18 14 7.0 Captan (ppm) 5000 5000a 200 600 439 73 477 80 413 87 64 13 123 20 26 21 9 4.5 50 150 95 63 112 75 94 84 18 16 38 25 1 3 7 14.0 �Table 6 TRANSLOCATION STUDY OF CAPTAN MOUSE LITTER SIZE DISTRIBUTION OF LIVE YOUNG DERIVED FROM FI GENERATION ADULTS--FIRST BREEDING Litter Size 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean (ji) Variance Standard Deviation () c Captan (ppm) 5000 5000a Control TEM 2500 5 4 4 3 0 4 9 0 3 0 2 12 5 1 0 4 17 6 5 1 6 1 5 21 8 11 5 8 4 7 8 6 11 3 14 35 37 73 80 89 72 32 16 7 1 0 0 17 13 19 4 25 35 17 2 31 40 53 16 53 77 77 14 61 75 85 15 52 87 93 17 27 54 55 14 21 36 19 13 8 14 11 5 1 7 7 1 1 2 3 0 1 0 0 0 0 1 0 0 11.72 10.24 11.73 11.56 11.88 8.13 13.59 8.68 7.40 7.20 2.85 3.69 2.95 2.72 2.68 4 Traumatized Fo females. 194 ��Table 7 TRANSLOCATION STUDY OF CAPTAN SUMMARY OF DEAD IMPLANTS PER FI MALE Number of Males with Females Having Captan (pom) 5000 5000a Control TEM 2500 0 Dead implants 68 37 69 49 20 44 31 68 36 13 18 11 7 2 18 9 3 1 Dead implant 54 36 2 Dead implants 30 3 Dead implants 18 8 2 7 25 16 4 Dead implants 5 Dead implants > 5 Dead implants 9 6 32 Traumatized FO females. 196 4 7 8 0 2 1 �Tsble 8 TRANSLOCAT::ON OTUDY OF CAPTAN SUMMARY OF DEj D ThPLANTS PER FEMALE Number of Dead Implants/Female Captpn (ppm) 5000 5000a Control TEM 2500 0 1 320 182 284 109 2 39 18 83 35 16 295 113 37 16 11 11 7 5 3 0 > 5 3 45 2 2 3 2 492 383 472 3 4 Total Pregnant Females Traumatized Fo female. 197 130 41 9 79 23 9 0 0 8 0 1 477 112 �Table 9 TRANSLOCATION STUDY OF CAFTAN SUMMARY OF PRESUMPTIVE TRANSLOCATION T MALES AFTER THREE BREEDINGS Control Total number of FI males Number of nonbreeder males Number of presumptive sterile males Number of partially sterile males VO 00 raumatized Fo females TEM 2500 200 1 1 6(3?) 200 4 14 31(6?) 200 5 1 2(1?) Captan (r>pm) 5000a 5000 200 2 1 5 50 0 0 2(1?) �Table 10 TRANSLOCATION STUDY OF CAPTAN INDIVIDUAL IDENTIFICATION OF PRESUMPTIVE F t MALES AFTER THREE BREEDINGS Treatment Control TEM Partially Sterile Presumptive Sterile _ 16? 65 122? 164? 189 190 72 215 216 232 235? 238 245 262 264 269 280 281 290 292 299? 300 314 315 202 231 251 256 268 273 288 317 321 326 339 343 369 389 Nonbrceder 220 , 344 345 350 359 360? 361 371 375 376 ' 388 390 399? 400? (Continued) 199 220 241 391 396 �Table 10 (Concluded) Treatment Partially Sterile Presumptive Sterile Nonbreeder Captan 2500 ppra 480? 526 474 449 479 496 523 583 Captan 5000 pom 635 657 760 779 780 736 634 733 Captan 5 0 ppma 00 805 837? Traumatized Fn females. 200 �Table 11 TRANSLOCATION STUDY OF CAPTAN BREEDING AND REBREEDING SUMMARY OK tRESUMPTIVE FI MALES (Live Implants Only) FI Treatment Male No. Control 11 First Breeding (3 Females) _ 16 17 24 39 43 50 5 9 / 65 67 72 86 102 122 129 139 144 152 164 179 189 190 192 200 .* * . . 9 4 0 4 . 2 . 3 . 9 - Totals TEM 202 215 216 220 226 227 228 ; 229 230 0 7 0 _ - 6 0 1 7 . _ 0 . . . 0 . 7 - _ 13 _ - 1 4 (14) 24 Third Breeding (3 Females) Second Breeding (3 Females) (13) . 6 5 _ ( ) U 13 9 0 6 . - . 0* 0 12 10 3 14 0 (12) 8 . 11 11 9 6 11 . (IS)* . 12 . • 7 16 14 14 9 10 10 8 13 10 0 0 0 1 12 13 11 - . U 0 () 9 4 () 9 0 - 4. 9 - 0 7 12 12 14 14 11 11 . 12 10 () 9 0 0 - . 12 . 12 4 1 1 0 1 14 0 0 8 12 0 - . -- 2 5 7 9 . 0 4 0 - 2 8 11 4 12 14 10 4 . . . 13 " (Continued) "0" indicates a plug was observed for a female that was not pregnant. ** "-" indicates a plug was not detected and the female was not pregnant. " ) indicates all implants were in early stages of development and impossible ( " to determine if they were live or dead upon gross observation. 201 �Table 11 (Continued) ; Treatment TEM Fl rfale No. 231 232 233 235 237 238 239 241 244 245 251 253 254 256 258 262 264 267 268 269 270 273 280 281 283 286 288 290 292 294 299 300 302 314 315 317 318 321 322 First Breeding (3 Females) ** 0* 3 4 8 7 9 5 3 0 . . ® 0 3 4 0 0 2 3 0 . 9 ® 9 - 0 5 6 8 8 6 9 0 0 4 0 9 . 0 0 10 . . — 1 6 8 1 . . I 0 . 8 5 5 . . . 7 2 - 6 9 - 0 5 3 (8 + 1) . (15) 3 0 4 0 - Third Breeding Second Breeding (3 Females) 8 0 4 0 9 . 0 0 3 (Continued) 0 8 11 7 10 1 13 . 10 1 0 14 13 . 13 5 ® 13 6 8 0 8 4 14 0 7 3 10 9 8 13 2 0 0 9 0 11 0 2 15 7 10 2 _ - 0 5 6 13 14 ®" 13 0 0 11 14 . 0 (3 Females) 11 3 0 . _ 11 4 5 ® 7 _ - 8 I 0 . 0 5 3 12 4 5 12 1 0 0 4 0 11 „ . 5 9 12 . . . 5 2 - 10 0 0 8 11 9 2 15 4 0 . 11 . - . "0" indicates a plug was observed for a female that was not pre "-" indicates a plug was not detected and the female was not pregnant. ( ) indicates all implants were in early stages of development and impossible " to determine if tney were live or dead upon gross observation. "®" indicates female was pregnant but had no live implants. 202 �Table 11 (Continued) n Treatment TEM Male No. First Breeding (3 Females) ** 326 327 333 334 339 343 344 345 346 349 350 355 356 359 360 361 363 367 0 0 6 0 0 2 6 4 . 0 5 4 9 (15) 4 5 0 369 371 372 (16) 375 3 376 381 382 385 387 388 389 390 391 396 397 399 400 5 Totals 4 - • -• 0 • - 5 - - (12) 2 (^ E 4 - 4 9 13 11 7 2 3 12 4 - . 0 - 8 1 6 (13) (11) 9 5 () 6 3 4 4 . 5 2 0* 0 10 0 0 5 4 12 10 3 6 - . 3 4 2 Third Breeding (3 Females) Second Breeding (3 Females) - • (10) t 0 - 0 0 t; 4 11 8 0 0 3 © 7 14 5 5 10 5 9 5 1 11 0 - 12 12 - • 11 7 10 0 0 5 3 10 1 . (15) 7 ® . 11 5 7 1 - 12 0 10 15 3 3 4 5 3 - 3 - 11 5 4 (14) (ID (12) 5 0 2 5 0 4 2 53 .8" 49 (Continued) "0" indicates a plug was observed for a femal e that was not pregr "-" indicates a plug was not detected and the female was not pregnant. " ) indicates all implants were in early stages of development and impossible ( " to determine if they were live or dead upon gross observation. "®" indicates female was pregnant but had no live implants. 203 �Table 11 (Continued) Male Treatment Captan ?500 ppm i / I 3. 430 432 436 449 450 451 452 455 461 469 472 474 479 480 484 489 496 518 523 526 5?8 546 561 568 569 582 583 585 =88 590 Totalf Captan First Breeding (3 Females) . .* * A 4 (7 f 1) _ 9 5 . 0* 1 7 0 9 10 8 () 3 4 11 . 7 0 8 9 8 7 8 8 635 10 . . 0 14 12 . . . 3 . (-) 9 . 10 0 "* 9 8 _. 14 10 . . . 9 . . .. 11 6 . . () 1 . (3 1) . . 12 (2 1) 9 12 1.1 11 _, 8 10 _ 12 10 0 13 10 13 0 11 13 0 0 . (1?) 11 9 . _ 0 - (11) 9 8 - () 8 9 () 9 - 8 - 12 11 . _ 11 _ _ 12 12 _ 13 13 10 13 11 9 11 _ 10 - - * - - * 0 _ 11 8 12 14 - _ 11 2 - 13 - 9 11 8 12 9 0 11 9 9 0 (2 1) 10 - 5 . . . 11 . 13 . 13 11 11 0 10 12 12 6 (py _ «• _ 0 2 1 9 1* . . 11 .. - 12 o 14 () g" . - . . - - "0" in-Jicates a plug was observed for a female that was not pregnant . "-" indicates a plug was not detected tad the female was not pregnant " ) indicates all implants were ir. ?arly stages of development and ( " to determine if thry were live or dead upon gross observation. "®" indicates female was pregnant but had no live implants. 204 . 0 _ 13 11 8 10 7 . 13 • (Continued) * * . . _ _ • 0 74 . 30 601 604 620 622 626 627 634 5000 ppm . Th rd "jreedir.g f3 Kenulest Second Breeding ( Females) 3 - �Table 11 (Concluded) First Breeding (3 Females) Male Treatment Captan 5000 ppm No. 638 646 633 657 660 668 671 672 679 722 732 733 734 736 743 760 761 765 767 769 779 780 10 10 0* 2 0 - (13)"* - 0 8 . . - 6 4 . . 0 7 9 9 10 8 2 4 Totals Captan g 5000 ppm 805 806 815 816 817 818 822 823 837 841 846 647 6 10 . 5 (4 1) Totals 6 _** 5 10 - . 5 10 0 0 1 30 . 9 (0 1) 6 . Third Breeding (3 Females) Second Breeding (3 Females) 6 1 8 6 (15) 8 - (2 1) 1 5 10 11 14 2 14 9 14 12 13 13 1 9 12 11 7 12 12 11 . 12 14 1 10 0 5 -.13 12 12 12 0 0 11 13 13 4 2 11 9 10 4 12 12 14 3 10 0 11 9 12 10 . - 12 13 9 7 11 6 13 7 13 10 10 - 2 10 10 0 -' 12 14 11 8 10 12 10 . . . 0 12 14 8 . . . 11 2 13 10 12 12 .- 4 12 - 4 9 . 7 (D I - - 12 2 Traumatized Fo females. "0" indicates a plug was observed for a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. " ) Indicates all implants were in early stages of development and impossible ( " to determine if they were live or dead upon gross observation. 205 �Table 12 TRANSLOCATION STUDY OF CAFTAN CYTOGENETIC EVALUATION OF Fl MALK MICE Testes Weight (mg) Classification-Based Upon Breeding Data 16 64.6 265 72 122 58.5 38.8 68.0 48.7 307 237 232 289 164 Control Body Weight (g) 17 65 Treatment Fl Male No. 55.8 314 189 190 , 2?2 262 290 345 361 449 474 479 480 49.6 47.5 54.6 41.4 62.6 54.1 50.4 60.2 56.5 65.1 58.7 245 222 276 243 222 294 278 256 175 287 307 496 523 526 583 60.9 50.7 54.1 50.7 242 348 295 297 Partially fterile (questionable) Nonbreeder Partially sterile Presumptive sterile Partially sterile (questionable) Partislly sterile (questionable) Partially sterile Partially sterile Partially sterile Partially sterile Partially sterile Partially sterile Partially sterile Nonbreeder Presumptive sterile Nonbreeder Partially sterile (questionable) Nonbreeder Nonbreeder Partially sterile Nonbreeder 10 O TEM Captan 2500 ppra Cytogenetlc Classification Normal Normal Normal Normal Normal Normal Nornia 1 Normal Positive Positive Positive Positive Positive Normal Normal Normal Normal Normal Normal Normal Normal reciprocal reciprocal reciprocal reciprocal reciprocal translocation translocation translocation translocation translocation �Table 12 (Concluded) Fl Male No. Body Weight (g) Testes Weight (ing) Classification-Based Upon Breeding Data Cytogeretlc Classification Captan 5000 ppra 634 635 657 733 736 760 779 780 57.1 56.0 61.0 62.2 57.9 55.9 34.8 53.0 Nonbreeder Partially sterile Partially sterile Nonbreeder Presumptive sterile Partially sterile Partially sterile Partially sterile Normal Normal Positive reciprocal translocation Normal Normal Normal Normal Normal Captan 500C ppma 805 837 70.0 56.9 299 312 231. 256 --56 298 224 295 26P 303 Partially sterile Partially sterile (questionable) Normal Normal Treatment KJ O -J Traumatized F0 females. �Table 13 TRANSLOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F HALES F. Male NUaber First breeding 11 Female Number Total Implantations • . (13) t . 1 17 2 3 1 2 3 24 39 43 50 59 65 67 72 86 102 122 1 2 3 4 8 1 2 3 7 1 2 3 0 .• • 1 2 3 5 0 5 . . 1 2 3 . . . . 1 2 3 1 2 3 ' . .? . . - 9* 0 . . 1 2 3 1 2 3 . ' . . 1 2 3 1 2 3 Initial Classification _** 1 2 1 16 Control Croup Dead Live Implantar ions Implantations 0 0 . . . ( ) H 16 4 13 Partially sterile (questionable) (13) . . Nonbreeder , . Nonbreeder 9 0 . 1 1 0 Nonbreeder \ 0 - Normal 4 7 6 - . 0 1 0 0 . . - - . . - • Nonbreeder . . - Partially sterile Presumptive sterile 4 0 5 . Partially sterile Nonbreeder . Nonbreeder . .- Nonbreeder . :^ •7 (11) 10 2 0 6 2 13 Partially sterile (questionable) Normal "0" indicates a pl«R was observed for a female that was not pregnant. * • "-" indicates a plug was not detected and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine if they were live or dead upon gross observation. 208 �Table 13 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES Control Croup " Male Fema le . Number Number First breeding (concl.) 129 139 144 152 j 164 179 189 190 192 200 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Implantations Lead Implantations .* * - - ; * Live Implantat ions - j Nonbreeder ' 4 3 0 Nonbreejer ™ Nonbreeder 7 12 9 Initial Classification 3 9 9 Partially sterile ™ - - Nonbreeder m 0* 1 0 1 4 6 7 (14) + Nonbreeder 0 0 0 0 0 0 0 0 I 0 Partially sterile I 4 6 Partially sterile 7 (14) - Partially sterile (questionable) Nonbreeder - Final Classification Second breeding 11 16 17 1 2 3 1 2 3 1 2 3 13 9 (15) 0 I 7 - i - - 13 8 (15) - Normal Rebredb Rebred - * 0" indicates a plug was observed for a female that was not pregnant. * "-*' indicates .•» plug was not detected and the female was not pregnant. " ) Indicates all implants were in early stages of development, and thus difficult to (" determine if they were 1ivc 01 dead upon gross observat ion. Sec third breeding for final classification. 209 �Table 13 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES F. Male Female Number Number Second breeding (cont.) 24 39 43 50 59 65 67 72 86 102 122 129 139 144 ** "0 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 I 2 3 I 2 3 1 2 3 Total Implantations Control Croup read Live Implantations Implantat ions 0* 12ww 0 1 0 0 - - . . . - - 12 11 12 0 0 0 0 3 10 12 3 6 2 IS 11 0 (12) + 10 6 16 14 14 2 . 15 9 10 0 0 0 1 0 0 - ? 0 11 • 0 . . 12 11 12 10 9 3 6 2 14 11 Final Classification Normal K Rebred Normal Normal Partially sterile Normal 0 - k ReLied Rebred h (12) - - I 1 9 0 7 7 8 16 1 0 0 14 h Rebred 5 7 7 4 9 10 Normal Normal k Rebred Normal Normal indicates a plug vas observed for a female that wasi not pregnant. " ) indicates all iirplants were in early stages of development, and thus difficult to (" determine if they were live or dead upon gross observation. Sec third breeding for final classification. 210 �Table 11 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number Second breeding (concl.) Female Number 152 1 2 3 164 179 189 190 192 200 Third breeding 16 17 39 43 72 86 Total Implantations 1 2 3 1 2 3 Control Cr:nn Dead Live Implantations Implantations 11 8 14 .* * 1 0 1 - 7 11 1 2 3 0 0 0 I 2 3 0 0 0 1 Normal Rebredb Normal Rebre'l" I 12 11 - 13 0 * ~ t, Rebred 13 - : - - . 1 2 3 12 12 11 1 2 3 . . - 10 1 2 3 0 0 I 1 1 2 3 1 2 3 0 0 0 - 0 0 13 12 - . () 9 10 1 . 0* 0 0 1 2 3 10 8 13 Final Classification . () 9t 1 2 3 1 2 3 MALES . 0 1 0 0 . -. 14 14 5 . . 2 2 1 Normal Nortial Partially sterile (questionable) . . ' 12 11 11 10 -' 12 12 ' NonL;-?eder Normal Normal Presumptive sterile * Normal 4 * "0" indicat.es a plug was observed for a female that was not pregnant, *'-" indicates a plug was not detected and the female was not pregnant act icatcs all implants were in early stages of development, -and thus d i f f i c u l t to observat ion'. Soc t h i r d breeding for f i n a l classification. 211 �Table 13 (Concluded) TRANS IDC AT ION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES Control Croup Dead Live Implantations Implantations F, Male Number Third breeding (oc. cnl) Female Number Total Implantations 129 1 2 3 1 2 3 1 2 3 1 2 3 14 ** 0 . 14 . . . . 0* 1 0 0 0 1 164 189 190 / 0 1 0 0 0 Normal Partially sterile (questionable) Partially sterile 0 1 1 ~"0" Indicates a plig was observed for a female that was not pregnant. " " Indicates a plug was not detected and the female was not pregnant. • 212 Final Classification Partially sterile �Table 14 TRANSLOCATION STUPY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male I/umber First breeding Female Number Total Implantat ions 202 I 2 3 0 215 1 2 3 0* 0 ** - - . TEH Group Dead Live Implantations Implantations 0 0 0 „ • . ? 1 2 3 () 9* 1 2 3 - 1 2 3 - 1 2 3 11 11 0 7 9 228 1 2 3 . () 9 - ? 229 1 2 3 8 0 10 1 0 1 1 2 3 - 1 2 3 1 2 3 12 9 10 1 2 3 - 1 2 3 15 11 11 4 1 2 3 5 ,12 12 216 220 226 227 230 231 232 233 2J5 237 - - 0 MALES . - 0 0 0 « . (-) 9 . . 0 4 2 . () 9 - Initial Classification Presumptive sterile Kcmbreeder Partially srrrlle (questionable) Nonbreeder Nonbreeder Partially sterile Partially sterile (questionable) 7 0 9 Normal - - Nonbreeder 0 - 0 Presumptive - - 0 3 4 1 Partially sterile - Nonbreeder partially sterile 5 8 7 6 0 3 4 5 9 8 Partially sterile - 9 5 9 . 7 sterile "0" indicates n plug w.is observed for a female that wa~» not pregnant. •:.••:: "•" inuit/.urf •"> pl»'P, was .tot detected and the female was not pre^.iaut. " ) indicates .til implants v^eie in early stage> of development , and thus difficult to (" determine i f they were live or dead upon gross observation. 213 �Table 14 (Contlnjed) TRANSLOCATION STUDY ^F CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES ?j Male Nurr.ber First breeding (cont.) Female Number 238 I 2 14 0* 9 -** . 239 3 I 2 3 241 244 245 251 253 254 T5C 258 262 264 267 1 2 3 268 269 llonbreeder . Nonbreedtr 6 0 3 . . Nonbreeder 0 0 Partially sterile 0 0 0 0 0 0 12 12 9 2 - 3 10 6 2 4 0 0 0 - - - 0 0 . 0 * 1 12 Presumptive sterile Normal - - - 1 2 3 1 2 3 Partially sterile 0 0 0 3 I 2 3 I 2 3 I 2 3 I 2 3 2 3 Initial Classification t 1 2 3 l ' . . 6 0 1 2 3 3 1 3 0 1 . . . . . 1 2 3 1 2 11 0 8 . . . — 2 8 Partially sterile Presumptive sterile Presumptive sterile 15 10 4 10 8 8 5 3 3 . . . . . . Nonbreeder . . Nonbreeder ' 12 ' 2 ** TEM Group Dead Live Implantations Implantations Total Implantations • - . - • - . . . - 5 "0" indicates a plug was observed for a iemale' that was not pregnant. ' / "-" Indicates a p l u g was not detected and the, female was not pregnant. 214 Partially sterile 5 Partially sterile NonbreeJer �Table U (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Kuaber First breeding (:n. •ot) 270 273 280 Female Number I 2 3 1 2 3 1 2 / / 281 283 286 288 290 292 294 299 300 302 314 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Xmplantatlons TEM Croup Dead Live Implantations Implantations 0* _** , 11 14 10 0 8 (18)t 11 11 . 9 (15) 0 . Presumptive sterile . . . . Nonbreeder - 9 7 2 0 6 It 5 1 4 6 12 11 9 , . U Initial Classification 0 . 13 12 3 4 12 0 0 0 9 MALES Partially sterile Partially sterile 3 2 1 . . Normal . Nonbreeder 0 0 0 • 9 9 8 _ 0 0 0 5 5 4 6 3 0 8 4 9 7 8 7 0 0 ? 2 3 . 3 ? (8 1) Presumptive sterile Partially oterlle Partially sterile Partially sterile (questionable) 9 8 6 (5 1) It . 2 . 9 . . . - - Partially sterile (questionable) . - Normal Normal Nonbreeder "0" Indicates a plug was observed for a female that was not pregnant. "-" Indicates a plug was not detected and the female was not pregnant. " ) Indicates all Implants were In early stages of'development, and thus difficult to (" determine If they were live or dead upon gross observation. 215 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE FI MALES TEH Group Dead Live Implantations Implantations F. Male Number First breeding (cont.) Female Number Total Implantations 313 I 2 3 0* 3 0 0 0 0 0 3 0 1 2 3 0 _** 0 0 . 317 318 321 322 1 2 3 I i 3 I 2 3 326 1 2 3 317 333 334 339 343 344 345 346 349 1 2 3 9 13 13 0 0 0 0 0 0 1 . 9 . _ - Normal . - Nonbreeder 10 . . 0 . . 0 - 0 . 0 0 0 5 . . 0 - - 1 2 3 0 0 0 0 0 0 0 0 0 1 2 3 10 12 14 10 10 . 7 7 - '3 5 - 1 2 3 1 2 3 1 2 3 Presumptive sterile 4 4 3 - 1 2 3 1 2 3 Partlall) sterile 5 9 10 -' 1 2 3 Initial Classification 11 . . - . ,(12) + Presumptive sterile Presumptive sterile 0 6 _ Partially sterile . Nonbreeder 0 8 6 5 . . 6 4 5 . . . . ? Partially sterile (12) Presumptive sterile Presumptive sterile Partially sterile Partially sterile Nonbreeder Partially sterile (questionable) "0" indicates a plug was observed for a female that was not pregnant. •ff "*" indicates a plug was not detected and the female was not pregnant. + * ( " indicates all implants were in early stages of development, and thus difficult to ') determine if they were live or dead upon gross observation. 216 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPT.W IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES TEM Croup F, Male Female Number Number First breeding (cont . ) 350 355 356 359 360 361 363 367 369 371 372 375 376 381 Vr 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 9 3 1 2 3 Total Implantations Dead Icipimttations 8 8 6 . . 12 11 8 .* * . . ' - • - (15) + 12 14 1) 9 . 0* 10 13 (16) . 14 11 0 10 . . 4 3 2 „ . . . 1 8 10 2 4 . - • 0 - ' 6 7 7 11 - G 0 4 - Initial Classification Partially sterile Nonbreeder Nonbreeder 9 4 8 (15) 6 8 « ?. 15 12 16 • Live Implantations . 4 4 9 5 0 4 6 (& ') 3 - 5 0 6 - Partially sterile Partially sterile (questionable) Partially sterile Partially sterile Nonbreeder Presumptive sterile Pai-tially sterile Partially sterile (questionable) Partially sterile Partially sterile Nonbreeder "0" indicates a plug was observed for a feiiale that was not pregnant. * " " * . indicates a plug wa.i not detected and the female was not pregnant. * ( " indicates all implants wort in »?arly stages of development, and thus difficult to ') determine if they v.erv live or dc«id upon gro.;s observation. 217 �Table 14 (Continued) TRANSLCv-Ai ION ..~"OY OF CAPTAN IMPLANTATION SUMMARY OF PRt-T'IMPTIVE TI MALES TEM C roup F. Mile Female Tot.l Number Nunhfc r Implantations 387 1 2 3 1 2 3 1 2 9 6 () 6 . . 388 3 1 14 2 3 1 6 389 First bieed Inj (concl.) 2 3 1 2 3 1 2 J 1 2 3 1 2 3 1 2 . 382 385 j 390 391 39C 397 399 400 3 1 2 3 (13) + (11) Dead Implantations Live Implantations 7 (13) (11) • 9 5 (6> . . ? „ * * 0* . - 11 12 14 . . . . _ . (14) 11 9 (11) 12 9 (12) 0 1 ? . . 11 5 0 . 7 o 8 . . . . ? 6 3 ? 10 7 ? Initial Classification Partially sterile quest lonable) Partially sterile Nonbreeder 3 4 0 Partially sterile 4 3 5 . Nonbreeder Partially stcri'- Non*>reedtr . Nonbreeder v!4) 5 4 (11) 2 2 (12) Partially sterile (questionable) Partially sterile (questionable) Part: illy sterile (questionable) Final Classification Second breeding 202 215 1 2 3 1 2 3 0 0 0 11 9 0 0 0 4 5 0 0 0 Presumptive sterile - Partially sterile 4 "0" Indicates a plug wa. observed for a female that was not pregnant. "-" indicates a plug "as not detected »nd the female was not pregnant. "O" Indicates all implants were i.i early stages of development, and thus difficult to determine if they were live or dead upon gross observation. 218 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES F. Male Number Second breeding (cont.) Female Number 216 1 2 3 220 226 227 228 229 230 Total Implantat ions TEM Group Dead Live Implantations Implantations *inal Classification -** I- . . • - -' -. - 2 3 12 2 Rebred . 1 2 3 1 2 3 14 5 - 1 2 3 12 11 8 0 7 0 12 4 8 I 2 3 14 12 1? 0 0 I 14 12 11 Normal 1 2 3 15 14 13 1 0 0 14 14 13 Normal 1 2 3 11 10 0 0 11 10 . - b Rebred Normal Normal Normal 232 CJ3 235 237 238 239 0 0 0 17 12 15 9 10 10 8 2 5 1 2 3 11 15 7 0 1 1 11 15 6 Normal 1 2 3 11 13 14 4 6 1 7 7 13 Normal 1 2 3 11 U 14 1 4 1 10 10 14 Normal 1 2 3 1 2 3 9 12 13 8 10 13 1 2 0 ^ 2 3 231 0 0 0 - 1 2 3 1 2 3 241 0* 0 0 0 . - 13 - - Partially sterile Normal - ""-" i n d i c a t e s a p l u g \7as not detected and the female was not p r e g n a n t . 219 Partially sterile 13 ' . "0" indicates a plug was observed for a female that was not pregnant, S t V t ! i i r J breeding for f i n a l c l a ^ s i f i c a t i o n . Presumptive sterile Q Rebred �Table 14 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number Second breeding (cont.) 744 245 251 253 254 256 258 262 264 267 268 269 270 273 280 Female Numbe r 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 I 2 3 Total Implantations TEH Croup Dead Live Implantations Implantations 2 0 0 0 0 4 12 13 11 1 0* 7 0 0 0 16 11 .* * 13 14 11 _ 10 13 11 1 0 0 0 0 2 1 0 0 0 14 11 0 0 0 13 14 11 . . . - 13 0 0 0 - 8 12 10 9 11 12 14 1 14 . 6 7 8 3 8 5 9 6 3 1 1 2 . . 13 0 5 4 5 0 5 9 13 0 12 . 0 8 0 . - 0 11 11 !«. - 3 3 5 8 8 5 for a female that was not pregnant. "-" Indicates a plug was not detected and the female was not pregnant. See third breeding for final classification. 220 Normal Partially sterile Presumptive sterile Normal Normal — 6 7 - Final Classification 3 0 0 0 "0" Indlc.r.es a plug was observed MALES Rebredh Normal Partially sterile Partially sterile Normal L Rebred Partially sterile k Rebred Rebred Partially sterile �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number Second breeding (cont . ) Female Number Total Implantations 281 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 TE!i Group Dead Live Implantations Implantations 11 12 283 286 288 290 292 294 299 300 302 314 315 317 318 321 MALES to 14 0* -** 0 0 0 13 8 0 9 14 11 11 12 12 14 12 12 13 9 12 13 13 IS 7 12 12 0 0 0 0 0 14 9 13 0 0 • 7 11 8 0 0 4 1 2 14 0 • 0 0 0 7 5 0 0 0 0 6 3 0 6 11 3 1 0 1 5 8 3 5 it 10 0 1 0 5 11 8 0 0 0 0 0 5 5 2 0 0 • 3 3 8 10 12 11 9 4 9 8 5 2 13 12 15 2 1 Normal k Rebred Presumptive sterile Partially sterile Partially sterile Normal Normal Partially sterile (questionable) Normal Partially sterile 0 0 Partially sterile 0 0 0 Presumptive sterile 9 4 11 0 0 ~ "-" indicates a plug was not Jetected and the female was not pregnant. 221 Partially sterile 4 "0" Indicates a plup was observed for a female that was not pregnant. See third breeding for final classification. Final Classification Normal Presumptive sterile �Table 14 (Continued) TRANSLOGATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE ?, F. Male Nuaber Second breeding (cont.) Female Number Total Implantations 322 1 2 3 TEM Group Dead Live Implantat ions Implantations 11 11 326 I 2 3 327 1 333 2 3 1 2 3 334 1 339 2 3 1 HALES 0 0 11 11 Final Classification Normal » * * 0 . 0* 13 (10) + .- 0 (0 1) 13 ? 12 0 . 0 . Presumptive sterile L Rebred - 2 0 10 0 Normal - - . • • • Rebred K 343 344 345 346 349 350 355 356 .- 2 3 0 0 0 0 0 0 0 0 0 Presumptive sterile I 2 3 0 0 0 0 0 0 0 0 0 Presumptive sterile 1 2 3 1 2 3 10 10 5 4 5 Partially sterile 11 5 6 6 6 12 4 2 8 1 4 4 3 Partially sterile 12 12 0 1 - 12 11 I 2 3 - 1 2 3 10 11 12 0 3 2 10 8 10 1 2 3 8 0 10 8 0 9 0 0 1 1 2 3 13 0 0 0 13 0 1 2 3 11 . ( 15) 0 . Normal Normal Partially sterile N jrr.3 1 . 11 . Normal (15) "0" indicates a plug was observed for a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. " ) Indicates all implants were in early stages of development, and thu.s difficult to (" determine if they were Use or dead upon gross observation. See third b-eeding for final classification. 222 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PREStWPTIVt F F. Male Number Second . breeding * (cont.) Female Number Total Implantations 359 1 2 3 1 2 3 1 2 3 1 2 3 1 TEM DeaJ * Implant at ions 10 9 16 10 13 -** 360 361 363 367 2 3 369 i7l 372 375 376 381 382 385 387 3S8 1 2 3 1 2 3 1 2 3 i 2 3 1 2 3 ! 2 3 1 2 3 1 2 3 1 2 3 1 2 3 7 3 7 2 0 ' 3 7 0 12 14 10 6 8 2 0 14 14 10 11 11 . 6 6 0 4 5 11 . 5 6 9 1 0 10 11 14 11 9 5 . 11 0 . 4 0 3 0 0 I 4 11 0* . Live Implantations 3 6 9 8 13 13 13 8 - ' - 0 0 0 0 10 12 7 15 - - 9 3 12 6 12 8 MALES 5 5 5 10 9 • Partially sterile (questionable) Partially sterile Nornia 1 Normal Rebred Partially sterile Normal Partially sterile 7 1 Partially sterile 1 11 0 Normal - Rebred 10 12 7 h Normal 15 3 3 4 "U" indicates a plus was obsorv-cd for a female th.it was not pregnant. "-" indicates a plug «as no', detected and the female w;is not pregnant. 223 Partially sterile 5 -ViV '. ?«• ililrd breeding for final classification. Final Classification Normal Partially sterile �Table 14 (Continued) TRANSLOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number Second breeding (concl.) Female Number 389 390 391 396 397 399 400 Third breeding 216 220 241 256 268 270 273 Total Implantations 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 TEH Croup Dead Live Implantations Implantations .* * 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 MALES — L 14 12 12 11 7 9 2 5 3 • 0 0 0 9 5 0 5 8 1 • 11 0* 0 14 7 4 9 13 3 9 - 11 0 0 5 2 4 4 5 2 4 . . . . . . . . . 0 0 9 . 0 . 0 9 . ., - Partially sterile Rebred b K Rebred Normal Partially sterile (questionable) Partially sterile (questionable) Partially sterile (questionable) Norbreeder . . - - Nonbreeder Presumptive sterile Presumptive sterile Normal . not pregnant . "0" indicates a i "-" indicates a p l u g was not detected and the female was not pregnant. See third breeding Rebred . 5 . Final Classification . for final classification. 224 Presumptive sterile �table 14 (Concluded) TRANSLOCAT10N STUDY OF CAPT \N IMPLANTATION SUMMARY OF PRESUMPTIVE F. HALES F. Male Number Third breeding (concl.) Fema le Number 286 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 327 334 369 382 389 391 396 Total Implantations TEM Croup Dead Live Implantations Iroplantat Ions 1 - 11 _** 11 10 1 () 9 12 12 ! • 10 7 () 9t . 14 2 0 n _ . 11 . -• 11 10 . . • - 10 0 0 - - - - ' - - _ Partially sterile (questionable) Normal Presumptive sterile Normal Presumptive sterile . . . Normal 0 0 0* Final Classification - - - - . . . - . Nonbreeder . Nonbreeder - "0" indicates a plug was observed for a female that was not pregnent. *'-" indicates a plug was not detected and the female was not pregnant. " ) indicates all implants were In early stages of development, and thus difficult to (" determine if they were live or dead upon gross observation. 225 �Table 15 TRANS LOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES F. Male Number Flr«t breeding 430 432 436 449 I 450 i 451 452 455 461 469 472 474 479 4BO 484 Female Number 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Implantations 2500 ppm Croup Dead Live Implantations Implantations Initial Classification .* * 4 4 4 f (7t 1) 9 14 7 11 . (17) 9 5 2 3 . . - • 0* 11 0 1 2 3 . . . . 0 4 0 4 0 10 12 13 . . 9 9 5 8 . 5 14 9 10 10 Partially sterile (questionable) Partially sterile Nonbreeder Normal Normal Normal . Nonbreeder . . . . Nonbreeder . . 1 1 3 . . . 8 10 _ 9 . . - . . 9 11 12 . . . . (3) 0 11 16 11 0 I 14 7 0 Nonbreeder . . () 3 0 7 0 7. 2 0 Nonbreeder Normal Nonbreeder Nonbreeder Partially sterile . 4 14 11 Normal "0" indicates a plug was observed (or a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine if they were Live or dead upon gross observation. 226 �Table 15 (Continued) TRANS J'lCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES F. Male Number First breeding (connl.) 489 496 518 523 526 528 546 561 Female Total Number : Implantations 1 2 3 1 2 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 568 1 2 3 569 1 2 3 582 583 585 588 590 14 13 B -** _ _ Initial Classification Normal Nonbreeder m . . . . - 7 4 ()+ 1 0 1 ? 7 3 () 1 0* 0 - . Nonbreeoer Nonbreeder 0 . . . 12 () 9 (13) . 1 2 3 - 1 2 3 - . . 4 ? 1 . . 1 2 3 10 11 15 . . (12) 1 2 3 11 0 9 1 2 3 2500 ppm Croup Dead Live Implantations Implantations \ 11 3 1 12 6 2 . . 1 1 3 . . * 3 0 0 . 8 () 9 (13) . Partially sterile Presumptive sterile Nonbreeder Nonbreeder Partially sterile (questionable) Nonbreeder . 9 10 U . (12) 8 0 9 Nonbreeder Nonbreeder Normal Partially sterile (questionable) Normal "0" Indicates a plug was observed for a female that was not pregnant. "-" indicates a plug uas not detected and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine if they were live or dead upon gross observation. 227 �Table IS (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F HALES F. Hale Number Second breeding Female Total Nur.be r Implantation 430 432 436 449 450 451 452 455 461 469 472 474 479 480 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Dead Implantations Live Implantations 0 0 2 1 1 1 . 1 0 0 „ . 12 8 12 11 10 12 11 12 13 . . 12 8 14 12 11 13 12 12 13 .* * . 11 13 14 • 0* 0 11 14 11 13 10 12 12 13 11 11 „ . 12 0 12 11 0 . 1 0 I 0 0 1 1 0 0 0 0 1 0 0 2 . . 1 0 1 1 10 13 13 0 0 10 13 11 13 10 12 11 13 11 9 . 11 0 11 10 0 - 0 - - - - (13) + - 7 8 0 (13) 8 - Normal Normal Normal | j Rebred Normal Normal Normal Normal Normal Normal Normal - • - • Final Classification Rebred Rebred Rebrea t, h t} * 0 Indicates a plug was observed for a female that was not pregnant. "" "-" indicate.; a oluc was not detected " ) Inulcates all Implants were in early stages of development, and thus difficult to (" determine if they were live or dead upon gross observation. See third breeding e - final classification. 228 �Table IS (Continued) TRANSLOCATION STUDY OF CAPTAK IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES 2500 ppra Croup F. Male Number Total Implantations Dead Im.-lantat ions live Implantations 484 1 2 3 11 0* 14 0 0 2 11 0 12 489 1 2 3 1 9 13 14 .* * . - 0 2 0 _ 49fc Second breeding (cent.) Female Number . 9 11 14 .. . 0 0 0 . 0 11 13 . 2 3 518 / 523 526 528 546 561 568 569 582 583 585 588 1 2 3 1 2 3 • 0 11 13 . . •-• I 2 3 1 2 3 1 2 3 0 5 0 1 15 . - ' -- 0 0 0 0 5 0 1 2 3 - - - - . Nor.tal Normal Rebred Normal K Rebred 14 - . . Final Classification . . . V. Rebred Normal h Rebred 5 Rebred - - - 1 2 3 11 11 8 2 0 0 9 11 8 Ncrmal 1 2 3 11 . 10 0 . 0 11 . 10 Normal 1 2 3 7 15 10 0 2 2 . - 7 13 8 Normal - Rcbred 0 0 Normal 0 12 13 13 9 0 9 * - ~ 1 2 3 1 2 3 1 2 3 . 12 13 13 * "0" Indicates a p l u g was observed for a female t h a t was not p r e g n a n t . f.& "-" indicates a plug was not detected and the female was not pregnant. See third breeding for final classification. 229 • K Rebred �Table IS (Concluded) TRANS1OCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number Second breeding (concl.) 590 Third breeding 449 Female Number Total Implantations 1 0* 13 12 2 3 474 479 480 496 523 526 546 561 583 588 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 MALES 2500 ppm Croup Dead Live Implantations Implantations 0 2 0 0 11 12 .* * . . . . . • . . . . 8 2 . . . . . . . . Normal . • . . Final Classification 10 2 1 0 9 13 15 11 . 12 . ^ — Nonbreeder Presumptive sterile 0 0 0 0 0 0 0 - 2 I 0 9 13 15 11 1 11 - - . - "0" indicates a plug was observed for a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. 230 Nonbreeder Partially sterile (questionable) Nonbreeder Nonbreeder Partially slerl'.e Normal Normal Nonbreeder Normal �Table 16 TRANSLOCATION STUDY OF CAPTAN IMPUWTATION SUMMARY OF PRESUMPTIVE I1 MALES 5000 ppm Croup F. Male Number First breeding Fera-<-.Number Total Implantations lid 1 2 3 .* * (ll).t () S 1 2 3 7 11 604 620 622 626 I 2 3 1 2 3 1 8 . . () 9 . •> 634 635 638 646 653 657 - 1 2 3 9 11 8 1 2 3 1 2 3 ? ? (ID () 8 0 2 5 7 9 9 0 . - 8 . 1 - (v> - - . . - . - ' . . - 1 2 3 12 2 . 1 2 3 2 3 I 2 3 9 13 8 0 11 8 . ' 7 8 8 8 . ; 0 0 0 0 1 2 3 - 1 3 10 6 6 0* (13) . . I 2 3 660 668 Live Implantations _ 14 3 627 Dsad Implantations _ 0 7 - 7 8 7 0 1 0 . . 1 - Initial Classification Partially sterile ( ques t ionable) No'-mal Normal Partially sterile (questionable) Nonbreeder Normal Nonbreeder ' 10 6 Nunbreeder Normal 6 10 0 (13) 2 5 1 0 10 8 . . 6 Normal Part is' ly sterile (questionable) / Partially sterile Normal Partially sterile **0M indicates a plug was observed f o • a femaK that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. " ) indicates all implant' wt-rc in early stapos of development, and thus difficult to (" determine if th*?y were 1 ivt. or dead upon gross ohservat ion. 231 �Table 16 (Continued) TRANSIOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male remale Total Dead Number Number Implantation* Implantations First bred Ing (cont.) 671 679 3 1 732 3 1 734 736 743 700 761 767 769 6 0 8 . . Nonbreeder 0 4 . Pjrt tally sterile » . Nonbreeder . . . . . 0 (15) + 8 5 9 12 . 2 7b5 0 0 4 » . 4 . _ - 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 3 1 2 3 1 2 3 1 2 3 . . . 2 733 . . 6 0* 12 _ * / / Initial Classification ^ -. . . 3 1 2 3 1 722 Live Implantations .* * 1 2 672 MALES 12 11 12 . ' 8 (12) • . . . 0 • ? 1 0 i 3 . 3 1 2 . . . Nonbreeder Nonbreeder . . . . 0 (13) 7 5 8 9 . 9 10 8 10 6 ? (12) Partially sterile Nonbreeuer . NonbrFeder Partially sterile (questionable) Partially sterile Normal Normal Normal • Partially sterile (question&ble) • "0" indicates a plu^ v&s obs<rved tor a female that was not pregnant. "-" indicates a plug was not detected and the fjaale was not p.egnant. " ) ind icaf es all implants ueiv in early stages of development, and thus d i f f i c u l t to (" determine if they w e i * l i v e or deud upon gross observation. 232 �Table 16 (Continued) TRANSLOCATION STUfW OF CAPTAS IMPLANTATION SUMMARY OF PRESUMPTIVE F 5000 ppm Croup Dead Live Implar tat ions Implantations F. Male Nuraber FirJt breeding (concl.) Female Number Total Implantations 779 1 2 2 0* 1 0 4 1 5 0 0 0 780 3 I 2 3 MALES 2 0 1 y 0 4 1 Initial Classification Partially sterile Partially sterile 5 Final Classification Second breed Ins 601 604 620 622 626 *27 634 635 638 64$ 6^3 1 2 3 1 2 3 9 10 15 1 2 3 10 1 2 3 1 12 0 .* * 0 12 11 0 - Normal 0 0 1 9 10 14 Normal 1 9 0 - .. -< 0 12 0 0 - . Normal Normal - 1 2 3 (12)t ? (12) 12 - 0 12 1 2 3 1 2 3 13 6 1 3 0 Normal 10 6 0 . 1 .• Normal K 1 2 3 1 - • - 1 2 3 10 1? 11 0 0 0 1 2 j 11 13 10 0 0 Normal 1 11 13 9 1 2 3 14 13 10 0 0 0 14 13 10 Normal - 1 - • Rebred • 0 10 12 11 b Rebred Normal " " indicates a plug was observed for a female that was not pregnant.. 0 » "-" indicates a plug was not detected .ind the fem.ile was not pregnant. • " ) indicates a l l implants were in early st,ie,es of development, and thus difficult to (" determine if they.were live.or dead upon Rross observat ioii. See third breeding for final classification. 233 �Table 16 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number Second breeding leant.) Female Number 657 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 660 668 671 672 679 722 732 733 734 736 743 760 761 765 Total Implantations MALES 500O ppro Croup Dead • Live Implantat ions Implantations 7 9 5 14 10 12 9 13 13 14 11 .* _* ?. I 4 14 9 12 9 12 12 5 8 1 0 1 0 0 1 1 U 11 . 0 0 . . 0 0 0 . 14 7 12 4 . 14 7 12 3 1 - 12 - 12 0 . . 11 12 . . .1 12 0 0 . 0 0 14 10 10 1 0 0 5 13 11 3 0 0 12 11 9 1 0 0 0 0 1 15 11 9 "0" indicates a pine was observed for a female that was not pregnant. % "-" indicates a plug was not d e t e c t e d and the female was not pregnant. See third brce-Hng for f i n a l classification. 234 Pdrtially sterile Normal Normal Normal Normal Normal K Rebred Normal h Rebred Normal ' 1 ... 14 10 11 1 0* 0 5 14 12 Final Classification b Reared Normal Partially sterile Normal Normal �Table 16 (Concluded) TRANSLOCATION STUDY OF CAPTAN _ IMPLANTATION SUMftAKY OF PRESUMPTIVE Fj MALES 5000 ppm Croup Dead Live Implantations Implantations F, Male Number Second breeding (concl . ) Female Numbe*" Total Implantations 767 1 2 3 14 13 12 2 0 0 1 2 3 12 13 10 0 0 0 769 779 780 Third breeding 634 635 722 733 736 1 2 3 0* A .* * 1 2 3 0 2 5 1 2 3 _ 1 2 3 - 1 2 3 1 2 3 1 2 3 0 0 0 0 1 12 13 12 12 13 10 Final Classification Normal Normal 0 4 - Partially sterile 0 2 4 Partially sterile . - - - 1 Nonbreedcr 12 _ . Partially sterile 13 . . - - - - / 0 0 - - 0 - Presumptive sterile "0" indicates a plug was observed . . - - - Cor a female that was not pregnant. * "-" indicates a plug was not detected and the female was not pregnant. 235 Normal Nonbreeder �Table 17 TRASSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F. Male Number First breeding S05 806 815 816 817 818 822 823 837 841 84b Female Number . _ 6 9 1 1 2 3 ' (10)t (11) 1 2 3 . . 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 | 1 2 3 10 6 . . ' 0 0 0 ? 7 . 0 0 - ' - ' . . 805 1 2 3 Nonbrceder Partially sterile - Partially sterile (questionable) (10) (11) . 10 6 _ Nonbreeder Nomia 1 ; . f . . _ . .. . . . . . 5 , . . 0 5 - - - - - 7 (14) ~ . Nonbreeder 6 9 7 ~ (14) - _ Second breeding Initial Classification ** 1 2 3 1 2 3 2 3 847 50 0 0 p?m Croup Dead Live Implantations Implantations Total Implantations 1 2 3 MALES . 2 Nonbreeder Nonb>-eeder Nonbreeder Nonbreeder Partially sterile Partially sterile (questionable) Final Classification h 0 - • Rebred 2 Indicates traumatized F« females. u & "0** indicates a plug was observed for a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. + ft "() indicates all implants were in early stages of development, and thus difficult to determine if they were live or dead upon gross observation. See third breeding for final classification, 236 �T a b l e 17 ( C o n t i n u e d ) TRANSlrtCATlON STUDY OF CAPTAN IMPLANTATION SIWIARY OF PRESUMPTIVE F E M A L E S F. Male Numbe r Second breeding (concl.) Female Nurtber 5000' ppni Croup Total IVad Live I f - ! an t a t ions Imp hint at ions I m p l a n t at ions 806 , , 2 3 1 2 3 822 823 837 Normal Normal 3 13 11 10 2 - 9 - 11 0* - 4 0 - 7 0 - Rebred 11 8 13 0 0 11 8 12 Normal 6 - 0 - 6 - Rebred 1 2 3 1 2 3 818 12 14 10 1 2 3 5 2 3 817 11 1 2 3 816 14 11 13 1 2 815 13 14 10 Final Classification 13 0 - 13 - Normal Rebred - 7 - 0 0 1 0 ** 1 - - - 8 - " 1 - Normal . 1 2 15 10 2 0 13 10 , 2 3 10 12 12 0 0 0 10 12 12 Normal 1 2 3 11 10 14 1 0 0 10 10 14 No'rma 1 „ - _ _ 1 2 3 - - - 1 2 2 7 0 11 2 13 Normal 3 13 9 13 . .-1 2 3 10 12 12 0 0 0 10 12 12 Normal 841 846 847 Third brerdl-n 805 817 822 I, • I n d i c a t e s traim.it i/.t-d F Normal Partially sterile • f cni.i ies . "0" i n d i c a t e : ; a p i n t - vas o b - t - r v e d for .1 fcTimle t h a t was nol p r e p n n n t . "-" i n d i c a t e s a p l u g W.TS not d e t e c t e d and the f e m a l o was not p r e g n a n t . See t h i r d brcoJ ing for f ino 1 c lass i f ic.it i o n . '237 . �\ Tahle 17 (Contludod) » - ' TRANSLOCATION ST'JDY OF CAPTAN IMPLANTATION SltMMARY OF PRESUMPTIVE F HALES SOflO F Male Number Third breeding (concl.) 837 Female Number 1 2 3 Total Implantations _** • ppm Croup Dead Live Implantations Implantations _ _ ~ . Indicates rraumatized F_ f - n a l e s . "-" indicates a plug was not detected and the female was not pregnant. 238 Final Clas.sit icacion Normal �� 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. 111 Folder The folder containing the original item. 3063 Series The series number of the original item. Series V 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 Simmon, Vincent F. Ann D. Mitchell Ted A. Jorgenson Description An account of the resource <strong>Corporate Author: </strong>Stanford Research Institute Date A point or period of time associated with an event in the lifecycle of the resource 1977-05-01 Title A name given to the resource Evaluation of Selected Pesticides as Chemical Mutagens: In Vitro and In Vivo Studies https://www.nal.usda.gov/exhibits/speccoll/files/original/355890c6af5da1f145114408fa767656.pdf b1c89d4bddbc724805f64a142582eca8 PDF Text Text Item ID Number: 00101 Author Corporate Author Simmon, Vincent F. Stanford Research Institute, Menlo Park, California Report/Article TltlB Evaluation of Seleclod Pesticides as Chemical Mutagens 'In Vitro' and 'In Vivo' Studies Journal/Book Title Year ™n Month/Day Ma v Color Number of Images 252 DOSCriptOn NOtBS Conlracl No. 68-01 -2458; EPA-600/1 -77-028 Friday, Doccrnbcr 08, 2000 Pago 101 of 106 �S immon , V . F . , 1977 F.va-luation oC selected pesticides as chemical mutagcns 'In vito' and 'In vivo1 studies I Technical Information Service •- *- *"*"^it" "'^ "^^ ^* "Iff™1* ™1* PL268&4Z _ Evaluation of Selected Pesticides as Chemical AAutagens 'In vitro' and 'In vivo' Studies a .- ' . • _ - . . _ . . _ . 1. Stanford.Research Institute, Menlo Park, Calif • - - • - - - r. • - c z .: - ' I "-- - Prepared for Health Effects Research Lab, Research Triangle Park, N C May 77 AEROM EDICAL LIBRARY APR 21 1980 DOCUMENT'S �:(:C!(,MiCAL REPOR1 •. A 1 ,:-asc read Instructions on the reverse be}u.'f completing) i REPORT NO J:PA-600/1-77-028 4. T I T L E AND S U B T I T L E 5 REPORT DATE May 197/ EVALUATION OK SELECTED PESTICIDES AS CHEMICAL MUTAGENS In Vitro and In Vivo Studies 6. PERFORMING ORGANIZATION CODE B. PtHFORMING ORGANIZATION RfPOHT NO. 7 AUTMOR(S) Vincent F. Silicon, Ann D. Mitchell and Ted A. .Torgenson LSU-3493 iO-'pROGHAM ELEMENT NO". 9>EFirORMlNG ORGANIZATION NAME AND ADDRESS Stanford Research Institute Menlo Park, California 94025 1EA615 TTCONtH'AflT^eHANi^NO. * 68-01-2458 12. SPONSORING AUKNCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED HeaJth Effects Research Laboratory - RTF, NC Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, N.C, 27711 iSTs u P PL'EMFNTARYIVIOTES" "" [^"ABSTRACT " " " 14. SPONSORING AGENCY CODE 600/1.1 ~ Twenty pesticides being reviewed as part of the EPA Substitute Chemical Program were studied for mutagenic activity by several _Ln vitro and in vivo test procedures. The pesticides reviewed were: monocrotophos, bromacil,, ca^codyllc acicf, captan, chlorpyrifos, dinoseb, DSMA, fenthion, folpet, azinphos-methyl, malathion, methomyl, monuron^ MSMA, parathion, parathion-methyl, quintozene (PCNB), phorate, simazine, and trtfluralin. Ten of the twenty compounds were evaluated in_vivp by the mouse dominant lethal test. All twenty compounds were tested in vitro. None of the ten compounds tested in the mouse produced a dominant lethal response. Ten of the twenty compounds were mutagenic in one or more, in vitro assays. Two were mutagenic in all of the in vitro assays: captan and folpet. KEY WORDS AND DOCUMENT ANALYSIS 7. b.lDENTIFIERS/OPEN ENDED TERMS DESCRIPTORS pesticides in vivo analysis in vitro analysis Diutagtms COS AT I Held/Group 06 F 06 T 19. SECURITY CLASS (This Keportl 19. DISTRIBUTION S T A T E M E N T 21. NO. __UNCLAS^IFIED RELEASE TO PUBLIC 20. SECURITY CLASS (Thispage) REPRODUCED EV EPA Form 2220-1 (9-73) c. NATIONAL TECHNICAL INFORMATION SERVICE U. S. DEPARTMENT OF COMMERCE SPRINGFIELD, VA. 22161 22. P'RICE ��NOTICE THIS DOCUMENT HAS BEEN R E P R O D U C E D F R O M T H E BEST C O P Y F U R N I S H E D U S B Y THE SPONSORING AGENCY. ALTHOUGH IT IS R E C O G N I Z E D THAT CERTAIN PORTIONS ARE I L L E G I B L E , IT IS BEING R E L E A S E D IN THE INTEREST OF MAKING A V A I L A B L E AS MUCH I N F O R M A T I O N AS POSSIBLE. �RESEARCH REPORTING SERIES Research reports of the Office of Research and Development, U.S. Environmental Protection Agency, have been grouped intc nine series. These nine broad categories were established to facilitate further development and application of environmental technology Elimination o< traditional grouping was consciously planned to foster technology transfer and a maximum interface in related fields. The nine series are: 1. 2. 3. 4. 5. 6. 7 8. 9. Enviroimenta! Health Effects Research Environmental Protection Technology Ecological Research Environmental Monitoring Socioeconomic Environmental Studies Scientific and Technical Assessment Reports (STAR) Interagency Energy-Environment Research and Development "Special" Reports Miscellaneous Reports This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RESEARCH series. This series describes projects and studies relating to the tolerances of man for unneaithful substances or conditions This work is generally assessed from a medical viewpoint, including physiological or osychological studies. In addition to toxicology and othe- medical specialities, study areas include biomedical instrumentation and health research techniques utilizing animals — but always with intended application to human health measures. This document is available to the public through the National Technical information Service, Springfield, Virginia 22161. �EPA-600/1-77-028 May 1977 EVALUATION OF SELECTED PESTICIDES AS CHEMICAL MUTAGENS in Vitro and In Vivo Studies By Vincent F. Simmon, Ann D. Mitchell, and. Ted. A. Jorgenson Stanford Research Institute Menlo Park, California 94025 Contract No. 68-01-2458 Project Officer Michael D. Waters Environmental Toxicology Division Health Effects Research Laboratory Research Triangle Park, N.C. 27711 U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF RESEARCH AND DEVELOPMENT HEALTH EFFECTS RESEARCH LABORATORY RESEARCH TRIANGLE PARK, N.C. 27711 10. �DISCLAIMER This report has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that tha contents necessarily reflect the views and policies of the U.S. Environmental Protection Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. it �FOREWORD The. many benefits of our modern, developing, Industrial society are accompanied by certain hazards. Careful assessment of the relative risk of pxisting and new man-made environmental hazards is necessary for the establishment of sound regulatory policy. These regulations serve to enhance the quality of our environment In order to promote the public health and welfare and the productive capacity of our Nation's population. The Health Effects Research Laboratory, Research Triangle Park, conducts a coordinated environmental health research proBr#:n in toxicology, epidemiology, and clinical studies using human volunteer Kiibjects. These studies address problems in air pollution, non-ionizing radiation, environmental, carcinogenesis and the toxicology of pesticides as well as other chemical pollutants. The Laboratory develops and revises air quality criteria documents on pollutants for which national ambient; air quality standards exist or are proposed, provides the data for registration of new pesticides or proposed suspension of those already in use, conducts research on hazardous and toxic materials, and Is preparing the health basis for non-ionizing radiation standards. Direct support to the regulatory function of the Agency is provided in the form of expert testimony and preparation of affidavits as well as expert advice to the Administrator to assure the adequacy of health care and surveillance of persons having suffered imminent and substantial endange.rment of their health. This report describes the testing of a series of twenty technical grade pesticide chemicals for genotoxic properties by use of a battery of JLn yjltro and .In vivo methods. The battery includes tests for gene and chromosomal mutations and primary damage to DNA as measured by effects on DNA repair recombination. Since DNA is chemically similar in all species, test results from a variety of cells and organisms are relevant in assessing the potential genetic hazard of pesticide chemicals in humans. John H. Knelson, M.D. Director, Health Effects Research Laboratory \ �ABSTRACT Twenty pesticides being reviewed as a part of the EPA Substitute Chemical Program were studied for mutagenic activity by several in vivo and lii vlcro test procedures. Ten of Che twenty compounds were evaluated in vivo by the mouse dominant lethal test. All twenty compounds were tested by the following _in_ vitro procedures: Unscheduled DNA synthesis (UDS) In human fibroblasts (WI-38 cells); reverse mutation in Salmonella typhlmur:Lum strains TA1535, TA1537, TA1538, and TA100 and in Escherlchla.coll. WP2; mitotlc recombination in the yeast Saccharomyces cereylslae D3; and preferential toxlclty assays In DNA repairproficient and -deficient strains of ]J. coH (strains VO110 and p3478, respectively) and Bacillus subtilis (strains H17 and MAS, respectively). None of the ten compounds tested in the mouse produced a dominant lethal response. Ten of the twenty compounds were mutagenic in one or more ^n vitro assays. Two were mutagenic in all of the in vitro assays: captan and tolpet. In a heritable translocate on study in mice, under the experimental procedures employed, captan at 5000 ppm in the diet of male mice for 8 consecutive weeks produced a heritable mutagenic event in F^ generation male mice. iv �CONTENTS LIST OF TABLES Hi SUMMARY 1 TWENTY PESTICIDES EVALUATED BY SRI FOR MUTAGENIC ACTIVITY 3 INTRODUCTION 4 DOMINANT LETHAL TEST IN THE MOUSE 6 General Experimental , , Animals anil Chemicals Determination of Acute Toxlclty Maximum Tolerated Dose Study Treatment Levels Administration of the Compounds Test Groups Necropsy and Evaluation Results and Discussion MAMMALIAN JEN VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS General Experimental Cell Culture Dilution of Compounds Controls UDS Assays Interpretation of Results MICROBIOLOGICAL ASSAYS 6 6 6 7 7 7 8 8 9 9 12 12 13 13 13 13 14 15 17 General 17 Experimental 18 Salmonella typhimurlinn Strains TA1535, ~TAi537," TA1538,"and TA100 18 JEscherlchia coll WP2 19 Escherlchla coU W3100/p3478 and Bacillus 'subtllls H17/M45 20 jjaccharomjrces cerevlslae D3 21 Aroclor 1254-Stlmulated"~MetaboHc Activation System . . . 22 Results and Discussion 22 DISCUSSION 25 REFERENCES 26 APPENDIX A: Mutageneals Studies of Pesticide Compounds - Mouse Heritable Translocation Test - Captan 17.1 �TABLES DOMINANT LETHAL TEST 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Chi-Square Test of the Fertility Index - Monocrotophos . . . . 28 Average Implants per Pregnant Female - Monocrotophos 29 Average Dead Implants per Pregnant Female - Monocrotophos. . . 30 Chi-Square Test of the Death Index - Monocrotophos 31 Number of Dead Implants per Total Implants - Monocrotophos . . 32 Chi-Square Test of the Fertility Index - Bromacil 33 Average Implants per Pregnant Female - Bromacil 34 Average Dead Implants per Pregnant Female - Bromacil 35 Chi-Square Test of the Death Index - Bromacil 36 Number of Dead Implants per Total Implants - Bromacil 37 Chi-Square Test of the. Fertility Index - Captan 38 Average Implants per Pregnant Female - Captan 39 Average Dead Implants per Pregnant Female - Captan 40 Chi-Square Test of the Death Index - Captan 4i. Number of Dead Implants per Total Implants - Captan 42 Chi-Square Test of the Fertility Index - Folpet. . . 43 Average Implants per Pregnant Female - Folpet 44 Average Dead Implants per Pregr.ant Female - Folpet 45 Chi-Square Test of the Death Index - Folpet 46 Number of Dead Implants per Total Implants - Folpet 47 Chi-Square Test of the Fertility Index - Azinphos-Methyl . . . 48 Average Implants per Pregnant Female - Azlnphos-Methyl . . . . 49 Average Dead Implants per Pregnant Female - Azinphos-Methyl. . 50 Chi-Square Test of the Death Index - Azinphos-Methyl 51 Number of Dead Implants per Total Implants - Azinphos-Methyl . 52 Chi-Square Test of the Fertility Index - Malathion T.3 Average Implants per Pregnant Female - Malathion 54 Average Dead Implants per Pregnant Female - Malathion 55 Chl--Square Test of the Death Index - Malathion 56 Number of Dead Implants per Total Implants - Malathion . . . . 57 Chi-Square Teat of the Fertility Index - Parathion 58 Average Implants per Pregnant Female - Parathion 59 Average Dead Implants per Pregnant Female 60 Chi-Square Test of the Death Index - Parathion 61 Number of Dead Implants per Total Implants - Parathion . . . . 62 Chi-Square Tast of the Fertility Index - Parathion-Methyl. . . 63 Average: Implants per Pregnant Female - Parathion-Methyl. . . . 64 Average Dead Implants per Pregnant Female - Parathion-Methyl . 65 Chi-Square Test of the. Death Index - Pp.rathion-Methyl 66 Number of Dead Implants per Total Implants - Parathion-Methyl. 67 Chi-Square Test of the Fertility Index - Quiritozene (PCNB) . . 68 Average Implants per Pregnant Female - Quintozene (PCNB) . . . 69 vi �43 44 45 46 47 48 49 50 Average Dead Implants per Pregnant: Female Qulntozene (FCWB) .... Chi™3quare Test, of the Death Index - Quintozene (PCNB). . . . Number of Dead Implants per Total fmplants Qulntosene (PCND) Chi-Square Test of the Fertility Index - Phorate Average Implants per Pregnant E'.--,wile - Phorate Average Dead Implants per Pregnant Female - Phorate Chi-Sqisare Test of the Death Index -• Phorate Number of Dead Implants per Total Implants - Phorate 70 71 72 73 74 75 76 77 UDS ASSAYS 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation. DNA Repair Synthesis Assay DNA Repair Synthesis Assay Metabolic Activation DNA Repair Synthesis Assay of Monocrotophos of Monocrotophos with of Bromacil of Bromacil with of Cacodyllc Acid of Cacodyllc Acid with of Captan of Captan with of Chloropyrifos of Chloropyrifos with of Dlnoseb of Dinoseb with of DSMA of DSMA with of Fenthlon of Fenthiori with of Folpet of Folpet with of Azinphos-Methyl of Azinphos-Methyl with of Malathion of Malathion with of Methomyl of Methomyl with , of Monuron vii 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 �76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation. ... DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay .of with Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of with Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation DNA Repair Synthesis Assay of DNA Repair Synthesis Assay of Metabolic Activation Monuron with MSMA MSMA with 1 Parathion Parathion with Parathion-Methy 1 Parathion-Methyl Qulntozene (PCNB) Qulntozene (PCNB) Fhorate Phorate with Simazine Simazine with Trifluralin Trifluralin with 103 104 105 106 107 108 109 110 Ill 112 113 114 115 116 117 MICROBIOLOGICAL ASSAYS 91 92 93 94 95 96 9? 98 99 .100 101 102 103 In Vitro Assays with Salmonella ^phjLmurium 118 Results" of Assays with Escerichia coli ~WP2 138 Mlcrobial Inhibition in~Egcerichia coll and Bacillus subtilis . ... 7~"."".. 148 .In Vitro_ Assays with Saccharomyces cerevisiae D3 Monocrotophos 150 In Vitro Assays with Saccharomyces Cjereyisiae D3 "Bromacil T .." . Y W "". " ' V 151 In Vitro Assays with .Saccharomyces cerevisiae D3 Cacodylic Acid. . . . . V ."."". ~. ."" T 152 1$. XiS,r,°- Assays with Saccharomyc.es .cereyj.si.ae D3 Captan 153 In Vitro Assays with .Saccharomyces cerevislAe D3 Chloropyrifos 154 _In Vitro Assays with Saiccharoiir/ces £ere\rlsiae D3 Dlnoseb 155 In Vitro Assays with Saccharoniy_ces. cejrwisiae D3 - DMSA . . . 156 In y_it:r<3 Assays with Saccjaromyces cerevisiae D3 Fenthlon ' 157 Iii Vitro Assays with jJaccharoiuyces cereyisiae D3 Folpet "."." . . " " . " . . .". . 7 158 In Vitro Assays with Saccharomyces. c.ej.evlsi3.?. D^ "Azinphios-Methyl . ."". .* - ". .""".".". ~~~~'~~ 159 In y_ltro_ Assays with Sa^cjtonniijrces cerevlslae^ D3 Malathion ".""".""."'. "7"."". ."".' ."".' 160 viii i �105 106 ^°7 108 109 110 111 112 113 114 Tn Vitrp_ Assays with ^ccharpmyces cer_eyiaiae D3 Methomyl ~. , In Vitro Assays with S&ircharqmyces £ereyisiae D3 Moiuiron , Jn Vitro Assays- with Sat^charqmjrces cerevlsiae D3 - MSMA . . . In Vitro Assays with Saccharom^cea cerevlBlae D3 Farathlon ~."".".. . . ." .7 In Vitro Assays with jtaccharomyces cerevlsiae D3 "" Parathlon-Methyl. . .".'. ~.~ .""."'.""." "i ~." "'. ."" In V_it.ro Assays with jJaccharomyces cerevlgiae D3 Quintozene (PCNB) . . . 7 . . ~ In Vitro Assays with 5a.ccl»arpmyces_ c_ereyl3iae_ D3 " Phorate T . . . . '." . ".". " In Vitro Assays with Saccharomyces cerevisiae_ D3 Simazine In X*-tr9 Assays with Saccharqmyces cerevirsiae D3 Trifluralin . , , . . " " . . . . " . T " " ' . " . " " " " " . ' ..' In Vitro Mutagenf.ais with .Salmouella tyjphlinur_ium. . . . . . . ix 161 162 163 164 165 166 167 168 169 170 �ACKNOWLEDGMENTS Stanford Research Institute wishes to thank the EPA project officers for their guidance and assistance during the course of this project. Dr. Robert E. McGaughy, Office of Research and Development, Washington, D.C., was Project Officer from the beginning of the project in June 1974 until July 1975. Project responsibility then was transferred to Dr. Ronald L. Baron, Health Effects Research Laboratory, Research Triangle Park, North Carolina, until January 1976. At that time, Dr. Michael D. Waters, now Chief of the Biochemistry Branch in the Environmental Toxicology Dlvlsi.cn, Health Effects Research Laboratory, Research Triangle Park, North Carolina, became Project Officer. Dr. Waters has continued in this project responsibility to the present. �INTRODUCTION The Federal Insecticide.. Fungicide, and RodenticJ.de Act designates Lhe Environmental Protection Agency as the governmental body responsible for the safety of all pesticides used in the United States. More recently, the Federal Environmental Pesticide Control Act (PL 92-516) strengthened EPA's regulatory responRihi.llt.les in the area of pesticides to include, ttitra- as well as Inter-state commerce. To be federally registered, a pesticide must h*ve beoii determined not to be hazardous to health or to the environment when used according to its labeling restrictions. Thus, relative to new law as well as to specific directives included in Public Law 9.1-135, 1973, EPA now is conducting a thorough review of rhe implications of using alternate chemicals, including older registered pesticides, for pest control. Ln the. pesticide review process, EPA emphasizes development of scientific criteria for evaluating the safety of compounds substituted for those penticides found to be hazardous. In addition to reviewing and evaluating the literature on pesticides and maintaining liaison with Industry and acadcmia, Che strategy program Includes laboratory studies to obtain additional data. One of these laboratory programs is directed toward gathering mutagenesis data on a selected -lumber of compounds. EPA's program is timely and responsive to one of the recommendations included in the President's Scientific Advisory Committee Report of September 1973, Chemlc a_ls_ and Health. In that document, the Committee recommended that "Regulatory agencies should take steps to insure that new scientific data raising the possibility of new or extended hazards from chemicals in use are subject to careful process of scientific, review for me.rir. interpretation." Development of methods for evaluating the rautagenic hazard of chemical compounds has advanced markedly in the last few years. In contrast to the undefined empirical tests used a short time ago, procedures now �available can detect chromosome breaks and other genetic changes caused by chemical stress. Mutant strains of microorganisms In cell culture and mammalian flbroblast cells In tissue culture are effective In vitro systems for .reliable detection of presumptive gene mutations, whereas the mammalian dominant lethal test is a recognized test for the assessment of chromosome damage to germinal cells.' Today many pesticide chemicals in commercial use have not been investigated adequately for their mutagenlc has:ard. With the public's increasing concern about possible pollution of our environment by chemicals, the widely used pesticides must be evaluated. In this project, SRI used test methods that are appropriate for these evaluations and that are in use by the scientific community. Under contract to EPA, SRI examined 20 pesticides for mutagenic activity using a combIntation of in vivo and in vitro mutagenicity assay systems. The 20 pesticides tested and their sources are listed in the ' following two tables. The assays used were the dominant lethal test in mice (only ten compounds); unscheduled DNA synthesis (UDS) in human fibroblasts (WI-38 cells); reverse mutation in SaJLmojiellji tvj^^miirium strains TA1535, TA1537, TA1538, and TA100 and in Esch^erlchia coll VF2; mitotic recombination in the yeast ^Sccharomyjce^ cerevisiae D3; and preferential toxicity assays in DNA repair-proficient and -deficient strains of IS. coll (strains W3110 and p3478, respectively) and Bacillus aubtjlls (strains H17 and M45, respectively. Based on positive responses in both Tier I (in vitro test) and Tier II (Droaophila) mutagenic studies, It was recommended that a heritable trenslocation test (Tier III) la the mouse be conducted �to further assess the matagenic potential of Captan. The results of these further studies are reported as Appendix A. The experimental procedures and results for the mammalian dominant lethal test, the UDS assay, and the microbiological assays are. described in the separate sections that follow. �IS VIVO MC IN VITRO MUTAGENES1S: SUMMARY DATA FOR E?ft PESTICIDES Positive Response, +; Negative Response, - Pesticide Mouse Dotiisanc Lethal* Salconella typhJJiuriiaat (His"1" Reversion) -HA +MA EacSerichia egli WF2 ( r '' Reversion) Ty1 -MA +MA Mocccrotophos bromacil Cacodylic. Acid Captan Chlor?yrlfos Dnos^S DbHA f<jnt!-.ion foipst Azlnphos-sechyl Halathion Konuron MSHA ?arathioE Parachlon-uechyl Qulntozene (FCNB) Phorate Sinazine Trifluralia • Only ten pesticides were tested by the dominant lethal procedure. t See page 170. T Marginally positive. Saecharomyces cerevlsiae (Mitotlc Recombination) -HA +MA Escheriehia coll (Selative Toxicicy) Bacillus surtills (Relative Toxicity) UDS (DMA Repair) -MA +MA �TWENTY PESTICIDES EVALUATED 3Y SRI FOR MUTAGENIC ACTIVITY Common Name* Monocrouophos Bromacil Cacodylic Acid Cap tar. Chiorpyrifos Dinoseb DSMA Fenthion Folpet Azinphos-methyl Malathion Me thorny 1 Monuron MSMA Parathion Par a t h ion-iae t hy 1 Quintozene (PCNB) Phorate Simazine Trifluralin Tradt Name of Compound Tested Manufacturer Batch or Lot Number Batch H, 9-SCL-77 E.I. DuPont de Nemours T80619/40 Hyvar Phyton 138 Phytar Ansul Chemical Company 5X640 Chevron Chemical Conpany Orthoslde 406 Dow Chemical Company MM-1 114-1 Dursban (603-D1) MM 200554 Dow Chemical Company PT emerge Ansul Chemical Company 8100 Ansar Chemogro 4-15-2026 Bay t ex Chevron Chemical Company SX579 Phaltan Chemogro 411-022S Guthlon American Cyanamid Company 40216006.300 Malathion 6602-82 E.I. DuPont de Nemours Latmate T-40817-20 E.I. DuPont de Nemours Telvar 170 H.C. Ansul Chemical Company Ansar Monsanto Chemical Company AD 1236 Niran Methyl Parathion Monsanto Chemical Company AD 0659 Technical Olin Mathieson Chemical Terrachlor Corporation American Cyanamid Company MC85 Thimet FL-740846 Primatol Ciba-Geigy Chemical Co. X-26290 Eli Lilly & Company Treflan Azodrin-5 Purity (%) Supplier 55.0 Manufacturer Shell Chemical Company 95.9 65.6 Technical 98.8 Battelle Baccelle Sattelle Battelle 97.7 Battelle Battelle Battelle Battelle Battelle Battelle 80.1 96.0 Technical Technical Technical 99.0 97.0 58.4 99.0 80.0 99.0 * Common name as approved by the International Organization for Standardization. 97.7 97.7 Battelle Battelle Battelle Battelle Battelle Battelle Battelle Battelle Battelle �DOMINANT LETHAL TEST IN THE MOUSE General In the dominant lethal test, the ten compounds under Investigation were fed in the diet to proven male breeder mice for 7 weeks. After this period, each male was mated with two adult virgin females for 7 days; these females were then replaced by two others for another breeding. The sequence was continued fof 8 weeks. This procedure emphasizes possible mutagenic effects on the male sperm, the normal female acting as a carrier to reveal in her offspring abnormalities that may have occurred in the male. We evaluated effects by examining the condition and state of fetal development during the middle to latter stages of gestation. Experimental Animals and Chemicals Adult ICR/SIM mice from a closed, random-bred colony were used for the acute toxiclty and maximum tolerated dose determinations as well as for the dominant lethal assay. These male and! female mice were supplied by Simonsen Laboratories, Gllroy, California. The males were 3- to 4-raonth-old proven breeders, and the females wore 10- to 12-week-oid virgin stock. At the direction of EPA, the Battelle Columbus Laboratories obtained the pesticides from the manufacturers and subsequently provided SRI with allquots for the studies reported here. Each pesticide was a "technical" grade product (or equivalent) and was provided in sufficient quantity for us to complete all aspects of the experimental program. Excess supplies were refrigerated or frozen, should they be needed fcr future reference. �Wo Investigated the solubility of each compound using water, propylene glycol, polyethylene glycol, corn oil, or carboxymeshylceLlulose to determine the. most appropriate vehicle for administration. Compounds were administered orally, by gavage for Che acute toxiclty (LD,-g) determinations, and via the diet Tor the maximum tolerated dose and dominant letha] studies. Determination of Acute Toxic ity_ Although acute toxiclty information on .some of the compounds was available in the literature, we conducted confirmatory tests on all to obtain an LD under our Laboratory conditions and for the 1CR/J1F.M strain of mouse. If no data were available, we conducted a preliminary range-finding test, followed by a determination of the oral Lft^Q. Maximum Tolerated Dose Study Based on the acute toxicity data and available information from the literature on dose levels known to cause adverse responses when administered in the diet, several dose, levels were selected and administered in the diet to adult male mice for 2 weeks. Treated males then were caged with two adult virgin females each for 7 days; these females were replaced by two others weekly for 2 weeks. The females were examined daily for the presence of vaginal (mating) plugs. At midterm of pregnancy, the females were sacrificed and examined for total implants, as well as for early and late fetal deaths. For this work, we defined a maximum tolerated dose as that dietary level which may produce up to a 20% weight loss, mild but transient clinical signs, no inhibition of breeding performance, and no mortality. Thus, these initial studies provided Information on changes in body weight, acceptability of the diet, clinical signs, mortality, and breeding performance. Treatment Levels For the dominant-lethal study, three dose levels were administered. The highest was the maximum tolerated dose or 5 g/kg (a maximum level �agreed on by EPA and SRI), whichever was lower. The intermediate and lower dosages were one-half and one-quarter of the highest dose, respectively. Administration of the Compounds Each pesticide was fed in the diet to adult male mice for 7 weeks. An appropriate amount of compound initially was dissolved or suspended In corn oil; then the compound-oil concentrate was added at a level of 3% to a finely ground commercial diet of known composition. The use of corn oil assured even distribution of the compound and prevented stratification of the tost material in an otherwise dry diet. Diets were pre- pared at 2-week Intervals and were refrlgeratwd at 4°C until fed to the animals. Fresh diet was placed in the. feed containers every other day to minimize the loss of compound through instability or volatility. Two reference control groups were included in this project. One was run at the beginning of each of the two dominant lethal series, five pesticides being run concurrently. In this manner, reference breeding and implant data were obtained at. two time periods, as was information on each shipment of research animals. Males In these groups were fed a finely ground commercial diet supplemented with corn oil at 3%. Control groups were treated in the same manner as the compound test groups. Two positive control groups were run concurrently wiLth each of the two series of five pesticide tests. For these groups, the known mutagen trlethylenemelamine (TEM) was administered as a single intraperitoneal inject. Ion of 0.2 mg/kg approximately 2 hours before the first mating. A commercial pelleted diet was available at all times. Each control and experimental test group contained 20 adult male mice. Af: the end of the 7 -week compound treatment period, each male was allowed to brer.d with Two virgin females over a period of 7 days. Femal.es wfico replaced weekly for 8 wpcks. 8 �Females wert sacrificed at midterm of pregnancy. A completu necropsy waa performed to determine If an Intercurrent infection was present; such a condition can induce preimplant&tion loss and early fetal deaths. At sacrifice, each female was scored for early fetal deaths, late fetal deaths, and living fetuses (all of which provide a LotaL implant score). The following parameters indicate effects in dominant lethal studies: Total implants (live Fetuses plus early and late fetal deaths), total <;.cad (early and late I'tif.al deaths), and dead implants per total Implants. Total imp]ants and dtad Implants were analyzed for significance by the t-test. Th« Jndex of dead implants per total implants was analyzed statistically by the t-test on arcsine- (or angular) transformed data, as described in Experimental Design (Theory_ and Apjgllcajclon) . index was computed for each female. Other parameters analyzed were the fertility and death indices. Rcsults and_ Discussion Single-dose oral acute toxicity data are as follows: Compound LD.50 Monocrotophos 17 mg/kg Bromacil 3.04 g/kg Captan > 15 g/kg Folpet > 10 g/kg Azinphos-methyl 15 mg/kg Malathion 1196 mg/kg Parathion This ' 17 mg/kg I'arathion-methyl 39 mg/'«.g Qulntozcne (FCNB) > 10 g/kg Phorate 6.59 mg/kg �After evaluating the acute toxicity data and those from subsequent maximum tolerated dose studies, we selected the following dosage levels for the dominant lethal studies: Compound Treatment Levels (E/kg_J: Dlet __ Monocrotophos Bromacil Captan Folpet Azinphos-methyl Malathion Parathion Parathion-methyl Quintozene (PCNB) Phorate 15, 30, 60 1250, 2500, 5000 1250, 2500, 5000 1250, 2500, 5000 20, 40, 80 1250, 2500, 5000 62.5, 125, 250 20, 40, 80 1250, 2500, 5000 5, 10, 20 Throughout the experiment, the biological criteria used to evaluate mutagenic effects in the mouse showed no consistent responses that could be attributed to treatment. Although we found occasional statistical differences between control and compound treated groups, they were random and did not suggest a time or dose-response effect. Summary data on the fertility index, implantations per pregnant female, dead implants per pregnant female, death index, and number of dead implants per total implants are presented by compound as follows: Tables 1 through 5, Monocrotophos; Tables & through 10, Bromacil; Tables 11 through 15, Captan; Tables 16 through 20, Folpet; Tables 21 through 25, Aalnphos-m«thyl; Tables 26 through 30, Malathion; Tables 31 through 35, Parathion; Tables 36 through 40, Parathion-Methyl; Tables 41 through 45, Quintozene (PCNB); and Tables 46 through 50, Phorate. Two copies of a description of the statistical analysis procedures used for dominant lethal tests -and computer printouts of the raw data and the statistical analyses are on file with the current Project Officer, Dr. Michael D. Waters, Environmental Toxicology Division, Health Effects Research Laboratory, EPA Environmental Research Center, Research Park, North Carolina 27711. 10 �The following statistical procedures were used: Chl-square cest of the fertility index; Armltage test Cor a linear trend in proportion for the fertility Index based on dose levels, based on .logarithms of the. dose Levels, and based on dose levels including the control group; t-test of the number of implantations in pregnant females; Regression fits of implantations on dose and log dose and with and without control group included; t-test of the (Freeman-Tukey transformed) preimplant.al.ion losses In pregnant females; t-test of the number of dead implants; Chl-square test of the death index; Arm.itage tost for a linear trend in proportion for the death index, based on dose levels with and without control group included and based on logarithms of Che dose levels; Probit analysis of the proportion of pregnant females with one or more dead implants; t-test of the (Freeman-Tukey transformed) number of dead implants (dead implants/total implants); Control group analyses of variances for number of pregnant females, number of implantations per pregnant female, preimplantation loss per pregnant female, number of dead implants per pregnant female, ratio of dead implants to total implants per pregnant ferwle; and t-test of the number of corpora lutea in pregnant females. Careful review and statistical evaluation of the data show that folpet, captan, parathion-methyl, parathion, phorate, malathion, bromacll, monocrotophos, quintozene (PCNB), and azinphos-methyl are not mutage.nLc in the. mouse by the dominant lethal test. �MAMMALIAN W VITRO UNSCHEDULED DNA SYNTHESIS ASSAYS General Many mutagenic and carcinogenic agents have been shown to induce unscheduled DNA synthesis (UDS) in an ill yitjro tissue culture system of mammalian coll:-;. UDS is a form of mammalian repair synthesis that involves at least two processes. The first is Interaction of the agent with DNA, resulting in damage of the DNA. The second, which follows, is incopor- atton oC micleotldes to repair the DNA. UDS may be considered a fal-cly universal system because it occurs In a wide variety oi' mammalian cell types and because it has been observed in all stages of the- cell cycle (Gg, GI, G?, and M) other than S_, the normal DNA synthetic phase.?l3 (UDS is not observed during S_-phase be- cause tin; high level of Incorporation of nucleotides during the scheduled DNA synthesis obscures the relatively Low level of incorporation of nucleotide.s during unscheduled DNA synthesis.) An additional feature of UDS is that It may detect a level of DNA damage higher than that revealed by examination of chromosomeal aberrations'4 because some DNA repair results in little or no detectable change in chromosome morphology. For each compound tested, an in vitro metabolic activation system should be incorporated for a parallel series of UDS assays since some compounds may be ineffective in producing DNA damage unlpss they arc first activated by a microsomal preparation from a mamiiiai.i.-'1 P liv-M homoftenatc. The- ['DS rystrm we have developed Is unique in that, at the end or fvich assay, ON.', in axtractt'.d from human diplold flbroblasts (WI-38 cells) so that, the extent of repair may be expressed per unit of DNA. We have found that this. UDS assay system affords sensitivity and precision without sacrificing efficiency or economy. Under separate contact, NCI approved our use and validation of this system for the prescreening of chemical ;arclnogpns. With the approval of the EPA project officer, we used this system for testing the* 20 substitute pesticides, with and wi thou l wo i :ibo 11 c. ac t i va t ion. �Experimental Coll Culture WT-38 cells grown In T-25 tissue culture flasks were used Cor the UUS assays. Kepllcdte cultures of chase cells were initiated in Eagle's Hrisal Medium (BME) containing 10% (v/v) fetal calt serum and aureomycin, an antibiotic, specific for PPLO*. For 1 to 2 weeks preceding rlie UDS assays, the cells were grown in medium containing 0,5% serum. This pro- duced contact-Inhibited cells in synchronous cultures in the 01 phase of the mitotic cycle. To reduce further the possibility of incorporation of -'H-TdR by an occasional S-phase cell that might escape the contactinhiblLJon synchrony and thus obscure measurements of UDS, the cultures 2 were prelncubated for 1 hour with 10" M hydroxyurea (HU) before each assay, and 10""' M HU was iidded during each subsequent step of the assays. Di 1 u t ion o f Cpmpou nds Chemicals to be tested were made up immediately before use and were dJLuted in appropriate solvents (water, echanol, or DMSO), the final concentration of solvent being one that did not produce a cytDtoxic effect after repeated testing. Soniflcation and pH adjustments were used to ensure maximum solubility or even suspension of the stock solutions of the compounds. The highest concentration was diluted further in solvent and then in culture medium to give several log dilutions of each compound. All compounds were in apparent solution and within the physiological pH range when tested, except as otherwise noted in the tables. Controls The positive controls were 4-nitroquinoline-N-oxidc (4NQO), a compound that induces UDS in the absence of a metabolic activation system, and dimethylnitrosamine (DMN), a compound that induces UDS only with metabolic activation. The negative controls were the solvents diluted in culture medium. * As, an additional check against the presence of PPLO, which could incorporate tritlated thynddine (3H-TdR) and thus obscure measurements of UDS, stock cultures were analyzed monthly for the presence of PPLO. The results of these analyses were consistently negative. 13 �UPS Assays The contact-inhibited WI-38 cells were incubated at 37°C with log dilutions of the substitute pesticides and with 1 uCi/ml of 3H-TdR (sp act, 6.7 Ci/nungle) . For testing in the absence of metabolic activation, the cells were exposed simultaneously to the substitute pesticide and to 3H-TdR for 3 hours. For testing with metabolic activation, the cells were exposed to the substitute pesticide, to 3H-TdR, and to 500 mg/ml of the 9000 x jj supernatant' fraction of a liver homogenate from adult male. Swiss-Webster mice, with appropriate cof actors,* for 1 hour; then the cells were incubated with only %-TdR for an additional 4 hours. The shorter exposure time for metabolic activation testing was used to preclude cytotoxic effects of the liver homogenate preparation. Both approaches included a postincorporation incubation with un labeled thymldine. UNA was extracted from the cells by a modification of the PCA-hydrolysis procedure;5 one aliquot of the DMA solution was used to measure the DNA content, after reaction with diphenylamip.e,6 and a second aliquot was used for scintillation counting measurements of the extent of incorporation of 3H-TdR. Results were expressed as incorporated per unit of DNA and were compared with the background rate of incorporation. We have defined ns an acceptable assay one in which the response of the positive control, compound is predicted, within the 95% confidence limits, by regressions of average dpm/pg DNA VEJISUS average dpm/ug for background. The regressions that follow are based on data that we have acquired in previous testing: Typti oi; Testing Without metabolic activat 1 -n With M»-cabjllr activation ____ R*yjT.£?-§lPJ?-+. _________ YI - 696 + 17.45 (X)* Y, - 263 + 1.83 (X)* Sample ! • : - -£n). **£ 48 13 Correlation Coefficient (r) 0.7668 0.9639 *Nicotinamidc, 3.05 mg/ml; glucose-6-phosphate, 16.1 mg/ml; 5.08 mg/ml; NADP, 0.765 mg/mi. "^Regressions over a range of background dpm/ug DNA of 0 to 450. *Y! - Average dpm/ug DNA for 10~5 M 4NQO (positive control). Y^ = AVI rate dpoi/ng DNA for 5 x 10":? M ! M (positive control). )N X = Average dpm/ g DNA for background (negative control). �If the observed avecage level of incorporation for Lh<? po&JtIve ronrr"I compound is ouCalrle the 95% confidence limits of uhe regression, we assume that, some variation has occured in the experimental procedutr-.i and repeat the test. Interpretation of Results^ In a report to the National Cancer Institute, ' we presented the results of tests performed without metabolic activation on 40 compounds of known carcinogenlcity. We have analyzed these results using either the parametric One-Way Classifiction Analysis of Variance or the nonparametric Rruskal-Wallis One-Way Analysis of Variance, depending on which was more appropriate.* At f.he 99% confidence limits, all the ultimate carcinogens significantly elevate the Incorporation of JH-TdR into the DNA. The itoncarclnogonic compounds, with one exception, fail to elevate significantly the incorporation of •'H-TdR at r.hiu level of confidence. Thus, the 99% confidence Units of these statistical analyses apparently can be used with reasonable accuracy to predict the biological significance of the response to a chemical. The number of compounds we have tested with metabolic activation is insufficient to establish a correlation between statistical significance and biological significance. Therefore, we assumed that the 99% confid- ence levels of the analyses of variance used without metabolic activation also apply for test'ng with metabolic activation. Resu1ts_and Discussi on Tables 51 through 90 present the results of the UDS testing, with and without metabolic activation, of the 20 substitute pesticides. Tables 51 an) 57, the DNA repair synthesis assays of nonocrotophos, include detailed summaries of the cell culture and experimental conditions for these assays. The assays presented In the following tables (53 through 90) were conducted under similar conditions. In routine testing in the *If there is reason to believe that tho variances of each of the treatments in a test are equal (I.e., Bartlett's test of the variance is negative), the parametric analysis is the appropriate one. If the variances are not equal, the nonparamtitric analysis is the appropriate one. �absence of metabolic activation, six samples each are used for five log concentrations of each test compound and for the negative and positive controls. However, because of the expense of the metabolic activation preparations, for all compounds except bromacil we tested three replicate samples in the presence of metabolic activation and used three concentrations of the rest compound (selected on the basis of the testing without metabolJc activation). Based on the criteria for positive responses, we observed significant Increases in unscheduled DNA synthesis In the absence of metabolic activation afi.er exposure of the cells to only two substitute pesticides, monocrotcphos and parathion. In the presence of metabolic activation enzymes, KJgnlv i.cantly increased UOS was detected for five substitute pesticides: monocrotcphoa, captan, folpet, azinphos-methyl, and monuron. Co-nr-art-d with those of negative controls, the levels of 3H-TdR incnrpuration were greatly reduced in the absence of metabolic activation at the highest concentrations tested for captan, folpet, azinphos-methyl, and mouuron, the same four compounds that induced UDS only in the presence of metabolic activation. The reduced levels of incorporation may be interpreted as cytotoxlc effects or as inhibition of repair caused by the highest concentration of the test compounds. A similar effect was observed in the presence of mefabollc activation for only one compound, captan, and this was observed at a higher concentration than had been tested without metabolic activation. SLich et al.e have discussed the problem of cyto- toxlcHiy and possible inhibition of DNA repair systems by some chemicals and liciv-: <jtr-;ns«d that , whereas such factors may obscure measurements of UDS, oin.:ii a close relationship exists between concentrations that induce UDS and n«ct-.iNat ions thiit are cytotoxic or that inhibit repair. llcr.ausc of the cy to toxic or Inhibitory effects of the substitute pesri i.lilt's, ).l should not; hv: assumed without further testing that monocrotophos and parathion would be carcinogenic without metabolic activation or that the other four substitute pesticides that induced UDS in the presence of metabolic activation are procarcinogens. The positive UDS ti>sul£>-, indicate that chnse Filx substitute pesticides should be tested moi:e iMinnsively, with the testing to Include evaluations of the effects of cli: s.e •: Jiemiculs In in yfvo Moat's*; j . �MICROBIOLOGICAL ASSAYS General SRI examined twenty pesticides for mutagenicity by in yitjro microbiological assays with Salmonella ty£him_uriuM_ (TA1535, TA1537, TA1538, TA100), Escherichia coll WP2, repair-deficient: and -proficient strains of Bacilly_s subtilis and E. coil, and with the yeast Saccharomy_ces cereylsiae D3. An Axoclor l25<V™3timulated, rat-liver-hoim>gfinate metabolic activation system was included in each procedure, except the relative toxlcity assays, to provide metabolic steps that the bacteria are either incapable of conducting or that thfy do not carry out under the assay conditions. The purpose of this study WAS to determine whether the compounds elicited a mutagenic response In. microorganisms. The assay procedure with S_. ^phlinurium has been proven to be 85 to 90% accurate in detecting carcinogens as mutagens, and it has about the q same accuracy in identifying chemicals that are not carcinogenic. The assay procedure with S. cerevisiae is about 5C% accurate In detecting carcinogens as agents that increase mltotic recombination. E, coll WP2 and the microbial sensitivity assay are two additional methods of detecting tnutagnns. The combination of these four assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. 17 �Experimental Salmonella, typhlmurium Strains TA1535, TA1537^ TA1538. and TA100 The S. ^yphjLmurjLiim strains used at SRI were obtained from Dr. Bruce Ames of the University of California at Berkeley.10'12 All are histidine auxotrophs (hls~) by virtue of mutations in the histidine operon. In addition to the mutations in the histidine operon, the indicator strains have mutations in the lipopolysaccharide coat Crfa ) and deletions that covor a gene lavolvod in the repair of uv damage (uvrB~). The rfa~ mutation makes rhe strains more permeable to large molecules, thereby increasing their sensitivity to these molecules. The uvr_B~ mutation decreases repair of some types of chemically damaged DNA and thereby enhances Hei'ssit Ivity to some aiutagenlc chemicals. Strain TA1535 is + reverted to histidine prototrophy 0»is_ ) by many mutagens that cause basepair substitutions. t'rameshlft mutagens. Strains TA1537 and TA1538 are reverted by many TA1537 is more sensitive than TA1338 to mutation by some acridine and benzanthracenes, but the difference, is quantitative rather than qualitative. TA1GO is derived from TA1535 by the intro- duction of the R factor plasmld pKMlOl.13 The introduction of this plasmid, which confers ampleillin resistance to the strain, greatly enhances the sensitivity of the strain to some base-pair substitution mutagens. We have shown that mutagens such as benzyl chloride and 2-(2-furyl)-3-(5-nitro-2-furyl) acrylainide (known as AF2) can be detected in plate -rtssays by TALOO but not by TA1535. The presence of this plasmid ill so !r.;jk<vj -."XT'I, T.MOO sensitive to some frameshift mutagens—e.g., 1CR-191 , ben£o(.i)pyrene, aflatoxin B,, and 7,12~dlmethylbcnz(a) anthracene. A l l r.!i*' indJrat.or strains arc stored at -80°C. For each experiment, an inoculum From frnsen stock cultures is grown overnight at 37°C in a nutrLftnr >•":<'-\i consisting of 1% tryf.tone And 0.5% yeast extract. Afterstationary overnight growth, the cultures are shaken for 3 r.o 4 hours to ensure, optimal growth. Each culture is checked for sensitivity to crystal vjul ;t. Th-; presence of the rfa mutation makes the Indicator strnLns .'.;*•!.fit. Ivc to this dye, whereas tha parent strain, rfa , is not senHlt: ivt to the dye. However, the mutation is reversible, leading to �the accumulation of rtji cells in the culture. Therufore. t.b: ,.••)!' must be tested routinely to ensure their sensitivity to crystal violet. Each culture, also is tested by specific mutageus known to revert each tost strain (positive controls). To a sterile 13 x 100 mm test tube placed in a '43°C heating block, we add in. the following order: Assays in agar (1) 2 ml of 0.621 agar* ( ) 0.1 ml of indicator organisms 2 (3) 0.5 ml of metabolic activation mixture (optional) (4) Up ro 100 pi oL a solution of the test chemical,*'11 For negative controls, we use steps (1), (2), arid (3) (optional) and 100 jjl of the solvent used fcr the test chemical . This mixture is utirred gently and then poured onto minimal agar plates.i After the soft agar has set, the plates are incubated at 37°C for 2 days. The number of his revertants (colonies that grow on plates lacking a sufficient- amount of histldine to support colony formation) are counted and recorded. Some of the revertants are routinely tested to confirm that they are hls_+, require biotin, and are sensitive, to crystal violet (r_fa~). Escherichi_a_ col 1 _WP2 The E. coll WP2 (iiyr_A~) used in this project, was given to us Dr. D. McCalla. ' by \ procedure similar to the one used with Salmonella is used to measure the reversion of WP2 to tryptophan independence. How- ever, instead of containing a trace of tryptophan in the top agar, the minimal agar plates contain 1.25 g of oxoid broth per liter to provide * 0.6% agar contains 0.05 mM histldine and 0.05 iriM biotin. t Minimal agar plates consist of 15 g of agar, 20 g of glucose, 0.2 g of MgSO,.7 H20, 2 g of citric acid monohydrate, 10 g of K2HP04, and 3.5 g of NaHNltyPO^.H;>0 per liter. **Solvents used as appropriate include: water, dimethyl sulfoxide, cthanol, and ben/.ene. 19 �Che trace of tryptcphan required for enhancement of any mutagenic effect of the test chemical. Alternatively, reversion of the mutated tryptophan gene, WP2 may undergo a forward mutation in a tryptophan tRNA gene to obtain tryptophan independence. We do not distinguish experimentally between the true revert ants and the phenotyplc revertants (although the latter tend t.i- form smaller colonies). _H17/_M45 Thi> _:•__. CA.U strains W3110 and p3478 were obtained from Dr. H. Rosiprikrai-.? . ' Strain p3478 is a polA" derivative of strain W3110. It carries .! single, revertable mutation in a gene for a DNA polymerase; " ~t Gross dn<l Gross showed that this mutation Ls involved in DNA repair synltiesj :.' This uiutatlou increases the sensitivity of strain p3478 to chemicals that lead to alterations (damage) of the DNA. Therefore, we ran assay for chemicals that damage DNA by comparing the relative sensitivity of the two strains (p3478 and W3110) to the test chemical. The IJ. subtilijs strains HI 7 and M45 were obtained from Dr. Kada. 18 Strain H17 (rec+) is derived from H17 but is deficient in the genetic recombination mechanism necessary to repair DNA damage. Cells deficient in r.hJs repair mechanism are killed more oasily by chemical mutagens than arc wild-type cells (rec+). If the chemical is toxic to rec~ cells, b:H at the same concentration is not toxic to rec_+ cells, the chemical ;;V(. 'ftt-1 v i s a i:;utap.en. fiv.i--- •l-iT*- from frozen stocks are grown overnight in nutrient broth* :it '<•"< .,:'•; .•.'irtViog. V<: ?. ml of nutrient broth containing 0.6% agar 1s Tidod r'. i mi ot the Lest culture. The suspension is mixed and poured cnL--i .1 1 '.'. , containing nutr i.<.=ni. broth and 2% agar. After the soft agar has solidified, a sterile filter disc impregnated with the test chemical is placed in the center of the plate. The r:hve.--; •, : .- abated at 37°C for 15 hours, and the width of the zone of * Trypr•.-'!»•, 1%, and 0,5% yeast extract, supplemented with 5 jig of tlij-'tii v-v's:1 to pruvent selection of t:liy+ revcrtants. 20 �!-oxLcir.y or Inhibition of growth is then measured. We usually must test several concentrations of chemical to detect accurately differences Ln i:he zones '••£ growth inhibition because higher initial concentrations load to steep concentration gradients that may reduce the differences in growth inhibition of the two strains. The positive control for this assay is 1 nil. of l-phenyl-3,3-dimethyltriazene placed on the disc. A zone of approximately 40-ram width is observed (52 and 61 mm, respectively). of chloramphenlcol placed on a disc. An additional control is 30 pg Equal zones of inhibition are oxpected in all four strains (approximately 30 mm) since the toxlcity of this chemical does not depend on a mechanism that leads to i)NA damage. All assays are performed at least three times. Saccharomyces cereyisiae D3 The yeast 3. cerevlsiae D3 is a dip]old heterozygous for a mutation ^n an adenine-metabolizing enzymes.*' Cells homozygous for this mutation produce a red dye when grown on medium containing adenine. Adenine- vcquiring homozygotes can be generated from the heterozygotes by mitotic recombination. nation. Many mutagens increase the frequency of mitotic recombi- Mitotic recombination is Indicated by the development of colonies with red pigmentation, and the degree of conversion to this pigmentad colony indicates the mutagenlcity of a compound or its on metabolite. The Saccharomyces test strain from the liquid nitrogen is grown overnight .it 30°C with aeration in 1.0% tryptone and 0.5% yeast extract. The cells are washed twJce In 0.067M P04 buffer (pH 7.4) and resuspended In the same buffer at a concentration of 10 cells/ml. The _ln vitro yeast mitotic recombination assay in suspension consists of 5 x 10 washed, stationary-phase yeast cells in 1 ml of 0.067M I'O^ buffer (pH 7.4) and 50 rag/ml of'the test chemical (or a fraction of the concentration required to give 50% killing).. Incubated at 30° for 4 hours. The suspension is After incubation., the sample is diluted serially in sterile saline and plated on tryptone-yeast-agar plates. 21 �Plates of a 10~^ dilution are incubated for 2 days at 30°C, followed by 2 days at 4°C to enhance the development of the. red pigment indicative oh" adenine-negative homozygosity. To detect red colonies or red sectors, we scan l.lie plates with a dissecting microscope at 10 x magnification. Plates of a 10 dilution are incubated for 2 days at 30°C for determi- nation of the total number of colony-forming units. The in Vitro yeast Itotic recombination assay in suspension with metabolic activation is conducted as above with the addition of the. metabolic activation system to the incubation mixture. ArocLor 125/t-Stimulated Metabolic_Actiyation System Some' •.•arcinoganic mutagens (e.g., dlmethylnitrosamine) are inactive unless they are converted to their active form by being metabolized. Ames et a I. ,21 hav? coderib«d the metabolic, activation systems we use. Adult mnle mice are given a single 500-mg/kg intraperltoneal injection of A poiychlorinated biphenyl (Aroclor 1254).^ Four days after the injection, the animals' food is removed. On the fifth day, the mice are killed. The liver are removed aseptically and placed in prewelghed, sterile glass beakers. The organ weight, is determined, and all subsequent operations to th« metabolic, activation step are conducted in an ice bath, [he organ is washed in an equal volume of cold, sterile 0.15 M KC1 (I snl/g of wet. organ), minced with sterile surgical scissors in three VO'MSK.' • ' O.l.S K.C1, and homogenized with a Potter-Elvehjem apparatus. 'fie h'jirn-v'-.i.it? Is centrifuged for 10 minutes at 9000 x £, and the. superi: ii .'.is;, i ; in .-"TfJ .ir.d stored J.n liquid nitrogen. To the postmito- ciu-niuia.k .jMp.v^nii'.f ate added MgCl2» KC1, glucose-6-phosphate, TPN, and (•odium oV'?spi!jr.e {pH 7,4). Results _and_ Dtscussion A.i i " 1 o«< ;>i>3! icides submitted to SRI for examination were tested at icj.--: (.hi>:c times in tho microbiological assays. 1 Hiifz'.ed ^"iii *. are an average cf those experiments. 22 The results pre- �T.'ib1'. 11 i> •-•.•;ii;. !K i r s i i l i 1 ; uf ag;ir with Snlmonol la _tyj»li1Lniui"iuin. • •> i . :. n i u g i v , 1 .i.^-.- v.i In this liisridlne reverse •:•'.• .; t l o i assay system, two pesl:icides—rnptan and folpct were hu.'tiig:-nii-. I'oi oach chemical, we observed an increase in the number ot his'-'.:! ine independent revertants on strains TA1535 and TA100 but not on Jiir.iiny. TA98, TA1537, or TA1538. These results suggest that these u«>st •:: ld<?.-. can alkyJate DNA, causing mutations of the base-pair substitution type. This conclusion is consistent wiLh Llie rjiitagbnic activity of these compounds in assays with E. coli WP2 (Table 9?), which is sensitive no base-pair substitution mutagons. Although liver homogcnate activation was not required for mutagcnic activity, the mut.igenic activity was enhanced somwhnt with activation .it some doses. A toxic effect: (reduction of the: number of mutants) wt;.i observed at 'doses of 100 pg ot" each compound. Table 92 presents I he results of jssiiys with E. roli WP2. Essentially, thi 1 results were i d e n t i c a l to (.hose- obtained with J>. fyp!.1H!!ui:'iU!!: TA1515 ,-md TAIOO; «-aptan and Tolpnt were mutagenic, but. none of the other pesticides was mutagenic. Table 93 piesents the results oE the assays for microbial inhibition in repair-deficient arid-proficient strains of _B. .su_b_tj_li_s and K. .cpj.i. Folpet, captan, chloropyrifos, and dinoseb all gave toxic zones that were larger on the repair-deficient strains than on the repair-proficLent strains, indicating a mutagenic response. Toxic chemicals that do not act by damaging DNA (e.g., chlorampheiiico]) should give equivalent zones of toxicity. However, many if not all nutagens damage DNA and, if the damage is not repaired, can result in cell death. Thus, a given ciinreiitration of mut.agon may be toxic for a repair deficient strain but not for a str.iin the effectively it-pairs its DNA. Tables 94 through 1 1 3 present the results of the assays fur m i t o t i r recombination ir Saccharomyras ccrevisiae D3. A p o s i t i v e - response in t h i s assay IK ind iru tc-d by an increase- of more llian three-fold in the .'ib'.olute number of mitoM'c reroiisbi runts per m i l l i l i t c r a.s w e l l as in lhi> relative nunibi-r of i n i l o l i c re< oi:ib iiicint s per 10 survivors. l-'olpei. �captan, monocrotophos, cacodylic acid, and azinphos-methyl increased mitotic recombination significantly and are considered positive by these procedures. Methyl parathion gave a marginally positive response. Our results indicate that 7 of the 20 pesticides examined give positive responses in one or more of the four microbiological assay ps o...••'J'srea. Although a mutagenic response in a microorganisms does r-i-'.na that a chemical is a mutagen in humans, the combination of four separate assay system greatly enhances the probability of detecting PO'-M Maily h&zardous chemicals. Folpet and captan are mutagenic in :>i •. : ou, assay procedures. Chloropyrifos and dinosftb are positive in U: I'.'crobial sensitivity. Monocrotophos, cacodylic. acid, and azinphos3>s-••••,••!. ;tre pofiitlve in the yeast assays. �DISCUSSION Of the 20 pesticides tested for mutagenic activity, 9 were clearly mutagonic in one or morr i.n vitro assays. Of these. 9, 2 ware mutagenic in all the in vitro assays, but none of them produced a dominant, lethal response in the mouse. In the Salmonella assays., these chemicals caused base-pair substitution mutations but not fraine.shi.ft mutations. The absence of activity in the dominant lethal assay may be due r.o a lack of sensitivity of the mouse to these types of compounds; for example, N-methyl-N'-nitro-N-nitrosoguanidine and other alkylating agent*: that cause base-pair substitution mutations do not all cause dominant lethality. Anotho.r explanation for the absence of activity may be flint these pesticides did not reach the gonadal tissues In sufficient amounts to cause a mutagenic event. None of the other 6 pesticides was mutagenic in all the in vitro assays. The combination of assays used in this program is one means of identifying those pesticides that may present a rautagenic health hazard. Those that show positive responses in several experimental systems should be evaluated more, thoroughly before they are substituted for ocher pesticides already considered as a risk to the environment. Also apparent is that no one assay system is uniquely capable of detecting the spectrum of mutagenic events that different chemical, structures may cause. 25 �REFERENCES 1. W. T. Federer. Experimental Design (Theory and Application). The M,i. Vi! ii:ui Company, 1955. 2. b. .„.; -«i•J',evlc acid L. Tolraach. Response of synchronized populations of "sieLn. cells to ultraviolet Irradiation at selected stages of the generation cycle. Radiat. Res. 32, 327 (1967). 7 . f<. T.. W,' -s .mssen and R. B. Painter. Radiation-stimulated DNA iils in cultured mammalian cells. J. Cell Blol. 29, 11 (1966). '- , il. Stich and B. A. Lalshes. DNA repair and chemical carcinogens. Ii> |fat Sobtology Annual. H. L. loachim (ecL), Apple ton-CenturyCroris, NP-W York, 1973, pp. 341-376. 5. K.. £lgjo, H. Hennings, D. Michael, and S. H. Yuspa. Natural synchrony of newborn mouse epidermal cells in vitro. J. Invest. Derm. 66, 292-296 ( 1 6 ) .96. 6. K. Burton. A study of the conditions and mechanism of the dipheny.lamJ.ne reaction for the coloriinetric estimation of deoxgrlbonucleic acid. Biochem. J. 62_, 3.1.5-323 (1956). 7. A. D. Mitchell. Unscheduled DNA National. Cancer LSU-2735 (April Potential Prescreens for Chemical Carcinogens: Synthesis, Task 2. Final report prepared for Institute under Contract N01/CP-33394, SRI Project 1976). 8. H. F. Scich. D. Kleser, R.H.C. San, and B. A. Laishes. The Use of ONA i-op/iir in the identification of carcinogens, precarcinogens, in j •• strnet rLssue. Jjs Canadian Cancer Conference: Proceedings of •-!.: .•.-•'! •"a"..i:.iian Cancer Conference, Vol. 10. P. E. Scholefleld •v«j.y. Jinlvqrsity of 1'oronto Presss, Toronto, Canada, 1973, pp. 83-110. . :!<•«'. :••"., 5. f,hoi. v. Yamasaki, and B. N. Ames. Detection of ;.ai.<- .',H^.-,:N-:-, ,!•> nutagffjs in the Sa^lmorKilla taicroaome test: Assay of .iOO ,-.f.vcii..,-ils. Pro,. N.-iir. Ac..id~." ScT/"uSA" 7_2, 5135-5139. 10. B. N. Amcs, I'., G. Curney, J. A. Miller, arid H. Bartsch. Carcinogens as frameshlft mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic araine carcinogens. Proc. Nat. Acad. Scl. USA 69, 3128-3132 (1972). IS.. Si. • • AMKS., F . D . L«e, and W. E. Durston. An improved bacterial '•-":( .-; v •. t t-iii for thu detection and c. JLossif ication of oiutagens and ..ir.-'..,Tv,»ons. Proc. Nat. Acad. Sci, 'JPA 7_0, 782-786 (1973). 26 i �12. R. N. Aro«-is, F.. W, Ours ton, K. Yamasaki, and P. !. I.PP. O., .--. IP--.;, n ) are mutagGiis: A simple test system combining llvor homogenous for activation and bacteria for detection. Proc. Nat. Acad. Sri. USA 7Ci, 2281-2285 (1.973). .13. J. Mcl'-ann, N. E. Spingarn, J. Koburi, and B, N. Au.es. Thi* deJi.'.r? Ion of carcinogens as muthgens: Bacterial tester strains with R fp.ctor plasmids. Proc. Nat. Acad. Sci. USA 72, 979-983 (197.5). 14. B. A. Bridges. Simple, bacterial systems for detecting mut.agenJc agents. Lab. Pract. 21, 413-416 (1972). 15. D. R. McCalla and D. Voutsinos. Mutation Res. 26_, 3-16 (1974). 16. M. D. Anderson, E. E. Slater, and H. S. Rosenkranz. Rapid detection of mutageus and carcinogens. Cancer Res. Tl, 970-973 (1971). 17. J. Gross and M. Gross. Genetic analysis of an E. coll strain with a mutation affecting; DMA polymerase. Nature 22i, 1166-1168 (1969). 18. T. Kada. MntagenLclty testing of chemical? in microbial systems. New Methods In Environ. Chem. Toxicology 127-133 (November 1973). 19. F. K. Zimmermann anr. R. Schwaicr. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nItro-l-nitrosoguanldine and other alkylatJng agents in Saccharomy_ces cereyisiae. Mol. Gen. Genet. UK), 63-69 (1967). " 20. U. J. Rrusick and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives ^, 83-96 (1973). 21. I.. D. Kier, E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensat.es. Proc. Nat. Acad. Scl. USA 7_l_, 4159-4163 (1974). On mutagenlcity of nitrofurans. 22. fl. N. Ames. J. McCann, and E. Yamasaki. Methods for Detecting Cnri-i.iogens and Mutngcn.s with the £Salmone : lla/MaminaL lan-Microsome Mutagenicity Test. Mut. Res. TJL... 34"7~364 ~(1975). 27 �TRST OF THE FEBTILITY INDEX - MOKOCftOTC.'HO?. .1 DEGREE OF FREEDOM i--. a V?.«ICLE CONTROL ft N P»5 MTO PERT. INDEX O5S3 N N Pftii MYO 1? 74-10 iFERT. :*OEx CriliQ N if PR6 HTO 30 MG/KG iFCST. 2NDEX KG/KG TFM ChiSO n PHi *t iFcBT. CHIS3 h ft PRG MTO FERT. INDEX CHISG MULTIPLE TNEA7MIENT I 28 ill .70 0.05 23 40 ,57 .7 6 16 40 .45 4.14 .a 40 .40 *,!l« 29 40 .72 0.30 2 24 .C .65 O.Cfl Ji •0 .? 7 .98 20 49 .0 6 1.28 16 40 5*0 4,&6 * 27 39 .9 6 .03 3 23 4S .? 5 0.36 3C 40 .75 19 «C .*T .45 22 40 .5 5 0.30 32 40 .ec 3.7? .6 0 00 « 3? 40 .67 0.00 2S 40 39 40 .5 5 .4 8 IS 4fi .38 6.07* 2? 40 .7 6 .5 8 4.79*: 25 40 .63 0.00 ?1 40 .5? .20 30 40 .5 7 1.42 .03 23 40 .7 5 .8 0 25 38 .6 6 0.00 27 36 .71 .24 !8 .l 21 40 .52 4.91* 26 38 .4 7 .07 27 36 ."> .02 20 40 .50 2'7S 24 38 .3 6 .4 2 26 36 .? 7 • 02 .3 6 i £4 «Q .60 C.Ofl 33 6 ?» 38 .63 C.93 2? *0 .7 6 7 30 38 .11 0.00 25 40 .3 6 9 27 38 .71 0*00 23 40 .0 7 * I SIGNIFICANT AT P LT 0.05 INCREASED ABOVE COHTSOL 2.01 .&» • 02 22 �Table 2 AVERAGE IMPLANTS PER PREGNANT FEMALE - SONOCROTOPHOS WEEK CONTROL 74-10 15 HO/KG 7*-10 30 MG/KG 74-10 60 .2 MS/KG TEH MG/KG M ULTIPLE TREA TKENT 1 28-11.39 249/ 2.08 31.3 189/ 18-10.50 ISO/ 2 16-11 . 5 316/ 29-10.90 2 to 319/ 303/ 26.11.65 332/ 31-10.71 242/ 20-12.10 ISO/ 2 16-11 . 5 *93/ 27-13.85 3 24S/ 23.10.65 356/ 30-11.87 239/ 19-12.58 "I 267/ 2£-12.14 *1 34a/ 52.I8.97 4 309/ 27.11.44 314/ 2.25 51.6 274/ 2 . 2 45 21. le,6/ 15-11 .0? 266/ 2 . 9.35* 7 5 2T4/ 24.11.42 372/ 33.11.27 270/ 25.10.33 248/ 2!«1S .81 e 302/ 24-12.58 327/ 27-12.11 275, 23-11.96 25S/ 25*10 .2'J 7 346/ 30-11.53 285/ 25.11.40 25S/ 2l>12. 14 316/ 8 292X 27-10.81 313/ 28.11.18 228/ 20-11.40 291/ • •• I SIGNIFICANT AT P LT SIGNIFICANT AT P LT INCREASED ABOVE CONTROL 0.05 0.01 3S4/ 30-11.8? 2V3/ 27.10.11** 2 28-11 . 9 306/ 27-J1.33 24-12 .12 322/ 26.12.3f>**! *" �Table 3 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - rtQNOCROTOPHPS MCEK ?*-10 CONTROL 15 M6/K9 74-10 30 MO/KG 74-10 60 .3 M6/K6 TEW r MUIL.TIPLE TftEATHENl 1 S3/ 28* .6 4 2B/ 23" 2 9X 26* .31 3X 31* .10 *D 3 9X 23* .39 ;6X 30* .53 4 2X 27* .? 0 ?X 25* .28 * 5 ilX 24* .«* 22X 33* .? 6 1.22 lex 18* .6 5 iOX 16* .3 6 62X 29* 2.14" 1GX 20* .50 2X 16* .13 77X 27» 2. 8 ' 5* 19* .7 * 9X 22* .! * 87X 32* 27" .2 26X 22* 1.16* 9X 15* .0* 6* 11X 27* .41* \il 25* .8 4 *X 2l« .3 4 ?2X 30* ,73 25* ,4* 2*D 9' , 6 2iX 24* .35 \6X 2T* .9 5 14X 23* • 61 6X 17X 27* .3 6 7 30/ 30* 1.00 \\f 25* .* 4 4/ 21* .19 ex 28* .9 2 IIX 27* .41 a 19X 27* 24X 28* .6 8 7X 20* ,35 9X 24. .8 3 ii/ 2S- ,2 * • •• D .70 SISNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELCM CONTROL �Table 4 CHI-SQUAR2 TEST OF THE DEATH 1SDES - MONOCROT3PHOS 1 OEGRSS 07 KSEEDOM •EcK VEHICLE CONTROL N N *i)I PRO DEATH CMIS3 74-10 N N SCI PWG IS MS/KG DEATH I NOES 74-10 N H "KG CKISO 30 MG/KG DEATH INDEX CHiSS 00 MG/KG N . 1 .S 0 'O DEATH C ,. 5 r s we I fi OEATM MULTIPLE TREATftEN T 11 1 10 28 .6 3 0.00 2 7 26 .7 2 0.00 3 9 23 .9 3 0.00 11 4 2 27 .0? o.oo 7 23 .8 4 .35 7 18 .39 .01 6 ,6 .39 .04 25 ?9 .So 13.27 ** 3 31 .10 1.84 7 20 .35 ,07 2 16 .13 .52 26 e.7 .6 9 24.26 « 30 .It .01 7 19 .37 .03 6 2? .27 .28 25 32 .79 ?.C5 * . 25 .6 2 2.54 11 22 .50 * IS .6C 11.21 ** a 2~ .10 3.6? 12 25 .8 4 .21 3 2: .33 .of 10 30 .33 ,CO 23 .8 4 .52 5 25 • 2G 7.48 10 27" .37 2.36 7 28 .25 .02 9 27 .33 »GT a 2, .33 .27 26 .36 .03 9.20** 5 t 24 .33 0*00 11 33 .33 .00 6 15 24 .63 0.00 13 2T .8 4 .6 5 11 .? 2 0.00 8 25 .32 .02 4 21 .19 .9 0 .4 4 0.00 IS 28 .4 5 .17 7 20 .5 3 .12 7 8 ** a 30 12 27 SIGNIFICA.NT AT ? LT 0.01 10 �Table 5 SWttSH OF DEAD IMPLANTS PER TOTAL DfPLAKTS - MONOCS01X)?HCS •CEK CONTROL ?4-10 15 we/US ?4-i3 30 KG/KG ?*.- SO 40 «fe/SG TEN .2 MG/KG *H.(VTIPtE TREATMENT 1 13/ 319- ,0« 2 ?•< 303* .03 3 <?/ 245* .04 M/ £49" .11 5/ 332« ! / 356* & ,6i .04 10/ 1B9* .OS. 10/ 160* .6 0 *>2/ 316* .20** 10/ 24S« . 4 0 2/ 180* .01 77/ 293* .26** 9/ 267* .03 B7/ 348* .25** ll/ 266* .04* 9/ 239* .0* .02 2 . 274. 6' .09** 9/ 166* .05** .04 ze/ 372* .e« 12/ 2?o* .04 9/ 246* .04 2?/ 356* .6 0 21/ 3C2* .07 16/ 327* .OS 14/ 2T5* .ns 6/ 2S5» .0?**D IT/ 273= .6 0 ^ 3.0 / 3*6* .09 ll/ 285= .04 4/ 255* .02 a/ 316* .03 ll/ 306* .04 B 14/ 292* ,' 0 24/ 313* .6 0 T/ <2B* .03 9/ 29S> .03 ll/ 322- .03 4 2/ 309.- 5 i*./ 274« 6 * •• D 7/ 314. . Oi SIGNIFICANT AT P LT 0,05 SIGNIFICANT AT P LT O.C1 DECREASED BELOW CONTROL �Taole 6 CHI-SW.-AHE TEST OF THE FERTILITY IKDEX - BROMAC1L 1 DEGREE OF FREEDOM VEHICLE CONTROL N N PERT. PRG MTO INDEX CHI SO 74=06 N N PRG MTO 1230 HG/KG PERT. INDEX CMISO 7.-06 N N PRG MTC 2500 PERT. INDEX r»-Gi CHiiG 5303 Mli/KG N N PRG MTD PERT. IHOEX CHIS9 KG/KG TEM K, N PRG KTQ PERT. FNCKX CMISO MULTIPLE TREATMENT 1 26 40 .70 0.00 21 40 .2 5 1.90 23 40 .57 .87 29 40 .2 7 0.00 29 40 .72 o.co 2 26 40 .5 6 0.00 24 40 .to .OS 26 38 .8 6 .01 23 40 .63 0.00 27 39 ,9 6 .03 3 2.3 40 .57 0.00 29 40 .72 1.37 19 38 .50 .19 25 40 .63 .05 32 40 .SO 3.78 40 .3 6 .05 22 38 .8 5 .! 4 23 40 .70 0.00 27 •0 .6? .6 0 20 .9 .8 5 1.4? 4 27 40 .67 0.00 25 S 24 40 .0 6 0.00 31 40 .7 7 22 38 .00 27 40 .7 6 .2 2 30 40 .5 7 6 24 38 .63 0.00 31 40 .77 1.30 25 38 .6 6 0.00 27 40 .7 6 .03 27 38 .71 .2* 7 30 38 .79 0.00 29 40 .72 .16 22 38 .8 5 2.98 29 40 ,72 .16 27 36 .75 .02 8 27 38 .71 0.00 213 39 .72 .03 24 38 .63 27 *C .67 .01 26 36 .7? .02 .24 �Table 7 AVERAGE IMPLANTS PEH PHEGHJUfT FEMALE - CONTROL 74-S6 74-06 Z500 HG/K6 74-06 5000 MS/KG TEH MO/KG MULTIPLE TREATMENT i ?,!<¥/ 28• 11 .39 235," 21-1 i. I, 2T8/ 23.12.09 32B/ 29.11.31 316/ 29.10.90 * C-Ci-.- 6 26 .11 . 5 253' 24BJ0.54 264/ 26.10 . 2 9 304/ 25.12.16 293/ 2.08 71.5 3 24S/ 23.10 . 5 6 33SX 29.il.5S 22S/ * 19.1 i . 4 319/ 2 . 2 /6**I 51. 348/ 32.10.87 4 30*-' 4 27-11 . 4 2?Q/ 25.11*60 2&8/ 22=12.18 34 6/ 28-12.36 266/ 87- 9.85 * * S7*/ S«-11 .42 3$\/ 31*11.65 26 I/ 22*11 .d6 523/ 27-21.96 356/ 30*11.67 .-. .10 a/ 5 24.12 . 8 363/ 31" 11. Sf 294/ 7 25-11 . 6 276/ 27«10.30** 273/ 2?-10.11 ** 7 34S/ 30.11 ,53 35&/ 29.12.24 2S3/ 22.11 .50 357/ 2«i.U.31 306/ 27.11.33 3 292/ 27«10.61 318X 28.11.36 5 277/ 24.11 . 4 307/ 27-11.37 322/ 26*12.38 **I • S I G N I F I C A N T AT P LT 0.05 ** SIGNIFICANT AT P LT 0.01 I INCREASED A30VZ CONTROL �Table 8 AVERAGE DEAD IMPLANTS PEH PREGNANT FEMALE - BROMACIL *EEK CONTROL 74-06 1250 MG/KG 74-06 2500 *S/ivG 74-06 5000 MS/KG TEH .2 MG/KG MULTIPLE TRE* TMENT 1 28= .46 8/ 21 = .38 10X 23= .43 17/ 29= .9 5 62/ 29= 2.14** 2 e/ 26= .31 12/ 24= .0 5 a/ 26= .31 5/ 25= .20 77/ 27= 2 8 * . S . 3 Ln 13/ 9/ 23= .39 6/ 29= .21 12/ 19= .63 10/ 25= .40 87/ 32= 2,72** 4 2/ 27= .07 7/ 25= .28 16/ 22= . 3 * 7 * 15/ 29« . * 5 * 1 I/ 27= .41* 5 ll/ 24 = .6 4 12/ 31 = .9 3 15/ 22= .8 6 B/ 27= .30 22/ 36= .3 7 6 2i/ 24= .8 8 7/ 31 = .23 **D 16/ 25= .* 6 21/ 27" .78 17/ 27» .63 7 30/ 30 = 1.00 ll/ 29= .3 3 14/ 22= .4 6 14/ 29= .8 4 ll/ 27= .41 8 19/ 2. 7 15/ 28- .4 5 8/ 2. 4 .33 9/ 27= .3 3 ll/ 26= .42 .TO • S I G N I F I C A N T AT P LT 0 . 0 5 *» S I G N I F I C A N T AT P LT C . C ! D DECREASED BELOW CONTROL �Table 9 CHI-SQf*RE TEST OF THE DEVTH INDEX - BROMAC !I. 1 DEGREE OF FfiEEDOM VEHICLE CQKT90L N N «DT PSKi OEATh INDEX CHISd 74-Ofc N fc aJl PRG 7»-06 1250 MS/KG DEATH INDEX CMisa N N 25CQ HG/KG DEATH WDI PRG INDEX CHI so *.-06 5000 MQ/KG H N DEATH riDI PRG INDEX CHIS3 .2 MG/KG 7EM h N «DJ[ PHG DEATH INDEX CHISg MULTIPLE TREATMENT 1 10 28 .6 3 0,00 7 21 .33 .2 0 7 23 .30 .01 2 7 26 «S7 0.00 10 24 .42 .4 6 a 26 .31 3 9 23 .39 0.00 » 29 .£1 1.32 9 19 .47 .us * 2 27 .07 O.CC 6 2S • 24 I.b2 11 22 .50 9.20** 5 5 S* ,38 O'OO 12 31 .9 3 .4 0 12 22 .5 5 . 4 7 9.00 14 29 .8 4 .48 25 29 .6 8 13.27** .96 24.26** 5 25 .20 .06 26 27 6 25 .2« .? 6 25 32 ,->B 7.05** 14 28 .so 10. a** a 27 .30 3.Q7 8 27 .30 10 30 .33 .00 10 sf 7 2-16 9 27 .33 .07 26 .38 .03 .09 b 15 24 .3 6 o.oo 5 31 • 16 10.65**D 13 25 .52 .21 10 27 .37 2.J6 7 8 30 .7 2 0.00 7 29 .4 2 .01 10 22 .5 4 1.2* 12 29 .41 .64 8 12 27 .4 4 o.oe 11 28 .9 3 .01 7 24 .9 2 .70 7 27 .26 1.30 ** D SIGNIFICANT AT F LT C.01 DECREASED EELOW CONTROL 10 «3 �Table 10 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS • EEK 74-06 CONTROL 5000 MG/K5 TEM .2 «G/R5 MULTIPLE TREATMENT 1 13/ 319= 2 3 i ' 8/ 3039/ 245* 4 2/ 309, a/ .0. 2* 77/ 293- .6. 2* 10/ 319- .03 87/ 3iiis. .25** . 15/ 34e> ,04«* ll/ 266- .04* 323- .02 ?£/ 35*= .06 .05 21,' 278 = .OS 1 I/ ?73 = .06 .06 ! / 357* * .34 K/ 306- .04 9/ 307- .03 ! / 32?* ! .03 5/ 304 = .02*0 12/ 225- .05 7/ 290. .02 16/ 268- ,06** .03 is/ .) 16 .02 **D 16/ 294* 14/ 253* .03 .3 0 .5 0 .04 6/ 335. .01 ll/ 274. .04 12/ 361 = 6 2l/ 302= .07 7/ 368* 7 30/ 346* .9 0 ll/ 355* .03 a 19/ 292* .07 IS/ 3ia- .5 0 SIGNIFICANT AT P LT SIGNIFICANT AT P !_T DECREASED BELOW CONTROL 62/ 316. .02 17/ 323- .) 13 .03 5 * •« D . OS .04 284- 235- 12/ 253- .4 0 0.05 0,0! 10/ 278- a/ a/ 261- 277- a/ �Table 1). CHI-S41MKE TEST OF THE FERTILITY INDEX - C'IP'SA.S 1 CEGREE OF FREEDOM VEHICLE C'J.',TS3i. * N P3C 1TO FES', INOCS ChlSi. MG/Ki 7*-0? «=as -'••vo INDEX CilSO i-u/'Ki.- ?*-62 S N PHG MTU ! tH 1a F IMOEX Ch;S3 7«-oe N u PR G "ilD fc PERT. INDE? CHI SO t. F£RT. PBG MTO IMDCX CHISQ HULTIPUE TREATMENT oo 1 2J *Q »57 o,OC 34 40 .as 6.10*1 31 40 .7 7 2.?9 29 40 .72 1.37 29 40 .7? 1.37 2 a7 39 ,&<5 i.OO 30 40 .75 .10 36 40 .0 9 4.07*! 30 40 .75 .10 27 39 .9 6 .6 0 3 2* »0 .5 6 0.03 23 *C .s-* .21 3U 40 .5 7 .54 31 40 .77 .8 9 32 41 .89 1.57 * 2? to «S7 ;-.''C 31 3« .2 8 1.35 33 40 .2 6 l.*7 32 »0 .80 i.os 27 *0 .7 6 .6 0 i ?<* *0 »''? O.CO 26 3S .6 6 .02 30 40 .5 7 o.oo 26 38 .9 6 .2 0 30 40 . M 0.00 *> 2* 40 »7g 0.0-3 27 .] 7 .91 34 40 .as 1.20 30 38 .79 .16 27 M .71 .01 .71 .01 30 40 .5 7 0*00 S* 38 .8 6 .32 27 36 .5 7 .00 38 .7] v*3 2* 36 .? 7 .6 2 ' S * I i"? 1J *0 *0 7 .? .60 O.CO 0.00 SICNIFICAKT AT ?LT O.C3 INCREASED ABOVE CONTROL 27 31 3« 39 35 .7 8 .6 2 34 40 .9 8 -09 27 �Table 12 AVERAGE IMPLANTS PER PREGNANT FEMALE - C4PTAN sit*. CONTROL 7<.-Cc 12--0 M S / K G 74-02 MG/K& 2500 HG/KG TEH MULTIPLE TREATMENT i 25' 6. 23*11.52 3B7/ 3«=11.3B 339/ 31 = 10. <,* 310/ 29=10.69 3S6/ 29=:0.9C 2 305/ 27=11.30 3?6/ 30=10.93 375X 36=10. *2 3*2/ 30=: l.*0 293/ 27«1C.H5 3 268/ 26=10. 11 2S2/ 23=10.96 313/ 30=10.4.3 3*5 / 31=11.13 34A/ 32=10. «7 * ?RB/ ?7=10.67 32»/ 31=10. *5 3S9/ 33= lO"*" 333/ 32=10. *1 2fci>' 27= 9 8 . 5 5 33»/ 29=11. S2 2fi2/ 26=: 0.65 348/ 30=11.b O 298/ 26= 11. * A 35is/ 30=11.87 6 323/ 29=11. ! • 312/ 27=11.56 361/ 34=10.h2 3*0/ 30=11.33 273/ 27=10.11 30=10.97 3G9/ 26=11. AA 30n/ 27*11.33 3»»11.^ 301/ 27=11.15 322/ 26=12.38 7 3?3/ 29=11.!* 30V/ 27=11.** 329/ r 3= If 32=11.*! 3B2> 33 = 11.S>» 383/ • SIGNIFICANT A7 P LT 0.05 «• SIGNIFICANT AT P L7 0 . 0 1 �Table 13 AVERAGE OS*D IUPLAWTS FER PREGNANT FEMALE - «fct.K T + -02 CONTROL 2500 MG/XG 7,-Cc 533C -li/tt-i Ifrt «WLT£f>LE TREATMENT 19/ 31- .fcl 15.' 2i* ^2 M, 24« 2.14 ** .0 5 J7/ 36- .<•? !»/ 30* ,*7 77/ 27s 2.85 *• .70 16/ 30- .53 15/ 31- .8 4 67/ 32« 2.72 »* 12/ 31ft .39 ]S/ 33s .5 * ll/ 32« .3* ll/ 27, • »1 ?*/ 26= .92 9/ 30s .30 ll/ 26- .42 ?2/ 30s .73 .4,3 21' 34* .t>2 18.' 3C* .60 i?/ 27- •63 27 = -.Si 14/ 30= .*7 a/ 2*= .31 ll/ 27- .41 33= .35 !/ ! 3s 4 .32 7/ 27= il/ 26- i 23* . ?5 2 ** O «/ :\S 27s .*! Is/ 30 = 3 io/ 26s ,*2 16/ 23- * ;&/ 2T- . =9 5 i5/ 24- . '2 . t 9/ ?^s ,^> 13/ J'l ? 1 Ji. 2<!s .72 IS./ d n/ 32s ,»l 12/ 17,' • SIONIFICAKT AV P LT O.OS •• EISNIFICftNT AT P LT 0.01 3» = .50 .26 .42 �Table 14 CHI-SQUARE TEST 0? Thl DEATH IM3EX - CAPTAK 1 DEGREE OF FREEDOM VEHICLE CONTROL N UDI N DEATH PRG INDEX CHISQ 74-02 N N »DI PRG 12SO HO/K6 DEATH INDEX CHISQ 74-02 N N tfDI PRG ?50C MG/KQ DEATH INDEX CHISQ 74-02 N N yOI PRG 5000 MS/KG DEATH INOE« .? KG/KG N PRG CHlSa N SEAT*. INDEX CHISC MULTIPLE TREATMENT 1 a 23 .5 3 0.00 10 34 ,9 2 .2 0 12 31 .39 .00 12 29 .41 .4 0 25 29 .«* IS.**" 2 9 27 .33 0.00 8 30 .7 2 .07 12 36 .33 .07 !C 30 ,33 ,OS 26 27 .* 9 20.79" 3 •0 24 .8 3 •0.00 12 23 .2 5 .6 4 11 30 .37 .32 13 31 ,*? .00 25 32 <78 7.85" 4 11 27 .41 0.00 9 31 .6 2 .6 5 7 33 .21 :.as 6 32 .25 1.02 8 27 . 3<3 .32 .38 .6 0 7 30 .23 .37 li 26 .42 .00 10 30 .33 ,G1 .03 15 34 .44 lag!} ! 0 30 .33 .0* 1C 27 .37 .23 .0] ^ 26 .27 .71 4 27 .33 .12 .19 5 27 .19 1.15 10 24 .38 .03 5 fc 11 29 .* 3 0.00 10 26 •-2* n.on 9 27 .33 .4 4 .00 11 30 .37 • 24 .9 3 9 34 .26 8 29 7 12 29 .! * 0.00 12 27 8 11 32 • 34 0.00 a 33 ** SIGNIFICANT AV PLT O.Oi �Table !5 NUMBER Of DEAD IMPLANTS PER TOTAL IHPLASTS - C*PT'.>. 74-02 C3NTROL 2500 MS/KG 74-02 n S«C/X3 TE'M .? MULTIPLE TREATMENT ; .03 IT/ 367* .3* 19/ 3 9 3. ,6 0 15/ 310* .CS **/ 316= .20** £ *•- d/ 2&5« 11.' 315s .Ofc iS/ 326s .05 IT/ 375- .5 0 14/ 34?> .04 77/ 293* .iff* 2 Si/ 26Gn .06 IS/ 25?B .06 !«./ 313* .US ! / 3*55 .0* 87/ 3 8 4. .25** -' ib/ 2AAc . &'• IS/ 359. •0* ll/ 333" .03 ll/ 266= ,04 5 IS/ 33*3 .3* 9/ 348= .03 11,' 29»= .0* 22/ 356 = .06 16/ 3*3= .05 17/ 273.-: .6 0 fsi 12'* 3£» = 2»/ 28?- .04 .39 b •>/ 323s ,ft3 i3- 312= .0* 21/ 3*1. 7 2i/ 323» .Ci7 ! / 3Q9= 5 .5 0 I*/ 329. .11* a/ 305. ,C3 1 S .-' 30&. .04 - !.' -•}«!= '. .03 j2' 382= .03 ll/ 383. .03 7/ 30i= .Og ll/ 322" .03 • SIGNIFICANT AT P LT 0.05 •* SlSNieiCANT AT P LT 0 . 0 1 .06* �Table 16 CH1-SQUAR2 TEST OF THE FERTILITY INDEX - FOLFZT 1 DEGREE OF FHSEDOM • EEK VEHICLE C O N T R O L N N PRG MTD PERT. INDEX CMISU 74-03 N N PHIi MTJ 1250 MG/K6 (•tRT. INDEX CHIaQ 74-03 h N PRG MTU 2500 H6/KI. FE.RT. INDtX CHIiO 74-OJ N N HRii MTU SOO'j Mto/KG TEN F£RT. INDtJt CrilSQ h w PRG HTD fEKT. INDEX CHI Si Ul MULTIPLE TREATMENT 1 23 40 .57 0.00 <J7 4Q .67 .»e 29 40 .72 1.J7 3\j. 4U ,T5 £.01 <><» «>J .7? 1.37 2 27 39 .9 6 0.00 2U 40 .70 .03 23 40 .37 ,fe 3S> «C .8 8 £.99 27 39 .9 6 .6 0 3 26 40 .5 6 0.00 i-'b 40 .63 O.UO 31 4U .77 .SIB 32 tLi .60 1.57 32 40 ,60 1.57 4 27 40 .7 6 0.00 30 40 .5 7 .tf* 18 40 .5 4 3.£5 30 40 .75 .24 jfr no .6? .0= Q.OO 26 40 .65 .<!3 33 4Q .8 3 j9) .! 30 40 .75 0.00 O.OC id 4o .7s U.OO ^ 36 •7i .01 .72 U.OO JO 4Q .75 40 .2 7 0.00 3U 4g .75 O.UO 28 4u • 7o 29 40 .72 0.00 28 40 .70 0.00 27 4Q .67 . 0* 31 32 40 .BO U.OO e~> 40 .67 1.03 29 40 = ?2 , JS j'J 5 29 6 29 7 a 40 4Q .77 .Q7 27 jo .75 .30 4Q ," j .07 ?t J6 .?2 -2B �17 IMPLANTS PEB PREC-NAST FEMALE 74-03 CONTROL 1 Xt-js 23*ilnb? JOB/ 2 7 x > l . «l J24X - j>i>00 Kfi/Kb 29*11.1? . ZHG/.V.; 32flX 30*10,93 316X 29*10.90 £ :i?5/ 27x11.36 JO-/ 2a*10.Bb 267/ 23»ll.i7 393X 3!i»lS.14 293/ 27x10.35 3 fat/ 26x10.31 2Ti/ 2bxl0.tt4 33«/ 31x10. 11 327/ 32x10.22 348/ 32.10.87 4 28Ui' 2?BH'» A '' 29V/ 30* 9.97 18J/ l«»i'J.l7 J20/ 30*10«b7 26fc/ 27* SUSS % 33*/ 29*11. Si 333/ 3U*il.l9 31a/ 26«12.<i3 3<*9/ iS)ill,*0 35a/ 30*11.87 i 323/ 2V>il.i* 33<r/ 3J=ilt13 294/ 2b«10.30 J12/ 28=11,14 273/ 27x10.11 7 323/ 24*11.14 30 f / 2Hai3.V6 311/ 27*11.32 3b6/ 3<cll.4b 306/ 27il!.33 322' 26«12.38 15 3fel/ 32*11.*! 30 i'' • SIGKIFICAMT AT P LT O.Oi •« SIGNIFICANT AT V LT U.01 27*il.l5 34!>X 1 2 ** 11.^9 J46X X 30 11.53 �Table 16 AVERAGE DEAD IMPLANTS PER PREGN»T FLSIALE - POPPET CONT3CL 7*-OJ 12!>0 KG/KG 74.03 2*33 MS/ISO 7<t-93 5000 MULTIPLt TKtATMtNT 1 8/ 23. .35 It)/ 27* .37 25/ 29. • a* 13/ 2 ll/ 27. .! * lb/ 2Us .4 6 20/ 23* .87* 3 16/ 2. 6 .2 6 U/ 25, .52 19/ 31. .01 4 it/ 2 . 7 .9 5 16/ 30s .b.3 a/ 18. b 15/ 29, .2 5 IS/ 30= ,40 9/ 6 «/ 29, .31 }7/ 3o» .b? 7 21/ 2. 9 .72 !<;/ 28. .41 ll/ 27. 8 13/ 32 = • S I G N I F I C A N T AT P LT O.C5 •• S I G N I F I C A N T AT P LT 0.01 0 DECREASED BELOW CONTROL 30» .43 2i/ 3&» .60 T7/ 27. 2,05** ll/ 32« .34 87/ 32. 2.72** .4 4 7/ 30- 26« .JS io/ 35. . **> 22/ ?Ca .73 10/ 2. 8 .67 i3/ £«• .* • !. »' jTa .63 .43 10/ 27» .37 le/ 3i> 08 H/ 27* .*1 .41 20/ 29. .9 0 13/ ll/ 26« .42 30« c c3*D .43 62/ 29" i.14** :i/ z?a .*i �Table 19 CH!-Si3TARB TEST OF THE DEATH INDEX - FOLPET 1 DECREE OF FREiDOK 74-03 ¥E*!Cl.i CONT^l-L N N • 01 i 1DEATH INDEX -41SU ft N »Ul PhG iL)E«TH !NOEK CnliiU N N «01 ORb 2500 7»-Q3 *<t/f\i DEATH iNUtX N *> 5000 Mfa/Ati IDEATH IN-JtK CHi^Q CHlSU TEH M N »l): f>nG ULATn INDEX CHtSU "ULTIPUE TMtATMENT I e 23 .35 0.00 a 27 .30 .ul 13 29 .6 4 .<•» 10 30 .33 .03 25 £1 .6 6 12.49** 3S .43 .5 3 26 <7 .6 9 20.79*. 7.85** e .3 9 27 .33 S.i-0 13 *S .»a .Si 12 23 .2 6 i.iS 1= 10 26 .8 3 0.00 V <!<9 .6 3 ,«1 4 31 .29 .22 d 32 ,2i .67 2b Je .78 4 I: 27 .41 O.i'O 11 20 .3? ,UO 7 la .39 ,iiJ 7 30 .23 1.27 a j-7 .3G .32 5 11 29 •38 0.50 10 39 • 33 ic 35 .34 :'.-U 10 JO .. 31 .01 6 0 29 •28 u*cs 11 30 .37 .«2 il 26 .39 .43 V 28 •32 .01 10 l7 .37 .22 7 12 29 .41 000 6 <;B .29 .S>4 4 27 .3 3 .12 13 31 .42 .05 t ft .33 .12 c* .8 3 .00 a *• 11 32 •34 0.00 SIGSIFTCAHT AT PLT 0.01 4 2r .33 >t«l '4 0 » 16 26 29 .Ji .5 5 .' « 1.H9 11 30 OT .01 10 �Table 20 NUMBER OF OEAO IMPLANTS PER TOTAL IMPLA.STS - FOLPET fettK 7*-03 CONTROL 1250 M6/KG 7«-03 /4-U3 5000 M u / r G ,2MG/«b Ttl MULTIPLE TREATMENT 1 a/ 265* .03 10/ JOB- .03 »/ 324* . J8 i3/ 328 • .04 62/ 316- .20** 2 ll/ 305* .4 0 le/ 304* .06 20/ 257- .06* 21/ 390 • .05 77/ 293* . 6 *K 2 3 16/ 268. .06 1J/ 271. .05 I1*/ 33*« ,U6 ll/ 327 • .03 87/ 3 8 .* .25 t* * 16/ 288* .0* J6/ 29V= ,Q5 5 6 e/ 1H3- ,y» 7/ 320- .02 ll/ 266* • C4 .04 Z£f 356* .36 .04 n/ £73* .06 .0* lit 333* .04 9/ 318- .03 16/ 399 * «/ 323 = .03 I?/ J3»= .05 4 16/ ^ . .ub i3/ 3i2 15/ 334- E 7 21/ 323* .0? li/ 307. .04 10/ 311- .03 IS/ 356* .05 M/ 306> .0* a 13/ 381* .03 ll/ 301- .04 20/ 345- ,U6 13/ 346* .4 0 ll/ 322- .03 • •• S I G N I F I C A N T AT P LT 8.C5 S I G N I F I C A N T AT H LT 0.01 �Table 21 ikl-SQUABE fSST OF THE FERTILITY INDEX - AZINPBO:--fETFYi. 1 DEGREE OF FREEDOM HS/K6 *E"SCLt * * PRO * ! T) PERT. IWE* •lisa P*3 »T() i ••*?.£* c«isa T4-09 f4 N PR6 MTD MS/KG PERT , CHISQ INDEX 74-09 H M PRO MTO BC KG./HG PERT. 1MJEX CnlSQ Kl PR6 N PERT. CHISQ MULTIPLE TREATMENT ^ 1 ?8 43 .70 C-,09 34 ?6 40 .5 6 c.eo 29 oo 2 J S3 40 .57 3.00 33 40 •as .2 7 40 .2 8 28 .23 40 .70 .06 27 40 .67 0.00 29 40 .72 0.00 27 40 .7 6 0,00 27 40 .7 6 0.00 27 39 .9 6 .03 .72 1.37 24 3* .63 .OS 32 40 ,SS 3,72 21 36 .8 5 .35 27 40 .7 6 .6 0 .02 30 40 .75 1.42 27 J« • 7i .24 • »M*I 29 40 * 2? »0 .7 6 5.00 30 42 .75 .24 29 40 .72 .06 5 2* *0 .60 o.co 2* *0 .72 .9 8 26 49 .5 6 .05 20 34 .53 6 2* 33 .3 6 3.00 JO 40 .5 7 .79 27 40 .7 6 • 03 28 34 .82 7 3?, i8 • 79 0.00 27 40 .7 6 .6 7 25 40 .3 6 1.81 24 34 .71 .30 27 36 .75 .02 8 27 J5 .71 0.00 26 40 .5 6 «n 26 40 .5 6 .11 22 34 .5 6 .10 25 36 .72 .02 * I SIGNIFICANT AT F LT D-lr. INCREASED ABOVE CONTROL 2.41 �Table 22 AVERAGE IMPLANTS PZR PREGNANT FEMALE - A7IN7HOS-METHYL CONTROL 74.09 20 MG/KG 74-09 MULTIPLE »C 74»0<5 80 MG/KG MS/KG TREATMENT 1 319/ 28*11.39 375/ 34.11 .03 300X 28*10 ,71 306/ 27.11 .33 31*/ 29.iC.90 2 303/ 26*11.65 335/ 29.11 . 5 5 312/ 2r«n .56 310/ e7.il .*3 293/ 2V. 10. (55 3 ?45/ 23.10.65 379/ 33.11 . 8 * 338X 29.11 . 6 6 272/ 2«.l!.33 348X 32.10.87 * 309/ 27.11.44 367/ 30.12 . 3 2 369/ 2 . 2 7 * ! 9 1 . 2 * 247/ 21.1! .76 ?66/ 27. 9 8 * .5 5 ?74/ 24-11.42 33?/ 29*11 . 2 6 310/ 26*11 .92 228/ 20=11 .40 35S/ 30»11.S7 t, 302/ 24*12.Sa 345/ 33*1 i .50* 2?6/ 2 . 10. 2 ? 2" 323/ 2a-H • S** 27">/ 27*10.11 ** T 346/ 30*11.53 288/ 27.10.67 273X 2S«11 .12 262/ 24.10 .42 30<S/ 27.11.3J 8 292X 27.10.81 306/ 26.11 . " 293/ 26.11 .27 2SO/ 22.11 .3* 322/ 26.S2.3a **I • SIGNIFICANT AT P LT O.OS •• SIGNIFICANT AT P LT 0.0! I INCREASED ABOVE CONTROL �Table 23 AVERAGE L'EAD 1MPIANTS PER FSEuNAtT =EMALE - £0 CONTROL "lu/Ms 74-3? K6/".G 40 .? Mr,'KG MULTIPLE TREATMENT 1 !3/ 26- .46 13/ 3-.« ,36 3*/ 23« 1.21 * 19/ J7- .•JO «/ 29" 2.1* ** 2 <3' 26" .31 16/ 2%» .5 5 15/ ?T« 19/ ?7, ,?0*« T7/ 2T« 2.85** e/ ll/ 24> .6 4 8T/ 32. 2.72** T/ 2i» .33* !/ ! 27" .41 * 3 •»A 23. .35 31X 3 . 3 .4 9 4 ?/ 27* .0'-' 2i/ 30* .TO** 5 ll/ 24- .4fc IS/ 29* .52 t £i/ 24m ,63 15.' 3C-» ,33**D T 3C.' 30* I.GJ 8/ S7s« .30 e/ 3 '*" ,. 27. 13/ 26- ,60 ia/ .TO * SIGMFICAhT AT P Lf 5.05 •' SIGNIFICANT AT P LT 9.01 D DECREASED BELCH CONTROL 31 / 2. 9 .56 .20 29" l.oT «* 6/ 2e," .23 ll/ 20= .5 5 22/ 3ft- .73 IT/ 27« .3 6 9/ 2fia ,32«. E IT/ 27. .3 6 2 . .32 5 19/ 2. * .79 li/ 27. .41 8/ £2i .36 11.' 26* .".2 2b. .e j �Table I-. CHI-SQUARE TEST OF THE DEATH INDEX - AZINPHOS-METHY'.. 1 DEGREE OF FREEDOM VEHICLE CONTROL MEEK M s hDI PRO DEATH INDEX CMisa 74-09 N N WOI PRG 20 74-09 HO/KG DEATH INDEX CHI SO N N tfOI PRG 40 MG/K6 DEATfi INOEX 74-09 HO NO/KG .2 K N .01 PRG OEUTH INOEx N PRO DE*Th INDEX 11 27 .41 .01 25 29 .flfi 13.27** 26 27 N CKiSO CMISQ C*iSG MULTIPLE TREATMENT 1 £ 10 2B .6 3 0.00 12 34 .5 3 .05 14 28 .27 0.00 11 29 .6 3 . 4 3 3 27 .0 3 .01 12 27 .4 * 1.09 ,9ft 24.26*" 16 33 .8 4 .18 5 29 .17 2.11 a 24 .33 .01 25 32 .78 7.05** 14 29 .6 4 9.53** 7 21 .33 3.65 a 27 ,30 3.07 6 26 .3 2 .* 6 7 20 .35 .02 10 30 .33 .00 9 it .33 3.25 £ 28 .9 2 10 27 ,37 2.36 .6 0 7 25 .8 2 ,0* iO 24 .*2 .6 7 9 27 .33 .07 .03 a 26 .31 .5 S 6 22 .36 .8 0 1G 26 ,3fl .03 2 7 26 3 9 23 .9 3 0.00 4 2 27 .07 0.00 13 30 .3 * 5 9 24 .8 3 0.00 13 29 .»5 6 15 2* .3 6 0.00 a 30 .7 2 .27 0.00 7 27 .6 2 .*» 0.00 10 7 8 30 a 12 27 * ** D SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED SELOV CONTROL 26 .8 3 7.70** .07 5.61 **D .0 5 .6 6 4.73 *D �Table 25 TCMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - AZIXPH.Oi-y.r.THl;L. «££* ?*-S9 CCfeSQOL 7*-09 20 74-09 KG/KG 60 MULTIPLE TREATMENT IS/ 31Q" ,0* 13/ 3TS. .03 34/ 300» .11 la/ 306" .36 62/ 316« .20" S 1,' 303- .03 ii/ 335, .OS IS/ 312- .OS 19/ 310* .06 ?7/ 293« .26** 3 */ 2*5. .0* 31/ 379S .8 0 .02 ll/ 272s .0* 67/ 348a . 5 2 " 4 iV 339* .01 2 I/ 367s .36 " .03* ll/ 266> .04* S 11 / 2?»« .0* IS/ 337» .0* .OS 22/ 356m .06 6 21/ 302« .07 \0/ 3*5» .33 **3 .03**S 17/ 873a .06 7 30 / 3*fes 8 l$/ £92» S0<» 7 .O 6/ 2ACSic .03 13' 306= . 0* • •• SIGNIFICANT AT *> LT O.U5 SIGNIFICANT AT P LT 0.51 I) DgCKFA.-3! BELOH COKTROL a/ 336* 31X 369* . 8 0 " ?/ 2*7« .02 ll/ 22Ea .6 0 9/ 323« 8/ 2 8 7. .03 19/ 262: .07 ll/ 306= .04 IO/ 293- .3 0 6/ J5o» .03 ll/ 322« -03 6/ 3iO» IT/ 276. �Table 26 CHI-SQUARE TEST OF THE FERTILITY INDEX - MALATHION i DEGREE OF FREEDOM V E H I C L E CONTROL N P»3 N FERT. NTo INDEX CHISQ 74.07 N N PRG MTD 1250 MG/KS FERT, INDEX CM I SCI 74-07 N N PRG MTD 2500 KG/K6 FERT. IN3EX CM 1 56 74-07 5003 MG/KG N N PRG MTD FERT, INDEX CM I SO MG/KG TEM N PRG MTO FERT, INDEX CHISU MULTIPLE TREATMENT 40 .5 6 .06 29 40 .72 0.08 29 40 ,7? o,c* 24 40 .0 6 ,05 31 39 .79 1.40 27 y* .1 6 .03 .00 24 40 .0 6 o.oa 33 40 .32 4.82*1 32 40 oflO 3.72 .6 6 .01 22 38 .8 5 .41 * ' 40 .67 .06 27 40 .? 6 .6 0 36 .8 5 .01 22 36 .61 • 01 34 *0 .5 8 S.08*I 30 40 .5 7 1.42 36 .8 7 19 34 .6 5 .15 35 40 .38 5.02*1 27 38 .71 .4 2 32 40 ,80 .03 27 36 .75 .92 37 40 .92 4.72*1 2* 36 .? 7 .2 0 0.00 31 40 .77 .6 2 ,65 0.00 30 40 .75 .4 5 4C .57 0.00 23 38 .61 27 40 .7 6 0.00 25 38 5 24 40 .C 6 G.OC 21 6 24 38 .3 6 0.00 28 1 28 2 26 40 3 23 4 40 7 30 38 s 27 36 ** I .8 7 .9 7 .71 0.00 30 36 0.00 30 36 SIGNIFICANT AT P LT 0.05 INCREASED ABOV3 CONTROL 1.26 26 .3 8 .03 19 34 .6 5 3.39 .3 8 .6 9 16 34 .47 3.36 �T » D l e 27 A.^HAftE IMPLANTS PER PREGNANT FSMALi CONTROL 74-Q? 12SO 74-07 2SOO MS/>6 530S MG/KS !£•( H'JLTIPLE TREATMENT iit,' 2 JliJ/ 3 *" 28*11.39 e-S/ * 3l^/ 29^10.90 3:^ii.*s 293S 27.10.85 33.12.18 *I 348/ 3.08 21.7 27.11.11 266/ 27. 9.85* 2.10 21.5 366 / 34.ll.jS 3So/ 30.11.87 1 . 11.89 9 391/ 35-^.17 •* 2T3/ 27.10.11" 19.18.37*1 352/ 32.il. C3 3I56/ 27all.33 16.11.69 ?;,3 397 / 3 . 0 7 32 ? / ?6.12.3B**l 31--IU16 26' 9. 2.16 61.5 363/ 3C»O2.10 267/ 24.11.12 356/ 23.J0.65 291/ 23.12.22 *T 305/ 2»«12.71-I 402/ 359v £7.11.44 304/ 25.12,16 256 / 22=11.6* 300/ 5 £7<i.' 2«.11,*£ 23G/ 91x11,33 2*3/ n JC-3^ 2»«12.5« J1S/ 2A<£S.3&* 226/ f 3*o/ 3.153 01.. 337' 30.il. 23 23S/ S £9^/ 2.06 71.i 323' 30»:0,T7 187/ * SlGNiFICANT AT P LT 0.05 •• SISKIF1CANT AT f LT 0.01 I IKCiLASED ABOVE COMTBOL 26.11.46 32 1/ 36' 4, £4.11.07 �Tahl*- 28 AVERAGE 3E*D IMPLANTS PER PREGNANT FEMALE - MAHTHIOS CONTROL 74-07 125i> MS/KG 7*-C7 2500 CS/KG 7»-07 5000 MGXKG TEM .? MULTIPLE TREATMENT 2. 6 .31 13/ 23* .45 «f/ 29= 2.14*' 2. * .& * 11X 31* .35 77X 2?^ 2.65" .61 e/ nx l*x 24* ,58 I4X 33. .? * 87X 32* 2.72** .6 5" 8X 22* .36* 7X 2. 7 .6 2 !1X 27a .*i* ax 22- .36 ex 34* .2* 22X 3C= .73 19* 1.05 23X 35* .6 6 l^X 27= .63 1 13/ 28* .6 4 lex 31. .8 5 2 8/ 26* .31 12/ 30. .0 * 3 9X 23* 14X 23* 2. 5 J4X 2«* .6 * */ 21X 24* .8 8 21X 28* .5 7 20X 7 3, 0' 39* i.CS lax 30* .33 6/ 19* .32 i5/ 32= .«7 iiX 27. .! * a 19X 27. )9X 30s .63 7X 16. .** 32X 37. .6 8 11X 26< .*2 2X 5 it/ 6 27. .39 .07 A .70 SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 21. .19 �Ti-ble 29 CHI-SQUARE TEST OF THE DEATH INDEX - HALATHIOli 1 DEGREE OF FREEDOM 7i-07 VErtlCLE CONT*0-_ N N dtl »RG OEAT* INCil H N 0£»TH ^ftc- £sie)i c*>ise C.iiStJ 74-07 -250 KG/KG N N SDI p»e 5300 2500 KG/KG i INDEX N P«S h CrU5G Ml m IMCES CM ISO DEATH INOEX CHISO MULTIPLE TREATMENT • 1C- 28 .( 3i (i.OO 13 31 .42 .05 T 26 .27 .16 JO 29 .34 .03 25 29 .6 8 13.27 " 30 .27 .8 0 9 24 .6 3 .5 2 a 31 .6 2 .4 0 26 27 .6 9 24.26 ** 11 24 .6 4 .03 9 33 .27 .41 25 32 .7fl 7.05 •* £ 7 iS .7 8 3.03 a 3 9 23 .39 S.OO 7 23 ,30 ,1G » ^ 3'f ,C> P. 06 10 25 .40 6.04* 3 22 .3 S 1.24 5 2' .19 .6 6 e 27 .30 3.07 = 9 2* .3 ,9 9. CO 3 Si .14 2.01 6 22 .27 .18 7 34 .21 1.36 10 30 .33 .0 0 2* .2 6 0,00 11 £8 .30 1.93 6 19 .32 Z.41 16 35 ,«6 l.Oi 10 27 .3? 27 , " .07 26 .38 .03 t> ' 8 • ** i% 9 ji 30 27 .2? .*<> 5.80 T 0. CO 1* SICHIFICAXT AT P LT fi.05 SICSIFICAXT AT f LI 0.01 30 30 ,Z3 .47 0. 90 .01 4 19 .21 .vil 12 se .3 3 .: * a / .44 ,C7 16 37 .43 .33 10 16 2.36 �T a b l e 30 KUIIBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - KALATHIOS ?*-07 5000 MG/KG MULTIPLE TREATMENT 1 13/ 319. .4 0 18/ 346, .05 3/ 298* .93 13/ 321* .04 62/ 316. .20" 2 e/ 303* .03 12/ 363* .03 ll/ 267= .4 0 ll/ 356« .3 0 ?7/ £93, . 6 2 " 3 9/ 2*5. .0* 14/ 281. .5 0 I*/ 305. .05 !*/ *02« .03 37/ 3 8 4 . .25** 4 2/ 309. .01 • I*/ 304. .OS" 256 . .03 7/ 300* .02 ll/ 266, .0»* 5 ll/ 27*. .0* */ 236= .02*0 ,03 8/ 386* .02 22/ 3 6 5 , .6 0 6 21/ 302. .07 21/ 31B> ,07 . 9 0 23/ 391- .6 0 ' 7 273, ,/ .06 7 30/ 3*6. ,09 10/ 337. .03 &/ 25 3. .03 IS/ 352, .04 ll/ 306, .04 3 19/ 292. .07 19/ 323. .6 0 7/ 187* .0* 32/ 397* .08 ll/ 322. .03 Ln • SIGNIFICANT AT P LT 0.05 •• SIGNIFICANT AT P LT 0.01 D DECREASED BELOW CONTROL e/ a/ 2 3 * . 20/ 226* �Table 31 CHI-S«?.i*aE T-iSY OF THE FERTILITY INDEX l DECRSE OF ?iu£.£.XHi *EEK N id •I: "TO '' 74-01 a.2.5 MQ/Kn VEHICLE *£RT. INGES Crfisa h N FERY> PRS HTO !*OE* CHI so N N 125). MS/KG FERT. ?4-01 H PRO NTO INDEX CHisa 230. Mt/KCi N MTO FEfi?. CnlSJ Hi N PMG MIR FEHT. : MDF. x CHISO CO MULTIPLE TREATMENT ?3 «0 .57 C.OC 2S 40 .63 .05 27 40 .AT »*e 22 40 .55 u.OG i:4 *0 .? 7 i.37 £ ?7 i9 .6% 3£ 40 .an .7i 23 40 .57 .72 23 •a .57 .72 27 jS .*<» .6 0 J 26 40 .6% £3 tO .i.7 ,ci 20 40 .50 1.29 76 40 .65 .OSs 32 *3 .HP 1.57 « 27 »0 .67 o.co B.OO e.oo 25 40 .63 .05 23 40 .7 5 .8 4 aa 40 .70 0.90 27 «0 .• '7 .06 5 S<> 40 .72 O.flS 25 40 .63 .51 25 40 .63 .SI 30 40 ,75 0.00 30 «1 .7S 0.00 6 2* 40 ,72 ».co s.os 26 40 .70 0.10 23 40 .7 8 1.3.' 32 40 .ao .2S 2F J'» .71 .01 >9 4n .•?? .0* 21 4G .52 2.61 2* 40 ,7* .6 0 c~> 36 .* 7 t.OI 33 49 .»2 t.ta ?<» 40 .72 .28 30 »0 ,*S ,0? if6 36 .7? .no .?(> ' • it 32 44 41 .? 7 .9 8 �T a b l e 3? AVEKWE IMPLANTS PER PHSGNANT FEMALE - PA3ATHION MEEK CONTROL 74-01 62.5 MG/KO 7*-01 125. M 5 / « 6 74-C1 250, M<;/KI; TF« MULTIPLE THEATNfcNT 1 23-11.52 266/ 25=10 . * 6 3!*/ 27-11 .63 22*/ 23=10 .IS* 316/ 29=10.90 2 tr 265/ 305/ 27x11.30 337/ 32=10 . 3 5 250/ 23-10 ,H7 23d/ 3 23--1C . 5 291/ 27-50. BS 26-10.31 25 7 / 23=11 .17 2«6/ 20-12 .30 **I 277/ 26=10 . 5 6 3»8/ 3iT=iO.S7 3 S68/ VD 4 298X 27-10.67 277/ 25-11 . 8 0 253X 23-11 .UO 311/ 2S=M .11 266X 27= W.85 5 33»X 29=11.52 299X 25=11 . 6 « 285/ 26-11 . 0 4 339/ 3 30=11 . 0 35*i/ 33=11.87 6 323/ 29-11.14 322/ 28=11 . 0 5 259/ 23-11 .it, 331/ 32-10 .3* 27?/ 27-10.11 7 323/ 29-11. !•» 322/ 29=11 .10 236/ 21*11 . * 2 339/ 29-11 . 9 6 30<i/ 27-11.33 3. 3o = ll . ? 33?/ 26=:2. 3« 8 9 •• I 38l/ 32-11.91 393/ S I G N I F I C A N T AT P LT 0.05 S I G N I F I C A N T AT P LT 0.01 INCREASED ABOVE CONTROL 33-11 .91 336/ 29=11 .<>9 35.5X �Table 33 AVERAGE DEAD IMPLANTS PER PREQ1AMT FEMALE - FAKATHIIW WEEK CONTROL 74-Cl fe2.S US/KG 74-01 12*>. MG/KG 74-01 250. «G/Ku TEM .2 «G/*C, HW.TIPLE TREATMENT 1 G.' 23= .35 IS/ 25- .72 ll/ 2 ll/ 2T- .*! !«/ 32« .44 3 it..' 26* .62 7/ 23- .30 10/ 4 16/ £7- .9 5 9/ 25s .36 5/ S !«/ 29» .53 IV ?5« S52 & 9/ 2. 9 .31 1G/ 2 . .36 6 7 2i/ 29« .72 35/ 29« a IS/ 32- .41 13/ • SIGNIFICANT AT P LT 9.OS <• SIONIFICANT »T P LT 0.01 D DECREASED BELOW CONTROL 33* 10/ 27» 2320- .41 8/ .3 4 IT/ .50 e/ .36 6?/ 29- iJ.!4** 23- .74 77/ 27* 2.85** 2b- .31 B7/ 32- 2.72** ll/ 27- 22- 42/ 28= 1.50 14/ 232. 5 .20 2* .6 5 22/ 2. 3 .39 303?= .37 */ ll/ I*/ .»4 21- 1.10 22/ 29- 6/ 3C« .52 23/ .9 3 9/ 2<». .31 .41 .73 17/ 30?T, .63 , 7e ll/ 27= .41 .20 I)/ ?*•= . 2 4 �X, Table 34 Cni-SQCAKE ITST ,r-F THE DEATH INDEX - PARATHlON 1 DEGREE OF FREEDOM VEHICLE CONTROL WEEK N N HOI PRO DEATH INDEX CHISB 74-01 N N MDI PRG 62.5 MS/KG DEATH INDEX CHISO 7*-01 N N WDI P«5 ?*-01 125. DEATH INDEX CMISQ 250. N >i HEATH • l!l PRO INDEX .? ChlSJ K N "Di P«S DEATH INDEX CHISU MULTIPLE TREATMENT 1 8 23 .5 3 0.00 6 25 .4 2 .5 2 9 27 .33 .4 0 5 22 .23 .32 25 24 .»<•> 12.49** 2 9 27 .33 0.00 11 32 .34 .04 B 23 .5 3 . 4 0 7 23 .30 .01 26 27 .9ft 20.7V** 7ft 7.8*3 ** 3 10 26 .8 3 0.00 7 23 .30 .08 8 20 .0 4 . 4 0 7 ?6 .27 .3-5 JS 3<» 4 4 11 27 .41 0.00 a 25 .32 .13 4 23 .17 2.^1 10 2B .36 .01 * tl .33 .32 25 29 .8 3 0.00 7 25 .8 2 .23 9 .6 3 .02 8 30 .27 .42 10 30 .11 .01 6 8 29 .8 2 0.00 9 28 .32 .01 7 23 • 30 01 10 32 .31 .00 10 27 .37 .22 7 12 29 .41 0.00 12 29 .41 .07 12 21 .7 5 .6 6 il 29 .38 0.00 9 2? •n .12 8 11 32 .34 0.00 11 33 .3 3 .1)3 9 29 .31 .uu 5 30 .17 1.70 10 ^fi .ia .nc 5 11 ** SIGNIFICANT AT PLT 0.01 �Table 33 3F OEAD IMPLANTS PER TOTAL IMPLANTS - PWlTHION 74-ui CONTBOL 74-0\ fc2.i 125. MG/KG 250. .? MULTIPLE TREATMENT 1 S/ 265* .03 IB' £66* .07 ll/ 314« .04 e/ 22*= .04 fc?/ 316= .20** ! / 337= 4 .04 10/ 250* .04 i7/ 23flr .07 77/ 293= .** 2.* 10/ 24fc. .4 0 B/ ?77« .03 97/ 34fl, . 5 2 " 42/ 311« .1* ll/ 266s .4 0 .6 3 Z v : s >• 305* .04 3 16/ ?68x .06 7/ 257. .03 ID/ 28Bs .06 */ 277, .03 5/ 253. . 02*D e S5/ 334. .04 13/ 299* .C4 14/ 2S5» .ns Jl/ 339= .03 2?/ 356s •- *' 323s .03 lu/ 322- .03 9/ 2 ^ 5 * .03 I*/ 331= .* 0 IT/ 273= .6 0 7 l\/ 323« .07 15/ 322= .5 0 23/ 236* .10 22/ 33<» .06 I!/ 30»i = .04 e 1 . 3313' .03 13' 393* .03 9/ 336> .3 0 6/ 355< .21 0*! ll/ 322= .03 * •• 0 SIGNIFICANT AT P LT 0.95 SIGNIFICANT AT P «.? 0.01 DECREASED SEUM COPT&OL �T a b l e 35 CHl-SQCAEUi TEST OF THE FERTILITY INDEX - PARATH ION-METHYL 1 3EGREE OF FREEDOM VEHICLE CONTROL -EEK M N PRG MTU PERT. INDEX CHISU 7»-OS PNG MTO 20 H6/KG FtRT. INDEX CNIbU 74-06 N N PRG MTU' »0 nb/Kb PERT. INDEX CHISU 74-Ci*; N N Prtfc KTD 80 Mlj/Ku TEW N i\ PKG M f D PERT, INCEX CHI SO .2 ' 1.37 29 iNOt* CH1SC* 40 .72 1.37 39 MULTIPLE TREATMENT 1 2 23 27 3 26 4 27 40 .57 0.00 29 40 .72 1.J7 24 40 .60 0.00 29 40 .9 6 0.00 33 40 .82 !.<* 23 40 .57 ,7«? 29 40 .72 .01 27 .9 6 .6 0 40 .5 6 0.00 30 40 .5 7 .4 3 32 40 .0 0 1.57 28 40 .70 .6 0 32 40 .80 1.57 40 .7 6 0.00 Jo 40 .5 9 26 37 .70 .00 £6 39 .67 .03 27 vu .o7 .06 7 39 e.,<i**i 5 29 4Q .2 7 0.00 .»2 »0 .0 8 .£8 2* *0 .6Q >b9 3j 4Q .75 B.CO 30 -»J • * 0.00 6 29 40 .2 7 0.00 33 40 .2 S .5 6 30 40 .75 O'bO 2» 4C .0 6 .9 3 £T 36 .71 .01 7 29 40 .72 0.00 36 40 .0 9 2.VS 32 40 .0 8 .c8 13 40 .2 8 .5 6 27 it, .75 .00 .5 8 .09 36 .72 .28 6 ** I 32 40 .0 6 0.00 SIGNIFICANT AT P LT 0.0! INCREASED ABOVE CONTROL 32 *0 .0 8 .6 0 35 40 .8 6 .7 3 34 40 26 �Table 37 AVEHAGE ISSPLASTS PER PREGNANT FEMALE - PAEATHION-iffill:«J. • ifcK 21) CONTROL 74-05 w.iLTIPLE 40 MG/KG Tt-O'S 80 .2 MG/K3 TRfcATHENT i £6S/ 23*11.52 30i/ 29-10,46* 26a/ 24.11.1? 303/ 29.10.45* 316/ 29=10.90 <J 33' 0. 27.11.3C 3»T/ 3.05 31.2 241/ 2 . 0 »8 31. 323/ 29.11.14 293/ 27=10.85 30s]0.20 3 ( 38' 6. 26*10.31 306/ 331/ 32.10. J4 297/ 28.10.61 348/ 32.10.87 -F 266/ 27« 9 8 .5 * 29S/ 27*10,67 41?.' 38«1«S9 27S/ 26-10.58 2Bi/ 26-10. Bl 5 334/ 29.11.52 39V 32*12.16 30C/ Z4.12.3U 36B/ 30^12.27 35&/ 30=11.87 27.10.11 6 323/ ! 323 / U 381/ * •• 29*11.14 2 . 1 1« 91. 32.11,41 366/ »0)/ 347/ SIGNIFICANT *T P LT 0.05 SIGNIFICANT AT P LT 0,01 334/ 30-11.13 279/ 24*11.62 273/ 36.11.14 357/ 32*11.16 349/ 33.12.09 306/ 27.11.33 32.10.d4* 407/ 3 . 1 63 S1. 402/ 34*11.62 322/ 26*12.38 33*11.39 �Table 33 AVERAGE DEAD IMPLASTS PER PREGNAitT rcMALE - PARATHrON-METHYi. 74-Ob CONTKOL 20 HG/Kli 74-05 »0 Mb/Kb 74-09 80 Mb/KG TtM MULTIPLE TREATMENT 1 8/ 23* 27. .35 9, 29- .31 .JO .! * 10/ 33= 2. 6 .62 8/ 30. .27 *D 16/ 27- .9 5 lb/ 3tl = 5 IS/ 29* .52 23/ a •*/ 2V. .oi 7 21/ 2. 9 e 13/ 32* 2 ll/ 3 16/ 4 8/ 24= 1.U4 * 23- . Jb 16/ .55 62/ 2<i= i | « .* .31 77/ 27 = 2.65** 2ea 1.21 S7/ 32. 2.72** 9/ 26= .35 ll/ 27= •41 12/ 30* .40 22/ 30* .73 .?<> 17/ 27= .3 6 9/ 29= 2<»» 22/ 32- .bS .7 4 b/ 26= . i« *D 32. .72 IT/ £4= .rl 20/ 33= .bl :o.-' 30= .' .i .72 7/ 36. .19 -3 ll/ 32. .34 lex 33* .48 ll/ ?7x .41 .41 ll/ 32= .4 3 18/ 35= .31 12/ 34- .35 ll/ 26= .42 Cn * •• D 25/ SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL 34/ fe/ 24= �Table 39 TEST OF THE DEATH INDEX - PABATHION-METHYL •itrt<. OLE ai.f.1. N N KOI PRO CONTROL DEATH IMOEX CM ISC 74-05 ft N *0i PRO 20 KG/KG C-£ATrt CM I in 74-Ob N h • 1 PR6 0 HG/Ku 40 DEATH iNDtX 7<t-05 * H SO YE* « «/!«-, ft N DCdlf CrUaG CnlSU .? M G /KG •ci P«G UtAT* INDEX CHI 51* NULTIPLE TREATMENT 1 $ 23 .5 3 C.09 t> £9 .S 2 .07 12 24 50 .8 5 9 29 .31 .30 25 29 .6 8 1 . 9 2 4 " 2 5 27 .3 3 0.00 9 33 .? 2 .5 0 b 23 26 .6 0 0 29 .28 .03 26 47 .6 9 20.79** a 33 3 10 26 .8 3 0.5C .27 .. 4' 14 32 »» .02 V <B .*<> .64 25 J2 .8 7 7.85** <• 11 27 .41 f,03 14 38 .37 .00 5 26 19 1.98 I 26 .27 .0 6 6 d7 .38 .32 * il 29 .8 3 0.00 11 32 .4 3 ,1)0 10 24 42 .00 10 30 .33 .01 1C 30 .33 .01 a 2* .28 0»00 11 33 .3 3 .5 0 6 30 27 .05 6 24 .5 2 • 01 10 £7 .37 .22 •41 0.00 6 36 •H 3. ' 4 B 32 25 1.18 13 33 .39 • 01 •i 27 .23 .12 .4 3 0,00 <* 32 .2* .JO 7 35 2u 1.10 & 34 .24 .9 4 1C 36 .6 3 .00 6 7 ; 2 a li •* C? 32 SIGNIFICAST iT f LT 0.01 �T a b l e 40 NUM3E3 OF DEAD [MPLANTS PER TOT^L IMFLWTS - PAH A T H I OS-METHYL «ttK CONTROL. 74-05 20 NG/KG 74-05 «0 KG/KG 74-0 = ItM 80 ,Z MULTIPLE TREATMENT 1 2 N B/ 265s ll/ 305s .03 9/ 301 = .0* 10/ 347. e/ 306. .03 6 25/ , 8 = .3V* id/ 303= .05 62/ 3lt>= .20** .03 B/ 241s .U3 9/ 323, .33 77/ 293» .2&*» .03 22/ 331. .a7 34/ 297s .11 8?/ 343. .5* 2. 9/ 281= .03 !>/ 266= .04 3 16/ 266s .06 4 16/ 288= .6 0 18/ 417= .04 5/ 275= 5 IS/ 334> .04 23/ 389= .06 17/ 300" .06 12/ 3fc8= .03 22/ 356= »C6 6 9/ 323= .03 20/ 366= .05 10/ 334 = .03 6/ 279= .02 17/ 2'3» .06 7 21/ 323= .07 7/ 401 = .02*D ll/ 357= .03 16/ 399s .04 ll/ 306s .34 0 13/ 381= .03 ll/ 347 = .03 18/ 407 = •<J4 12/ 402= .03 11 / 322= -03 J * •* D SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL «u2*D �Table 41 CHL-SQL'ARE TEST O? THE FERTILITY INDEX - QUISTOZ1&E. (PCKS) 1 DEGREE OF FREEDOM :c5C MS.'XG •tf.ti * »« FEBT. P3G ffj INDEX CMIS3 N M PAS HTQ IKOfc* CHtSa 7*-0» N PSS N MTO 2500 MG/KG FERT. INDEX CHISO ?<t-oe H N PRO MTO soco "M; TEM N CrllSQ Mfi/KG N PflG MTO fEBT. SHOP* c*'.sa MULTIPLE TREATMENT g , oc i 2 i f 4 0 . 0 7 .72 0.00 29 ( . j l - l 34 .62 .CO 40 .75 .6 0 31 40 .77 .6 2 29 *0 .? 7 0.00 22 40 .5 5 .7 4 26 40 .5 6 .5 0 27 39 .9 6 .03 30 2 ?6 40 . 65 3 23 »3 .57 O.OC 25 38 .6 6 .27 26 38 ,8 6 ,58 24 40 .72 1.37 32 40 .0 8 3.72 » f 43 .67 0.60 33 33 .87 3.09 30 40 .75 .2* 28 4C .0 7 0.09 27 *0 .7 6 .6 0 5 24 40 .60 0.00 19 39 .50 .4* 28 40 .0 7 .9 4 33 40 .2 8 3.91*1 30 *Q .5 7 1.42 6 2c 38 .63 0.00 33 38 .61 0.00 2 6 40 .5 6 .00 36 40 .90 6-47*1 27 38 .71 .4 2 7 *u 38 .79 0.03 27 38 .71 .26 27 40 .7 6 .8 7 28 40 .0 7 .2 4 27 36 .5 7 .02 31 40 36 .72 .02 e I 27 33 .71 C , 00 40 21 A. 00 SIGHIFICAHT AT F LT 0.05 INCREASED ABOVE COMTSDL JO 3n .T9 .8 2 .7 7 .IS 30 40 .5 7 .02 26 �T a b l f -,2 AVERAGK LMPLAOTS HER PREGSAST FEMALE - JUIYTOZESE (FCNE) CONTROL 74-OH 1350 MS/KG 74-08 2500 74-08 SOOC riu/KG TEW MULTIPLE TREATMENT 349/ 29*12.03 338/ 30 = 11. ,?7 361/ 6 31 = 11 . 5 316/ 29=10.90 303/ 26=11.65 222X 21=10.57 235/ 22=10.68 2S9/ 26= 9 .96* 293/ 27.10.65 3 ?*S/ 23=10.65 296/ 25=11.64 326/ 26=12. -54 «*I 353 / 29=12 .17*1 34B/ 32-10-87 4 309/ 27=11.44 392/ 33=11.88 349/ 30=11.63 317/ 28*11 .32 266X 27= 9.05* 5 27«/ 24=11.42 214/ 19=11. 26 342/ 28=12. <?1 376/ 33.11 . 9 3 356/ 30=11,87 6 302/ 24S12.S8 267/ 23=11.«,! 291/ 26=11.19 ** »00/ 36=11 . 273/ 27=10.11 ** 306/ 27«11.33 322/ 26»12.36"I 1 28:11.39 2 \o 319/ 7 8 346/ 292/ 30=11.53 27=10. 81 26// 3S4/ • S I G N I F I C A N T AT P LT 0.05 «• S I G N I F I C A N T 4T P LT 0.01 I INCREASED ABOVE CONTROL i:- 2T«10.63* 292/ 27=10. «1 30£/ 28=10 .79 30=11.80 336/ 31=10. H4 333/ 30 = 11 .10 �Table ii DEAD IMPLANTS PER P5EGSANT FEMALE - qiiIHIt.-7.ESz ...-Ys "<G/K& 74-08 2500 M6/K6 .2 Mfi/KG TTM 7»-Os MULTIPLE TREATMENT 31" .35 62/ ?<»* 2.1* ** fr/ 26» .23 77/ 2 . 2. 85 ** 7 29» .52 87/ 32» 2.72 ** 10/ 2e= .36* ll/ 27« .41 * .75 lb/ 33= .5 4 2?/ 3. 0 = 71 26= .35 **D 16/ 36s .44*0 I?/ 2. 7 .3 6 !/ & 27= .<-,<) ?0/ 28» .71 ll/ 27* .41 10/ 31= .32 )3/ 30: .43 ll/ 26* .2 4 i 13/ 28* ,*6 ll/ 2S> .38 12/ 30= . tO 2 5/ S6r .31 :a/ 21 = .5V 15/ 22= .8 6 9/ 23* .39 25= .52 7/ 26= .gl 15/ ^ 2/ 27* .07 15/ 33» . 5 * 4 * 7/ 30= .<-3 5 ll/ ». . .6 4 111/ 19. .53 ai/ 28= S SI/ 24s .88 9/ » JO/ 30* 1.00 1 1/ 27s .! * a 5<>/ ?T" i?/ 30 = .S7 ll/ 3 ^ 0 * •• D .70 ti/ 23= SIGNIFICANT AT i» LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELCH COXTROL ^35*0 �Tail? ii CHI-SQ'JARE TEST OF THE DEATH INDEX - QUIST'W.RN* (PCN3) 1 DECJREE OF Frl VEHICLF CONTROL U&E.K N •ni N PRG DEATH INDEX CHISO 74-06 N N • DI PKG 1250 MG/KG DEATH INDEX CHISO 74-03 N N WOI PR'J 74-oa 2SOO MG/KO DEATH INDEX sooo . 3 Mr,/KG N PHG N CiIbQ flEATh INDEX CnlSU N N •DI PKG DEATH INPF* CHJSO MULTIPLE TREATMENT 1 10 28 .3* 0.00 7 29 .24 .44 10 30 .33 .01 •* 31 .2^ .07 25 ^4 .8* 13.27 ** 2 7 26 .27 0.00 10 21 .4B 1.35 9 22 .! * .51 2 26 .08 2.15 26 £T .9ft 24.26** 3 q 23 .9 3 0.00 11 25 .44 .00 6 26 .3 2 .82 11 29 .38 .04 25 32 ."« 7.05** 4 2 27 .07 0.00 12 33 .36 5.44 * 6 3« .20 .97 7 28 .25 i.96 « 27 .3" 3.07 28 .6 4 .1* 13 33 .39 .02 10 JO .31 .00 8 26 .31 3.B6*D 12 36 .33 3.84*D 10 £7 .•*7 2.34 27 .2 5 2.01 10 28 .36 .21 9 £7 .IS .07 9 31 .9 2 .89 12 30 .40 .00 2ft .33 .03 5 9 24 .3fl 0.00 9 19 .47 6 15 24 .63 0.00 6 23 .26 7 8 30 .27 0.00 10 27 .37 .31 a 12 27 .4 4 0.00 13 30 .43 .03 * ** D SIGNIFICANT AT P LT 0.05 SIGNIFICANT AT P LT 0.01 DECREASED BELOW CONTROL .12 4.91 *D 13 14 10 �Table 45 .Tfflat; OF DEAD IMPLANTS PER TOTAL IMPLANTS - QUINTiSZiiN? ,.'£ f.«. 74-08 1250 COKTROL 2500 MG/KQ 74-88 ,2 MG/SG TF- 5000 MULTIPLE TREATMENT 1 !3/ 319- .04 ll/ 3*9= .03 12/ 33»> 2 »/ 303- .03 1 . 222= 2' .OS IS/ 235. 3 9/ 245- .04 13' 296- .4 0 7/ 326= 4 2/ 309- .01 IS/ 392- .04** 7/ 349« .03 t,gf 316* 1 .20** 6/ 259- .02 77/ 293= .26** • 1 2 »D ) 15/ 353- .04 B7/ 34«- .* 2* .02 10/ 317- .03* ll/ 266* .4 0* .6 0 16/ 376s .4 0 22/ 3^6* .Oft .03 *D 16/ 400- .* 0 IT/ ?73= .6 0 .6 0 ll/ 2?»» .04 10/ 214- b SI/ 302- .07 fl/26?- 7 30,' 3 «6- .09 ll/ 287- .04 16/ 292* .05 20/ 302= .07 ll/ 306 = .04 .0? I*/ 354 = .5 0 10/ 33«> •03 13/ 333= .04 ll/ 322- • 03 a I?/ 2?2 • SIGNIFICANT AT P LT 0.0«> •* S I G N I F I C A N T AT ° LT 0.01 D DECREASED BELOW COHTXOL .02*0 21/ 342= ll/ 361- S e .OS .4 0 9/ 291 = �Table 46 CHI-SQUARE TEST OF THE FERTILITY INDEX - PHOBATE 1 DEGREE OF FREEDOM • EEK VEHICLE. CONtHUL N N PRO HTO FF.RT. INDEX CHISU 74-04 K N PKb MTU MG/Mi •> FtUT. INDEX CHI SO 74-u* N N PHG MTU 10 Mb/Kb FEBT. INDEX CnlbQ MO/KG 20 N N PHG MTO PERT. IfcDE.lt CHISU N PHli H F U fEKT. INDEX CHISU U) MULTIPLE THEATNENT 1 23 40 .57 0.00 2J 40 .57 .US. 21 40 .52 .t>5 . 4 40 .0 6 o.oo 29 40 .72 1.37 2 27 39 .9 6 0.00 30 40 .75 .10 2J 40 .i>7 .72 29 40 .72 .01 27 J* ,(r,9 .Ob 3 26 40 .5 6 0.00 2b 40 .65 .US 24 40 .60 .liS 31 40 ,77 ."« 32 40 .0 8 1.57 4 27 40 .7 6 0.00 26 40 .65 0. 00 26 40 .65 O.UO 32 40 .0 8 l.OJ 27 40 .*" .06 .72 0.00 #> 40 .3 6 .» !! 27 40 .7 6 .6 0 31 40 .77 .07 30 40 .75 25 40 40 .5 6 .3 4 33 40 .2 8 .S 6 21 3* .71 27 Jb .75 .01' 20 JO .72 .21* b 29 40 b 29 40 .72 O.OU .3 6 .31 26 7 29 40 .72 0.00 30 40 .75 O.UO 29 40 .72 .06 Ji 40 .0 8 B 32 40 .9 8 0.00 26 40 .65 l.»7 29 40 .72 «<B 30 40 .75 1.95 .07 0.00 .01 �Table 47 AVBRAGE IMPLANTS PER PREGNANT FEMALE - PHOIiAT2 7«-0* CCK'SOL 10 NO/Rti .2 TtM 7»-0« MULTIPLE TRLATHtNT 1 ?65/ 5 23-11 . 2 2* I/ 23»i0.te* 221/ 24-11 .21 316/ 29-10.90 30i/ 27-11 .30 326/ 30*10.37 2SS/ 10 .52* 212 .11 . 9 3 0 269/ i 3U7/ 29-10 . 9 5 293/ 27-10.05 3 268/ 2 . .31 6 10 29«/ 26-11.50*1 257/ 2 .10 .71 * 34i/ 31-li .03 3«B/ 32=10.87 * 6 288/ 27.10. 7 2S>1/ 26-11.19 277/ 353/ 32-11 .03 266 / 27- 9 8 .5 S J3*/ 2 .ii .52 * 2?B/ 2b=S1.12 31Z/ 10 6 26- . 5 27.il eb6 6 j2 )V 2 . .1* 9 11 305/ 7 32j/ 29.11 .1* J36/ B .»Bi/ 32- il .91 jib/ AT P I.T 0.05 ** SJG'v^ICANT AT f LT C.C1 1 IHCBEASED ABOVE CONTROL 25.12.20*1 30.11.20 2.:.2 6'21 295/ 3l?/ 33b/ 3 26- li . 5 369/ 31-11 .90 356/ 30-11.87 384/ 33.11 .79 273/ 27.10.11 27.11.33 26-12.3B 10 . 3 * »07/ 35-11 . 3 6 306/ .5 6 J24/ 30*10 .do* 32^/ **- 29- 11 �Table 48 AVERAGE DEAD IMPLANTS PER PREGNANT FEMALE - PHORATE MG/KG CONTROL r<t-Ut 10 Mti/nG f»-0» 20 Mb/Kb .2 FtM MuLTIfLE THEATHfcNT 1 a/ 23* .35 9/ 23" .39 10/ 21* .*H 3/ 2** .13 6?/ 29= 2.1* ** 2 ll/ 27* ,»1 lt>/ 30* .53 «/ 23* .U 17/ 29* .9 5 77/ 27» 2.85 ** 3 16/ 26* .62 1J/ 26* .50 U/ 24* .SO IS/ 31s .B * 87/ 32- 2.72 ** * 16/ 27s, .59 15/ 26s .SB 26/ 26s l.DO 12/ 32* .6 3 ll/ 27= .41 3 IS/ 29s .52 13/ 25s .S2 ll/ 27* 13/ 31* ,»2 22/ 30* .73 6 9/ 29s .31 13/ 25s .52 7/ 26* .27 7/ 33* .21 30* .43 2i/ 29* .76 7/ 35* 26* .38 26/ 29. .90 ll/ 33* 7 21/ 29* .72 U/ 8 13/ 32s ,»1 10/ • •• D S I G N I F I C A N T AT f LT 0.05 S I 6 N I F I C A H T AT P LT 0.01 DECREASED BELOW CONTROL .! * .37 17/ 27s .63 IS/ 27* .41 ll/ 26* .4? �Table 49 CHI-SQl'ARE TEST OF THE DEATH INDEX - PHORA7E 1 DECREE OF FREOXtt i?Et* ^i.'ilCLE CONTntn. N N »DI Pkb QEATrt INOex V«. ~%K 5 MCi/Kb f. PMC CH;Sw SftDEx CHIiU 74-U* N N • 01 PR6 10 HG/Ku 0£ATH 1NUEX T -.;» 20 N N CH1SU N OE4TM iHliCa .?. HG/KG TEM W<!/KB " N CniSu UEATH INDEX CHI SO HOUTlPLt TREATMENT 25 <:.-> .86 12.49 ** .37 26 ^7 .96 20.7*** .32 .04 25 32 .78 7.b5** 34 .34 .05 8 ^7 .30 .32 1 a 23 .35 w.OO d 23 .J5 .10 7 21 .33 .US 2 24 .00 3.45 2 9 27 .33 U.tO 13 30 ,»3 ,«!S 2 23 .09 3.07 13 2V .45 3 10 « .33 3.00 T s!fe .27 .JS U £4 .50 .,49 10 31 4 11 27 .*k a. 00 11 26 .42 .03 IS) 2b .5tt .»2 11 1 5 il 2<s .38 I . CO Id 25 .48 ,£2 7 2T .26 .46 11 31 ,3S .01 10 JO .33 .01 6 0 2S .28 0.00 10 25 * 40 .46 7 26 .27 .Ob 7 33 .21 .00 10 <*7 .37 .22 7 12 <!9 .41 IJ.CO S 30 .30 .41 15 29 .52 ,*B » 3* .!« 4.66*D 9 e? .33 .12 b,UO 4 26 ,35 .07 14 29 .48 .71 a 30 1C ft, .38 .00 a * ** D ii 3£ .3* SIGNIFICANT AT F Lt 0 0 . 5 SIGNIFICANT AT P LT 0 0 . 1 DECREASED BELOW CONTROL .27 .19 �T a b l p 50 NUMBER OF DEAD IMPLANTS PER TOTAL IMPLANTS - PRORATE • EEK MG/Ku CONTROL /4-0* 74-04 10 20 Mb/Ho MG/KQ ICM MULTIPLE. TRtATMtNT .01 *D 62/ 316= .20 ** 17/ 307 = .06 77/ 293= . 6 ** 2 .) 15 IS/ 341 = .04 67/ 348 = .25 ** 26/ <iT7« PU 9 12/ 3S>3= .03 ll/ 266= .0* .05 ll/ 312= .04 13/ 369= .04 Z2/ 356= .06 13/ 3ub = .0* 7/ 29j= ,o2 7. 1-9= .' .02 )7/ 77.1= .6 0 .07 13/ 336= .04 i'i./ 317= .' 0 7/ 407 = .02*0 ll/ 306= .4 0 .3 0 10/ 31b= .03 26/ 33b= . l)B ll/ 324= .03 ll/ 322= .03 .04 10/ 221= .U= 3/ * , . .* lb/ 32b= .05 4/ 2b5= .0^ .Ob 13/ 299= .04 12/ 257= 16/ 288= .06 lb/ 291 = .OS S IS/ 334= .04 13/ 278= 6 9/ 323 = .03 7 21/ 323= 8 13/ 361* 1 8/ 265 = .3 0 2 ll/ 305» .04 3 6. 16/ 2 8 4 • •• D 9/ 241= SIGNIFICANT AT P UT 0.05 SIGNIFICANT AT P UT 0.01 DECREASED BELOW CONTROL �Table 51 DMA REPAIR SYNTHESIS ASSAY OF Monocrotophos (dpm/u« DNA) 4NQO (M) Monocrotophos (M) Sample 1 _£_. tt 65 io-7 io-6 io-8 IO"4 lO-3 46 31 54 70 64 1354 46 46 86 1273 10-5 2 35 42 3 39 39 25 40 36 69 1306 4 34 34 25 40 40 51 975 5 30 26 53 64 64 972 6 20 32 -f 38 46 _.t 32 37 50 92 71 1135 —f 47 1169 SD 7 7 10 7 14 15 168 SE 6 3 5 3 & 6 69 Mean 9* Sample deleted from calculations because of low DNA value. Only five samples used. Cell culture and exporiosontal condj.t_ions T-25 flask cultures of passage 24 WI-38 cells were initiated in medium containing iO% serum. The medium was replaced with medium containing 0.5% sarv.m on day 5 following initiation and subsequently on days 11 and 15. Tho assay was conducted on day 22. Hyriroxyurea (10~*M) preincubation = 1 hour. Compound exposure time = 3 hours. 3 li~TdR added with compound. 3 H-TdR incorporation = 1 uCl/ml (S.A. ==6.7 Ci/mmole), 3 hours. Postincorporation incubation = medium containing TdR, 3/4 hour. Cells were removed with IN NaOH. 1 minute, 70°C. DNA was oxtfflcted by the PCA-hydrolysls procedure and measured following reaction with dtphenylamlne. S«j;aUvu control and compounci solvent -~- 0.5% EtCH. 78 �Table 52 DMA REPAIR SYNTHESIS ASSAY OF Monocrotophoa WITH METABOLIC ACTIVATION (dpm/ng DMA) _Monocrotop_ho8_ OjO DMN (M) 0_ 10-^ 10-J j°Jll_. Sample 1 55 67 43 87 206 2 54 66 48 84 220 3 52 43 52 82 223 54 59 48 84 216 2 14 4 2 9 1 8 2 1 5 Mean SD S E 5 X 10"' Cell £ulture_and experimental conditions T-25 flask cultures of passage 24 WI--38 cells were initiated in medium containing 10<jfe serum. The medium was replaced with medium containing 0.5% serum on day 4 following initiation and subsequently on day 10. The assay was conducted on day 16. Hydroxyurea (10~2M) preincubation - 1 hour. Compound exposure time = 1 hour, with the 9,000 g fraction of a mouse liver homogenaie. 3 H-TdR added with compound. 3 H-TdR incorporation = 1 p,Ci/ml (S.A. =6.7 Ci/mraole), 4 hours. Post.incorporation incubation = medium containing TdR, & hour. Cells were removed with IN NaOH, 10 minutes, 22°C. DNA was extracted by the PCA-hydrolysis procedure and measured following reaction with diphenylamine. Negative control and compound solvent = 0.5^ EtOH. 79 �Table 53 DNA REPAIR SYNTHESIS TESTING OF BROMACIL (dpm/ug DNA) Broroacll (M) _4NOO_(M) -- J>*. IP"7 10~6 10"5 ID-'*1" 10-3t 195 153 212 230 207 201 148 129 212 215 121 248 178 179 231 144 152 204 144 137 200 165 28 12 Sample ! 2 3 4 5 6 Mean SD SE 217 298 218 48 19 156 187 182 251 199 32 13 218 184 220 193 38 15 240 178 209 34 14 Negative control and compound solvent =0.5% DMSO. Slight precipitate observed at 10~3 M and 10™1* M 80 2670 2850 107 2688 2702 80 2438 74 87 50 20 2662 2668 105 134 55 �Table 54 DNA REPAIR SYNTHESIS ASSAY OF BROMACIL WITH METABOLIC ACTIVATION (dpm/pg DNA) Bromacll (M) DMN (M) 0 10~7 10-6 10-5 10-* ID'3 113 102 141 191 112 174 91 102 120 161 400 117 110 102 110 178 397 102 149 131 158 189 82 91 135 218 152 104 167 136 170 96 126 190 145 165 100 119 165 141 SD 41 30 13 37 23 SE 17 12 5 15 9 185 141 133 155 24 10 5 X 10"2 Sample 1 2 3 6 Mean * vample lost. 81 * 529 645 448 484 105 50 �Table 55 DNA REPAIR SYNTHESIS ASSAY OF CAOODYLIC ACID DNA) Cacodylic Acid (If) 4NQO (H) o* io-7 io-3 10-s io-4 1 . at 0 Sample 1 61 41 47 _-* 31 36 1891 2 31 48 27 -_* 27 19 1681 3 32 59 25 63 30 45 2418 4 L8 32 68 29 19 2245 5 25 39 38 __* 23 22 38 1430 35 22 __* 29 28 36 2275 Mean 34 40 34 46 28 32 1990 SD 15 13 10 23 3 11 387 SE 6 5 5 11 1 4 158 6 Negative control and compound solvent =0.5^ DMSO. t Slight lowering of pH at 10~3 M. 4' Sample lost. 82 10-6 �Table 56 DMA REPAIR SYNTHESIS ASSAY OF CAOODYL1C ACID WITH METABOLIC ACTIVATION ONA) Cacodylic Acid (M) DMNjM) 0* io-5 10- « io-3 Sample 1 44 25 21 29 381 2 30 33 22 25 384 3 23 39 28 21 339 Mean 33 32 24 25 368 SD 11 7 4 4 25 SE 6 4 2 2 15 Negative control and compound solvent =0.5$ EtGH. 83 5 X 10~2 �Table 57 DMA REPAIR SYNTHESIS ASSAY OF CAFFAN DMA) Gaptan (M) 8 io- 4MQO(M) fl --12LL io- 10~ 5 10-" IP"5 Sample l 37 43 41 74 81 8 924 2 64 58 53 60 81 6 947 3 76 50 68 52 57 7 1106 4 76 60 73 37 51 5 801 5 63 61 56 50 72 5 760 6 66 44 66 82 65 6 884 Mean 64 51 59 59 68 6 904 SD 14 8 12 16 12 1 122 SE 6 3 5 7 5 Negative control and compound solvent = 0.5$ DN5O. 0.4 50 �Table 58 DNA REPAIR SYNTHESIS ASSAY OF CAPTAN WITH METABOLIC ACTIVATION DNA) )_ io~5 10-* 5**)l.l*?i io-3 5 x io-a Sample 1 30 53 89 7 i 2 40 __t 50 73 5 323 48 71 5 384 35 50 77 6 353 SD 7 2 9 1 43 SE 5 1 5 0.6 31 3 Mean Negative control and compound solvent =0.5$ DMSO, Sample lost. 85 �Table 59 DNA REPAIR SYNTHESIS ASSAY OF CHLOROPYRIFOS (dpm/,ig/DNA) Chloropyrifos 4NQO (M) io-5 JL _10-7_ 115 2801" 282* 2 1 143 " 129 93 110 67 3 98 102 84 60 <t 72 95 64 110 _„* 52 —* 68 5 97 89 99 98 64 37 1208 6 78 - 98 85 86 79 49 1209 Mean 92 103 85 101 64 66 1339 SO 17 15 13 11 9 31 220 SE 7 7 6 5 4 15 98 Sample i 10-6 TO-5 KM __* 6.1 HT3 109 __* Negative control and compound solvent = 0.5^ DMSO. Sample deleted from calculations because of low DNA value. Sample loKt. 1337 —* 1721 1220 �Table 6G DNA REPAIR SYNTHESIS ASSAY OF CHLOHOPYRIFOS WITH METABOLIC ACTIVATION DNA) DMN (M) Chloropyrifos (M) 10- 10-4 10-" 5 X 10~2 Sample 1 72 79 70 52 355 2 75 65 75 71 349 3 55 67 63 79 384 Mean 67 70 69 67 363 SD 10 8 6 14 18 SE 6 5 4 8 11 Negative control and compound solvent =0.5$ DMSO. 87 �Table 61 DEN REPAIR SYNTHESIS ASSAY OF DINOSEB (dpm/^g DNA) Dinoseb (M) 4NQO (M) _°: .10-' 115 101 67 103 106 2 143* 101 68 100 100 3 96 112 54 116 79 1721 4 72 61 58 62 66 1220 5 97 57 63 60 67 1208 6 78 58 73 60 76 1209 Mean 92 82 64 84 82 1339 SO 17 26 7 25 17 220 SE 7 11 3 10 7 98 Sample 1 _L2I± ' ! • 11 •• • _iP±+ Negative control and compound solvent = 0.5$ DMSO. Suggestion of precipitate at 10-" M. Sample deleted from calculations because of low 'DMA value. § Sample lost. 88 HlL. 1337 __ § �Table 62 DMA REPAIR SYNTHESIS ASSAY OF DINQSEB WITH METABOLIC ACTIVATION (dpm/pg MA) - «M»._.n«, 0* Dinoseb (M) , J. _v 1 * . . w- _ _ __ . 10-5 10~" 10-3 DMNjM) 5 X 10-a Sample 1 72 93 76 71 355 2 75 81 80 64 349 3 55 51 58 89 384 Mean 67 75 71 74 363 SD 10 22 12 13 18 SE 6 12 7 8 11 Negative control and compound solvent =0,5$ DMSO. 89 �Table 63 DNA REPAIR SYNTHESIS ASSAY OF DSMA (dpm/>g DNA) DSMA j>! (M) io-7 ig-g 1Q-S 10- y io-3 Samp Le I 67 51 79 86 100 62 2 58 54 67 64 155 49 3 44 36 99 64 35 58 4 55 36 400* 45 44 59 5 79 62 74 53 55 68 6 105 32 75 107 89 69 Moan 68 45 79 70 80 61 SD 22 12 12 23 45 7 SE 9 5 5 9 18 3 Negative control and compound solvent = H2O. Sample deleted from calculations because of low DNA value. 90 �Table 64 DNA REPAIR SYNTHESIS ASSAY OF DSMA HTITH METABOLIC ACTIVATION (dPm/|ig DNA) DSMA DMN__(M) (M) io-5 10 -< 10-3 5 X 10" Sample 1 44 28 25 38 381 2 30 36 29 36 384 3 23 21 32 28 339 Mean 33 29 29 34 368 SO 11 8 4 5 25 SE 6 4 2 3 15 Negative control and compound solvent - 0.5$ EtOH. 91 �Table 65 DNA REPAIR SYNTHESIS ASSAY OF FENTHJON (dprn/u-g DMA) ', L Fenthion (M 0* Sample 1 89 2 3 43 107 4 10-6 10-5 4NQO (M) 10-j 10~3t io-s 65 105 154 37 64 2983 337* 34 67 2272 83 85 40 36 2552 62 46 54 63 63 4059 5 61 34 102 31 44 1728 6 94 51 44 —§ 33 1893 Moan 76 64 88 41 51 2583 SO 24 26 44 13 15 857 SE 10 11 18 5 6 350 •a Negative control and compound solvent ~ 0.5$ EtOH. Precipitate observed at 10"3 M. ' Sample deleted from calculations because of low DNA value. § Sample lost. 92 �Table «6 DNA REPAIR SYNTHESIS ASSAY OF FENTHIOH WITH METABOLIC ACTIVATION DNA) Fenthion J.M) _0?. SSLUtiL 10-JL 10~ * 10 ~JL 5 X 10~a Sample 1 55 54 30 51 206 2 54 50 46 64 220 3 52 42 64 63 223 54 48 57 60 216 SD 2 6 10 7 9 SE 1 4 6 4 5 Mean Negative control and compound solvent - O.S^t EtOH. 93 �Table 67 DM REPAIR SYNTHESIS ASSAY OF FOLPET DNA) Folpet (M) 4NQO(M) * Sample 1 37 43 63 45 82 29 924 2 64 58 62 54 58 25 947 3 76 91 108 52 92 31 1106 4 76 83 91 92 65 26 801 5 63 107 72 70 85 31 760 o 66 60 84 104 107 29 884 Mean 64 73 80 71 82 28 904 SO 14 24 18 25 18 2 122 SE 6 10 7 10 7 1 50 * Negative control and compound solvent =0.5% DM80. 94 �Table 63 DNA REPAIR SYNTHESIS ASSAY OF FOLPET WITH METABOLIC ACTIVATION (dpm/yg DNA) DMN JM^ _F°lP_et ($ 0* 1 0~" ID-- ID'3 5 X 10~2 30 49 49 —_ T 40 __t 54 63 82 40 323 58 384 35 52 49 353 SD 7 9 43 SE 5 3 2 98 81 18 10 5 31 Sample 1 2 3 Mean 54 Negative control and compound solvent Sample lost. 95 m 0.5% DMSO �Table 69 DNA REPAIR SYNTHESIS ASSAY OF AZINPHOS-METHYL DNA) Azinophos-raethyl (M) JL! 1^ 2 10-" 102 145 99 99 .115 3 77 4 4NQO (M) io-5 10- « 12± 264* 51 55 804 103 99 105* 39 924 96 71 82 55 33 629 97 125 100 __§ 80 85 34 856 5 85 93 79 57 97 761 6 111 n 72 56 68 35 897 95 108 89 79 63 49 822 12 25 16 15 14 25 87 5 10 7 7 6 10 36 Sample 1 Mean SD SE 10- * Negative control and compound solvent - 0.5% DMSO. t Precipitate observed at .10"3 M. t Sample delated from calculations because of low DNA value. 5 Sample lost. 96 �Table 70 DNA REPAIR SYNTHESIS ASSAY OF AZINPHOS-METHYL WITH METABOLIC ACTIVATION (dpm/yg DNA) E>MN (H^ _0_* -!E1 -.19.7.1 JLSZi 5__x_ur_* --1" Sample 1 30 70 75 42 2 40 66 65 55 323 —f 35 78 41 54 384 71 60 50 353 SO 7 6 18 8 43 SE 5 3 10 4 31 3 Mean itNegative control and compound solvent =0.5^ DMSO. Sample lost. 97 �Table 71 DNA REPAIR SYNTHESIS ASSAY OF MALATHION (dpm/pg DNA) Mala thion (M) 4NQO (M) _o*. 10- lOTi 10-5 10-" 1C! 110 128 124 SE 6 9 4 29 12 90 126 67 113 91 156 107 31 13 33 130 116 127 143 128 144 78 74 119 132 127 112 SD 123 125 106 100 114 138 118 14 Sample 1 2 3 A 5 6 Mean 111 86 133 116 110 111 15 6 * Negative control and compound solvent • 0.5% EtOH. 98 34 23 44 39 40 35 7 3 1943 1626 1538 1264 1737 1651 1626 225 92 �Table 72 DNA REPAIR SYNTHESIS ASSAY OF MALATHION WITH METABOLIC ACTIVATION (dpm/jig-HJNA) Halathion (H) 7o-r DHN (Mj_ 0* io-5 Sample 1 55 48 46 38 206 2 54 49 52 48 220 3 52 62 55 223 54 51 37 41 5 6 9 3 4 5 Mean SD 2 53 8 SE 1 5 1 0™ * Negative control and compound solvent =0.5^ EtOH. 99 5 X IO-2 216 �Table 73 DNA REPAIR SYNTHESIS ASSAY OF METHOMYL DNA) Methomyl (M) 0* io-7 Sample 1 135 2 133 3 165 —f 100 4 72 S 6 io-« 10~s 4MJO (M) io-« ID'3 10"6 116 117 126 88 1442 133 108 116 69 1544 129 97 122 69 1518 98 97 115 109 69 1385 104 85 103 109 70 1423 103 95 93 116 139 130 61 118 94 112 118 71 SD 32 6 17 115 14 f — 1462 9 9 67 SE 13 3 7 6 4 4 30 Mean 95 Negative control and compound solvent =0.5^ DMSO. Sample lost. LOO �Table 7-\ DNA REPAIR SYNTHESIS ASSAY OF METHOMYL WITH METABOLIC ACTIVATION DNA) Methpmyl (M) DMN(M) 0* 10-° 1C-4 ID'3 Sample 1 44 26 22 24 381 2 30 25 20 25 384 3 23 22 26 30 339 Mean 33 25 23 26 368 SO 11 2 3 3 25 SE 6 1 2 2 15 ft Negative control and compound solvent - 0.5$ EtOH. 101 5 X 10-a �Table 75 DMA REPAIR SYNTHESIS ASSAY OF MDNUUON DMA) Monuron (M) _°! Sample 1 135 2 133 3 165 4 io-7 113 __* io-6 4NQO (M) io-5 10-' 86 97 83 48 1442 93 88 72 47 1544 10-3' ^ » ^ W « * H 89 77 81 46 1518 72 129 __* 83 75 82 49 1385 5 104 118 92 77 84 45 6 103 131 110 109 85 38 1423 _J 118 123 92 87 81 46 1462 32 8 9 14 5 4 67 13 4 4 6 2 1 30 Mean 3D SE Negative control and compound solvent -'• 0.5% DMSO. Precipitate observed at 10"3 M. t Sample lost. 102 , �Table 76 DNA REPAIR SYNTHESIS ASSAY OF MONURON WITH METABOLIC ACTIVATION DNA) Monuron (ll) .£ 10-" 10- «_. _12I',. 5 x 10~a -t Sample J. 30 78 81 74 2 40 88 68 74 323 3 ~r ' 92 63 88 384 35 86 71 79 353 SD 7 7 9 8 43 SE 5 4 5 5 31 Mean w Negative control and compound solvent =0.5^ DHSO. Sample lost. 103 �Table 77 DNA REPAIR SYNTHESIS ASSAY OF MSMA (dpm/jig UNA) MSMA < k 4NQO (M) 01 ) 10-8 10-4 io-3 10-" 108 60 68 902 84 93 53 93 52 39 1241 44 114 68 61 66 61 1380 4 55 1 235 " 50 63 66 67 990 5 79 75 75 48 60 1087 8 105 53 66 __* 50 59 75 971 Mean 68 73 77 60 64 62 1095 SD 22 29 23 9 16 12 182 SE 9 13 9 4 7 5 74 0* 10-' Sample 1 2 67 38 58 3 10-" Negative control and compound solvent = H20. Sample deleted from calculations because of low DNA value. Sample lost. 104 �Table 78 DNA REPAIR SYNTHESIS ASSAY OF MSMA WITH METABOLIC ACTIVATION (dpm/^g DNA) MSMA (M) -.0! DMN (M) JL°:L 10~5 io-3 JLJLlfiL1. Sample 1 44 27 21 32 381 2 30 25 35 25 384 3 23 21 34 24 339 Mean 33 24 30 27 368 SD 11 3 8 4 25 SE 6 2 5 3 15 •ii- Negative control and compound solvent =0.5^ EtOH. 105 �Table 79 DNA REPAIR SYNTHESIS ASSAY OF PARATHION (dpm/pg DNA) _Q^ Sample 1 2 3 4 5 6 Mean SD SE iQlL Parathlon (M) IP"6 10-5 123 127 151 221 125 106 100 114 138 118 14 6 129 135 124 137 145 133 7 3 155 135 200 160 210 169 29 12 129 157 137 155 126 154 35 14 4NQO (M) IP"5 io± 10- 102 94 90 104 93 116 102 1626 1538 1264 1737 93 100 10 4 1651 1626 225 92 83 72 93 71 86 13 5 * Negative control and compound solvent - 0.5% EtOH. 106 3 1943 �Table 80 DNA REPAIR SYNTHESIS ASSAY OF PARATHION WITH METABOLIC ACTIVATION DNA) Parathion £M) DMN (H) 0* 10-s 10- « 10-3 Sample 1 44 33 42 20 381 2 30 33 32 23 384 3 23 35 30 30 339 Mean 33 34 35 24 368 SD 11 1 6 5 25 SE 6 1 4 3 15 Negative control and compound solvent -0.5% EtOH. 107 5 x 10- * �Table 81 DNA REPAIR SYNTHESIS ASSAY OF PARATHION-METHYL DNA) Parathion-Methyl (M) . £ 4NQO (M) 10-7 10~8 10~B 10~4 10~3 t 10~5 Sample 1 36 36 33 34 44 40 2 34 38 32 .55 44 23 411* 781 3 8G 49 28 31 53 28 782 4 94 56 27 52 41 29 858 5 53 49 35 43 40 29 1296 6 112* 46 41 44 27 ,1103 Mean 61 .6 4 33 85 50 44 ,2 .8 964 SO 28 8 5 20 .5 6 227 SE 13 3 2 8 2 3 102 T,* Negative control and compound solvent = 0.5% EtOH. Precipitate observed at 10~3 M. * Sample deleted from calculations because of low DNA value. 108 �Table 32 DNA REPAIR SYNTHESIS ASSAY OF PARATHION-METHYL WITH METABOLIC ACTIVATION DNA) Parathion-Methyl (M) •M- DMN (M) 0 10- 10-* 10"" "^ Sample 1 55 44 65 51 206 2 54 54 52 48 220 3 52 57 37 45 223 54 52 51 48 216 SD 2 6 14 3 9 SE 1 4 8 2 5 Mean Negative control and compound solvent =0.5$ EtOH. 109 5 X 10~a �Table 83 DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB) DNA) PCNB (M) j£ Sample 1 2 61 3 32 4 18 5 25 6 35 31 Mean 34 SD 15 SE 6 JO"7 io-« IP-6 10-* 35 43 33 21 27 23 22 21 18 37 44 30 31 23 18 19 30 16 18 21 38 20 27 32 22 27 20 39 20 27 35 9 4 27 23 20 26 5 8 4 6 2 3 2 3 Negative control and compound solvent =0.5$ DM90. Precipitate observed at 10~3 M. 110 . 10"3 �Table 84 DNA REPAIR SYNTHESIS ASSAY OF QUINTOZENE (PCNB) WITH METABOLIC ACTIVATION DNA) 10 5 X 10 Sample 1 2 72 75 95 106 355 75 79 71 72 349 3 55 84 73 49 364 Mean 67 79 80 76 363 3D 10 5 13 29 18 SE 6 3 8 17 11 Negative control and compound solvent = 0.5% DHSO. Ill �Table 85 DNA REPAIR SYNTHESIS ASSAY OF PHORATE DMA) . Phorate (M) io-3t 1' 0" 27 56 411* 17 50 45 781 22 42 55 782 26 107 _.§ 43 43 50 858 53 40 44 52 37 61 1295 112* 56 26 77 39 59 1103 Mean 61 43 52 39 40 55 964 SD 28 13 32 23 8 6 227 SE 13 5 14 9 3 2 102 0* 1 36 58 38 25 2 34 45 44 3 86 31 4 94 5 6 Sample 10-7 J£± 1Q-8 J£± Jf. Negative control and compound solvent = 0.5^ EtCH. Precipitate observed at 10"3 M. * Sample deleted from calculations because of low DNA. value. § Sample lost. 112 �Table 86 DNA REPAIR SYNTHESIS ASSAY OF PHOKATE WITH METABOLIC ACTIVATION DNA) Phoraice. (ll) DMN (M) _wrf_ 5 X 10-' J>! _™L* Sample 1 55 45 43 37 206 2 54 59 41 35 220 3 52 63 39 38 223 54 55 41 37 216 2 10 2 1.4 9 1 6 1 08 . 5 Mean SD SE -12™ Negative control and compound solvent =0.5^ EtCH. 113 �Table 87 DNA REPAIR SYNTHESIS ASSAY OF SIMAZItiE (dptn/ug DHUO Slmazlne (M) 4NQO (M) _ai Sample 1 2 3 4 5 6 Mean SD SE io-7 IP"6 10"5 iEi IP"3 10"5 195 153 212 230 217 298 218 48 19 165 91 131 138 152 113 132 27 11 151 171 152 146 179 213 169 25 10 195 190 312 290 237 305 255 55 22 177 193 253 281 166 161 205 50 20 369 208 233 165 205 t 2670 2850 2688 2702 2435 2662 2668 134 55 * Media control and compound solvent » 0.5% DMSO. t Sample lost. 114- 236 78 32 �Table 38 DNA REPAIR SYNTHESIS ASSAY OF SIMAZINE WITH METABOLIC ACTIVATION g MA) Slmazlne __(M) 5 X 10"* Sample 1 72 57 64 59 355 2 75 58 60 58 349 3 55 64 61 76 384 Mean 67 60 62 64 333 SD 10 4 2 10 18 SE 6 2 1 6 11 Negative control and compound solvent = 0.5^> DHSO. 115 �Table 89 DMA REPAIR SYNTHESIS ASSAY OF TRIFLURALIN DHA) Trlfluralin (M) 4NQO (M) 0* io-7 10-e io-» io-« 10-' 10- • Sample 1 56 42 53 51 71 639 2 68 29 26 _J' 152* 97 123 3 51 78 48 68 158* 112 570 4 45 51 76 89 78 83 894 5 50 47 79 97 56 80 663 6 29 41 59 57 43 53 986 Mean 50 48 58 73 62 87 812 SD 13 17 22 19 28 26 222 SE 5 7 10 9 12 11 91 Negative control and compound solvent =0.5$ EtCH. Sample lost. Sample deleted from calculations because of low DMA value. 116 1125 �Table 90 DNA REPAIR SYNTHESIS ASSAY OF TRIFLURALIN WITH METABOLIC ACTIVATION DNA) Trifluralin (M) 0* DMN (M) 10-' 10-* 10-' 5 X 10~a Sample 1 2 72 67 79 51 355 75 58 55 74 61 .._t 349 3 64 79 Mean 67 66 74 56 363 SD 10 8 9 7 18 SE 6 5 5 5 11 * Negative control and compound solvent =0.5% DM30. Sample lost. 117 384 �Table 91 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM Compound Metabolic Activation yg Compound Added/Plate 95 113 Negative control + Positive control, 4-o-tolylazo-l-toluidine v- oo Mofioerotephos — + _ — + + + + + + + Average Number of Histidine-Positive Revertants /Plate TA1538 TA100 TA1537 TA1535 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 13 13 8 10 7 10 87 101 93 107 97 95 126 9 22 14 23 13 17 14 10 9 9 7 11 10 10 6 183 11 10 13 10 9 7 6 101 89 75 79 71 78 113 16 20 16 14 16 19 18 14 13 12 10 15 9 10 12 12 11 10 11 15 10 o n o ft o •o �Table 91 (continued) Compound Metabolic Activation pig Compound Added/Pla te Negative control Positive controls p-Propiolactone AF-2 2-Anthramlne Bromacll ^ . Average Number of Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA1535 145 154 + - + + + + + + + 50 p,l 0.05 50 22 25 25 24 16 30 756 372 63 338 50 1 5 10 50 100 500 1000 120 129 123 117 16 3 10 4 101 23 17 31 29 40 30 14 24 13 22 16 18 15 6 26 14 13 15 1 9 15 11 1 5 1 0 5 0 1 0 0 500 1000 118 131 157 136 138 145 162 35 28 33 26 30 21 8 21 1 6 21 14 12 20 5 16 20 19 13 15 20 16 �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Cacodylic Acid Average Number, of Histidine-Positive Revertants/Plate TA100 TA1537. TA1535 TA1538 56 72 15 14 12 9 7 15 15 15 15 10 11 11 8 14 15 12 11 15 14 13 10 150 5 11 8 8 9 9 8 18 8 18 12 19 15 13 25 25 1 5 10 50 100 500 1000 48 42 42 39 43 44 44 1 5 10 50 100 500 1000 69 53 64 50 64 54 59 17 15 12 18 22 15 16 17 15 16 15 18 21 14 S? n a ^j p™ HO fr £ �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Captan Average Number of Histidine-Positive Revertants/Plate TA1537 TA100 TA1535 TA1538 72 98 2 2 18 14 7 3 8 25 2 5 0 0 0 0 2 2 2 0 0 1 7 14 16 26 6 22 3 100 211 532 822 820 720 1 5 10 15 25 50 Killing 1 5 10 15 25 50 141 210 285 340 330 704 29 80 76 104 SO Killing 20 60 113 55 71 143 19 22 26 21 46 44 •o ft �Table 91 (continued) Compound Metabolic Activation lig Compound Added/Plate Negative control Average Number of . Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA153S 92 80 18 14 12 16 16 16 Positive control, 4-o-tolylazo-o-toluidine Chloropyrifos 5 10 50 100 500 1000 66 92 65 SB 67 87 79 20 22 14 26 20 17 13 12 15 21 15 17 18 20 15 168 22 28 24 25 22 17 22 1 5 10 50 100 500 1000 67 71 87 77 77 72 77 11 9 11 16 11 13 14 15 15 15 18 14 14 11 14 16 20 13 16 30 22 1 3 M O O O CD �Table 91 (continued) Compound Metabolic Activation Vi g Compound Added/Plate 97 30 Negative control Positive control, 4-o-tolylazo-o-toluidine Dinoseb ro Average Number of Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA.1535 15 15 12 16 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 21 17 • 69 59 57 67 82 91 12 12 17 17 12 7 Killing Killing 79 82 94 83 87 104 Killing 13 14 12 14 13 12 Killing 21 16 18 1.7 16 17 Killing 19 18 17 15 17 10 Killing 15 168 15 14 20 17 15 19 Killing 15 14 15 17 14 7 Killing CD s- �Table 91 (continued) Metabolic Activation yg Compound Added/Plate 56 72 Negative control Positive control, 4-c-tolylazo-o-teluidine DSMA Average Number of - *Histidine-Positive Revertants/Plate TA1538 TA100 TA1537 TA1535 15 14 7 15 12 9 . 25 25 10 250 1 5 10 50 100 500 1000 50 56 51 66 71 53 43 12 10 20 15 16 13 12 7 10 10 11 7 12 7 4 5 3 8 8 7 9 1 5 10 50 100 500 1000 54 85 50 55 50 60 53 22 7 16 17 13 15 14 8 8 8 15 3 5 7 10 8 11 9 5 2 o (A �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Azinphos-methyl Average Number of Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA1538 101 102 26 26 6 3 25 25 13 24 13 78 1 5 10 50 100 500 1000 66 74 74 73 75 107 104 39 22 23 30 49 30 31 1 5 10 50 100 500 1000 76 69 68 81 65 84 119 23 23 24 22 24 :io 24 4 3 2 3 4 2 3 1 2 3 3 2 0 0 10 11 9 11 10 10 13 20 25 28 21 19 24 23 o CD �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-taluidine Fenthion Average Numb en of ;. ^c, -Histidine-Positive Revertants/Plate TA1537 TA1538 TA100 TA1535 94 80 36 20 10 9 12 12 15 168 25 25 1 5 10 50 100 500 1000 64 97 105 112 100 107 90 31 34 32 36 38 42 32 8 11 15 15 16 10 6 13 17 12 14 14 14 12 1 5 10 50 100 500 1000 114 97 81 90 98 86 89 15 17 9 16 14 22 20 10 12 9 12 7 8 10 12 10 17 21 13 10 15 (D 3 O �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Average 'Number of Histidine-Positlve Revertants/Plate TA100 TA1537 TA1535 TA153S 72 93 Negative control Positive control, 4-o-tolylazo-o-toluidine 1 5 10 25 3 7 25 25 Folpet 19 15 50 100 500 10GO 1 5 10 25 50 100 500 1000 127 150 244 300 550 286 20 35 39 48 111 110 Killing Killing Killing Killing 112 173 241 420 720 532 20 51 79 70 218 216 Killing Killing Killing Killing 7 0 1 0 2 0 0 0 2 1 5 6 10 3 0 0 8 20 6 183 5 8 11 14 7 2 Killing Killing 30 26 35 36 45 48 Killing Silling •<B �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control 39 92 Positive control, 4-e-tolylazo-o-tol.uidine OS 1 5 10 10 10 8 10 25 25 Malathion IsJ Average Number of Hiscidine-Positive Revertants/Plate TA1537 TA100 TA1535 TA1538 7 7 6 183 500 1000 54 48 85 99 81 82 61 8 7 7 8 7 12 10 11 12 10 6 7 5 7 3 3 5 7 5 7 4 1 5 10 50 100 500 1000 65 61 99 92 75 90 66 5 6 9 8 9 6 8 7 7 6 12 10 9 5 4 6 5 4 4 50 100 a 7 gf h0) ft HO a �Table 91 (continued) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Methomyl N> vO Average Number of Kistidine-Positive Revertants/Plate TA1538 TA100 TA1537 TA1535 128 149 18 14 33 20 1 5 10 50 100 500 1000 123 112 98 109 110 119 105 28 35 28 27 34 23 24 1 5 10 50 100 500 1000 145 115 125 129 132 133 122 19 20 16 18 21 17 13 12 10 12 10 13 10 14 17 22 17 206 14 10 18 14 24 26 21 18 18 21 19 20 20 24 15 15 13 20 19 18 14 25 25 �Table 91 (continued) Compound Metabolic Activation Mg Compound Added/Plate Negative control Positive control, 4-o-colylazo-o-toluidine Monuron UJ o Average Number of Hisiidine-Positive Revertants/Plate TA100 TA1535 TA153V TA1538 128 149 17 12 17 15 25 25 17 22 6 177 1 5 10 50 100 500 1000 126 . 98 108 122 114 122 125 .15 24 17 19 19 20 22 15 12 12 11 15 18 14 1 5 10 50 100 500 1000 125 142 119 116 108 104 123 10 13 17 15 15 15 11 15 15 13 19 16 15 15 19 18 22 21 29 29 19 21 15 18 21 19 12 17 1 o 9 �Table 91 (continued) Compound Metabolic Activation li g Compound Added/Plate 56 72 Negative control Positive control, MSMA Average Number of Histidine-Positive Revertants/Plate TA100 TA1537 TA1538 TA1535 4-o-tolylazo-o-toluidine 15 14 12 9 7 15 10 250 25 25 1 5 10 50 100 500 1000 79 69 62 52 41 53 48 1 5 10 50 100 500 1000 79 64 65 67 53 66 68 15 15 14 17 15 17 13 11 15 7 7 12 14 10 7 13 11 11 12 8 12 6 4 6 6 7 5 5 11 8 10 14 8 7. 10 10 10 9 7 8 8 10 �Table 91 (continued) Compound Metabolic Activation pg Compound Added/Plate Negative control Positive control, 4-o-toIylazo-o-toluidine Parathior Average Number of Histidine~Fositive Revertants/Plate TAiOO TA1537 TA1535 TA1538 95 114 19 21 6 13 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 94 138 85 98 87 110 107 56 75 69 76 88 105 103 12 12 13 13 15 1314 11 16 14 14 17 15 12 7 7 4 4 4 4 3 12 7 5 6 7 8 6 6 7 6 177 8 7 7 8 6 8 13 15 15 19 8 5 9 12 (B rt HO �Table 91 (continued) Compound Metabolic Activation ug Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Phorate Average Number of Histidine-Positive Revertants/Plate TA100 TA1535 TA1537 TA1538 96 118 15 17 8 11 25 25 1 5 10 50 100 500 1000 70 65 85 65 72 70 58 17 15 17 17 11 14 14 i 5 10 50 100 500 1000 101 103 79 89 79 59 70 14 11 13 15 15 16 19 8 8 7 5 7 6 7 11 8 10 9 6 6 8 8 11 6 177 16 11 11 8 9 6 9 15 10 12 11 7 11 5 I �Table 91 (continued) Compound Metabolic Activation wg Compound Added/Plate Negative control Positive control"-," 4-o-tolylazo-o-toluldine Siaazine .&> Average Number *of' Histidine-Positive Revertants/PlaLe TA1537 TA1538 TA100 TA1535 98 106 10 7 8 8 83 72 73 87 85 71 6S 84 90 82 S3 87 89 120 7 5 7 7 8 3 4 15 10 20 10 12 7 11 25 26 22 266 22 20 20 22 17 25 22 9 4 8 9 8 4 2 11 11 16 9 11 16 10 22 22 15 14 15 15 18 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 ( 4 H- �Table 91 (concluded) Compound Metabolic Activation yg Compound Added/Plate Negative control Positive control, 4-o-tolylazo-o-toluidine Trifluralln Average Number of Histidiae-Positive Revertants./Plate TA1537 TAiOO TA153S TA1535 96 80 15 15 17 20 14 8 18 24 29 23 25 22 18 15 168 15 14 14 16 15 11 15 13 16 18 14 16 13 15 9 9 14 13 15 11 10 25 25 1 5 10 50 100 500 1000 1 5 10 50 100 500 1000 73 81 74 93 86 76 95 72 80 78 90 81 79 81 11 12 16 19 18 18 13 10 13 14 15 8 12 12 l-l p> �Table 92 RESULTS OF ASSAYS WITH ESCHERICHIA COLI WP2 Compound Metabolic Activation Negative control . -f- Positive control, AF-2 + - yg of Compound Added per Plate Moncrotopbos - • + + + + + + Bromacil — B — B . . ^ — + + + + + + 0.05 0.05 1 10 50 100 500 1000 i 10 50 100 500 1000 1 10 50 10 0 500 1000 1 10 50 100 500 1000 Average Number o± Tryptophan Positive Revertants per Plate 68 73 204 220 89 83 76 77 61 70 90 88 76 73 75 95 65 74 71 70 70 67 71 73 66 70 70 71 �Table 92 (continued) Compound Cacodylic Acid Metabolic Activation - + + + + 1 10 50 100 500 1000 1 Average Number of TryptophanPositive Revertants per Plate m 102 89 82 91 85 95 + - + + + + + + 10 50 100 500 1000 j 5 10 15 25 381 733 1358 1755 50 + Captan yg of Compound Added per Plate 76 79 89 81 85 2600 1 5 10 15 25 50 89 182 423 699 955 1712 124 �Table 92 (continued) of TryptophanMetabolic Activation of Compound per Plate^ Dindseb 1 10 50 100 500 1000 57 57 49 71 52 42 1 10 50 100 500 1000 Chloropyrifos 60 73 53 49 61 49 1 10 50 100 500 1000 64 73 5fr 55 44Toxic 1 10 50 100 500 1000 73 69 63 65 49 Toxic �Table 92 (continued) Compound Metabolic Activation DSMA Average Number of TryptophanPositive Revertants per Plate 1 10 50 100 500 1000 + + + + + •i- Fenthion Mg of Compound Added per Plate 86 81 67 68 68 71 1 10 50 100 500 1000 69 62 67 73 81 79 1 10 50 100 500 1000 59 63 62 56 70 71 1 10 50 100 500 1000 64 50 67 64 64 81 �Table 92 (continued) Compound Folpefc Metabolic Activation -^g of Compound Added per Plate 1 5 10 25 50 100 Azinpbos-methj'l 1 5 10 25 50 100 1 10 50 100 500 1000 1 10 50 100 500 1000 Average Humber'Wf "1'ryp^lbphanPositive Reveftantfl "per Plate 65 162 170 424 720 1260 74 167 202 900 16SO 1880 92 87 83 89 68 88 83 74 87 86 73 79 �Table 92 (continued) Halathion Methomyl Metabolic Activation wg of Compound Added per'Plate - 1 10 50 100 500 1000 62 54 60 60 54 48 + + + + + + Compound 1 10 50 100 500 1000 58 50 55 59 75 64 - 1 10 50 100 500 1000 61 76 83 57 68 71 + + + + + + 1 10 50 100 500 1000 70 81 78 83 63 74 . Average Number of TryptophanPositive Revertants per Plate �Table 92 (continued) Compound Metabolic Activation pg of Compound Added per'Plate Average Number of TryptophanPositive Revertants per Plate Momuron - 1 10 50 100 500 1000 72 68 57 63 65 63 MSMA + + + + + + - 1 10 50 100 500 1000 i 10 50 100 500 1000 60 59 47 71 50 61 55 64 57 76 60 63 + + + + + + 1 10 50 100 500 1000 55 71 73 71 61 72 �Table 92 (continued) Compound Parathion Metabolic Activation ]jg of Compound Added per Plate 1 Average Number of TryptophanPositive Revertants per Plate 10 50 100 500 1000 1 10 50 100 500 1000 Parathion-methyl 71 64 66 70 64 64 69 53 76 57 72 66 1 10 50 100 500 1000 1 10 50 100 500 1000 53 56 60 68 63 52 64 83 60 65 53 71 �Table 92 (continued) Compound Metabolic Activation Average-Number of TryptophaaPositive Revertantsv.per Plate 1 10 50 100 500 1000 Qulntozene (PCNB) + + •s+ Phorate ug of Compound Added per Plate 70 60 57 62 1 10 50 100 500 1000 78 67 54 57 59 62 1 10 50 100 500 1000 63 64 65 49 71 60 1 10 50 100 500 1000 78 86 83 73 90 70 54 :-s �Table 92 (concluded) Compound Simazine Metabolic Activation pg of Compound Added per Plate 1 Average Number of TryptophanPositive Revertauls per Plate 10 50 100 500 1000 1 10 50 ICO SCO 1000 Trifluralin 55 51 73 54 54 53 64 66 72 56 71 83 1 10 50 100 500 1000 75 73 81 86 69 60 1 10 50 100 500 1000 58 63 63 65 70 70 �Table 93 MICROBIAL INHIBITION IN ESGHERICHIA COLT AND BACILLUS SUBTILIS Diameter of Zone of Inhibition (mm) • ... \ • Compound TCg of Compound Added to Disc E. coli W3110 p3478 B. subtilis HI 7 m45 Positive control, "i-plieiiyl-3,-.' diiriethyitriazene 1.0 37 52 40 61 Negative control, chlcracsphenacol 0.03 34.5 34 32 31 Monocrotophos 1 6 . 6 6 6 Bromacil 1.0 6.5 6.5 6.5 6.5 Cacodyiic acid I 6 6 6 6 Cap tar. 0.1 6.5 11 9 19 Chloropyfif os 2.5 6 10 6 11 Dinoseb 1 10 17 8.5 11 DMSA 1 6 6 6 6 Fenthion 1 6 6 6 6 Folpet 0.1 6.5 6.5 7.5 Azinphos-methyl 1 6 6 6 CD 10 6 �Table 93 (concluded) Diameter of Zone of Inhibition (mm) Compound mg of Compound Added to Disc E. coli p3478 W3110 B. subtilis H17 m4 5 Malathion 1 6 6 6 6 Me thorny 1 1 6 6 6 6 Monuron 1 6 6 6 6 MSHA 1 6 6 5 6 Parathion 1 6 6 6 6 Parathion-methyl 1 6 6 6 6 Qulntozene (PCNB) 1 6 6 6 6 Phorate 1 6 6 6 6 Simazins 1 6 6 6 6 Trifluralin 1 6 6 S 6 �Table 94 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - SwSiOCRCTCPEOS Metabolic Compound Percent Concentration (w/v or v/v) Mitotic Recombinants Survivors per 105 Cells/ml per ml Percent of 3 7 Control ,. . (x 10- ) Survivors (x 10- ) EXPERIMENT 1 5.7 100 4.5 7.9 5.8 100 4.5 7.8 0.1 5.8 102 1,650 2,845 0.1 46 . 79 1,435 3,120 5 5.7 10O 44 77.2 5 47 . 81. 30 63.8 91 . 100 8 8.8 86 . 100 9.5 5 6.3 69 26 41.2 5 48 . 56 40 83.3 Negative control Positive control 1,2,3,4-Diepoxybutane Monocrotophos EXPERIMENT 2 Negative control Uonocrotophos 11.0 �Table 95 IN VITRO ASSAYS WITH SACCHAROHYCE3 CEREVISIAE 03 - BROHACIL Compound Metabolic Activation Percent Survivors Percent of Concentration Cells/ml (w/v or v/v) (x 10 •>) Control Mi t otic Recombinants per I0a per ml fx 10 3) Survivors EXPER1HHJT 1 4.8 Negative control 4.7 Positive control 1,2,3.4-Dlepoxybutane 100 100 3 3 71 74 104 94 104 92 745 683 3.4 3.5 005 .0 0.01 0.05 01 .0 05 .0 50 . 4.5 5.0 005 .0 0.01 00 .5 0.10 05 .0 Bromacil 0.04 0.04 4.4 3.9 4.3 3.8 2.4 94 83 91 81 51 5 4.5 4.2 00 .4 00 .4 00 .5 01 .0 0.25 4.4 2.0 42 3 2 3 1 1 6.3 6.4 2191 1951 6.0 4.4 6.0 2.3 10.0 3 1 11.4 7.7 7.0 2.6 3 12.5 10 0 10 0 5 11.1 3 7.1 4.5 4.2 100 10 0 80 7 653 5.7 54 . 5.5 49 . 126 120 122 108 7 5 5 1 4 .9 47 . 48 . 4.9 117 112 114 117 4 5 6 1 3 EXPERIMENT 2 Negative control Positive control 1,2,3,4-Dlepoxybutane Bromacil 0.50 0.05 01 .0 0.25 05 .0 1933 1555 12.3 9.3 9.1 20 . 8. 2 10. 6 12.5 2.0 �Table 96 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CACODYLIC ACID Compound Metabolic Activation . Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10~7 ) Control Mitottic Recombioants per 10a per ml 3 (x ID' ) Survivors EXPERIMENT 1 Negative control ; 74 . 7.5 7.7 10 0 5 5 5.6 76 20 35.7 5 6.3 82 11 17.5 7.1 100 3.5 49 . 6.5 100 3 4.6 5 15.5 217 1,187 766 5 17.5 270 1,159 662 + Cacodylic acid so 10O + 10.1 6.5 EXPERIMENT 2 Negative control + Cacodylic acid + �Table 97 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - CAPTAN Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Recombinar.ts per 105 per ml 3 (x lO" ) Survivors EXPERIMENT 1 Negative control 7.1 100 3.5 49 . 6,5 100 3.0 4.6 0.003 60 . 84 205 342 0.0 03 9.1 140 145 159 7.5 100 1.5 2.0 60 . 10 0 4 67 . 10 37 481 85 58 114 + Captan + Wl EXPERIMENT 2 _ Negative control + Captan 003 .0 + 0. 0 03 .7 7 5.1 �Table y8 IN VITRO ASSAYS WITH SAGCHAROMYCES CEREVISIAE D3 - CHLOROPYRIFOS Compound Metabolic Activation Percent Concentration (w/v or v/v) Mitotic Recorabinants Survivors Cells/ml ,per 10s .„ Percent of per ml 7 •* (x 10~ ) Control ",,(* 10 -.».) ^urviVorssf EXPERIMENT 1 7.5 100 1.5 2.0 6.0 100 4 6.7 5 7.7 103 7 9.1 5 8.0 133 10 12.5 6.3 100 1.5 2.4 7.4 10O 3.5 4.7 0.1 3.8 60 1,045 2,750 0.1 5.2 70 903 1,737 5 7.9 125 3 5 7.4 100 10 Negative control + ChloropyrifoB + •-J-- EXPERIMENT 2 Negative control + Positive control, 1,2,3, 4-diepoxybutane + Chloropyrifos + 3.8 13.5 �Table 99 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DINOSE3 Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per ml per 10s (x 10-') Survivors EXPERIMENT 1 - 6.3 100 1.5 2.4 + 7.4 100 3,5 4.7 0.1 3.8 60 1,045 2,750 0.1 5.2 70 903 1,737 0.2 62 . 98 9 14.5 0.2 40 . 54 1 2.5 0.3 Negative control . 3 5 5 0.3 2.4 32 5 5.5 100 2.5 4.5 5.2 100 2.0 3.8 0.1 4.4 80 3 68 . 0.1 40 . 77 4 10.0 02 . 4.1 75 8 19.5 0.2 4.3 83 8 18.6 Positive control, 1, 2 ,3 ,4-diepoxybutane + in in Dinoseb + + 167 20.8 EXPERIMENT 2 _ Negative control + Dinoseb + + �Table 100 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - DSMA Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of Control (x 10-') Mitotic Recombinan,£s 5 per ml per 10 (x 10j3) Survivors: EXPERIMENT 1 Negative control 7.4 100 7.5 10.1 7.7 100 5 6.5 4.5 1.1 15 0 4.5 5.2 68 0 7.1 100 3.5 4.9 6.5 100 3 4.6 5 4.7 66 3 6.4 5 3.4 52 7 + DSHA + Ul a-. EXPERIMENT 2 _ Negative control + DSMA + 20.6 ! �Table 101 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FENTHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10"7) Control Mitotic Recombinants oer ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 6.3 100 1.5 2.4 7,4 100 3.5 4.7 0.1 3.8 60 0.1 5.2 70 903 5 6.6 105 9 5 7.5 101 5 6.7 7.5 100 1.5 2.0 6.0 100 4 6.7 5 7.S 104 4 5.1 5 7.1 118 6 8.5 + Positive control, 1 , 2 , 3 , 4-d iepoxybutane Ul •vj + Fentbicn + 1,045 2,750 1,737 13.6 EXPERIMENT 2 Negative control + Fenthion + �Table 102 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - FOLPET Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Percent of Cells/ml Control (x 10-7) ^MJ-.totic Rjgcombinants | "* ' per ml * "~p~e*" 10s . U io-3) Survivors EXPERIMENT 1 _ 7.5 100 1.5 2.0 60 . 100 4 67 . 003 .0 40 . 53 119 298 0 .0 03 3.8 63 65 171 6.3 100 1.5 2.3 74 . 100 3.5 47 . 0 .0 03 9.5 151 89 94 0 .0 03 9.1 123 82 90 Negative control + Folpet + EXPERIMENT 2 _ Negative control + Folpet + �Table 103 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 -AZINPHOS-METHYL Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Percent of Cells/ml Control (x 1 " ) 0' Mitotic Recombinants per ID5 per ml 3 (x 10- } Survivors EXPERIMENT 1 _ 5.7 100 4.5 7.9 5.8 100 4.5 78 . 0.1 5.8 102 0.1 46 . 4.5 4.5 Negative control + Positive control 1,2,3,4-Diepoxybutane + 79 1,435 3,120 5.3 93 15 28.3 5.8 100 15 25.9 100 8 8.8 8.6 100 S.5 5 Azinphos-methyl 2,845 S.I + 1,650 5.7 63 68 119.3 5 6.2 72 80 129 EXPERIMENT 2 _ Negative control -i- Azinphos-methyl + 11.0 �Table 104 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MALATHION Compound Metabolic Activation Percent Concentration (w/v or -v/v) Survivors Cells/mi Percent of Control (x 10~7 ) Mi tot ic Recorabinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 Negative control • . -. . i \ --. • * •. • Positive control, 1 , 2., 3 , 4-diepoxy butane o* o 5.7 100 4.5 7.9 5.8 10 0 4.5 78 . 0.1 5.8 102 1,65O 2,845 0.1 4.6 79 1,435 3,120 5 7.8 137 11 14.1 5 6.3 109 7 11.1 9.1 100 8 8.8 8.6 100 9.5 5 8.1 89 13 16.0 5 7.6 88 8 10.5 + + Malathion + EXPERIMENT 2 Negative control + Malathion + 11.0 �Table 105 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - METHOMYL Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 6.6 5.8 100 100 0.1 0.1 1.8 27 266 1,478 1.5 29 184 1,227 2.0 5.0 76 4 2.0 2.7 50 0 3.0 Positive control, 1,2,3, 4-diepoxybutane + Negative control 3.7 56 8 21.6 3.0 4.1 76 6 14.6 5.5 100 2.5 4.5 5.2 100 2.0 3.8 3 4.7 85 15 31.9 3 4.4 85 10 22.7 + Methomyl + + 4.5 2.5 6.8 4.6 80 . EXPERIMENT 2 _ Negative control + Methomyl + �Table 106 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MONURON Compound Metabolic Activation Percent Concentration (v/v or v/v) Survivors Cells/ml Percent of Control (x 10~7) Mitotic Recorabinants per 105 per ml Survivors . (x 10-») EXPERIMENT i 66 . 100 45 . 68 . 5.4 100 2.5 4.6 0.1 1.8 27 266 148 ,7 0.1 1.5 29 184 1,227 5 3.5 53 3 8.6 5 3.8 70 1 2.6 5.5 100 2.5 4,5 5.2 10 0 2.0 3.8 5 6.9 125 2 2.9 5 6.2 119 9 14.5 Negative control + Positive control, 1,2,3, 4-diepoxybutane + Monuron + EXPERIMENT 2 Negative control + Monuron + �Table 107 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - MSMA Compound Metabolic Activation Percent Concentration >/v or v/v) Survivors Cells/ml Percent of (x 10~7) Control Mitotic Recombinants per ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 Negative control 7.4 100 7.5 7.7 100 5 6.5 5 4,3 58 1 2.3 5 5.4 70 3 5.6 7.1 100 3.5 4.9 6.5 100 3 4.6 5 4.9 69 10.2 20.8 5 5.8 89 10.4 17.9 + MSMA -f- 10.1 EXPERIMENT 2 Negative control + MSMA + �Table 108 IK VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PARATHION Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Percent of Cells/ml Control (x 10-') Mitotic Recombinants per 10s per ml 3 (x 10- ) Survivors EXPERIMENT 1 _ Negative control 5.7 Positive control, 1 , 2 , 3 , 4-diepoxybutane 4.5 7.9 5.8 + 100 100 4.5 7.8 2,845 3,120 1,650 46 . 79 1,435 6.5 3 4.6 5 5.8 114 100 5 8.6 100 8 88 . 8.6 100 9.5 5 ••! 102 9.1 Parathion 5.8 5 + 0.1 0.1 8.8 96 4 4.5 5 8.2 95 5 6.1 EXPERIMENT 2 _ Negative control + Parathion + 11.0 �Table 109 IN_ VITRO ASSAYS WITH SACCHAROMYCES rEREVISLAE D3 - PARATHION-METHYL Compound Metabolic Activation Percent Concentration {w/v or v/v) Survivors Cells/ml Percent of Control (x 10-7) Mitotic Reconibinants per ml per 10s 3 Survivors (x ID' ) EXPERIMENT 1 Negative control 5.7 100 4.5 7.S 5.8 100 4.5 7.8 0.1 5.8 102 1,650 2,845 0.1 46 . 79 1,435 3,120 5 7.7 135 16 20.8 5 5.4 93 15 27.8 - 91 . 100 8 S.8 + 8.6 100 9,5 5 7,4 81 19 25.7 5 7.2 84 25 34.7 + Positive control, 1,2,3, 4-diepoxybutane + Parathion-methyl + EXPERIMENT 2 Negative control Parathion-methyl + 11.0 �Table 110 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - QUINTOZENE (PCNB) \— f Compound Metabolic Activation Percent Concentration '',w/v or v/v) Survivors Cells/ml Percent of (x 10-7) Control Mitotic Recombinants per ml per 105 3 (x 10- ) Survivors EXPERIMENT 1 _ 5.7 100 4.5 7.9 5& . 100 4.5 7.8 0 .1 5.8 102 1,650 2,845 0.1 4.8 79 1,435 3,120 2 3.7 65 3 8.1 2 4.2 72 4 9.5 9.1 100 8 8.8 8.6 100 9.5 1 58 . 64 4 69 . 1 7.0 81 7 10.0 2 68 . 75 3 44 . 2 7.5 87 10 13.3 Negative control + Positive control, 1,2,3, 4-diepoxybutane + Quintozeue (PCNB) + EXPERIMENT 2 Negative control + Quintozene (PCNB) + + 11.0 �Table 111 IK VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - PHOKATE Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10~7) Control Mitotic Recombinants per ml per 10s (x ID'3 } Survivors EXPERIMENT 1 9.1 100 8 8.8 100 3.5 11.0 5 8.7 96 9 10.3 5 7.5 87 3 40 . 5.7 100 4.5 7.S 5.8 100 4.5 7.8 5 7.5 132 4 5.3 5 7.2 124 7 9.7 Negative control -!- Phorate + 8.8 EXPERIMENT 2 Negative control + Phorate + �Table 112 _IS VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - SIMAZINE Comoound Metabolic Activation Percent Concentration : w/v or v/v) Survivors Cells/ml Percent of Control (x 10~7 ) Mitotic Recombinants per ml per 10 3 (x 10" ) Survivors EXPERIMENT 1 _ 66 . 100 4.5 6.8 5.4 100 2.5 46 . 0.1 1.8 27 266 1,478 0.1 1.5 29 184 1,227 5 3.8 58 3 7.9 5 2.0 37 1 5.0 5.5 100 2.5 4.5 5.2 100 2 3.8 5 7.0 127 7 10.0 5 7.0 135 4 5.7 Negative control ; Positive control, 1 , 2,3,4-diepoxybutane + + Siraasine + EXPERIMENT 2 _ Negative control + Simazlne + �Table 113 IN VITRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 - TRIFLURALIN Compound Metabolic Activation Percent Concentration (w/v or v/v) Survivors Cells/ml Percent of (x 10"7 ) Control Mitotic Hecombinants per 10* per ml (x 10-3) Survivors EXPERIMENT 1 Negative control 7.5 100 1.5 2.0 6.0 100 4 6.7 5 8.4 112 5 5.9 5 6.0 100 2 3.3 6,3 100 1.5 2.4 7.4 100 3.5 4.7 0.1 3.8 60 1,045 2,750 0.1 5.2 70 903 1,737 5 8.7 138 7 80 . 5 8.4 114 3 3.6 + Trifluralin + EXPERIMENT 2 VO Negative control + Positive control, 1,2,3, 4-diepoxybutane + Trifluralin + �Table 114 III VITRO MUTAGENESIS WITH SAIMONELIA TYPHIMURIUM SUMMARY DATA FOR EPA PESTICIDES Positive Response, +; Negative Response, - Pesticide Hottocrotophos BroAacll Cucodylic Acid Captaa ESMA Fen tb Ion Folpet Malathlon Methonyl MS4A Parathloa Parathlon-Bsethyl tpilntozene (PQtB) Phorate Slaazine Trlfluralln TA100 - Metabolic + Metabolic Activation Activation TA1535 - Metabolic + Metabolic Activation Activation TA1S37 - Metabolic + Metabolic Activation Activation TA1538 - Metabolic + Metabolic Activation Activation �APPENDIX A MUTAGENEST.S STUDIES OF PESTICIDE COMPOUNDS MOUSE HERITABLE TRANSLOCATION TEST CAPTAN 171 �SUMMARY SRI conducted a heritable translocation study of Captan in mice to investigate whether heritable mutagenic events occur when the compound is ingested repeatedly over an extended period. For 8 weeks, adult male mice were administered Captan in their diet; 60 mice received 2500 ppm, and 61 received 5000 ppm. A control group of 60 adult male mice received an untreated diet during this time. A positive control group containing 66 adult male mice was treated as a control group for 4 weeks and then received the known mutagen triethylenemelamine (TEM) Jn the drinking water for 4 weeks. After treatment, all males were bred with two virgin females each to produce an F generation, the males of which were raised to maturity. Selected (200 per group) F males were bred to three virgin females each, and presumptive translocates were rebred to three additional females each. A third breeding was conducted with selected nonbreeder and/or presumptive males. Evaluation of the data on fertility, breeding, and litter size distribution for F. and F generations does not suggest the presence of translocation heterozygotes in control or Captan-treated male mice. Data on dead implants and rebreeding did, however, suggest the presence of translocation heterozygotus in the group treated with 5000 ppm Captan. Meiotic cell preparations of the testcs of the presumptive males were evaluated cytogenetically. Normal meiotlc chromosomes were found in the following numbers of F males derived from the. group specified: 8 of 8 controls, 8 of 8 from the 2500 ppm Captan group, 8 from the 5000 ppm Captan group, and 2 of 2 from the 5000 ppm Captan-treated group derived from traumatized F females. Five of 5 TEM-treated F males and i. of 8 from the. 5000 ppm Captan-treated males showed reciprocal translocations. 172 �The results of this study show that under the experimental procedures employed, Captan at. 5000 ppm In the diet of male mice for 8 consecutive weeks can produce a heritable mutagenic event in F generation male mice. 173 �' INTRODUCTION The EPA is reviewing and evaluating the health hazard of pesticides and of substitute candidate, pesticides according to available data. Additionally, the Agency is obtaining supplemental laboratory data. The objective is to enable the EPA to select those chemicals that are minimally hazardous when used according to labeling restrictions. SRI is participating in this Substitute Chemical Program by investigating the mutagenic potential of selected materials by in vitro and in vivo procedures. Captan has been shown to respond in a positive manner in Salmonella typhimurium, Escherichia coll WP2, Saccharomyces cerevisiae, J£. coll (relative toxicity), Bacillus subtilis, WI-38 unscheduled DNA synthesis (UDS) with metabolic activation, and Drosophlla melanogaster experiments. It was not positive in a mouse dominant-lethal test. Based on positive responses in both Tier I (in vitro test) and Tier II (Drosophila) mutagenic studies, It was recommended that a heritable translocation test (Tier III) in the mouse be conducted to further assess thie mutagenic potential of Captan. In this study, young adult male JCR/SIM mice from a closed, randombred colony were administered Captan in the diet for 8 weeks. After treatment, each male was mated to two virgin females to produce an F generation, the males of which were raised to maturity and bred to three virgin females each. Pregnant females were evaluated against pre- determined selection criteria for Identification of suspect F.J males, which were rebred and evaluated again. Presumptive F.. males were examined cytogenetically. Through this procedure, a heritable mutagenic response can be detected. Potential mutagenic effects were Identified by examination of fetuses during the middle to later stages of gestation. 174 Cytogenetic �examinations were made of meiotic cell preparations of the testes from suspect males for confirmation of findings obtained front the breeding studios. Reported here are the results of the heritable translocatlon study of Captan. 175 �MOUSE HERITABLE TRANSLOCAT10N TEST Human populations frequently are exposed to man-made chemicals, often at barely detectable levels, for extended periods. To evaluate the genetic hazards of such chemicals, a prudent approach is to study them In mammalian systems so as to maximize detection of a mutagcnlc response. The study reported here was such an investigation of Captan for its potential to produce heritable genetic defects. Chemical induction of chromosomal aberrations in the mouse is a valuable and Important experimental aid in understanding the many genetic defects due to chromosomal anomalies in humans. To date, mammalian evaluations of chemically induced chromosomal aberrations have been attempted with the dominant-lethal test and cytogenetic studies of somatic and germinal cells. Although these procedures can provide useful information, they do hot measure heritable genetic effects, the most important mutagenlc occurrences ttiat are permanent and transmissible. A need exists for a method to reliably identify compounds that cause heritable chromosomal aberrations in mammalian systems. The mouse translocatlon procedure appears to be such a system. A well-defined translocation test will demonstrate the fertility of an F male population derived from F males created with a tost agent. Confirmation of a nonbreeder, sterile, or partially sterile response, can be obtained by cytological examination of the germ cells from suspected males. Sterility and partial sterility are closely correlated with the induction of translocation heterozygotes. The procedure used in conducting this translocatlon test was based on experimental techniques described by Leonard and DeKmidt,1 Cattanach et al.,'1 Falconer et al.,J and Generoso.4 We modified this approach, in consultation with government and industry scientists actively engaged In mutagenesls research. 176 �Materials and Methods Animals Male and female ICR/S1M mice were purchased from Simonsen Laboratories, Cilroy, California. The FQ males were 8 to 10 weeks old. The females used in the breeding phases were 10- to 12-week-old virgin stock. Chemical_ Supply A supply of Captan sufficient for all aspects of the experimental program was received from Battelle CoJumbus Laboratory and F.PA-RTP. Lot number SX-640, Chevron Chemical Company, was used for all treatment periods. The excess material has been placed in storage in case it is needed for future reference. Dosage Selection and Compound Administration SRI and EPA staff selected the two dosage levels of Captan Lo be used in this experimental program. For 8 weeks, Captan was fed in the diet at 2500 and 5000 ppm. An appropriate amount of Captan was dissolved and/or suspended in corn oil. Then the compound-oil concentrate was added at a level of 3% to a finely ground commercial diet (Purina) of known composition. The use of corn oil assured even distribution of Captan and prevented its stratification in an otherwise dry diet. Diets prepared at 2-week intervals were refrigerated at 4°C until fv>d to the animals. The diet was replaced in the feed containers twice weekly to minimize the possibility of compound loss. Body weights and food consumption were recorded weekly during the 8-week exposure period. Reference Control Males in the reference control group were fed the Purina diet with only corn oil added at a level of 3%. These mice were treated In the same manner as those in the compound test groups. Body weights were recorded weekly, as was food consumption. 177 �Positive Control For the positive control group, the known mutagen triethylenemelamlne (TEM) was administered in the drinking water at 0.32 ing/liter for 2 weeks and then at '0.124 mg/liter for 2 weeks. TEM treatment was initiated after the males had been on the control diet for 4 weeks. Body weights and food consumption were recorded weekly. TEM is one of the chemical mutagens that have the demonstrated effect of inducing translocations in the F progeny of F_ treated males. Genetic Tests After 8 weeks of treatment, the males in each treatment group were mated to two adult virgin females each, After 1 week, each female was housed individually and allowed to deliver its litter. The F males were discarded. All litters were raisod to weaning age, at which time the females were discarded. The F males were raised to maturity. At maturity (10 to 12 weeks of age) , 200 I*' males from each experimental group were selected randomly and housed individually. Three adult virgin females were housed with each F male for the first breeding. plugs. They were examined daily for the presence of vaginal These females were sacrificed 14 days after mating, and a uterine analysis was performed for determination of the number of total, live, and dead implants. Males bred to females that produced litters fitting our criteria for presumptive classification as sterile, partially sterile, or nonbreeder were rebred to three new virgin females each. The same evaluation was made for the second breeding. Our criteria for presumptive classification of a male as "partially sterile," "sterile," or "nonbreeder" are: • ^Partia 1J_y_ S, t er II e^jto 1 e each must have 9 or fewer live implants, with at least I. having h or fewer live implants. - _If^only_ 2^f_3._fema.JLjes_ .are pregnant, both must have 9 or fewer live Implants, with 1 having 6 or fewer live implants. 178 �. . L £ . . . ? e ? : ? ? l . l ' ? gl-- : > i . l - ? - .S J . , _ 5 J I . . I a n ! . 6 or fewer live implants. tnis female must have • "Sterile" Male - None of 3 females pregnant—previously identified by presence of vaginal plug. • "Nonbreeder" Male - None of 3 females pregnant—not previously identified by presence of vaginal plug. Any F, male that did not fit one of these descriptions waa considered "normal" and was discarded. For each F male in the control and compound-treated groups suspected of being a translocate or nonbreeder after 2 or 3 breedings, a cytogenetic evaluation was made of meiotic cell preparations of its testes. Five males from the positive control group were also subjected to cytogenetic evaluation. I]yaluat^ipn_qf Breeding n_ata F I males were identified as sterile, partially sterile, or nonbreeders by the methods outlined above. Individual data were totaled to give the number of observed (presumptive) translocaticns per treatment group, using a data base of 600 to 800 females per group. Also, for an accurate review of such findings, the F breeding and litter data were thoroughly evaluated. The various measured evaluated included percentage of pregnancies, average litter size, average number of males and females, average number of males with females having zero to five or more dead Implants, average number of females having zero to five or more dead Implants, percentage of females with plugs, and percentage of pregnancies with and without plugs. Meic tic_.Cell Cytogenetic Studies Cytogenetic examinations were made of the testes of 31 F mice, with the two testes from each mouse being examined separately. 179 The procedures �used for the cytogenetic preparations arc as follows. C0« was used to sacrifice the mice. The testes Were removed, weighed, and placed In an Isotonlc solution of 2.2% sodium citrate. The tunica of each testls was punctured to release the tubules, which were then rolled on a glass plate to release the cell contents into the isotonic solution. The resulting cell suspension was centrifuged at 800 rpm for 5 minutes; the supernatant was removed, and each pellet of cells was resuspended in 5 ml of 1% sodium citrate hypotonic and held at room temperature for 15 minutes. The cells were centrifuged again at 800 rpm for 5 minutes and the supernatant was discarded. The cells were then treated with Carney's fixative (3 parts methyl alcohol and 1 part glacial acetic, acid) to give a total volume of 5 ml, and. immediately centrifuged again at 800 rpm for 5 minutes. This procedure was performed twice, Then the cells, suspended In an appropriate amount of fixative, were dropped onto clean, wet microscope slides and allowed to air-dry. The slides were stained with 2% buffered Ciemsa for 5 minutes. Coverslips were attached with Permount. The slides were coded to preclude bias on the part of the scorers. 180 �RESULTS AND DISCUSSION Table 1 presents the average body weights for mice In the various groups. The body weights of the control, TEM, and 2500 ppm Captan group were within normal limits and comparable throughout the experiment. The 5000 ppm Captan-treated group showed a depressed body weight for 5 weeks before demonstrating a recovery trend during Weeks 6 to 8. This body weight depression appeared to be due to the inability of the male mice to acclimate to such a high level of compound in their diet. Table 2 summarizes the average food consumption by treatment group. Both Captan-treated groups (2500 and 5000 ppm) showed a lower average weekly food intake than did the control arid TEM-treated groups. During the week immediately following (..he 1-week F generation rating, one animal rack holding some females that had been mated with the mice given 5000 ppm Captan was accidentally tipped and some cages were sptiled onto the fllor, resulting in our inability to identify which males had been mated with these females. There were, however, sufficient numbers of F, generation males from those females that were not traumatized to allow us to randomly select 200 F males for use in subsequent F generation breedings. In addition, we held all females that had been traumatized by the tipping of the rack and maintained them throughout the remainder of the study is a separate group. From this separate group we selected 50 F males (at least one. per female retained) for use in the F generation breedings and evaluations. F Generation Information on the breeding performance, litter size, sex distribu- tion, and clinical effects of the Ffi generation should be Included in the evaluation of translocation data, because it may provide valuable reference data. 181 �Table 3 .summarizes the breeding .mcl Litter performance of the F generation. NQ adverse effects were observed in the control and 2500 ppm Captan groups. The two 5000 ppm Captan groups showed a reduced pregnancy ratej the rate for the traumatized females was 29% below that of the controls. Litter sizes for the 5000 ppm groups were slightly below control values. As expected, the TEM group had a reduced pregnancy rate and a litter size 47% below the control level. Table 4 presents litter-size distributions of live young from the F generation mating. Although the distribution patterns for the control and Captan-treated animals were within normal ranges for our strain of mouse, there was evident a definite pattern of decreasing litter size and increasing variance and standard deviations between the different experimental groups. As exnected, the TEM-treated animals showed the classic shift toward smaller Jitters. Figure ] graphically presents the data on the F- generation litter-size distribution. J_i Generation Table 5 summarizes breeding data from the first mating of the F generation male mice. In the females mated with TEM nales and with males from the 5000 ppm Captan group of traumatized F_ mothers, there were 10% fewer females with mating plugs, and an increased percentage of nonpregnant females in comparison with control values; also, these two male groups had an increased percentage of males with no pregnant females. Results from males in the 2500 and 5000 ppm Captan groups were within normal limits for this strain of mouse and comparable with t values from control males. Litter-size distributions of live Implants derived from the first mating of F I generation males are presented in Table 6. Responses of control and Captan groups were within normal limits and readily comparable. The TEM group showed approximately a 13% reduction in litter size. Mean litter sizes were 11.72 for the control group, U.73 for tin1 2500 ppm Captan group, 11,56 for the 5000 ppm Captan group, 11.88 for the 5000 ppm (traumatized F 182. female) group, and �10.24 for the TEM-treatcd group. The data on the F generation littersize distribution are. presented graphically in Figure 2. Tables 7 and 8 summarize the data on dead implants per F, male .and dead implants per femaLe, respectively. The 2500 and 5000 ppm Captan groups showed a slight increase in total dead Implants for both males and females at the 4, 5, and >5 levels when compared with controls. TEM animals showed significant increases in dead implants for both males and females. Table 9 summarizes the breeding results by treatment of those FI males classified as presumptive sterile, partially sterile, or nonbreeders after three breedings. Table 10 identifies these F males individually by number and treatment. Details of the breeding and rebreeding data for presumptive F males are presented in Table 11. In the reference co:itrol group, 24 of the 200 males were considered as presumptive translocates. When rebred, 12 males remained in this classification. A third mating of selected questionable and/or nonbreeder males reduced the number of presumptive males to eight: 1 nonbreeder, 1 presumptive sterile, and 6 partially sterile (3 of which were questionable partially sterile). For the TEM group, 83 of 200 F males were identified as presumptive mutants after the first breeding. When rebred, 53 still met the original criteria. A third breeding reduced this number to 49; 4 continued to be nonbreeders, 14 were presumptive sterile, and 31 were partially sterile (6 of which were questionable partially sterile). In the 2500 ppm Captan group, 30 of 200 F males were identified as presumptive mutants after the first breeding. When rebred, 11 still met the criteria: 5 were nonbreeders, 1 was a presumptive sterile, and 2 were partially sterile (1 cf which was questionable partially sterile). The 5000 ppm Captan group also had 30 of 200 F^ males identified as presumptive mutants after the first breeding. The second mating reduced this number to 9. After a third breeding, 8 males still met the 183 �original criteria: 2 were nonbreeders, 1 was a presumptive sterile, and 5 were partially sterile. For the group of F. males derived from traumatized F females and males treated with 5000 ppm Captan, 12 of 50 FI males were identified as presumptive mutants after the first making. A second breeding reduced this number to 4 and a third breeding further reduced to 2 the number of F males that still met the original criteria; both were partially sterile, with one of them being questionable partially sterile. The data on the F. and F.. generation.?' fertility, breeding, and litter-size distribution'as well as the data on the F generation's dead implants and rebreeding show that Captan tends to induce doserelated effects on the reproductive performance of male mice. The data also suggest the presence of translocation heterozygotes in the 5000 ppm Captan group. Review of the data on dead implants, breeding, and rebreedlng for the F- generation of the TEM-treated group showed, as expected, the potential for the presence of translocation heterozygotes in 24.5% of the FI males. Cytogenetic Studies Table 12 presents the findings from the cytogenetic evaluation of meiotic cell preparations from F males in the Captan groups characterized as nonbreeder, presumptive sterile, or partially sterile. Also, eight control males and 5 of 49 TEM males were evaluated. v, Whenever possible, 25 spermatocytes per testls were scored. The slides were decoded only after all scoring was completed. The results are summarized as follows: • All eight males examined In the control group were cytogenetically normal. • The five TEM males all showed positive reciprocal translocations. • All eight males in the 2500 ppm Captan group were cytogenetically normal. • Seven males in the 5000 ppm Captan group were cytogenetically normal; however, the eighth male (No. 657) showed as a positive reciprocal translocation. 184 �• The two males examined In the 5000 ppm Captan group derived from traumatized F iemales were cytogenetically normal. Discussion Increased use of the translocatlon procedure has revealed that a meaningful relationship exists between the Incidence of dead Implants in F matings and the occurrence of a heritable translocatlon event. Previous experiments at SRI and at Oak Ridge National Laboratories have demonstrated this correlation. The following paragraphs discuss occurrences of dead implants in this study. When females had a total implant count of less than six or when all their implants were identified as dead, we generally considered thin to be a result of first breeding or of some factor other than compound treatment (such as background IncJ-denc.c) and excluded those females from this evaluation. Tables 13 through 17 present the total, dead, and live implantation data for suspect translocates of the control, the TEM group, and the three Captan groups. The control group (Table 13) showed a normal implant distribution, with the exception of one F male (No. 122) for whom the number of implants was high. In the TEM group (Table 14), the expected Increase in dead implants and the resultant decrease in live implants occurred, although the numbers of tot.al implants were generally normal. This pattern occurred during all breeding periods. The 2500 ppm Captan group (Table 15) contained seven males in the first breeding with females having high dead implant counts but normal live litter size, according to the criteria. Also, 3 males showed high dead implant counts during the first breeding, with live implant counts fitting the criteria for partially sterile males. This Increase in dead implant occurrence was not repeated in subsequent breeding, and all FI males were classified as normal after the breeding phases. 185 �(1 •'< -I I*-} Five FI males in the 5000 ppm Captan group (Table 16) showed an increase in dead Implants during the first breeding. In the rebreeding of these males, only male No. 657 continued to show the increase in dead implants and the resultant decrease in live implants. All other FI males in this group had a normal distribution of dead and live implants for all breedings. Table 17 presents the implant data for F. males derived from traumatized females in the 5000 ppm Capcan group. With the exception of an occasional female showing an increase in dead implants, the distribution of total, dead, and live Implants was normal. The numbers of total implantations were generally within normal limits for all experimental groups in all breedings. 186 �REFERENCES 1. A. Leonard and G. H. DeKmidt. Mutation Res. 9, 127 (1970). 2. B. M. Cattanach, C. E. Pollard, and T. H. Isaacson. Mutation Res. J>, 297 (1968). 3. D. S. Falconer, D. M. Slizynskl, and C. Auerbach. 81 (1952). J. Genet. 51, 4. W. M. Generoao. Proc. Second Annual Environmental Mutagcn Soc., p. 9 (March 1971). 187 �Table 1 AVERAGE BODY WEIGHTS IN GRAMS FOR MICE RECEIVING VARIOUS LEVELS OF CAPTAK IN THEIR DIETS Week of Test Initial 1 2 3 4 5 6 7 8 Control TEM Dietary Levels of Captan Cppm diet) 2500 5000 33.8 32.5 33.7 34.8 34.6 34.8 37.3 37.7 39.2 33.4 33.8 33.6 32.9 33.3 31.8 34.6 33.7 31.4 35.4 34.4 32.1 35.8 34.4 31.4 35.9 36.0 33.0 38.1 37.5 35.3 38.9 39.3 35.8 39.8 38.9 36.9 188 �Table ?. AVERAGE FOOD CONSUMPTION FOR MICE RECEIVING VARIOUS LEVELS OF CAPTAN IN THEIR DIETS (Grama of Food Consumed/Mouse/Day) Week of Test 1 2 3 4 5 6 7 8 TEM Control Dietary Levels of Captan (ppm diet) 2500 MOO 4.01 4.05 3.74 3.25 4.82 5.00 4.72 4.38 4.91 5.08 4.90 4.40 5.28 5.33 4.45 5.08 5.55 4.95 5.44 5.36 5.56 4.95 4.75 5.81 6.14 5.63 5.27 5,55 5.74 5.13 4.83 189 4.95 �Table 3 TRANSLOCATION STUDY OF CAPTAN F0 GENERATION MICE SUMMARY OF BREEDING AND LITTER DATA Parameter Control TEM 2500 ppm 5 0 ppm 00 5 0 ppma 00 60 66 60 31 30 120 132 119 61 59 Number pregnant 93 77 91 38 33 Percent pregnant 77.5 58.3 76.5 62.3 55.9 8 5 13.3 16.1 11.68 Number of FQ males Number of F females Number of nonbreeder males 8 19 Percent nonbreeders vO o 13.3 Average live litter size 12.30 6.55 12.16 5.84 3.26 5.68 Average number of males weaned/litter 28.8 6.29 — -11.33 5.25 Group of females accidentally tipped off rack following 1 week of mating with 5000 ppm-treated mates. These traumatized females, and their offspring, most of which could not be identified back to a particular FQ male, were considered as a separate group for the remainder of the study. �Table 4 TRANSLOCATION STUDY OF CAPTAN MOUSE LITTER-SIZE DISTRIBUTION OF LIVE YOUNG DERIVED FROM F GENERATION ADULTS 2500 Captan (ppm diet). _5000. jooof 1 0 0 0 0 2 0 I 0 3 0 3 0 0 0 4 0 8 0 1 5 0 10 0 1 0 0 6 0 15 2 1 1 7 8 I 0 0 1 2 12 8 0 0 9 9 8 3 3 1 3 10 6 2 8 6 3 11 8 7 18 9 24 5 12 3 1 8 5 13 22 0 20 9 8 14 18 1 10 6 1 15 6 0 6 1 3 16 3 0 3 0 0 17 0 0 2 0 0 18 0 0 0 0 0 Mean (p,) 12.30 6.55 12.16 11.68 11.33 3.89 5.84 4.97 5.73 6.09 1.97 2.42 2.23 2.39 2.47 Litter __Si*£__ Control 1 0 2 Variance (o3) Standard Deviation () a TEM Traumatized FQ females. 191 �24 \ I .— •in I I I I I I I I I i TO- Control 22 ___«,.. 2600 pprn Captan —»' • — • « 6000 ppm-Ceptan « • 6000 ppm Captan 20 (Traumatized Females) 18 16 ui 12 10 / ,>\ .•""••' -l5^ 8 10 12 14 LIT TEH SIZE FIGURE. 1 LITTER SIZE DISTRIBUTION FO GENERATION 192 16 IB �Table 5 TRANSLOCATION STUDY OF CAPTAN FL GENERATION MICE SUMMARY OF BREEDING DATA- -FIRST BREEDING Parameter Number of Fl males Number of females Number of mating plugs Percent mating plugs Number pregnant Percent pregnant Number pregnant with plug Percent pregnant with plug Number pregnant without plug Percent pregnant without plug Number not pregnant Percent not pregnant Number not pregnant with plug Percent not pregnant with plug Males with no pregnant females Percent males with no pregnant females a Traumatized Fo females. Control 200 600 450 75 492 82 434 88 58 12 108 18 16 15 13 6.5 TEM 2500 200 600 376 63 383 64 326 85 57 15 217 36 50 23 39 19.5 200 600 442 74 472 79 419 89 53 11 128 21 23 18 14 7.0 Captar. (ppm) 5000 jQOOa 200 600 439 73 477 80 413 87 64 13 123 20 26 21 9 4.5 50 150 95 63 112 75 94 84 18 16 38 25 1 3 7 14.0 �Table 6 TRANSLOCATION STUDY OF CAPTAN MOUSE LITTER SIZE DISTRIBUTION OF LIVE YOUNG DERIVED FROM FI GENERATION ADULTS--FIRST BREEDING Litter Size 1 2 3 4 5 6 7 8 9 10 11 12 13 . 14 15 16 17 18 19 20 Control TEM 2500 5 4 2 4 5 8 • 7 14 35 37 73 80 4 4 0 5 6 4 89 >. 72 32 16 7 1 0 0 9 12 .; , 17 '•'•' - 2 1 11 8 17 25 31 53 61 52 27 21 8 1 1 1 0 5 6 13 35 40 77 75 87 54 36 14 7 2 0 1 Captan (ppm) 5000a 5000 3 0 3 0 1 5 0 1 6 1 8 4 11 3 19 4 17 2 53 16 77 14 85 93 0 0 15 17 14 13 5 1 0 0 0 55 19 11 7 3 Mean (JA) 11.72 10.24 11.73 11.56 11.88 Variance Co*) Standard Deviation 8.13 13.59 8.68 7.40 7.20 2.85 3.69 2.95 2.72 2.68 (CO traumatized Fo females. 19A �120 Con 11 ol 110 — TEM 2500 mm GOOO ppm Capinn 100 — 5000 ppm Capinn (Ti,ium,ili/rd Females) in — 1. ..I._!...L.J....L10 12 LITTEfl SIZE FIGURE 2 LITTER SIZE DISTRIBUTION F, GENERATION 195 — FIRST MATING �Table 7 TRANSLOCATION STUDY OF CAPTAN SUMMARY OF DEAD IMP!ANTS PER Fl MALE Number of Males with Females Having Cap tan Copm) 5000 5000a Control TEM 2500 0 Dead implants 68 37 69 49 20 1 Dead implant 54 36 44 68 11 2 Dead implants 3 Dead implants 30 18 25 16 31 13 36 18 7 2 4 Dead implants 8 9 18 9 0 5 Dead implants 2 6 4 3 2 > 5 Dead implants 7 32 7 8 1 Traumatized FQ females. 196 �Table 8 TRANSLCX;ATION STUDY OF CAPTAN SUMMARY OF DEAD IMPLANTS PER FEMALF. Number of Dead Implants/Female Control TEM 2500 0 320 182 295 28^ 79 1 109 83 113 23 2 39 35 37 130 41 3 18 16 16 9 0 4 11 7 11 2 3 o 0 5 3 0 0 > 5 3 45 2 8 1 492 383 472 477 112 Total Pregnant Females Traumatized Fo female. 197 Cap tan (ppjn) „ 5000 j>QO.pJ 9 �Table 9 TRANSLOCATION STUDY OF CAPTAN SUMMARY OF PRESUMPTIVE TRANSLOCATION T MALES AFTER THREE BREEDINGS Control Total num&er of FI males TEM 2500 200 200 200 Number o f nonbree.der males Number o f presumptive sterile males Number of partially sterile males VO 00 Traumatized Fo females Captan 5000 1 1 6(3?) 4 14 31(6?) 200 5 5000a 50 2 1 1 2(1?) 5 0 0 2(1?) �Table 10 TRANSLOCATION STUDY OF CAPTAN INDIVIDUAL IDENTIFICATION OF PRESUMPTIVE AFTER THREE BREEDINGS MALES Partially Sterile Presumptive Sterile Nonbreeder Contro L 16? 65 122? 164? 189 190 72 220 TEM 215 216 232 235? 238 245 262 264 269 280 281 290 292 299? 300 314 315 327? 344 345 350 359 360? 361 371 375 376 388 390 399? 400? 202 231 251 256 268 273 288 317 321 326 339 343 369 389 220 241 391 396 Treatment (Continued) 199 �Table 10 (Concluded) Treatment Partially Sterile Presumptive Sterile Captan 2500 ppm 480? 526 474 449 479 496 523 583 Captan 5000 ppm 635 657 760 779 780 736 634 733 Captan 5000 ppma 805 837? ^Traumatized Fo females. 200 Nonbreeder �Table 11 TRANSLOCATION STUDY OF CAPTAN BREEDING AND REBREEDING SUMMARY OF PRESUMPTIVE MALES (Live Implants Only) Treatment Control Male No. 11 16 1.7 24 39 43 50 59 65 67 72 86 102 122 129 139 144 152 164 179 189 190 192 200 First Breeding (3 Females) .** 202 215 216 220 226 227 228 229 230 1.3 6 (15)* - (LI) 9 4 0 4 6 3 0 1 7 - Totals TEM Third Breeding (3 Females) Second Breeding 13 Females) _ 0 7 0 .2 9 1 /t (14) - . 6 5 (ID 13 9 0 6 - _ 0* 0 12 10 3 14 0 11 11 9 6 11 _ (12) 1 5 7 ~ 10 8 0 0 12 13 - 9 7 14 9 10 10 0 0 .4 (9) 0 - 0 (9) 2 9 - 0 7 12 12 14 14 11 0 U 2 4 12 14 10 11 11 - 12 _ 2 . 16 14 _ 12 12 4 - 14 13 (9) 0 0 0 0 1 1 0 1 - 12 2 '4 0 0 7 . 11 12 1.0 8 0 ._ 4 - 8 11 13 - (Continued) "0" indicates a plug was observed for a female that was not pregnant. ** •-" indicates a plug was not detected and the female was not pregnant. t,, ( ) indicates all implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. 201 �Table 11 (Continued) Treatment TEM First Breeding (3 Females) Females) Wo, 231 232 233 235 237 2218 239 241 244 245 251 253 254 256 258 262 264 267 268 269 270 273 280 281 283 286 288 290 292 294 299 300 302 314 315 317 318 321 322 ** . 0* 3 8 5 3 4 1 7 9 0 6 8 1 _ _ _ © 0 3 4 0 0 2 3 _ 0 9 © 9 0 5 6 8 8 6 9 0 0 4 0 9 0 0 10 8 5 _ Third Breeding (3 Females) Second Breeding (3 Females) Femalea) 1 0 5 _ 7 6 9 2 8 0 5 3 (18) t (15) 3 0 4 0 - 0 4 0 9 0 0 3 (Continued) 0 8 11 7 10 1 13 0 2 15 7 10 2 - 0 5 6 13 14 ©" - 10 1 0 14 13 13 5 © 13 13 0 0 11 14 0 4 5 © 11 3 0 11 5 9 12 _ 6 8 0 8 4 14 7 8 1 0 0 7 3 10 9 8 13 2 0 0 9 0 11 0 5 3 12 4 5 12 1 0 0 4 0 11 - . 5 2 - 10 0 0 8 11 9 2 15 4 0 11 .- "0" indicates a plug was observed for & female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. "( ) indicates all implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. "®" indicates female was pregnant but had no live implants. 202 �Table 11 (Continued) Male First Breeding Q Females) Treatment TEM 326 327 333 334 339 343 344 34e, 346 349 350 355 356 359 360 361 363 367 369 371 372 375 376 381 382 385 387 388 389 390 391 396 397 399 400 Totals ** 0 0 6 - .* 0 0 10 (10) t 0 1 12 4 9 (15) 4 5 0 4 (16) 3 5 (13) 9 . 3 0 5 4 ) 4 4 .6 .. .. 0 (11) 5 4 4 3 - 4 2 0 0 5 4 12 10 8 9 - () 6 0 13 11 7 2 3 12 4 5 10 5 1 11 10 15 3 5 -. 6 (^ F 0 0 4 4 11 8 0 0 3 © 7 14 5 . 5 9 0 12 12 11 10 0 0 5 3 10 I (15) 7 () P 11 5 7 1 . 3 7 4 3 5 3 11 5 4 0 2 5 0 4 2 - 5 2 „ 0 5 (12) 2 () 9 - _ 0 0 3 6 - Third Breeding Second Breeding 13 Females)} (14) (ID 1.1 12 53 83 49 (Continued) "0" Indicates a plug was observed for a female that was not pregnant. ** , '-" Indicates a plug was not detected and Che female was not pregnant. "( ) indicates all Implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. " ( ) " indicates female was pregnant but had no live implants. ? 203 �Table 11 (Continued) n Treatment Captan 2500 ppm Captan 5000 ppm Male No. 430 432 436 449 450 451 452 455 461 469 472 474 479 480 484 489 496 518 523 526 528 546 561 568 569 582 583 585 588 590 Totals 601 604 620 622 626 627 634 635 First Breeding (3 Females) .* * b 4 (17)' _ _ 9 5 Third Breeding (3 Females^ Second Breeding (3 Females) — " 9 8 12 11 _ 11 8 10 _ 12 12 12 13 10 0 13 10 13 0 0 13 0 11 12 11 . 11 _ 0 11 _ 11 13 10 13 11 9 11 10 _ - 5 . 11 13 13 11 0 _ . 8 10 7 13 12 — 0* 7 9 8 (3) 4 11 7 0 8 - 9 8 7 8 . 8 - 1 0 10 14 10 10 - 9 . - 11 6 . _ - 0 14 12 . 3 () 9 10 0 30 (U) 9 . 8 - (1) . . (13) . 12 (12) 9 () 8 9 () 9 8 - . (13) 11 _ 9 _ 0 0 14 9 11 8 12 9 0 11 9 9 0 (12) 10 - 11 0 10 12 12 6 <B 8 12 _ 14 _ 13 9 15 13 11 U 14 (P1 - (Continued) ** "0" Indicates a plug was observed for a female that was not pregnant. "-" Indicates a plug was not detected and the female was not pregnant. "( ) indicates all Implants were in early stages of development and Impossible " to determine If they were live or dead upon gross observation. " ? " Indicates female was pregnant but bad no live implants. () 204 �Table 11 (Concluded) Treatment Captan 5000 ppra Fl (tale No. 638 646 653 657 660 668 671 672 679 722 732 733 734 736 743 760 761 765 767 769 779 780 First Breeding (3 Females) 10 10 0* 2 0 . 6 4 0 7 9 9 10 8 2 4 805 806 815 816 817 818 822 823 837 84L 846 847 (13)f 5 10 . 0 - 5 10 0 0 1 6 1 8 _ 6 8 (15) 8 (12) 1 5 10 11 14 2 14 9 14 6 10 5 (14) Totals 9 (10) 6 - 12 13 13 1 9 12 11 12 11 9 10 4 12 12 14 3 12 12 11 12 14 1 5 12 12 12 0 0 10 0 13 11 13 13 4 2 30 Totals Captan 5000 ppm 6 -** Third Breeding Second Breeding J 3 FemaJjBs) 10 0 11 9 12 10 - 4 9 7 (11) - . 12 13 9 7 11 6 13 7 13 10 10 14 11 0 8 10 12 10 2 10 10 . 12 - 11 2 13 10 12 12 12 14 4 12 Traumatized ?o females. "0" indicates a plug was observed for ; ** "-" indicates a plug was not detected and the female was not pregnant. "( ) indicates all implants were in early stages of development and impossible " to determine if they were live or dead upon gross observation. 205 �Table 12 TRANSLOCATION STUDY OF CAPTAN CYTOGENETIC EVALUATION OF FI MALE MICE Fl Testes Weight (ma) 16 64.6 265 Partially sterile (questionable) Normal 58.5 38.8 68.0 48.7 307 237 232 289 Nonbreeder Partially sterile Normal Normal Normal Normal 164 Control Body Weight (g) 17 65 72 122 Treatment Male No. Classification-Based Upon Breeding Data 55.8 314 Partially sterile Presumptive sterile Partially sterile Cytogenetic Classification (questionable) N> o Normal (questionable) TEM Captan 2500 ppm 189 190 232 262 290 345 361 49.6 47.5 245 222 Partially sterile Partially sterile Normal Normal 54.6 41.4 62.6 54.1 50.4 276 243 222 294 278 Partially Partially Partially Partially Partially Positive reciprocal translocation Positive reciprocal translocation Positive reciprocal translocation Positive reciprocal translocation Positive reciprocal translocation 449 474 479 480 60.2 56.5 65.1 58.7 256 175 287 307 Nonbreeder Presumptive sterile Nonbreeder 496 523 526 583 60.9 50.7 54.1 50.7 242 348 295 297 sterile sterile sterile sterile sterile Partially sterile Normal Normal Normal Normal (questionable) Nonbreeder Nonbreeder Partially sterile Nonbreeder Normal Normal Normal Normal �Table 12 (Concluded) Male No. Body Weight (g) Testes Weight (mg) Classification-Based Upon Breeding Data Capcan 5000 ppm 634 635 657 733 736 760 779 780 57.1 56.0 61.0 62.2 57.9 55.9 54.8 53.0 299 312 231 256 256 298 224 295 Nonbreeder Partially sterile Partially sterile Nonbreeder Presumptive sterile Partially sterile Partially sterile Partially sterile Normal Normal Positive reciprocal translocation Normal Normal Normal Normal Normal Captan 5000 ppma 805 837 70.0 56.9 268 303 Partially sterile Partially sterile (questionable) Normal Normal Treatment ro o Traumatized Fn females. Cytogenetic Classification �Table 13 TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES local F Male Female Dead Live Number Number ^plantations Implantations Implantations First breeding 11 16 1 2 3 1 2 .* * - - 24 39 43 50 59 65 67 72 86 102 122 -.v 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 (13)t . . - 3 17 13 • ? . •• . . - . (13) . - 9 0 . 9* 0 . 4 B 7 0 0 . 0 i 1 4 7 6 0 0 - 4 0 5 . - Nonbreeder Partially sterile (questionable) Nonbreeder Nonbreeder 0 5 0 5 . . . •• (11) If, Initial Clas sj.ficatloi Normal Nonbreeder Partially sterile Presumptive sterile u 1 0 0 . - . . . 7 10 2 0 . (11) 6 2 13 Partially sterile Nonbreeder Nonbreeder Nonbreeder Partially sterile (questionable) Normal "0" indicates a plug was obgtfrVod for a f e m a l e t h a t u.is not pregnant. "-" indii:ar.?s a piu); was not dt-tuctb'd and Che female uis not p r e g n a n t . "()" indicates all Implants vrere in e a r l y stages of development, aric! thus d i f f i c u l t to determine if they were live or dene! upon gross 'observation. 208 �Table 13 (Continued) I'RANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MA1JES F Male Female Total Number Number lmp_l anjt ationa First breeding (concl.) 129 139 IV* 152 16/t 179 189 190 192 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 I 2 3 1 2 3 ! 2 1 I 2 _ ..Contrpljjroujg Dead Live Implant a_ti on s Imp lantBliona ** - . - . - 7 12 9 - - 4 3 0 . .. - 0* 1 0 0 1 0 It 0 0 0 6 0 7 0 ? - - . - 2 3 200 (L4) + - 3 1 - - . . 3 9 9 . . 0 1 0 1 It 6 7 (14) - - Initial Claaslficatio Nonbreeder Nonbreeder Nonbreeder Partially sterile Nonbreedei Nonbreeder Partially sterile Partially sterile Partially sterile (questionable) Nonbreeder Final Classification Second 11 breeding 16 I 2 3 1 2 17 3 1 2 3 13 9 (15) - 0 1 •; - - - - - 13 H (15) - Normal Rebred Rebred h b "0" Indicates a plug was observed for a female Ihut was not pregnnnt. "-" iinlic.ii.es .1 plug was ni<t delected and the fern .Tic was not pregnnnt. " ) incllc.itps .ill implants wrrc in cnrlv stages of development, and thus difficult to (" determine if Llicv were l i v e or dead upon gross observation. See t h i r d b r e e d i n g f o r f i n a l i - L a s s if t e a t ion . 209 NOT REPRODUCIBLE �Table 13 (Continued) TRANSLATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE T F Male Female Total Number Number Implantations Second breeding (cont.) 24 39 43 50 59 65 67 72 66 102 122 129 1 2 3 I 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 139 I 144 2 3 i 2 3 MALES Control Croup Live Dead Implantations Implantations 0* 12 tafr 0 12 11 12 10 12 3 6 2 15 11 0 . (12) + 0 1 0 . 0 0 0 0 3 0 0 0 1 0 0 - - ? - - - 10 1 1 6 16 14 14 2 . 15 •• 9 10 0 7 7 8 1 0 0 0 11 • 0 12 11 12 10 9 3 6 2 14 11 - Final Classification Normal Rebred Rebred h K Normal Normal Partially sterile Normal 0 - (12) 9 5 16 7 7 4 14 9 10 Rebred b Rebred b Normal Normal Rebred b Normal Normal "0" indicates a plug was observed for a female tluit was not pregnant. ;( "-" Indicates a plug wns not detected and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine 11 they were live or dead upon gross observation. See third breeding for final classification. 210 �Table 13 (Continued) TRANSLOCATION STUDY OF C APT AM IMPLANTAIION SUMMARY OF PRESUMPTIVE F MALES _ ._..._ F, "Male" Female " "ToTaL™ Dead Live Numhe r Number Lni£].ant_a,tlon8_ IniE.lantatJ.ons. lEJRL™!:8.1-!??.! -_F. Second breeding (cone] .) 152 164 179 169 190 192 200 Third 16 breeding 17 39 43 72 1 2 3 1 2 3 1 2 3 1 2 1 1 2 3 1 2 3 1 2 3 1 2 1 2 3 1 2 3 1 2 3 1 11 8 14 _*•« (9)t 11 - 1 10 0 1 . 13 ? 1 - () 9 10 - 0* 0 0 0 0 1 0 0 13 12 13 .. _ . 12 12 11 10 - 0 0 0 0 I 1 - 0 - . _ . 0 1 0 0 . - - e . . 0 0 0 Normal Rebred Normal Retired 1 0 0 12 11 13 . _ 12 11 11 10 - Rebred Normal Normal Partially sterile (questionable) Nonbreeder Normal Normal Presumptive sterile 86 3 1 2 3 - - 14 14 5 2 2 1 12 12 4 Normal "0" Indicates J plug was observed for a female tliac wjs not pregnant. "-" indicates a plug was not decocted and the female was not pregnant. " ) indicates all implants were in early stages of development, and thus difficult to (" determine 1C they were live or dead upon gross observation. 'SRC rhird breeding for final classification. 211 �Table 13 (Concluded) TRANSUOCAXION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F HALES Control Croup Dead Live F. Male Female Total Number Number 1Implantations Implantations Implantations Third breeding (concl.) 129 164 189 190 1 2 3 1 2 3 1 2 3 1 2 3 14 ** . • 0* 1 0 1 1 "0" indicates a plug vas observed 0 . . 0 0 0 0 0 14 . . • 0 1 0 1 1 for a female that was not pregnant. "-" indicates a plug was not detected and the female was not pregnant. 212 Final Classification Normal Partial ly sterile (questionable) Partially sterile Partially sterile �Table 14 THANSLOCATION STUDY OF CAPTAN TMPLANFATION SUMMARY OF PRESUMPTIVE F, MALES __ __TEM Oroujs Total Dead Live F. Male female Number Number jjpg 1 an tail on s. Implantations .lmp.1 ant a First breeding 202 215 216 220 226 227 228 229 230 231 232 233 I 2 3 1 2 3 1 2 3 1 2 3 I 7. 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 2J5 3 1 2 217 3 1 2 3 0* 0 0 kit - . (9)* . 0 11 II - () 9 - 8 0 10 . 0 0 0 0 . .. 7 . . 0 7 9 - 7 1 0 1 - 0 - - - - 12 9 9 5 9 - 10 . - - 15 11 11 7 4 5 b 0 3 12 12 0 0 Q . () 9 . .. .0 4 2 . () 9 7 0 9 . . 0 3 4 I - PisaumptLve sterile Nonbreedcr Partially sterile (questionable) Nonbr eerie r Nonbrceder Partially sterile Partially sterile (questionable) Normal Nor.breeder Presumptive sterile P a r t i a l l y sterile Nonbreeder e 7 Partially s t e r i l e 6 5 9 6 P.irtlally sterile "0" lndic«ti* r < ••' pl"K w.is observed [or n fcmalr tli.u was not prepnant. i. •/: "-" ind itiitr i .1 pliiR u,tq nol di-rortrd and Lhc C entitle w,is not pregnant. "()" i n d i c a t e s ,1.1 i m p l . m l s uorr in e a r l y stnges ol d e v e l o p m e n t , and thus d i f f i c u l t to i l c t c n n i n c i f t h e y were 1 L v o or dead upon gross o b s e r v a t i o n , 213 �Table 14 (ContInucd) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRKSUMPTIVE F. MALES F, Male Female Number Number First breeding (cont.J 238 239 241 244 245 251 253 254 256 258 262 264 267 266 269 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Implantations 14 0* 9 .* * . . 6 0 4 0 0 0 12 12 6 0 0 TEM Group Dead Live Implantations Implantations 11 0 8 . . . . . 6 0 1 3 0 I . . _ 0 0 0 9 2 2 0 0 Initial Classification 0 - - 12 12 15 8 8 . 10 4 10 5 3 . - . - - - Nonbreeder _ Nonbrcedcr . - Nonbreeder 0 0 1 0 0 3 10 4 0 - Partially sterile Presumptive sterile Normal Partially sterile Presumptive sterile 0 2 0 5 3 5 . _ . . . "0" indicates a plug wan observed for a female t;<at WAS not pregnant. t- "-" indicates a plug was not detected and the fe.ua le was not pregnant. 214 Partially sterile Presumptive sterile Partially sterile P rtially sterile Nonbreeder Nonbreeder Nonbreeder �Table 14 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES TEM_Groi;ji Total F, Male Female Number Number Implantations First breeding (cont.) 270 273 280 7.81 283 286 288 290 292 294 299 300 302 314 1 2 3 I 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 0* .* * - 13 1? .1 4 12 12 11 9 . - 0 0 0 9 14 11 14 10 0 8 (18)t 11 11 9 (15) 11 . . - Dead Implantations 0 4 5 1 4 6 3 2 1 . 0 0 0 4 9 7 8 7 0 0 9 2 3 3 1 2 . - Live Implantations Initial Classification 0 . 9 7 2 0 6 9 <! 8 .. 0 0 0 5 5 4 6 3 0 8 (18) 9 8 6 (15) 9 . - Presumptive sterile Nonbreeder Partially sterile Partially acerUe Normal Nonbreeder Presumptive sterile Partially sterile Partially sterile Partially sterile (questionable) Normal Partially sterile (questionable) Normal Nonbreeder "0" Indicates a plug was observed for a female that wan not pregnant. "-" Indicates a plug was not detected and the female was not pregnant. "()" Indicates all Implants were In early stages of development, and thus d i f f i c u l t to determine If they were live or dead upon gross observation. 215 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES TEM Group F. Male Female Number Number First breeding (cont.) 315 317 318 321 322 326 327 333 334 339 343 344 345 34fi 34S 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 Total Dead Live Implantations Implantations Implantations Initial Classification 0* 3 0 0 0 0 0 5 9 10 0 0 1 . 0 - - 0 3 0 0 4 4 3 0 0 9 . . . 0 - 0 0 0 - 5 - 6 . - 0 .* * 9 13 13 0 0 10 . . 0 11 . - 0 0 0 0 10 12 14 10 10 . <!'-) + 0 0 0 0 7 7 8 6 5 . . 7 Partially sterile Presumptive sterile Partially sterile Presumptive sterile Normal Nonbreeder Presumptive sterile Partially sterile Nonbreeder 0 - Presumptive sterile 0 0 0 Presumptive sterile 3 r j - Tart tally sterile 6 4 5 . - Partially sterile Nonbreeder Partially gtcrllo (quest lonable) . (12) "0" indicates a wns observed Cor a female rli.it was not pregnant. ""'',.,, ' • • i •% . i - < : . . . » . . '-" indicates a phiR was not Jctp.ct.ed and the female was not. prcgndht . "()" indicates all implants nere in e a r l y stapes of development, and thus d i f f i c u l t to determine if they were live or dead upon gross observation. 216 �Table 14 (Continued) TRANS I/ICATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE I' F "Male _Njumb;" r Flvst breeding (cont.) Fema ' c Number rc'tdl Im£lantat lona 350 I 2 3 TKM Grou P IV ad Live Impi.int.'itloiis Imp Ian t u t lone 12 11 H _.h, 355 356 359 360 36L 363 36 / 369 371 MALES Initial Classification P a r t i a l l y sterile . - . - Nunbreeder . - I 2 3 4 3 2 - 1 2 3 8 8 6 . - - Nonbrceder ft 8 8 9 /i 8 P.irl t a l l y 3 t « r l IP r, 1 2 3 12 16 1 2 3 (l'j) + - •] • - 1 2 3 12 14 11 8 10 2 4 <i 9 1 2 3 9 - 4 - s - I'artUilly sterile i 2 3 . - . - . - Nonbreudur I 2 3 ()'•'' - 0 - - (15) - - P a r t i a l l y start If (rjur.it ioriahle) P.u-tially sterile 0 - Presumptive s t e r i l e - /, I 2 3 LO 13 - 6 7 - 372 1 2 3 (16) - •> - 375 1 2 3 14 .. - 11 . - 3 - P,irti.al Iv s i e r i l e 1 „! 3 11 0 10 6 0 Part i.illy s t e r i Lr '• 5 0 6 1 - . - 17(> 381 6 - (16) - P a r t i a l l y sterile. P a r t i a l l y sterile (questionable) Nonbret'iler "0" i n d i c a t e s a p l » t r ft-1. - w "'! ohsi-r^cil FIT ,1 ( r m a l c t h a t w j s n o i prf-|;iianL . i nil 11.'Hi", ;i p l u j > , was not d i ' l i ' C tod nnd the f o i i ' i l c was not p r e g n . i n t . j "()" Lii(ltc,ii.cs a l l Linplniun u r r o in ''.'ii-lv 'itajies cl dcvi loju'it'il , <uid thus d i f f i c u l t d c i i ' r m i n o i l i h o v won 1 1 i v o IT .lead upon BIMSS n l i s o r v i i l i n n . 2.1.7 to �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES TEM Group F. Male Female Number Number First breeding (concl . ) 382 . 38$ 387 (13) + (11) _** 1 2 3 1 9 6 () 6 . . 14 9 0* . 11 12 14 . . 3 1 2 3B9 390 391 396 397 399 400 Implantations 1 2 3 2 388 Total 3 1 2 3 I 2 3 I 2 3 1 2 3 I 2 3 1 2 3 1 2 3 . (14) 11 9 (ID 12 9 (12) Dead Implantations Live Implantations 7 7 0 1 (13) (ID 9 5 () 6 7 5 0 .. - . 3 4 0 - 7 3 5 . . Partially sterile (questionable) Partially sterile 4 9 8 . - Initial Classification 11 . ? 6 5 7 10 7 7 - Nonbrecder Partially sterile Nonbrecder Partially sterile Nonbrceder Nonbrceder Partially sterile (questionable) (14) 5 4 (ID 2 2 Partially sterile (questionable) Partially sterile (questionable) (12) Final Classification Second breeding 202 2li I 2 3 1 2 0 0 0 11 1 0 0 0 4 5 0 0 0 Presumptive sterile 7 4 Partially sterile 3 "0" indicates a plug was observed for a female chat was not pregnant . * "-" indicates a plug was not detected an;! the female wns not p "(}" indicates; all implants were In early stages of development, and thus difficult to determine if they were live or dead upon gross observation. 218 �Table 14 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES TKM[..Group F. Male Number Second 'iroedlng '.cont.) 216 220 226 227 228 220 230 231 232 Female Number I 2 1 L 2 1 I 2 3 1 2 J 1 2 3 L 2 3 1 2 3 1 2 3 1 7. 233 235 237 ?18 239 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 Tot nl Imj)1 an tat Ions Imp lantatlons .* * . 14 5 12 11 a i/i 12 12 15 14 13 11 10 0* 0 0 17 12 15 11 15 7 11 13 . - 2 3 0 7 0 0 0 1 I 0 0 0 0 0 0 0 9 10 10 0 I 1 Live implantations ImpJLanta - 4 Normal a 14 12 11 Normal 14 14 Normal n 11 10 - 8 2 5 11 15 6 1 2 0 . - 3 - 7 13 Normal Normal Norm.il Partially sterile 13 - Normal - Rebred "0" I n d i c a t e s a p l u j ^ was observed for n f o m a l c that was nor p r e g n a n t . "-" i n d i c a t e s a p l u g was not d e t e i t c ' d and clip female was not pregnant. 219 Partially sterile 7 ,'. Si'e clii.nl breeding for f i n a l c l a s s i f L e a l ion. Presumptive sterile 0 8 10 13 0 . . - Normal 0 0 9 12 13 - Rebrodb Normal LU 10 14 n Rebred 12 2 12 11 14 14 14 Final Classification . 4 (> I 1 4 1 2 241 Dead �Table 14 (Continued) TRANSLATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES TEH droop Totnl F. Male Female Number Number Implantations Second breeding (cont.) 244 245 251 253 254 1 2 3 I 2 3 1 2 3 1 2 3 1 2 256 258 262 264 267 268 269 270 273 280 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 I 2 3 1 2 3 Dead Implantation Ltve Implantations 12 13 11 1 0* 7 0 0 G 16 11 .* * 2 0 0 0 0 4 0 0 0 2 1 10 13 11 1 0 3 0 0 0 14 11 13 14 11 . 13 0 B 12 10 9 11 12 14 I 14 _ 0 0 0 - 13 L4 11 13 0 - .. 6 7 - 8 0 11 11 10 0 0 3 8 5 9 6 3 1 L 2 _ . 0 0 0 - - 0 - 5 4 5 0 5 9 13 0 12 _ 6 7 B - - - 3 3 5 8 B 5 "-" Indicates .1 plug was not detected and the female was not pregnant. Sec t h u d breeding fci f i n a l classi.ficdti.ori. 220 Normal Partially sterile Presumptive sterile Normal Normal b Rebred Normal Partially sterile Partially sterile Normal Rebredb Partially Rebred 0 "0" I n d i c a t e s a plug was observed Tor a fe.uale Lli.-it was nut pregnant.. :> Final Classification Rebred sterile b b Partially sterile �T a b l e 14 (Continued) TPANSLCCATION STUDY OF (.A.PTAN IMPLANI'ATrON SUMMARY OF PRESUMPTIVE F, MALES JNk'T.bs.1. •Second breeding (cont . ) 281 (in1???!?..!?. 1 1. 3 1 2 Total i3lP':anJ:1tA(-nilc T.KM_Gruup Dead Live !mP'j?,n.Ln..!LLPnl?. .-!S.P.!-Ji1?.a!:-^yP..5. 11 I? 10 7 11 8 4 1 2 Final C.lagsIficatton Partially sterile 14 3 14 0* .** 286 1 . . .. - - - 28S 2 3 1 0 0 0 0 0 0 0 0 0 6 3 0 7 5 0 (i 11 3 3 3 1 0 1 5 8 1 8 10 2H3 2 290 292 294 3 1 'f. 1 1 "I 3 ;. 2 :i 299 ; 2 300 :i 1 302 2 3 1 2 314 315 :> i i 3 1 2 317 3 i 11 H 0 9 14 11 11 12 12 1/4 321 Retired Presumptive sterile Partial ly sterile P-ITI islly sterile No rma I 9 5 4 10 8 Partially sterile 5 (questionable) 0 1 0 13 7 12 1? 5 11 Part tally sterl le 8 2 I 4 0 0 0 0 0 0 f) 0 0 Partially storlle n 0 0 0 0 =, b 2 9 4 11 0 0 0 i) - 13 q 12 13 n 15 \ 14 9 1 2 3 12 11 Normal No rma 1 0 2 1 0 - 4 9 12 12 L 318 0 0 - n 0 n - - 2 12 15 "0" I n d i c a t e s a plup. was observed for n female thai w.ii, not p r e g n a n t . r.-* "-" indicates i plug uda nut doii'clod and thr female wns not Si.'u third breeding for f i n a l classification. 221 pregnant. Normal Presumptive .sterile Normal Presumptive sterile �Table 14 ( C o n t i n u e d ) TRANSLOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES TEH Group F, Male Nurtb'er Second breeding (cent.) 322 326 327 333 334 339 343 344 345 346 Female Number Total Im plantations Dead Implantations 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 I 2 3 1 11 11 _** 0 0 0 - 2 349 3 1 2 350 3 1 2 355 3 I 2 356 3 1 2 3 0* - Live Imp Ian tat ion a Final Classification 11 11 0 0 (10) Normal Presumptive sterile 13 (10)t 12 0 - 13 •) - 0 0 0 0 Presumptive sterile 0 0 0 0 0 0 Presumptive sterile 0 2 0 - 0 0 0 0 0 0 10 5 10 6 6 2 8 I 0 1 - 11 6 1? 4 12 12 10 11 1? a 0 3 2 0 10 8 0 9 n 0 - 0 0 - IL . (1^) 0 . •> b Rcbred 10 0 No rnifl 1 . - Uebrecl n - 0 5 4 Partially sterile 5 4 4 3 12 11 - Partially aterile Normal 10 8 10 0 0 I 11 0 Normal Partially sterile Normal - 11 . Normal (15) "0" Indicates a plug was observed for a female that was nor pregnant. *i - indicates a plug was not detected and the female was not pregnant. "()" indicates all implants ucrc in early stages of development, and thus difficult, to determine if they were live or dead upon gross observation. Sue third breeding for final c lassification. 222 �Table 14 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE f } I'EM C r o u p Dea.l Live Implantations Implantations F, Mali? Number Second breeding (cont .) Female Number Total Implantations 359 1 2 3 LO 9 16 1 2 3 10 13 _** 1 2 3 13 13 8 I/. K 10 10 360 361 363 367 369 371 372 375 376 381 3H2 J65 387 3KH "0" I 2 3 1 2 3 1 2 3 3 6 9 8 13 - Partially (questionable) 2 0 - 12 14 - Norma 1 f> 6 0 11 11 . - 4 5 11 Normal 10 11 14 1 2 3 11 9 - 1 0 - 1 2 3 5 11 1 l, 11 0 4 0 - 3 0 0 0* - sterile Partially sterile 8 -3 1 2 3 1 2 3 7 3 7 2 0 Final Class.If Icatlc 3 7 0 *) 6 9 1 2 3 MALES - - 5 5 5 10 9 5 7 1 1 11 0 - Partially sterile Robred ' Partially sterile Normal Partially sterile Partially sterile Normal 1 2 - - - - " Rcbrrd 1 2 3 10 17. 7 0 0 0 10 12 7 Normal 1 2 3 I'j 12 b 0 15 - Normal 1 2 J 9 3 R i;> intl i rates <i |il"R uas observed for 3 3 4 a [(.male Lli.-u VMS not pregnant. -.• "-" IndicaiTb n plui' war. not ilererred and the female* was not pregnant. Sec tlilnl brucilinK for f i n a l *. l a s s i fi c a t i o n . 223 b Partial ly sterile �Table 16 ( C o n t i n u e d ) TRANSLOCAIION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRF.SUMPTIVE F MALES TEM Group F Male Female Number Number Second breeding (concl.) 389 1 2 390 391 396 397 399 400 3 I 2 3 1 2 1 2 3 1 2 3 I 2 3 1 2 3 256 1 2 3 I 2 3 1 2 3 1 268 Third breeding 2 3 l 216 220 241 Total Imp lao tat ions bead Implantations .* * 14 12 12 • . 11 0* 0 11 7 9 • Live Implantations Final Classification 2 5 3 ~ 273 b Partially sterile Rebred b . 0 0 0 9 3 0 5 8 1 . 11 Pebrcdb 0 Normal 0 5 2 4 Partially sterile (questionable) 4 5 2 Partially sterile (questionable) 4 . . . Partially sterile . 5 . . - - - - 0 0 0 14 7 4 9 13 3 9 . - (questionable) Nonhrecder Nonbreeder Presumptive sterile 2 270 Rebred **r Presumptive sterile 1 2 9 - 0 - 9 - 1 2 . . „ - - - Normal Presumptive sterile 3 "0" Indicates a plug was observed Cor a fomale tliir was no I pregnant. t; "-" indicoLes a plug was nor. detected and the fc'in.i'.c was not pregnant . Set- third breeding for final classification. 224 �Table 14 (Concluded) TRANSLOCATION STUDY OF CATTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MAIES TEM Croup F. Male Number Third breeding (cone I.) Ftfma IE Number 286 1 2 3 1 2 3 1 327 334 2 369 3 1 2 332 389 391 1 2 J 1 2 1 1 2 396 3 I 2 3 Total Iro plantations 11 .* * 11 (9) t - L'ead Jjnplantnt ion.j I 10 •i - Live il.1Pla.n.?flI_'-5n,?. Final Class if icat Ion 10 1 (9) - 14 12 . - 2 0 . - 12 12 . - 11 10 - 0 f) 0 0 - .- - - - • . - - - - . . - - - Partially sterile (questionable) 11 0* Normal - 10 - Normal Fresumpt ! v sterile Normal Presumptive sterile Nonbrccdcr Nonbreeder "0" indicates a plug was observed for a femulc that was not pregnant. "-" Indicates a plug was not detected and the Ecmalc was not pregnant. " ) indicates all implants were In early stages of development, and tl'-us difficult to (" determine if they were I Ive or dead upon gross ibsrrvation. 225 �Table 15 TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F L MALES F. Ma,le Female Number Number First breeding 430 432" 436 1 2 3 1 2 3 1 2 449 450 451 452 455 461 469 472 474 479 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 480 464 3 1 2 3 I 2 3 Total Implantations 2500 ppm Group Dead Live Implantations Implantations .* * - . - 4 4 (17)t 9 14 7 11 . 9 0* 5 14 11 0 - 10 12 13 . . , _ 9 11 12 . . . (3) f) 11 16 11 9 5 2 3 . - 4 (17) 9 9 5 8 . . 4 0 1 0 7 0 9 10 10 . Nonbreeder Partially sterile (questionable) Partially sterile Nonbreeder 14 4 Initial Classification 0 0 1 2 3 .. .. 1 1 '3 - . . No nn a 1 Norm.! I Normal Nonbreeder Nonbreeder Nonbreeder 8 10 Normal 9 . . - 7 0 • (3) 0 - 7 2 0 14 11 Nonbrecjdef Nonbreeder Partially sterile 4 Normal "0" indicates a plug was observed for a female that was not pregnant. :• - indicates a plug was not detected and the female was not pregnant. "O" Indicates all Implants were l.i early stages of development, and thus difficult to dutprmine if they were live or dead upon gross observation. 226 �Table 15 (Continued) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F. MALES F, Male Female _Nfunibe_r_ Number Firs I breeding (concl .) 489 1 2 3 496 1 i 518 3 I 523 3 I 2 2 526 3 1 2 1 528 546 561 1 2 3 1 2 3 1 2 568 3 1 2 569 582 583 585 588 590 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 1 I 2 3 Total Dead I.«!]p..l_ant_at_i._ons. Implantations )4 13 8 .* * . . - 3 1 2 . . . - Live Initial Classification 11 12 6 Normal - Nonbreeder - Nonbres-der - Nonbi'p.edei 7 7 4 (l)t 0 1 • ) 3 (I) 0* - 0 0 - . . . . - - L2 () 9 (13) . 4 8 () 9 .. . - - - . - 9 I . . - - - 10 11 1 15 (12) 11 0 9 - 1 3 . i 3 0 0 Partially sterile Presumptive sterile Nonbreeder Nonbreeder Partially sterile (questionable) (13) . . 9 10 12 - Nonbreeder Nonbreeder Nonbreedur Normal Partially sterile (questionable) (12) 8 0 Normal 9 "0" indicates <n pluK was observed for a female chat van not pregnant. * - indicates a plug was not decoded and the Eemjlc wus not pregnant. 4- " ) indicates all implants were in early stages of development, and thus difficult Lo (" determine I f they were l l v r or deaf I upon Rrosa observation. 227 �Table 15 (Continued) TRANSLOCAT10N STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES F, Male Female Number Number Second breeding 430 432 436 449 450 1 2 3 1 2 3 1 2 3 1 2 3 1 2 451 452 455 4f.l 469 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 472 474 3 I 2 3 I 2 479 480 3 1 2 3 1 2 3 Total Implantations 2500 ppm Group Live Dead Implantations Implantations Final Classification 12 e 14 12 11 13 12 12 13 .* * 11 13 14 0* 0 11 14 11 13 10 12 12 13 11 11 . . 12 0 12 11 0 (13)t 8 I 12 8 12 11 10 12 11 12 13 .10 13 13 0 0 10 L 0 0 0 0 1 13 11 13 10 12 11 0 0 13 11 9 .. 11 0 0 2 1 1 1 1 0 0 1 0 1 0 0 2 . . I ! 0 11 10 0 - Normal Normal Retired 0 1 t Normal Norm ill Normal Normal Normal 0 Normal Rcbred - b Normal 0 - Normal Rcbred b b - (1J) 9 Rcbred b "0" indicates a plug was observed for a female that was not pregnant. .-* - indicates a plug was not detected and the female VMS not pregnant. "()" Indicates a l l implants were in early stages of development, and thus difficult to determine If they were live or dead upon press observation. Sec third breeding for final classification. 228 �Table 15 (Continued) TRANS LOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F, MALES ? 5.QP.. PJ"P . F, M a l e jluinbot Second breeding Fundlc Number 48'. 1 2 3 n 1 2 3 9 13 14 ( con t . ) 489 496 518 523 526 528 546 561 568 569 582 •>B1 505 588 I 2 3 1 2 3 1 2 3 1'Otlll Tmplan in t tons. Dcnd ImjjJ an t a t. limn Live Imp ) an ta tlcna 0 0 2 11 0 12 Normal 0 9 11 14 No rma 1 0* 14 'i 0 - .** . - . - Kchrcd n 15 . . . - I 2 3 No nn a 1 - 0 r 1 2 3 0 11 IJ - n • 1 2 3 > 0 0 0 0 •j b Rcbred b b 0 14 - Normal - Rob red - i . . - - Rcbred 1 2 3 11 11 H 2 0 0 9 11 8 Normal 1 2 3 11 - n - 11 - 0 10 in Normal 1 2 3 7 0 7 !') 2 > 13 8 1 ? 3 - - - Rrbri'd I 2 3 12 11 13 0 12 13 13 Normal 1 2 11 <) - 0 - 9 - Rehriid r> (1 0 - "0" indicate."-, a p l " H was observed 1'or a Crm.ilc 111,11 u is ncl p r r K M ' U i L :.-v< v Rebred" 0 0 0 0 11 1 2 3 . - Final C l a o s i f l c "-" i n d i c a t e s a plug was noi d e l e c t e d and die f o n m l e \im nol p r e g n a n t . hire r h i r i l b r e a d i n g for f i n . ' j l c l a s s i f L c u c i o n . 229 Normal b b �Table 13 (Concluded) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES 2500 ppm Croup F, Male Female Number Number Second breeding (concl.) 590 Third breeding 449 474 479 480 1 2 3 1 2 3 I 2 3 1 2 3 1 2 496 523 526 546 561 583 588 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total Dead Implantations Implantations 0* 13 12 .* * . 10 . •• . 2 I 0 9 13 15 11 - 0 2 0 . , . B . . 0 0 0 0 0 0 0 - - - 12 . - - 1 Live Implantations Final Classification 0 11 12 . . Nonbreeder - Presumptive sterile 2 - Nonbreeder . 2 I 0 9 13 15 11 11 - "0" indicates a plug was observed for n fcmnU* chat was not pregnant. ft "-" (nilicat.es .1 p l u g was not detected and the fur.ialo was not pregnant. 230 Normal Pdrtially sterile (questionable) Nonbreeder Nonbreeder Partially sterile Normal Normal Nonbreeder Normal �T a b l e 16 TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F F^ Male" Female Number Number First 601 breeding 604 620 622 1 2 3 1 2 3 1 2 3 1 2 626 3 I 2 3 627 634 635 1 2 3 1 ? 3 1 2 3 638 1 2 3 646 653 657 660 1 2 3 1 2 3 1 2 3 1 2 668 3 1 2 3 Total Implantations .* * ( 1 I) + (8) 7 11 14 H . - CM . 9 11 R . •• 10 6 6 12 - 5000 ppin Grou P_ Dead Live IseJ.SPiat.lonS. Jj)J tial Classification . 9 7 0 2 •i 0 '! _ - 0 ) 8 8 . - . Partially sterile (questionable) Normal Normal Partially sterile (questionable) Nonbreeder Normal R . - 0 0 0 2 - Nonbreeder Nonbrocder 10 6 0* 0 ? - 9 7 8 7 0 11 H 7 . (H) (8) 7 9 ') 8 -. . - 1 3 0 - (13) 13 8 MALES 0 1 0 . 1 6 Normal 10 0 (13) - Normal Partially sterile (questionable) 2 5 1 0 10 Partially sterile Normal H . - Partially sterile 6 "0" i n d i c a t e s a plug was observed for a female t l u t u.-is not p r e g n a n t . V: "-" indir.-itcs a plug was not detected an<i rhc female uas not pregnant. t '( ) i.ndical.es all Implants were in early stcigrs of development, and tbus difficult to determine if chpy were live or dead upon gross observation. 231 �Table 16 (Continued) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PRESUMPTIVE Fj MALES F. Male Female Number Number First breeding (cont . ) 671 672 679 722 732 733 734 736 743 700 7ft 1 765 767 I 2 3 1 2 3 1 2 3 1 2 3 I 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 Total Implantations .* * . 6 0* 12 . 4 . . -. 0 - (15)t 8 5 9 12 12 11 - 5000 ppm Group Dead Live Implantations Implantations _ .. . 6 0 0 0 8 4 . . 4 0 . . . . 0 0 t (15) 1 7 5 0 8 1 9 3 . 9 3 1 10 - 1 8 (12) 8 •t Nonbreeder Nonbreeder Partially sterile Nonbreeder Partially sterile Nonbreeder Nonbreeder Nonbrseder Partially sterile (questionable) Partinlly sterile Normal Normal 10 - 2 3 769 2 3 12 .. - Initial Classification 8 (1?) Normal Partially sterile (questionable) "0" indicates a plug was observed for a female thnr was not pregnant . "-" indicates a plug was not detected and tlic female was not pret-nanL. " ) indicates all implants wen1 In early stages of devulopmont, and thus difficult to (" determine if thoy were live or dead upon Rro.i.9 obsctvation. 232 �T a b l e 16 TRANSLOCATION STUDY OF CAP! AN IMPLANTATION SUMMARY OF PRF.SUMPTIVE F ] MAJ,ES F" Male _Numher_ First breeding i.concl .) 'Female fjumbfii: 779 1 2 3 780 I 2 3 _ __5000_ ppni G r c u p Tor.al Dead Live I m p l a r t a t iona Imp Ian tat iona .Implantations 2 0 1 0 4 1 0 0 0 n* 2 0 1 4 1 ;_ I n i t i a l Classlilcatic P a r t i a l l y sterile P a r t i a l l y sterile Final C l a s s i f i c a t i o n Second breed ing 601 604 620 622 626 627 634 635 <>38 '>46 M3 1 2. 3 I 2 J n 1 0 11 0 Normal 10 0 0 I 9 10 1'. Normal 12 1 2 3 1 2 10 1 2 3 (12) t 12 1 2 3 I 2 3 13 1 2 3 I 2 1 2 J 1 2 J 0 1 Normal 0 0 0 12 Normal 0 (12) 12 Normal 1 10 6 0 No rma 1 - 0 I 9 Rebred " - 1 1 0 Rebred 10 12 11 0 0 0 10 12 11 Nonndl 11 0 0 1 Norm al 10 1I 13 9 n 0 0 0 14 13 10 b Normal n 10 "0" i n d i r a r c s a p l n p w.is obsorvod for J f e m a l e I b a i was n o t p r e g n a n t . !• "-" i n d i i . i t , " ; .1 p l u R was n n t d o t c c t r d find tliv f c m . i l r wds IIOL p r e g n a n t . * " ( ) " i n d i r d t o - j . i l l i m p K n U i wc'rr i n p . i r l y s t a p e s oC i l l v c l o p m c n L , .mil t h u s d i f f i c u l t i l j l v r m l n c L f Liu1)1 were l i v e nr dcjd upon i>ross n ' . ) ? o r v a t ion. Sec r'.urd h r c c d i n n f;n l n u l c \ .i.->s i f ic.ii ion . 233 to �Table 16 ( C o n t i n u e d ) TRANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF PRESUMPTIVE F , MALES F. ,Mj»lc Number Second breeding (cont . ) Female Number Total Implantations 657 1 2 3 1 ? 3 I 2 3 1 2 3 1 2 3 1 5000 pp<n Group _ Dead Live Implantations Imp! an t a t lo n 9 Final Classlf Lcat ion 7 9 5 14 10 12 9 13 13 14 11 -** .660 668 671 672 679 2 722 732 733 734 736 7'3 , 760 761 765 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 '3 5 2 1 8 1 4 0 1 0 14 9 12 Normal 0 9 12 12 Normal 1 1 0 0 . - - U» 0 7 12 ' ' 0 0 14 11 - - - - 14 7 12 3 - 12 0 - - Normal Normal 12 - Partially sterile 1 - - - 0 0 11 12 _ - Normal Rebred Normal - Re b red 11 12 _ . - - - 14 10 11 0 0 10 1 0* 0 0 1 0 0 0 5 14 1.2 0 0 5 13 11 12 11 9 1 ! 3 0 0 13 11 9 h Normal LO "0" indicates a pliifl was observed for a female that was not pregnant. "-" indicates n plug was not di'toclcd and rhu female was inn. pregnant. 234 Rebred 14 .1 Soc 1'iird breeding for final classi Cication. 1) Normal - 1 b Partially sterile Normal No nn LI 1 �T.ihlo 10 ( C o n e l u l l e d ) IKANSLOCATION STUDY OF CAFTAN IMPLANTATION SUMMARY OF I'RESUtlPTIVE F MALES SpOOjpm F Male; Number Second brcudlnK (cone I .) 767 ?r,9 Total female Numbc r Implant actions 1 ? 3 I 1 2 0* 4 2 0 0 ll 0 0 0 0 1 0 2 I 0 0 1 . - . 1 . 0 2 ) 771 /HO 2 3 Tlur.1 breeding 634 I 2 3 635 722 •'33 1 2 3 1 2 3 1 2 3 736 Dead Implantations I. 1 2 3 14 13 12 12 13 10 13 . .. - 0 12 13 12 Normal 12 13 10 Normal 0 4 P a r t i a l l y s«ti>rilt! Nonbrccder 1'artlally sterile 12 - - "0" indicates ti plug was observed for a female thai was not pregnant. I: "-" indicates a plup, was not detected and the female was not pregnant. 235 Partially sterile Norma 1 Nonbrecdcr Presumptive sterile �Table 17 fRANS LOCATION STUDY OF C APT AN IMPLANTATION SUMMARY OF PRESUMPTIVE F F Male Female Number Number First breeding 805 806 815 816 8L7 Total Implantations I 2 3 1 2 3 I 2 3 1 2 3 1 2 818 822 823 837 3 I 2 3 1 ? 3 1 2 3 I 2 3 841 84(> 847 1 2 5000 pp.n Group Dead Live Implantations Implantations ** . 6 9 7 - . 0 0 . . 6 9 0 - (10)t (U) . •J - - 10 - 0 . . - . . - - - . - 0 5 - - 1 (14) - •; - Nonbrceder Nonbreeder Partially sterile Partially sterile. (questionable) 10 '.' 0 . - - (10) (U) . . - 6 . - Initial Classification 7 1 2 3 2 3 MALES - - 6 . 5 (14) - Nonbreed?r Normal Nonbrceder Nonbreeder Nonbreoder Nonbreeder Partially sterile Partially sterile (questionable) - Final C l a s a i f i c a t l o n Second breed! n i>. H05 Bebred ' i n d i c . u o s t [V.umiiLl/.c'd F., f e m a l e s . ,v ,v-\ t "0" i n d i c a t e s <i pl'ip, was observed tor a Ornate t l u L was not. "-" indic.'ilc's ,1 plug was n''t detected jnd rht; fomnlc was nol procnjnt. "()" indicate 1 . 1 ; J l l i m p l a n t s wcro in r j r l y suigos o f d e v e l o p m e n t , and thus d i f f i c u l t t o d e t e r m i n e if they were l i v e or dead upon gross o b s e r v a t i o n . 'Sue I ' . i i r d brci-dinp, f o r f i n j l classification. 236 �T a b l e 17 ( C o n t i n u e d ) TRANSLATION STUDY OF CAPI AN IMPLANTATION SUMMARY OF PRESUMPTIVE F MALES _ _ _ .5000' ._|jpm (-rpu[)_ _ _ Dead Total Live rmg lap tat ions Implant a t :um:j Implantations Final C l a s s i f i c a t i o n F. Male Niinibe r Second brooding (concl.) Female Number 806 1 2 3 13 14 10 1 0 0 12 14 10 Normal 1 2 ') I'l 11 13 1 0 3 IJ 11 10 Normal I 2 i; * Normal I 2 3 11 n* n 4 0 - 7 0 •• Kobr«-d h 0 0 11 811 816 817 818 822 823 837 841 846 847 1 2 3 805 817 B22 8 1) 2 n 1 0 Normal 12 6 1 2 b 1 2 3 n - 1 2 1 8 1 - - - I1) 2 0 - 13 10 - Normal 0 0 0 10 12 12 Noniia 1 1 10 10 14 Normal I 2 3 1 2 3 1 2 3 Third b reed inn ** 1 2 Kobred'' in in 12 12 ;i in 1/1 . n 0 - 0 0 „ Retired _ 1'artially sterile - 2 7 1I 2 11 Normal in i? i? Normal 1J n 1 2 3 10 12 12 0 I n d i c ' i i l c s tr.jum.it izcd F _' Normal - I 2 3 4 13 - n 0 Inn.ili";. "0" i n d i c a t e s ,1 I ' l n p was n b s c i v e d lor a Connie f i a r vjni MOL prep.n.ini. "-" i n d i c a t e s n pluj; was not d i ' l p c t r d .irnl the female u;is mil p r e g n a n t . Sot t h i r d breeding for l i n j l c la-ii if i r a t i o n . 237 �Table 17 (Concluded) TRANSLOCATION STUDY OF CAPTAN IMPLANTATION SUMMARY OF PKESUMPTIVF, f} MALTS F. Male Female Number Number Third breeding (concl.) 837 1 2 3 Total Implantations -** - 5000a ppm Uroup Dend Live Implantations Implantatlona - - 'Indicates traumntizod F. females. '-" indicates a p l u g was nol. detected and the female was not pregnant. 238 Final Classification Normnl �a a: a. V ,- � 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. 012 Folder The folder containing the original item. 0101 Series The series number of the original item. Series II 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 Simmon, Vincent F. Ann D. Mitchell Ted A. Jorgenson Description An account of the resource <strong>Corporate Author: </strong>Stanford Research Institute, Menlo Park, California Date A point or period of time associated with an event in the lifecycle of the resource 1977-05-01 Title A name given to the resource Evaluation of Selected Pesticides as Chemical Mutagens 'In Vitro' and 'In Vivo' Studies Subject The topic of the resource animal testing pesticide testing