1 15 5 https://www.nal.usda.gov/exhibits/speccoll/files/original/3eb21aeedec7655e768f2dd325145aed.pdf 85569229284257be4204655bf9da2c87 PDF Text Text Item ID Number: Author Corporate Author 001 oe Cockerham, Lorris G. U.S. Air Force, Air Force Systems Command, Frank J. Seiler Research Laboratory Report/Article Title Histopathological and Ultrastructural Studies of Liver Tissue from TCDD-Exposed Beach Mice (Permyscus Polonotus) Journal/Book Title Year 198 Month/Day Marcn Color ° D Number of Images 61 Descriptor) Notes Project 2303 Thursday, December 28, 2000 Page 106 of 157 �FRANK J. SEILER RESEARCH L A B O R A T O R Y FJSRL TECHNICAL REPORT - 80-0008 MARCH 1980 HISTOPATHOLOGICAL AND ULTRASTRUCTURAL STUDIES OF LIVER TISSUE FROM TCDD-EXPOSED BEACH MICE (PERQMYSCUS PQLIQNOTUS) LORRIS G, COCKERHAM ALVIN L, YOUNG CHARLES E, THALKEN APPROVED FOR PUBLIC RELEASE; PROJECT 2303 DISTRIBUTION UNLIMITED. AIR FORCE SYSTEMS COMMAND UNITED STATES AIR FORCE �FJSKL-TR-80-0008 . '• '- * This document was prepared by the Faculty Research Division, Directorate of Chemical Sciences, Frank J. Seiler Research Laboratory, United States Air Force Academy, Colorado. The research was conducted under Project Wnrk Unit Number 2303-F1-83, "Ultrastructural Evaluation of Tissues Removed from Animals Exposed to TCDD". Lt Colonel Lorris G. Cockerham was the Project Scientist in charge of the work. When US Government drawings, specifications or other data are used for any purpose other thar a definitely related Government procurement operation, the Government thereby incurs no responsibility nor any obligation whatsoever, and the fact that the Goverrroent may have formulated, furnished or in any way supplied the said drawings, specifications or other data is not to be regarded by implication or otherwise, as in any manner licensing the holder or any other person or corporation or conveying any rights or permission to manufacture, use or sell any patented invention that may in any way be related thereto. Inquiries concerning the technical content of this document should be addressed to the Frank J. Seiler Research Laboratory (AFSC), FJSRL/NC, USAF Academy, Colorado 80840. Phone AC 303-472-2655. This report has been reviewed by the Chief Scientist and is releasable to the National Technical Information Service (NTIS). At NTIS it will be available to the general public,* including foreign nations. This technical report has been reviewed and is approved for publication. Major, USAF, BSC Project Scientist Director of Reeearch/DFCBS KENNETH E. Colonel, USAF Director Directorate of Chemical Sciences SIURU, JR., Lt Colonel, USAF Commander Copies of this report should not be returned unless return is required by security considerations, contractual obligations, or notice on a specific document. Printed in the United States of America. Qualified requestors may obtain' additional copies from the Defense Documentation Center. All others should apply to: National Technial Information Service 5285 Port Royal Road Springfield, Virginia 22161 �UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 1. REPORT NUMBER 2. GOVT ACCESSION NO FJSKL-TR-80-0008 4. RECIPIENT'S C A T A L O G NUMBER 5. TYPE OF REPORT A PERIOD COVERED TITLE (and Subtitle) Histopathological and Ultxastructural Studies of liver Tissue fron TCDD-Expqsed Beach Mice (Peroroyscus polionotus) 7. 3. a Final Report 1974-1977 PERFORMING O<3G. REPORT NUMBER B. AUTHORfs.) 6. CONTRACT OR GRANT NUMBERfsJ Lt Colonel Lorris G. Cockerham Major Alvin L. Young Lt Colonel Charles E. Thalken 9. PERFORMING O R G A N I Z A T I O N NAME AND ADDRESS Department of Chemistry and Biological Sciences 10. PROGRAM ELEMENT. PROJECT, TASK AREA * WORK UNIT NUMBERS USAFA/DFCBS-R USAF Academy, Colorado 80840 II. 12. CONTROLLING OFFICE NAME AND ADDRESS REPORT DATE I'.arch 1980 Department of Chemistry and Biological Sciences USAFA/DFCBS-R 13. NUMBER OF PAGES USAF Academy, Colorado 80840 14. 51 MONITORING AGENCY NAME ft ADDRESS^? different tram Controlling Office) 15. SECURITY CLASS, (of this report) UNCLASSIFIED 1S«. 16. DECLASSIFICATION/DOWNGRADING SCHEDULE DISTRIBUTION STATEMENT (at this Report) Approved for public release; distribution unlimited 17. DISTRIBUTION S T A T E M E N T (ol the abstract entered In Block 20, It different from Report) 8. SUPPLEMENTARY NOTES 9. KEY WORDS (Continue on reverse side It necessary and Identity by block number) Animal Survey; Beach mouse (Peroroyscus polionotas); Bioaccumulation; Ecological Effects; 2,4-Dichlorophenoxyacetic acid (2,4-D); Herbicide, Hepatic Parenchymal Cells; Histopathology; Morphometric Data; TCDD; Teratogenic; 2,3,7,8-Tetrachlorodibenzo-p-diaxin (TCDD); Test Area C-52A, Eglin AFB Reservation; 2,4,5Trichlorophenoxyacetic acid (2,4,5-T). 0. A B S T R A C T (Continue on reverse side II necessary and Identity by block number) Quantitative ultrastructural studies were conducted on liver tissue from beach mice, Peromyscus polionotus, exposed to the toxin 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) in field and laboratory environments. Hepatic tissue from 52 animals was examined for changes in smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and mitochondria. Fifteen of 30 animals were collected from a unique military test site in northwest Florida where they had been continuously exposed to soil levels of 10 to 710 parts per trillion (ppt) DD , F O R M73 1473 JAN EDITION OF 1 NOV 65 IS OBSOLETE UNCLASSIFIED �Jff CLASSIFIED SECURITY CLASSIFICATION OF THIS PAGEfHTiwi Data Entered) 20. Abstract (continued) TCDD. Twelve of 22 animals were exposed 10 times in 28 days to 2.5 parts per billion (ppb) TCDD applied as a dust to their pellage. All remaining animals were from either a field control site or not exposed to TCDD in the laboratory. All tissue was examined both nistopathologically and by an ultrastructural stereological technique. The levels of TCDD in composite liver samples from mice collected in the field varied frcm 960 ppt for females to 1300 ppt for males, while a composite liver level of TCDD for the laboratory animals was 125 ppt. The levels of TCDD in the livers of the beach mice collected from the field substantiated bioaccumulation of TCDD but not food chain biomagnification. Although the levels of TCDD in the livers were greater than those found in the soil, TCDD was not detected in the portion of the food chain consisting of seeds. The laboratory dusting study confirmed that ingestion of TCDD can occur as a result of body contact with soil containing TCDD and subsequent grooming by the burrowing animal. No significant histopathological or ultrastructural changes were found in hepati parenchymal cells after long term, low level exposure to TCDD in the field, or after short term, low level exposure in the laboratory. Statistically significant differences in liver weight to body weight ratios concurrent with the absence of cellular changes following exposure to TCDD in the field is explained by the low level of exposure. This study has demonstrated the application of the analytical technique of stereology to field studies of toxicity. The modified technique, as used in this study, may be a valuable tool for characterizing quantitative cellular responses to injury. UNCLASSIFIED �FJSRL-TR-80-0008 HISTOPATHOLOGICAL AND ULTRASTRUCTURAL STUDIES OF LIVER TISSUE FROM TCDD-EXPOSED BEACH MICE (PERCMYSCUS POLIONDTUS) By Lt Colonel Lorris G. Cockerham Major Alivin L. Young Lt Colonel Charles E. Thalken Department of Chemistry and Biological Sciences USAF Academy, Colorado 80840 Prepared for: Headquarters Air Force Logistics Ccraaand Wright-Patterson Air Force Base, Ohio 45433 TECHNICAL REPORT FJSRL-TR-80-0008 March 1980 Approved for public release; distribution unlimited. Directorate of Chemical Sciences Frank J. Seller Research Laboratory Air Force Systems Command US Air Force Academy, Colorado 80840 ��PREFACE The authors gratefully acknowledge the support of the Frank J. Seller Research laboratory for providing the electron microscope facility required for this project. Appreciation is also expressed to Air Force Logistics Comtiand (AFLC/LO) for providing funds for field work and analysis of the tissues. The authors also wish to thank the Interpretive Analytical Services Division, Dow Chemical U.S.A., Midland, Michigan, and the Armed Forces Institute of Pathology, Washington, D.C., for competent and consistent analytical and histopathological support. �TABLE OF CONTENTS Title Page Introduction 1 Materials and Methods 5 Soil and Seed Analysis Animal Description laboratory Study Animal Preparation and Examination Hepatic Ultrastructural Study TCDD and Histopathological Analyses Statistical Analysis Results 5 5 6 7 8 11 11 15 Soil and Seed Analysis Beach Mouse Grooming Habits Analysis of Livers and Pelts Body Weight and Organ Weight Analysis Histopathology Hepatic Morphometric Analysis General Cellular Observations Discussion 15 15 15 17 29 30 37 39 Field Study 39 Laboratory Study Methods 43 45 Conclusions 46 Literature Cited 48 11 �LIST OF TABLES Number 1 Tissue Preparation Schedule to Prepare Liver Sections for Ultrastructural Study 2 3 4 5 6 7 Page 9 Concentration (ppt) of TCDD in Soil From Grid I and From the Control Area 16 Concentration (ppt) of TCDD in Liver and Pelt Samples from Beach Mice, Peromyscus polionotus, Collected from Control and TCDD-Exposed Field Sites, 1974 16 Body Weights and Organ Weights of Control Peromyscus polionotus Obtained in June 1974, Test Area C-52A, Bglin AFB, Florida 18 Body Weights and Organ Weights of Treated Peromyscus polionotus Obtained in June 1974, Test Area C-52A, Bglin AFB, Florida 19 Organ Weights, Expressed as Percent of Body Weight, of Control Peromyscus polionotus Obtained in June 3.974, Test Area C-52A, Eglin AFB, Florida .21 Organ Weight, Expressed as Percent of Body Weight, of Treated Peromyscus polionotus Obtained in June 1974, Test Area C-52A, Eglin AFB, Florida 22 8 Initial and Final Body Weights of Peromyscus polionotus Dusted with Alumina Gel Containing 2.5 ppb TCDD (Test) ... 23 9 Body Weights and Organ Weights of Peromyscus polionptus Dusted with Alumina Gel Containing No TCDD (Control) T ... 24 10 Body Weights and Organ Weights of Peromyscus polionotus Dusted with Alumina Gel Containing 2.5 ppb TCDD (Test) ... 25 11 Organ Weights, Expressed as Percent of Body Weight, of Peromyscus polionotus Dusted with Alumina Gel Containing No TCDD (Control) 12 27 Organ Weights, Expressed as Percent of Body Weight, of Peromyscus polionotus Dusted with Alumina Gel Containing 2.5 ppb TCDD ( e t Ts) 13 28 Hepatic Morphonetric Data of Control Peromyscus polionotus Obtained in June 1974, Test Area C-52A, Eglin AFB, Florida. . 32 111 �LIST OF TABLES (Continued) Number Page 14 Hepatic Morphometric Data of Treated Peromyscus polionotus Obtained in June 1974, Test Area C-52A, Bglin AFB, Florida. . 33 15 16 17 Hepatic Morphometric Data, Expressed as Ratios, of Control and Treated Peromyscus polionotus Obtained in June 1974, Test Area C-52A, Eglin AFB, Florida ..... . ....... 34 Hepatic Morphometric Data of Peromyscus polionotus Dusted with Alumina Gel Containing No TCDD (Control) .... 35 Hepatic Morphometric Data of Peromyscus polionotus Dusted with Alumina Gel Containing 2.5 ppb TCDD (Test) .... 36 18 Hepatic Morphometric Date, Expressed as Ratios, of Peromyscus polionotus Dusted with Alumina Gel Containing No TCDD (Control) or with Alumina Gel Containing 2.5 ppb TCDD (Test) ..... . ........... ....... IV .38 �LIST OF FIGURES Number Page 1. Hepatic Parenchymal Cell Prior to Printing with a Dot Grid Overlay (x5726) 12 2. Hepatic Parenchymal Cell Printed with Dot Grid Overlay for Morphcmetric Analysis (x5726) 13 3. Acute Necrosis and Inflammation in the Liver of a Beach Mouse Captured from Grid I. Hematoxylin and Eosin 31 4. Microscopic Appearance of Venous Ectasia in the Kidney of a Beach Mouse Captured from Grid I. Hernatoxylin and Eosin 31 ��lOTRQDUCTION Dioxin (2,3,7,8-tetracMorcdibenzo-p-dioxin; TCDD) has been called the most toxic chlorine-containing onmpound. It may occur as a contaminant of wood preservatives, pesticides, and medical and industrial chemicals produced from chlorinated phenols ( ) The acute oral LD5Q 6. is reported in the range from 0.6 yg TCDD/kg body weight in male guinea pigs to 115 yg TCDD/kg of body weight in rabbits (16,38). Sublethal doses have produced pathological changes in liver, spleen, intestine, thymus, lymph nodes and adrenal glands in laboratory studies (11,21,35,38) Data, however, have indicated that the liver is the major target organ for the effects of TCDD (4,11). Rarely, if ever, in nature are men and animals subjected to massive exposures to TCDD. The few incidences of known exposure (5,20,34) are thought to have been to minute quantitites (picograms) for relatively short time periods (3-6 weeks). Most recently, the presence of TCDD as a contaminant in the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) precipitated concern over the use of this herbicide in the United States ( 9 . Under present conditions of application of 2,4,5-T 1) herbicide, the estimated concentration in the soil would be less than one part per trillion (ppt) (19). Nevertheless, data are needed on the potential effects of low level, long-term exposure to TCDD. The experiments reported here were designed to quantitatively assess the effects of low level exposure to TCDD on the ultrastructural hepatic morphology in animals living in the field. The goals of this study, then, were to (1) determine what ultrastructural changes occur �in hepatic parenchyraal cells in response to low level, long-term exposure to TCDD in the field, (2) determine what ultrastructural changes occur in hepatic parenchymal cells in response to low level, short-term exposure to TCDD in the laboratory, (3) determine if ingestion, and hence liver accumulation, of TCDD can occur as a result of body contact and grooming and not necessarily through the food chain, and (4) demonstrate the use of stereology in the quantitative assessment of toxicity in a field environment as well as in the laboratory. A suitable field site for this study must necessarily (1) be contaminated with significant (i.e., readily detectable) quantities of TCDD, (2) have an endemic animal population present, and (3) be isolated from human activity, yet available for investigation. A unique site in northwest Florida possessing these criteria has been reported by Young (42). In support of programs testing aerial dissemination systems, Test Area (TA) C-52A, Eglin AFB Reservation, Florida, received massive 2 quantities of military herbicides. This approximately 2.6 km test area received approximately 73,000 kg of 2,4,5-T and 76,790 kg of 2,4dichlorophenoxyacetic acid (2,4-D) herbicide during the period 1962-1970. Significant levels (10-710 ppt) of TCDD were found in 1973 within the top 15 on of the test area soil. Test Area C-52A is principally a grassy plain surrounded by a forest stand dominated by longleaf pine (Pinus palustris), sand pine (Pinus clausa), and turkey oak (Quercus laevis) (42). The portion used in the present study was a cleared area occupied mainly by broomsedge (Andropogon virginicus), switchgrass (Panicum virgatum), woolly panicum (Panicum �lanuginosum), and low growing grasses and herbs. Of major interest in o this study was an 0.4 km plot located in the southern portion of the testing area. Although dissemination of herbicides at this site was discontinued after two years, it received the heaviest application. From 1962 to 1964, this site (called Grid I) received 39,547 kg of 2,4-D and 39,547 kg of 2,4,5-T. By 1969 only traces (parts per billion; ppb) of 2,4,5-T were detected (42) while TCDD was detected at significant levels in 1973 in analysis of soil samples from the top 15 cm of soil. Analysis of soil cores at 15 cm increments to a depth of 90 cm indicated no detectable TCDD (lower limit of detection was 10 ppt) below the 45 cm level. A more detailed description of TA C-52A, its history and present status, may be found in reports by Young (42) and Young, Thalken, and Ward (43). These reports are available from the Defense Documentation Center, Defense Supply Agency, Cameron Station, Alexandria, ^ 22314. The most common mammalian species reported on TA C-52A is the beach mouse, Peromyscus polionotus (30,42). This was the animal of choice for investigating long term, low dosage effects of TCDD in the field because mice have been used extensively in toxicological studies of TCDD (9,11,35,38) and thus provide known indicators of toxicity. Concurrently with the field studies, a laboratory experiment was conducted to simulate contact of the rodent's pelage with TCDD contaminated soil. The objective of the study was to determine if ingestion of TCDD can occur in the field as a result of body contact and grooming and not necessarily through the food chain. The accumulation of TCDD in the liver would implicate grooming as a means of contact while �histopathological and ultrastructural studies of the liver would assess the effects of a low level, relatively short-term exposure to TCDD. Thus a comparison of long and short-term effects on the same species could also be accomplished. �MATERIALS AND METHODS Soil and Seed Analysis To establish the actual levels and the persistence of TCDD in the soil in June 1974, samples of the top 0-15 cm of soil were taken from six sites on Grid I. One of these sites was also sub-sampled at increments of 0-2.5, 2.5-5.0, 5.0-10.0, and 10.0-15.0 cm. These soil samples, along with soil samples from four designated control areas approximately 800 to 1600 meters east of Grid I, were later analyzed for TCDD concentrations. To eliminate the food chain as an intake route for the TCDD, seed samples were taken from living plants adjacent to burrows on Grid I. These living plants were of the same species as the soil contaminated plants found in the burrows. The composite seed samples were also later analyzed for TCDD content. Animal Description The beach mouse is a small rodent weighing about 13 g, approximately 120 mm in length, with brown (adult) or dark gray (juvenile) fur on the back, and pale gray to white fur on the ventral region and legs (43). It may be found in old field habitats and in areas of 5% to 60% vegetative cover, preferring sandy areas. Field work for this study was conducted in June and July 1974. Havahart traps (Havahart Traps, Dept 1, P.O. Box 551, Ossing, NY 10562), sizes 0 and 1, for small animals, were used to trap the rodents. The traps were baited with a mixture of peanut butter and oatmeal and then �randomly placed on areas of the test grid where 20% to 80% vegetative coverage was present, or near openings to mouse burrows. The four designated control areas approximately 800 to 1600 meters east of Grid I were trapped in the same manner as was Grid I. Traps were checked daily and were moved to other locations within the test and control areas after four days failure to catch an animal. Fifty-three live mice were captured and taken to .the laboratory for histopathologic examination, hepatic ultrastructural study, and chemical analysis of the tissue. Fifteen of the mice captured from Grid I were designated as treated field animals and the first 15 mice captured from the control area were designated as control field animals. The remaining 23 mice from the control area were selected to be used as subjects in a laboratory dusting study. Laboratory Study When it was observed that the mice spend much of their active hours grooming, another route of contact with TCDD besides the food chain was proposed. As the rodents enter and leave their burrows, they pass through the TCDD laden 15 cm of soil. This soil adheres to their pelts and as a result of the grooming habits of the beach mouse, the TCDD could be ingested in this manner. With this thought in mind, a laboratory experiment was designed to simulate a probable source of contact for the beach mouse. Twenty-three of the beach mice captured from the designated control areas were brought into the laboratory and individually placed in separate Iso-cages (Carworth, Division of Becton, Dickinson and Co., �New York) and maintained on laboratory chow (Ralston Purina Company, General Offices, Checkerboard Square, St Louis, MD). The 23 animals were weighed, sexed, and randomly divided (using a random numbers table) into a "control" group of 11 animals (four female and seven male) and a "test" group of 12 animals (five female and seven male). These animals were observed for two to three weeks (depending on date captured) in the laboratory to determine grooming habits and to allow for metabolic stabilization after change in diet before dusting was initiated. The fur on the ventral thoracic and abdominal regions, sides, back and tail on each test animal was dusted with 100 mg of alumina gel containing 2.5 ppb TCDD by analysis. Control animals were dusted in the same areas but with alumina gel alone. All dusting was accomplished using a camel hair artist's brush. The 100 mg application per animal resulted in an approximate exposure of 60 mg of gel at each application per animal (based on average weight of recovered residue following dusting). The dusting procedure was repeated every third day for a total of 10 applications during a 28 day period. On the 29th day the 22 mice (one control animal died apparently as a result of handling) were sacrificed and prepared for examination. Animal Preparation and Examination The 30 mice selected for the field study and the 22 mice from the laboratory study were prepared for examination using a cervical dislocation procedure to accomplish humane euthanasia. All animals were then weighed, skinned and systematically examined for gross developmental �defects such as cleft palate, cleft lip and polydactyly. Body and organ weights were recorded, internal organs were examined for gross lesions and representative sections of each tissue were placed in neutral 10% buffered formalin and processed for histopathological examination. A representative section of the liver was also processed for ultrastructural studies. All remaining liver tissues and pelts were pooled according to the study, sex and treatment, placed in glass jars, frozed and submitted for TCDD analysis. Hepatic Ultrastructural Study After the liver was removed from the 52 beach mice, and weighed, a section approximately one ram thick was taken from across the central lobe (Lobus centralis). This section, to be used for the ultrastructural study, was minced and transferred to containers of the primary fixative, 2% glutaralydehyde, buffered to a pH of 7.2 with Sorensen's phosphate buffer solution. Fixation of the minced tissue was allowed to continue for two hours at 4°C prior to rinsing with buffer solution to remove any excess fixative. The small pieces of tissue were then post-fixed for one hour at 4°C with phosphate-buffered 1% osmium tetroxide. The tissue was rinsed again with buffer solution prior to dehydration with a graded series of acetone. After dehydration, the tissue was transferred directly from 100% acetone to a graded series of solutions of acetone and the embedding medium, Epon-812, and eventually to the embedding medium alone in BEEM capsules. An outline of the preparation procedure is presented in Table 1. 8 �TABLE 1. TISSUE PREPARATION SCHEDULE TO PREPARE LIVER SECTIONS FOR ULTRASTRUCTURAL STUDY SOLUTION Glutaraldehyde (2%) Buffer (Phosphate) Buffer (Phosphate) Buffer (Phosphate) Os04 (1%) Buffer (Phosphate) Buffer (Phosphate) Buffer (Phosphate) 30% Acetone 60% Acetone 90% Acetone 100% Acetone 100% Acetone 100% Acetone Acetone/Epon mixture ( : ) 11 Acetone/Epon mixture (1:3) 100% Epon mixture 100% Epon mixture 100% Epon mixture 100% Epon mixture Cure TEMP TIME 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 4°C 2 hrs 1 hr 1 hr 1 hr 1 hr 1 hr Rm Rm Rm 35 °C 45°C 60°C Rm 1 hr 1 hr 15 min 15 min 15 min 15 min 15 min 15 min Overnight 12 hrs Overnight 12 hrs Overnight 3 days 6 days �After the epoxy resin blocks had cured for a ndnimum of six days, the tissue was then sectioned with glass knives on a Sorvall "Porter-Blum" MT-2 ultxamicrotome. Tissue sections of approximately 75 rra thickness were placed on uncoated copper grids and stained with uranyl acetate and lead citrate using procedures outlined by Hayat ( 4 . 1) A Zeiss EM-9 electron microscope was used to examine and photograph the tissue. To insure unbiased results, a minimum of five electron micrographs were taken from radomly chosen sections. The cells selected to be photographed displayed a large cross-section of the nucleus, thereby guaranteeing that a representative cross-section of the cell was recorded. Data for analysis was obtained from the electron micrographs through a technique known as stereology. This method of quantitative analysis of the cell ultrastructure uses morphometric procedures based on the techniques developed by Weibel et al. (39) and Weibel (40), then modified and used by Buckanan (3). This modified technique employs a method of extrapolating from areas to volumes using a system of "point counting." A grid overlay of points to be counted was constructed by marking a grid of dots spaced five ran apart on a sheet of clear acetate. The resulting transparent grid overlay was then randomly placed over the photographic paper as the eel] image (Figure 1) was printed on the paper. This produced an electron micrograph of the cell with a grid of white dots superimposed over the image (Figure 2). All of the dots lying over the mitochondria (METO), the damaged (swollen and ruptured) mitochondria (d.MITO), the smooth endoplasmic reticulum (SER), the rough endoplasmic 10 �reticulum (HER), and the total area of the cytoplasm were then counted visually using a push-button counter. The volume fraction of each structure is considered to be the ratio between the point count of that structure and the total point count of the cytoplasm (24). After the volume fraction was determined for each structure of each cell photographed, the means of the volume fractions or ratios were then computed for each animal. In this manner, the ratio of mitochondrial volume to cytoplasmic volume of the hepatic parenchymal cell was determined for each animal as was the ratio of damaged mitochondrial volume to total mitochondrial volume, RER to cytoplasm, SEE to cytoplasm and RER to SER. These volume fractions or ratios were used as quantitative measurements of the structures to compare the hepatic parenchymal cells from treated animals with those from control animals. TCDD and Histopathological Analyses To support the ultrastructural studies, analysis of the soil, seed, liver, and pelt samples for TCDD content, as well as the determination of the TCDD concentration in the alumina gel used in the dusting study, was conducted by Interpretive Analytical Services, Dow Chemical USA, Midland, MI 48640. Histopathological examination of internal organs was accomplished by the Veterinary Pathology Division, Armed Forces Institute of, Pathology, Washington DC 20305. Statistical Analysis The Wilcoxon Rank Sum Test was used to analyze statistically the body weight and organ weight data as well as the hepatic morphonetric data. This statistical procedure is designed to test the hypothesis that 11 �^mmmw^m ^vfe<%f^$i .*,* -ffim^ M\v J". ,-..> > -.'• . V Figure 1. Hepatic Parerich^nal Cell Prior to Printing with a Dot Grid Overlay. (x5726) 12 * f - 'i^L. * �Figure 2. Hepatic Parcnchymal Cell Printed with Dot Grid Overlay for Morphometric Analysis. (x5726) 13 �that tado random samples have been drawn from populations have identical distributions. In addition, the body weight and organ weight data were statistically analyzed by Regression Analysis using linear, double logarithmic, and semi-logarithmic correlation, and by Analysis of Covariance. The Analysis of Covariance was performed using the current or final body weight as a covariate- thereby eliminating the variations in organ weight caused by variations in body weight. This method has proved superior to the analysis of relative organ weights (36). 14 �RESULTS Soil and Seed Analysis There were wide fluctuations in TCDD concentrations in the mixed soil from Grid I, with TCDD concentrations of 10, 25, 70, 70, 110, and 710 ppt (Table 2). The unmixed 15 cm core, obtained from the site having 110 ppt TCDD, showed that TCDD was stratified within the top 15 cm of soil. Concentrations of 150, 160, 700, and 44 ppt TCDD were detected at depths of 0-2.5, 2.5-5.0, 5.0-10.0, and 10.0-15.0 cm, respectively. TCDD was not detected in soil samples taken from the designated control area. No TCDD was found in any seeds taken from Grid I (mindjnum detection limit of one ppt TCDD). Beach Mouse Grooming Habits It was observed that beach mice have meticulous grooming habits, spending as much as 50% of their active hours in the process. Areas of the body that received the most grooming attention were the ventral thoracic and abdominal regions, sides, back, and tail. Analysis of Livers and Pelts Livers, as well as the pelts of beach mice captured from Grid I, where significantly high soil levels of TCDD were found, displayed evidence of accumulation of TCDD (Table 3). The male beach mice from Grid I displayed a hepatic TCDD level of 1300 ppt while the level for the females was 960 ppt. The pelt levels were 130 ppt and 140 ppt for the male and female mice, respectively. 15 �TABLE 2. COSiCEIOTRATION ( P ) OF TCDD IN SOIL FROM PT GRID I AND FROM THE CONTROL AREA LOCATION Grid Grid Grid Grid Grid Grid Grid Grid Grid I I I I I I I I I Grid I Control Control Control Control DEPTH (CM) OMWTRATION ( P ) PT 0-15.0 0-15.0 0-15.0 0-15.0 0-15.0 0-15.0 10 25 70 70 110 710 150 160 700 44 ND3 ND ND ND 0-2.5 2.5-5.0 501. .-00 10.0-15.0 0-15.0 0-15.0 0-15.0 0-15.0 detected at a lower detection limit of 6 ppt TCDD TABLE 3. CONCENTRATION ( P ) OF TCDD IN LIVER AND PT PELT SAMPLES FROM BEACH MICE, PEROMYSCUS POLIONOTOS, COLLECTED FROM CONTROL AND TCDD-EXPOSED FIELD SITES, 1974 TREATMENT SEX LIVER PELT Control Male Female Male Female 51 83 1,300 960 <40a Grid I winimum level of detection. <40a 130 140 �The livers of both male and female mice from the control area also contained TCDD, but at a much lower level than those from Grid I, with the males having a TCDD level of 51 ppt and the females 83 ppt. For the males this was only 3.9% of the level found in the test animals and for the females only 8.6%. With the minimum level of detection at 40 ppt, TCDD was not detected on the pelts of either the control males or the control females. No TCDD was found in the livers and pelts from beach mice dusted 10 times in a period of 28 days with alumina gel containing no TCDD. The animals dusted with alumina gel containing 2.5 ppb TCDD had detectable levels on their pelts of 45 ppt for males and 89 ppt for females. The pooled sample of liver tissue contained 125 ppt TCDD. . (Due to the small amounts of liver tissue available, analysis by sex for TCDD in the liver was not possible.) Body Weight and Organ Weight Analysis The basic body weight and organ weight data for the field study are shown in Tables 4 and 5. An analysis of body weights per se was not attempted since the ages of the beach mice were not known and the animals could only be classified by sex and treatment. The data were first examined using regression analysis followed by a two-tailed test of the normal distribution to determine whether the correlation coefficients differed significantly between the control and test groups. For this analysis, the animals were divided into groups according to treatment and sex, and were then examined for linear correlation, semi-logarithmic correlation, and double logarithmic 17 �TABLE 4. BODY WEIGHTS AND ORGAN WEIGHTS OF CONTROL PEBQMYSCUS POLIONOTUS OBTAINED IN JUNE 1974, TEST AREA C-52A, EGLIN AFB, FLORIDA SEX SPECIMEN # BODY WT (GM) LIVER WT (GM) SPLEEN WT (MG) ADRENAL WT (MG) KIDNEY WT (MG) HEART WT (MS) LUNG WT (MG) M L-118 12.75 .530 20 14 174 105 102 M L-148 14.65 .811 17 32 226 108 119 M L-194 10.44 .580 16 17 174 84 94 M L-230 12.62 .778 12 20 183 93 68 M L-499 11.72 .726 15 28 211 90 110 M L-841 11.70 .537 16 11 195 130 92 M L-886 12.59 .495 14 23 207 96 112 M L-917 12.66 .524 21 18 199 113 108 M L-932 11.45 .548 20 21 171 100 96 F L-322 10.23 .679 25 12 170 84 88 F L-473 9.93 .730 30 26 168 64 75 F L-661 16.40 .6 84 24 26 253 102 120 F L-666 12.96 .831 24 20 195 94 106 F L-671 11.61 .642 26 17 171 77 99 F L-744 7.77 .303 14 12 125 53 82 12.29 .614 16.78 ±3.03 20.44 ±6.58 193.33 ±19.22 102.11 ±13.87 100.11 ±15.05 23.83 ±5.31 18.83 ±6.34 180.33 ±42.20 79.0 ±18.35 95.0 ±16.61 Male ±1.17 ±.122 Female 11.48 ±2.97 .675 ±.201 18 �TABLE 5. BODY WEIGHTS AND ORGAN WEIGHTS OF TREATED PEROMYSCUS POLIONQTUS OBTAINED IN JUNE 1974, TEST AREA C-52A, EGLIN AFB, FLORIDA SPECISEX MEN # BODY WT (GM) LIVER SPLEEN ADRENAL KIDNEY HEART WT WT WT WT WT (GM) (MG) (M3) (MG) (MG) LUNG WT (MG) M L-051 11.49 .824 29 30 201 113 103 M L-249 10.06 .529 14 18 187 73 80 M L-529 11.09 .635 9 22 174 84 81 M L-555 10.05 .436 12 18 204 149 124 M L-579 11.74 .797 45 22 191 70 85 M L-611 13.63 .696 11 27 196 97 112 M L-729 11.63 35 27 204 82 84 M L-751 9.24 .750 1.017 17 10 203 90 78 M L-805 12.25 .696 31 234 97 124 M L-959 9.32 .725 16 37 15 168 80 95 - F 13.49 8.63 .922 11 16 249 114 130 F L-009 L-251 .493 17 10 147 91 82 F L-538 16.32 1.044 55 24 241 108 82 F L-558 L-797 9.46 15.57 .828 54 16 163 81 83 .926 17 19 216 111 90 Male 11.05 ±1.39 .710 ±.160 22.5 ±12.84 22.00 ±6.83 196.2 93.5 96.6 ±18.32 ±23.27 ±18.08 30.8 .843 + .210 ±21.78 17.00 ±5.10 203.2 ±46.00 F Female 12.69 ±3.50 19 93.4 101.0 ±14.30 ±20.73 �correlation of the absolute organ weight to absolute body weight. The correlation coefficients were not significantly different at the 0.05 level. The absolute organ weight data were then converted to display the organ weights as percent of body weight. These converted data are presented in Tables 6 and 7. Using the Wilcoxon Rank Sura Test to examine the groups that have been separated according to treatment and sex, differences in the converted data for the kidney and liver were noted between the two male groups. However, when it was noted that the data from one animal (L-751) for the liver and kidney deviated from the mean by 2.5 or more standard deviations, the data were reexamined, emitting the data from that animal, and no significant differences were seen at the 0.05 level. Again emitting the data from one animal (L-751), the organ weights were reexamined with an anlysis of oovariance using the body weight as the covariate. At the 95 percent level of confidence, using this procedure of analysis, the only difference between exposed and controlled field groups was in liver weight. The exposed field group had a significantly greater liver weight than did the control group. The initial body weight data for the beach mice used in the laboratory dusting study were compared with the final body weights in Table 8. Ignoring sex of animals, the data indicated that the control animals exhibited a slight weight gain during the 28-day study (+0.17 g) while the test group showed a slight decline (-0.45 g). Statistical analysis of the weight change using the Wilcoxon Rank. Sum Test (p=0.05) 20 �TABLE 6. ORGRN WEIGHS, EXPRESSED AS PERCENT OF BODY WEIGH1, OF CONTROL PEROMYSCUS POLIONOTUS OBTAINED IN JUNE 1974, TEST AREA I-52A, EGLIN AFB, FLORIDA SPECISEX MEN # M M L-118 LIVER SPLEEN ADRENAL KIDNEY HEART LUNG 0.16 0.12 0.11 1.36 0.82 0.80 L-148 4.16 5.54 0.22 1.54 0.74 0.81 M L-194 5.56 0.15 0.16 1.67 0.80 0.90 M L-230 6.16 0.10 0.16 1.45 0.74 0.54 M L-499 6.19 0.13 0.24 1.80 0.77 0.94 M L-841 4.59 0.14 0.09 1.67 1.11 0.79 M L-886 3.93 0.11 0.18 1.64 0.76 0.89 M L-917 4.14 0.17 0.14 1.57 0.85 M L-932 0.17 0.18 1.49 F L-322 4.79 6.64 0.89 0.87 0.24 0.12 1.66 0.82 0.86 F L-473 7.35 0.30 0.26 0.64 0.76 F L-661 5.27 0.15 0.16 1.69 1.54 0.62 0.73 F L-666 6.41 0.19 0.15 0.73 0.82 F L-671 5.53 0.22 0.15 1.50 1.47 0.66 0.85 F L-744 3.90 0.18 0.15 1.61 0.68 1.06 Male 0.14 5.01 ±0.88 ±0.03 0.16 ±0.05 1.58 ±0.13 0.83 0.82 ±0.12 ±0.12 Female 5.85 0.21 ±1.22 ±0.05 0.16 ±0.05 1.58 ±0.09 0.69 0.85 ±0.07 ±0.12 21 0.84 �TABLE 7. ORGAN WEIGHTS, EXPRESSED AS PERCENT OF BODY WEIGHT, OF TREATED PEROMXSCUS POLIONOTUS OBTAINED IN OUNJ3 1974, TEST Al C-52A, EGLIN AFB, FLORIDA SPECI- SEX MEN # LIVER SPLEEN ADRENAL KIDNEY HEART LUNG M L-051 7.17 0.25 0.26 1.75 0.98 0.90 M L-249 5.26 0.14 0.18 1.86 0.73 0.80 M L-529 5.73 0.08 0.20 1.57 0.76 0.73 M L-555 4.34 0.12 0.18 2.03 1.48 1.23 M L-579 6.79 0.38 0.19 1.63 0.60 0.72 M L-611 5.11 0.08 0.20 1.44 0.71 0.82 M L-729 6.45 0.30 0.23 1.75 0.71 0.72 M L-751 11.01 0.18 0.11 2.20 0.97 0.84 M L-805 5.68 0.13 0.25 1.91 0.79 1.01 M L-959 7.78 0.40 0.16 1.80 0.86 1.02 F L-009 6.83 0.08 0.12 1.85 0.85 0.96 F L-251 5.71 0.20 0.12 1.70 1.05 0.95 F L-538 6.40 0.34 0.15 1.48 0.66 0.50 F L-558 8.75 0.57 0.17 1.72 0.86 0.88 F L-797 5.95 0.11 0.12 1.39 0.71 0.58 0.86 ±0.25 Male 6.53 ±1.88 0.21 ±0.12 0.20 ±0.04 1.80 ±0.22 Female 6,73 ±1.21 0.26 ±0.20 0.14 ±0.02 1.63 0.83 ±0.19 ±0.15 22 0.88 ±0.16 0.77 ±0.22 �TABLE 8. INITIAL AND FINAL BODY WEIGHTS OF PERGMYSCUS POLIONOTUS DUSTED WITH ALUMINA GEL CONTAINING 2.5 PPB TCDD ( E T * TS) CONTROL GROUP WEIGHTS (GRAMS) TEST GROUP WEIGHTS (GRAMS) INITIAL FINAL +0.44 12.69 12.07 -0.62 16.80 +3.30 16.10 15.72 -0.38 11.00 11.43 +0.43 13.12 12.77 -0.35 13.40 12.60 -0.80 17.15 18.02 +0.87 15.25 14.23 -1.02 13.71 13.65 -0.06 12.50 12.72 +0.22 14.48 13.20 -1.28 14.01 14.38 +0.37 14.90 15.57 +0.67 13.12 13.10 -0.02 12.36 11.78 -0.58 14.10 13.26 -0.84 14.03 12.61 -1.42 13.40 12.97 -0.43 16.00 14.94 -1.01 13.90 13.77 -0.13 15.25 14.12 -1.13 INITIAL FINAL 17.06 17.55 13.50 DIFFERENCE a Data on sex of animals are shown in Tables 9 and 10. 23 DIFFERENCE �TABLE 9. BODY WEIGHTS AND ORGAN WEIGHTS OF PEROMYSCUS POLIONOTUS DUSTED WITH ALUMINA GEL CONTAINING NO TCDD (CONTROL) SPECISEX MEN # BODY WT (GM) LIVER WT (GM) SPLEEN WT (MG) ADRENAL KIDNEY HEART WT WT WT (MG) (MG) (MG) LUNG WT (MG) M 069 12.97 .718 14 27 190 158 118 M 323 14.38 .686 13 26 207 81 125 M 626 12.72 .1 60 10 22 186 75 95 M 628 13.26 .698 19 43 118 100 101 M 655 12.60 .577 10 26 199 115 95 M 669 13.10 .645 23 30 197 130 79 F 112 16.80 .8 90 24 49 255 112 106 F 274 14.23 .825 20 28 230 132 95 F 591 11.43 .606 14 46 201 92 80 F 696 17.55 .951 26 41 258 156 112 13.17 0.656 ±0.64 ±0.055 14.33 ±4.32 29.00 ±7.32 182.83 ±32.59 109.83 ±31.29 102.17 ±16.81 Female 15.00 0.840 21.00 ±5.29 41.00 ±9.27 236.00 ±26.50 123.00 ±27.39 98.25 ±14.06 Male ±2.77 ±0.170 24 �TABLE 10. BODY WEIGHS AND ORGAN WEIGHTS OF PEROMYSCUS POLIONOTUS DUSTED WITH ALUMINA GEL CONTAINING 2.5 PPB TCDD ( E T TS) SPECISEX MEN # BODY WT (GM) M 221 18.02 M 296 M LIVER SPLEEN ADRENAL KIDNEY HEART WT WT WT WT WT (GM) (MG) (MG) (MG) (MG) LUNG WT (MG) 156 123 39 225 202 119 92 22 27 226 116 109 .805 19 34 246 105 88 12. 77 .542 20 32 189 122 90 742 15.57 .723 33 59 214 127 90 M 966 15.72 .953 37 25 226 144 107 F 054 13.77 .832 9 31 243 123 88 F 073 28 219 101 112 17 30 195 98 84 F 224 444 .751 .714 14 F 12.61 12.07 11.78 .593 17 20 196 117 80 F 641 14.99 .912 14 35 279 126 113 14.72 0.758 ±1.84 ±0.124 25.71 ±6.78 36.86 ±11.48 218.29 127.00 99.86 ±18.59 ±17.44 ±13.33 Female 13.04 0.760 14.20 ±3.27 28.80 ±5.54 226.40 113.00 95.40 ±35.38 ±12.79 ±15.87 25 42 13.20 .790 .713 24 372 14.12 .779 M 446 13.65 M 528 M Male ±1.33 ±0.121 25 �indicated no significant difference. No significant difference in weight change was found when the arumals were compared according to sex. The post-mortem body weight and organ weight data for the laboratory dusting study are shown in Tables 9 and 10. For satistical analysis the organ weight and body weight data from the laboratory animals were also grouped according to treatment and sex before examination for linear correlation, semi-logarithmic correlation, and double logarithmic correlation of the absolute organ weight to absolute body weight. A two-tailed test of the normal distribution was used to determine whether .correlation coefficients of control and treated groups differed significantly from each other. At the 0.05 level a significant difference was noted between the spleen weight to body weight coefficients of the control female and treated female beach mice. The organ weight data from the laboratory study were also converted to be expressed as percent of body weight. These data are presented in Tables 11 and 12. After separating the groups according to treatment and sex, significant differences attributable to treatment could be seen in spleen to body weight ratios for the control male and treated male groups (p=0.05). Sex differences were also noted in the data for kidney, liver, and spleen for the treated male/treated female, control male/control female, and treated male/treated female groups respectively. Examination of the organ weight data with an analysis of covaiiance, using the body weight as the covariate, revealed none of the previously found differences. Indeed, this statistical analysis showed there were 26 �TABLE 11. ORGAN WEIGHTS, EXPRESSED AS PERCENT OF BODY WEIGHT, OF PEROMYSCUS POLIONOTUS DUSTED WITH ALUMINA GEL CXWEAINING NO TCDD (CONTROL) SPECI- SEX MEN # LIVER SPLEEN ADRENAL KIDNEY HEART LUNG M 069 5.54 0.11 0.21 1.47 1.22 0.91 M M 323 4.77 0.09 0.18 1.44 0.56 0.87 626 4.80 0.08 0.17 1.46 0.59 0.75 M 628 5.26 0.14 0.32 0.89 0.75 0.76 M 655 4.58 0.08 0.21 1.58 0.91 0.75 M 669 4.92 0.15 0.23 1.50 0.99 0.60 F 112 5.83 0.14 0.29 1.52 0.67 0.63 F 274 5.80 0.14 0.20 1.62 0.93 0.67 F 591 5.30 0.12 0.40 1.76 0.80 0.70 F 696 5.42 0.15 0.23 1.47 0.89 0.64 Male 4.98 ±0.36 0.11 ±0.03 0.22 ±0.05 1.39 ±0.25 0.84 ±0.25 0.77 ±0.11 Female 5.59 ±0.27 0.14 ±0.01 0.28 ±0.09 1.59 ±0.13 0.82 ±0.12 0.66 ±0.03 27 �TABLE 12. ORGAN WEIGHTS, EXPRESSED AS PERCENT OF BODY WEIGHT, OP PEROMYSCUS POLIONOTUS DUSTED WITH ALUMINA GEL CONTAINING 2.5 PPB TCDD(TEST) SPECISEX MEN # LIVER SPLEEN ADRENAL KIDNEY HEART LUNG M 22.1 4.38 0.14 0.23 1.25 0.87 0.68 M 296 5.40 0.18 0.30 1.53 0.90 0.70 M 372 5.52 0.16 0.19 1.60 0.82 0.77 M 446 5.90 0.14 0.25 1.80 0.77 0.64 M 528 4.24 0.16 0.25 1.48 0.96 0.70 M 742 4.64 0.21 0.38 1.37 0.82 0.58 M 966 6.06 0.24 0.16 1.44 0.92 0.68 F 054 6.04 0.07 0.23 1.76 0.89 0.64 F 073 5.96 0.11 0.22 1.74 0.80 0.89 F 224 5.92 0.14 0.25 1.62 0.81 0.70 F 444 5.03 0.14 0.17 1.66 0.99 0.68 F 641 6.08 0.09 0.23 1.86 0.84 0.75 Male 5.16 0.18 ±0.04 0.25 ±0.07 1.50 ±0.18 0.87 ±0.07 0.68 ±0.06 0.11 ±0.03 0.22 ±0.03 1.73 ±0.09 0.87 ±0.08 0.73 ±0.10 ±0.74 Female 5.81 ±0.44 28 �no significant differences in the organ weights of the control and testgroups in the laboratory dusting study (p=0.05). Histopathology The supporting histopathological studies were performed by the Veterinary Pathology Division, Armed Forces Institute of Pathology on both test and control mice with no distinction being made between the animals from the field study and the animals from the laboratory (dusting) study. A series of histological examinations were performed on the heart, lungs, trachea, salivary glands, thymus, liver, kidneys, s±onach, pancreas, adrenals, large and small intestines, spleen, genital organs, bone, bone marrow, skin, and brain. Initially, the tissues were examined on a random basis without the knowledge of whether the mouse was a control or test animal. All microscopic changes, including those interpreted as minor or insignificant, were recorded. Following the recording of all microscopic findings, the tissues were reexamined on a control and test basis. Results of both studies determined that the test and control mice could not be distinguished on a microscopic basis. Significant lesions were found in only one mouse, a test mouse from the field study. The liver displayed moderately severe, raultifocal, necrotizing hepatitis (Figure 3). Sections from the liver of this animal were stained from a variety of stains in attempts to identify an etiologic agent. Neither bacterial or funal organisms were demonstrated and the lesions were considered viral induced as they resembled the lesions seen in viral hepatitis of laboratory mice. 29 �The gross lesions observed in the kidney of one test mouse from the field study proved to be severe ectasia of renal veins. Microscopically, the vascular dilatation was interpreted as beircj of little functional significance (Figure 4). All other lesions observed in both control and test mice were minor and insignificant and of the type normally observed when a large group of animals are examined at the microscopic level. Hepatic Morphometric Analysis The hepatic raorphcmetric data for each animal in the field study are presented as mean values in Tables 13 and 14. Since morphometric analysis is concerned with the volume fraction of each structure in question or the ratio between the point count of that structure and the total count of the cytoplasm, and since the count for each structure could vary with cell size, only the total cytoplasm count was statistically analyzed for differences. Using the Wilcoxon Rank Sum Test to examine the total counts (p=0.05), no significant difference was seen between the control and treated field animals. After the volume fraction was determined for each required structure of the photographed cells, the means were computed for each animal by sex and treatment and presented in Table 15. There were no significant differences between field control and field treated animals for any of the cellular structures in question (p=0.05). The hepatic morphometric data for the laboratory study animals were treated the same as the data from the field study. The mean values are shown in Tables 16 and 17. After being separated according to sex and treatment. The total cytoplasm count (indicating cell size) showed 30 �Figure 3. Acute Necrosis and Inflammation in the Liver of a Beach Mouse Captured from Grid I. Ilematoxvlin and Eosin. Figure 4. Microscopic Appearance of Venous Ectasia in the Kidney of a Beach Mouse Captured from Grid I. Heraatoxylin and Eosin. 31 �TABLE 13. HEPATIC NDRPHOMETRIC DATA OF CONTROL PEROMYSOJS POLIONOTUS JNED IN JUNE 1974, TEST AREA C-52A, EGLIN AFB, FLORIDA SEX SPECIMEN # TOTAL COUNT MITO COUNT M L-118 549.0 140.3 M L-148 438.4 100.2 M L-194 321.0 M L-230 M DAMAGED MITO COUNT RER COUNT SER COUNT 6.0 52.4 273.9 0 89.6 192.8 63.2 6.2 95.2 138.0 434.4 63.6 9.8 65.2 201.2 L-499 378.8 59.6 2.4 78.6 146.6 M L-841 374.6 102.2 1.8 94.2 110.6 M L-886 353.8 105.4 10.2 59.0 135.8 M L-917 273.9 71.4 9.6 49.9 116.4 M L-932 225.3 47.9 1.6 67.9 58.9 F L-322 506.0 119.0 14.8 115.8 206 1. F F L-473 275.2 41.6 7.8 120.6 L-661 324.9 92.0 9.9 91.6 59.7 122.1 F L-666 308.8 67.5 1.5 58.5 136.2 F L-671 445.0 81.4 1.0 140.6 128.8 F L-744 170.9 73.2 0.4 23.7 58.4 Male 372.1 ±96.02 83.76 ±29.85 5.29 ±3.98 72.44 ±17.63 152.69 ±62.33 338.5 ±120.47 79.12 ±25.84 5.9 ±5.87 81.65 ±42.70 129.45 ±48.60 Female 32 �TABLE 14. HEPATIC MORPHQMETRIC DATA OF TREATED PEROMYSCUS POLIONOTUS AIMED IN JUNE 1974, TEST AREA C-52A, EGLIN AFB, FLORIDA DAMAGED MITO COUNT RER COUNT SER COUNT 16.2 27.2 156.2 58.0 0.2 40.8 108.0 405.2 94.4 1.8 76.2 182.4 L-555 265.5 102.5 0 41.0 97.0 M L-579 390.5 67.5 1.5 34.0 230.0 M L-611 451.0 74.4 3.2 75.8 139.4 M L-729 277.8 56.4 0 55.0 128.0 M L-751 439.3 104.3 9.3 54.7 226.7 M L-805 211.8 49.6 0.2 39.2 102.6 M L-959 353.8 81.3 8.2 78.3 132.7 F L-009 418.0 82.8 2.5 84.5 225.0 F L-251 185.0 52.7 6.0 39.3 67.3 F L-538 333.0 87.6 5.0 77.2 144.0 F L-558 410.2 75.5 0 60.2 223.8 F L-797 400.0 93.8 1.8 73.0 175.2 Male 339.1 ±81.74 77.12 ±19.39 4.06 ±5.45 52.22 ±18.88 150.3 ±48.40 Female 349.2 ±97.80 78.48 ±15.89 3.06 ±2.43 66.84 ±17.75 167.06 ±65.43 SEX SPECIMEN # TOTAL COUNT MITO COUNT M L-051 326.2 82.8 M L-249 269.8 M I/-529 M 33 �TABLE 15. HEPATIC lyDRPHOMETRIC DATA, EXPRESSED AS RATIOS, OF CONTROL AND TREATED PEROMYSCUS POLIONOTUS OBTAINED IN JUNE 1974, TEST AREA C-52A, EGLIN AFB, FLORIDA LOCATION SEX M M Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated M M M M M M M F F F F F F M M M M M M M M M M F F F F F Control Male SPECI- MITO/ d.MITO/ MEN # TOT MITO RER/TOT L-118 L-148 L-194 L-230 L-499 L-841 L-886 L-917 L-932 L-322 L-473 L-661 L-666 L-671 L-744 L-051 L-249 L-529 L-555 L-579 L-611 L-729 L-751 L-805 L-959 L-009 L-251 L-538 L-558 L-797 .272 .228 .199 .4 18 .6 10 .272 .297 .264 .212 .228 .150 .286 .223 .192 .428 .255 .1 21 .234 .372 .7 18 .162 .198 .238 .231 .226 .201 .270 .258 .183 .242 SER/TOT REISER .044 .0 01 .075 .5 10 .4 00 .017 .088 .5 11 .032 .125 .213 .121 .2 01 .1 01 .005 .201 .002 .019 .0 01 .022 .059 .0 01 .082 .003 .067 .037 .1 11 .052 .001 .022 .0 15 .207 .287 .5 10 .1 21 .249 .6 16 .8 15 .303 .232 .340 .182 .182 .313 .138 .083 .164 .8 17 .158 .8 04 .7 11 .9 19 .122 .8 10 .219 .213 .1 28 .234 .147 .197 .481 .436 .432 .9 40 .377 .299 .385 .2 41 .6 27 .409 .435 .376 .443 .294 .343 .476 .410 .452 .376 .589 .304 .457 .524 .488 .1 51 .358 .434 .549 .424 .226 .476 .666 .334 .529 .878 .454 .438 1.258 .578 .837 .487 .419 1.102 .1 45 .7 16 .409 .424 .421 .143 .576 .438 .238 .389 .594 .435 .4 60 .550 .271 .489 0.228 0.066 ±0.052 ±0.055 0.207 ±0.064 0.399 ±0.076 0.584 ±0.314 Control Female 0.251 0.083 ±0.098 ±0.084 0.231 ±0.080 0.383 ±0.058 0.640 ±0.275 Treated 0.230 0.046 ±0.057 ±0.062 0.157 ±0.046 0.446 ±0.081 0.381 ±0.153 0.231 0.045 ±0.037 ±0.042 0.202 ±0.033 0.455 ±0.075 0.477 ±0.138 Male Treated Female 34 .386 �TABLE 16. HEPATIC TOKPHCIMETRIC DATA OF PERDMSfSCUS PCIJONOTUS DUSTED WITH ALUMINA GEL CONTAINING NO TCDD (CONTROL) SEX M M M M M M F F F F SPECIMEN # TOTAL COUNT MTTO COUNT 069 323 386.0 445.6 455.0 359.4 524.4 386.2 400.0 280.8 376.6 477.8 84.2 115.2 137.0 91.4 112.6 110.2 74.2 69.8 95.2 124.6 626 628 655 669 112 274 591 696 Male 426.10 108.43 ±60.87 ±18.76 Female 383.80 90.95 ±81.16 ±25.02 35 DAMAGED MITO COUNT 10.2 7.2 44.4 16.8 35.2 46.6 15.0 13.3 27.4 30.0 RER COUNT SER COUNT 71.8 57.4 62.8 49.6 84.4 57.4 47.2 39.5 55.8 75.0 131.4 181.4 183.0 126.8 229.4 140.2 155.8 107.3 150.2 155.6 26.73 ±17.50 63.90 165.37 ±12.43 ±39.86 21.42 ±8.50 54.38 142.22 ±15.28 ±23.43 �TABLE 17. HEPATIC MDKPHOMETRIC DATA OF PEROMYSCUS POLIONOTUS USTED WITH ALUMINA (3L CONTAINING 2.5 PPB TCDD (TEST) DAMAGED MITO COUNT HER COUNT SER COUNT 91.8 22.2 85.4 168.2 96.4 28.8 67.4 183.6 88.4 19.4 69.2 115.4 97.4 27.8 55.0 133.2 120.4 18.6 54.6 159.0 84.2 22.4 69.0 134.2 134.0 35.4 59.8 143.4 SEX SPECIMEN f TOTAL COUNT MITO COUNT M 221 M 296 M 372 M 446 M 528 M 742 M 966 F 054 F 073 F 224 F 444 F 641 432.0 437.0 349.8 343.6 379.0 333.0 388.2 284.5 462.7 358.2 435.6 473.4 66.7 9.8 60.2 102.8 125.8 38.3 60.0 170.0 71.6 13.2 48.0 150.2 102.6 14.8 73.8 170.4 109.2 35.4 57.6 204.6 Male 380.37 101.80 ±41.77 ±18.35 24.94 ±6.02 65.77 ±10.70 148.14 ±23.42 Female 402.88 95.18 ±80.05 ±25.28 22.30 ±13.44 59.92 ±9,22 159.60 ±37.30 36 �no significant difference between the control and treated laboratory animals. (As with the field animals, this was the only data, not expressed as ratios, analyzed statistically.) The mean volume fraction, or ratio for each required cellular structure of each animal in the laboratory dusting study are shown in Table 18. The volume fractions or ratios from treated laboratory animals were conpared with those from control animals using the Wilcoxon Rank Sura Test (p=0.05). No significant differences were noted between animals dusted with alumina gel containing no TCDD (control) and animals dusted with alumina gel containing 2.5 ppb TCDD (test). General Cellular Observations Concentric membrane arrays (myelin figures) mitotic figures, and multinucleated hepatocytes were not observed during viewing of the tissue for photograph. However, occasional binucleated cells were seen and two basic types of parenchymal cells were differentiated on the basis of staining intensity. 37 �TABLE 18. HEPATIC MORPHQMETRIC DATA, EXPRESSED AS RATIOS, OF PEROMSfSCUS POLIONOTUS DUSTED WITH ALUMINA GEL CONTAINING NO TCDD (CONTROL) OR WITH ALUMINA GEL CONTAINING 2.5 PPB TCDD (TES1 TREATMENT SEX M M Control SPECI- MITO/ MEN # TOT 069 323 626 628 655 669 112 274 591 696 221 296 372 446 528 742 966 054 073 224 444 641 d.MITO/ RER/TOT SER/TOT RER/SER MITO .219 .257 .295 .257 .210 .278 .183 .253 .256 .264 .216 .222 .252 .281 .318 .254 .340 .231 .266 .200 .238 .234 .4 16 .083 .294 .139 .269 .349 .226 .174 .286 .244 .249 .278 .231 .285 .153 .213 .199 .1 19 .298 .175 .146 .321 .189 .128 .4 12 .138 .162 .150 .122 .151 .4 19 .155 .197 .154 .200 .160 .143 .208 .155 .215 .136 .135 .170 .123 .340 .410 .0 40 .350 .436 .364 .394 .374 .400 .324 .392 .423 .328 .383 .421 .396 .369 .367 .370 .423 .394 .425 .566 .323 .359 .403 .382 .418 .328 .1 49 .371 .471 .1 50 .367 .614 .420 .346 .540 .422 .586 .369 Control Male 0.253 ±0.033 0.213 ±0.105 0.152 ±0.022 0.383 ±0.038 0.408 ±0.084 Control Female 0.239 ±0.038 0.232 ±0.046 0.144 ±0.015 0.373 ±0.034 0.397 ±0.062 Treated Male 0.269 ±0.047 0.230 ±0.046 0.174 ±0.027 0.387 ±0.033 0.460 ±0.098 Treated Female 0.234 ±0.023 0.212 ±0.092 0.156 ±0.037 0.396 ±0.028 0.400 ±0.117 Control Control Control Control Control Control Control Control Control Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated Treated M M M M F F F F M M M M M M M F F F F F 38 .320 .431 .293 �DISCUSSION A factor of concern in interpreting the data was the sample size for both the field study anri the laboratory study. The number of beach mice in each group, when separated by sex and treatment, ranged from five to ten in the field study and from four to seven in the laboratory study. In such small samples the deviation of one individual will strongly influence the data for the entire group. For this reason, caution roust be used in the interpretation of the results. Field Study • The soil samples from the test area displayed wide fluctuations in TCDD concentrations, probably as the result of unequal distribution of the herbicide during aerial dissemination. Three major flight paths intersected at Grid I and the soil samples were taken from areas thought to be on the flight paths. However, if the samples were obtained from an area outside the flight paths or from the intersection of all three flight paths, the TCDD levels would be expected to vary considerably. Nevertheless, analysis of the soil samples did show that the beach mice from Grid I were exposed to concentrations of TCDD up to 710 parts per trillion (ppt) in the soil. In contrast, the soil from the control areas did not contain TCDD at a minimum detection level of six ppt and therefore did not provide a source of exposure for the control animals. Since the seed samples from Grid I did not contain TCDD at a minimum detection level of one ppt, seeds from Grid I were probably not a source of TCDD. The mice continually contaminated themselves with soil containing TCDD by repeated movement in and out of their burrows. It was observed 39 �that the mice plug their burrows with about 15 cm of soil after they enter and then must burrow through this plug when they exit the tunnel. This recurrent burrowing activity in increased exposure to the contaminated soil. The levels in the pelt samples from mice trapped on Grid I confirm this method of contact. In contrast, TCDD was not detected in pelt samples from control animals. Since the seeds from Grid I were probably not a source of TCDD and the contaminated soil was confirmed as a source of contact, there were no data from this study to support biomagnification of TCDD. However, the level of TCDD detected in the livers of beach mice collected from Grid I confirms uptake by the animals and substantiates bioaccumulation by the liver. In general, levels of TCDD in the livers were somewhat greater than the most concentrated zones of TCDD in the soil. In the years 1962 through 1964, enough TCDD was applied to Grid I to accumulate to the concentration of 12,267 ppt in the top 15 cm of the soil (43). By 1974 the level had declined about 94 percent to approximately 700 ppt. This level, although far greater than the estimated 0.1 ppt concentration in the soil after normal application of the herbicide 2,4,5-T (19), is much less than that normally used in laboratory experiements (1,7,11,17,18,21,22,25,27,29). Although the beach mice were exposed to soil levels of TCDD as high as 700 ppt, it is highly doubtful that the level ingested through grooming would even approach the levels given to animals via gavage in laboratory experiments; consequently, the accumulation of TCDD in the liver was much less than that reported in laboratory studies. Kociba et al. (22), in a chronic, two year study showed that rats given 0.01 yg TCDD/kg/day had an average TCDD content of 5100 ppt in 40 �the liver. Rats given 0.001 yg TCDD/kg/day had an average of 540 ppt in the liver. The livers from beach mice collected from Grid I in this study had a TCDD content of 1300 ppt for males and 960 ppt for females. Extrapolation of the data would then give the beach mice a daily TCDD intake dose of approximately 0.0012 ijg/kg. Although extrapolation between species is not always advisable, Pries and Marrow (8) did state that total retention of TCDD was closely related to total intake. TCDD was also found in the livers of the beach mice collected from the control area, although at a much lower level. The presence of TCDD in these pooled samples may have been due to high levels in one or more mice that could have migrated from the test area to the control area. A previous trapping study in this area (42) reported the longest randan travel distance observed to be slightly over 900 meters. A travel distance of this magnitude was considered rare but could account for the presence of TCDD in the control animals. Nevertheless, even though the levels in the control mice were low compared to the levels in the test animals, the use of these mice as true controls must be viewed with caution. Satistically significant differences in organ weight to body weight ratios were noted between control and exposed beach mice. The increase in liver weight found in this study is in agreement with other investigators (8,10,13,21,25,27,28,29,37); however, the lack of additional changes can be explained only by the level of exposure, which is considerably lower than in these experiments. Kociba et al. (22) found changes in liver and thymus weights in rats given 0.1 or 0.01 yg TCDDAg/day for a two year period but no change in organ weights due to treatment with 0.001 yg TCDD/ kg/day. With an exposure rate of approximately 0.0012 yg TCDD/kg/day, 41 �the exposed mice in this study could be expected to display data falling between the two lower exposure groups of the chronic study by Kbciba et al. (22). This, in fact, was the case with all the data reported in this field study. The histopathological examination of the field animals affirmed the absence of significant differences between the beach mice taken from Grid I and those taken from the control area. Except for one report of viral hepatitis and one of renal vein ectasia, all lesions were of the minor or insignificant type normally observed in microscopic surveys of large numbers of field animals. Neither of the more serious lesions observed were considered to result from exposure to TCDD. This is in agreement with investigators using comparable exposure levels (22). The binucleated cells observed during electron microscope photography were considered normal since two nuclei have been reported in 25 percent of hepatic parenchymal cells ( 1 . The appearance of two types 4) of parenchymal cells differing in electron density has not been fully explained (1) but may represent a transition between parenchymal and ductal cells as Hampton suggests ( 2 . Kbciba et al. (22) observed 1) both multinucleated and swollen hepatocytes in groups of rats given 0.1 or 0.01 yg TCDD/kg/day while the group given 0.001 yg/TCDDAg/day displayed neither of these findings. No mention was made by these investigators of parenchymal cells differing in staining intensity. The lower exposure level seen in this study, although much higher than that anticipated in an environment following normal herbicide application (19), may account for the absence of histopathological and ultrastructural changes that were seen in other experiments with 42 �TCDD (7,11,18,21,22,28). The results of the chronic toxicity study on TCDD in rats by Kociha et al. (22) substantiate a lack of adverse effects at such a low dose level. The lack of adverse effects from TCDD seen in mice from the test area may indicate the presence of some mechanism for physiological adaptation not necessarily present in the mice from the control area. Berry (2) has shown that mice from neighboring populations separated by distances of one to 2.5 km may differ considerably in their genetic composition. Since the distance separating the control and test areas falls within this range, genetic variation may be considered as an explanation. Indeed, several investigators (26,31,32,33) have shown that certain inbred strains of mice are nonresponsive in the detoxification of TCDD. To determine if this is indeed the situation with these beach mice would require a much more exhaustive experiment beyond the scope of this study. Laboratory Study The laboratory dusting study confirmed ingestion during grooming as a possible method of contamination of the beach mice livers. Although the TCDD levels in the liver and pelt samples from the treated animals in the dusting study were not as high as from mice collected from the test area, TCDD was not detected in samples from the laboratory control animals, giving a clear treated/control comparison. The relatively short exposure time (28 days) was probably responsible for the laboratory treated animals having lower TCDD levels than the field treated animals. The findings of this dusting study are in agreement with those reported by Kociba et al. (22) in the group of animals given the lowest 43 �dose of TCDD. The one exception is in spleen weight as compared to terminal body weight. An increase in spleen weight was found in males and a decrease was found in females dusted with TCDD. Although histopathological examination of the spleens, as well as of the other organs, failed to support any differences between the control and test animals, the change in spleen weight tends to agree with previous investigators (11,13,35,37,43) who suggest that the spleen may be the most sensitive organ by which to assess exposure to TCDD. While these investigators agree in a loss of spleen weight with exposure to TCDD (11,37) there is seme disagreement on whether the male or the female is more sensitive (13,35). However, no explanation is given for the sex difference in sensitivity. The 125 ppt TCDD found in 'the livers of the treated animals of this study falls far short of the 540 ppt TCDD in the livers of rats given 0.001 ug TCDD/kg/day.by Kociba et al. (22), a dose level that caused no cellular effects considered to be of any toxicologic significance and within the limits of variation seen in the controls. Although the actual oral dose in this dusting study could not be determined, it was probably well below the 0.001 yg TCDD/kg level. The liver TCDD level of 125 ppt associated with this apparent low dose level resulted in histopathological findings and hepatic morphometric data which showed no significant differences between the control and treated animals. Again, as in the field study, binucleated cells were observed but were considered normal. Light and dark staining cells were also noted but their significance could not be determined. 44 �Since the dose level of TCDD in this study could not be determined, it is difficult to compare the results from the laboratory dusting study with those presented by other investigators. However, this study does demonstrate a possible method of contamination of the beach mice livers. Msthods Previous investigators such as Weibel (40) have incorporated computer processing and stereological techniques to evaluate data and determine actual volumes of cell organelles. Buchanan (3), however, modified these techniques to determine relative values rather than absolute. It is this modified stereological technique that is used in the present study to compare cellular ultrastructure of control and treated groups. These stereological techniques, also known as morphometric analysis or morphometry, have not been applied in a quantitative assessment of TCDD effects prior to this study (1,7,11,17,18,22,23, 27,28,29). Therefore, this study is the first to present data derived from actual measurements of TCDD effects on ultrastructural hepatic morphology rather than from microscopic observations and estimations. 45 �O3NCLUSIONS The results of this study indicate that TCDD persisted for long periods of tijne in the soil of Test Area C-52A, Eglin Air Force Base, 2 Florida. Soil samples taken fron the 0.4 km of Grid I confirm that leaching does not occur and that the TCDD remaining in the soil after 10 years is stratified within the top 0-15 cm of the soil. Persis- tence of the TCDD in the soil-is thought to be related to the massive application rates r&ther than to the absence of chemical or biological degradation. Although the levels of TCDD in the livers are slightly greater than those found in the soil, TCDD was not detected in the portion of the food chain consisting of seeds. The laboratory dusting study confirms, however, that ingestion of TCDD can occur as a result of body contact and subsequent grooming. The results of this study indicate no significant ultrastructural changes in hepatic parenchymal cells in response to long term, low level exposure to TCDD (field), or in response to short term, low level exposure to TCDD (laboratory). The levels of TCDD encountered in this study, up to approximately 700 ppt in the soil and an average of approiraately 1,000 ppt in the livers, are much less than those normally found in most laboratory experiments, but far greater than the estimated concentrations in the environment, or in anjmal tissues after normal application of the herbicide 2,4,5-T. In addition, this study demonstrates the application of the analytical technique of stereology to field studies of toxicity. The 46 �modified technique, as used in this study, combined with tissue processing found in many modern pathology laboratories can produce usable data in 36 to 48 hours, rendering sterology a possible tool for characterizing quantitative cellular responses to injury. 47 �LITERATORE CITED 1. Allen, J.R. and L.A. Carstens. 1966. Electron raicroscxpe alterations in the liver of chickens fed toxic fat. Lab. Invest. 15:970979. 2. Berry, R.J. 1978. Genetic variation in wild house mice: where natural selection and history meet. fta. Sci. 66(1):52-60. 3. Buchanan, G.M. 1973. Effect of high dietary molybdenum on rat adrenal cortex. Unpublished thesis. University of Colorado, Boulder. 4. Buu-Hoi, N.P., P.H. Chanh, G. Sesque, M.C. Azum-Gelade, and G. Saint-Ruf. 1972. Organs as targets of "dioxin" (2,3,7,8-tetrachlorodibenz»-p-dioxin). Naturwiss. 59:174-175. 5. Carter, C.D., R.D. Kimbrough, J.A. Liddle, R.E. Cline, M.M. Zack, Jr., W.F. Barthel, R.E. Kbehler, and P.E. Phillips. 1975. Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188:738-740. 6. Firestone, D., J. Ress, N.L. Brown, R.P. Barren, and J.N. Damico. 1972. Determination of polychlorcdibenzo-p-dioxins and related compounds in commercial chlorophenols. J. Assoc. Offie. Anal. Chem. 55(1):85-92. 7. Fowler, B.A., G.W. Lucier, H.W. Brown, and O.S. McDaniels. 1973. Ultrastructural changes in rat liver cells following a single oral dose of TCDD. Environ. Health Perspect. 5:141-148. 8. Fries, G.F. and G.S. Marrow. 1975. Retention and excretion of 2,3,7,8-tetxadhlorodibenzo-p-dLoxin by rats. J. Agr. Food Chem. 23(2):265-267. 9. Greig, J.B. and F. DeMatteis. 1973. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on drug metabolism and hepatic microsomes of rats and mice. Environ. Health Perspect. 5:211-219. 10. Greig, J.B., G. Jones, W.H. Butler, and J.M. Barnes. 1973. Toxic effects of 2,3,7,8-tetrachlorcdibenzo-p-dioxin. Fd. Cosmet. Toxicol. 11:585-595. 11. Gupta, B.N., J.G. Vos, J.A. Moore, J.G. Zinkl, and B.C. Bullock. 1973. Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Perspect. 5:125-140. 48 �12. Hampton, J.C. 1964. Liver. Pages 41-58 in S.M. Kurtz, Ed., Electron Microscopic Anatony, Academic Press, New York. 13. Harris, M.W., J.A. Moore, J.G. Vos, and B.N. Gupta. 1973. General biological effects of TCDD in laboratory animals. Environ. Health Perspect. 5:101-109. 14. Hayat, M.A. 1972. Basic Electron Microscopy Techniques. Van Nostrand Reinhold Co., New York. 15. Je^e^uel, A.M. 1974. Ultrastructural basis of drug metabolism. Israel J. Med. Sci. 10:380-385. 16. Johnson, J.E. 1971. The public health implications of widespread use of the phenoxy herbicides and picloram. Bioscience 21(17): 899-905. 17. Jones, G. and W.H. Butler. 1973. Hepatotoxicity of dioxin (2,3, 7,8-tetrachlorodibenzo-l,4-dioxin). for rats. J. Pathology of G. Brit, and Ireland. 109:Pxv. (Abstr.). 18. Jones, G. and W.H. Butler. 1974. A morphological study of the liver lesions induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. J. Pathol. 112(2):93-97. 19. Kearney, P.C., E.A. Woolson, A.R. Isensee, and C.S. Helling. 1973. Tetrac^orodibenzodioxin in the environment: sources, fate, and decontamination. Environ. Health Perspect. 5:273277. 20. Kimbrough, R.D. 1972. Toxicity of Chlorinated hydrocarbons and related compounds. A review including chlorinated dibenzodioxins and chlorinated dibensofurans. Arch. Environ. Health 25(2):125-131. 21. Kociba, R.J., P.A. Keeler, G.N. Park, and P.J. Gehring. 1976. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 35(3): 553-574. 22. Kociba, R.J., D.G. Keyes, J.E. Beyer, R.M. Carreon, C.E. Wftde, D.A. Dittenber, R.P. Kalnins, L.E. Erauson, C.N. Park, S.D. Barnard, R.A. Hummel, and C.G. Humiston. 1978. Result of a two year chronic toxicity and onccgenicity study of 2,3,,7,8tetxachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46:279-303. 23. Lucier, G.W., O.S. McDaniel, G.E.R. Hook, B. Fowler, B.R. Sonawane, and E. Faeder. 1973. TCDD-induced changes in rat liver microsonal enzymes. Environ. Health Perspect. 5:199-209. 49 �24. Meek, G.A. 1976. Stereology. Pages 409-410 in Practical Electron Microscopy for Biologists, 2nd Ed. John Wiley and Sons, New York. 25. McConnell, E.E., J.A. Moore, and D.W. Dalgard. 1978. Toxicity of 2,3,7,8-tetxac:hlorodibenzo-p-dioxin in rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicol. Appl. Pharroaool. 43:175-187. 26. Niwa, A., K. Kumaki, D.W. Nebert, and A.P. Poland. 1975. Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse. Arch. Biochetn. Biophysics. 166:559-564. 27. Norback, D.H. and J.H. Allen. 1969. Morphogenesis of toxic fat induced concentric membrane arrays in rat hepatocytes. Lab. Invest. 20:338-346. 28. Norback, D.H. and J.R. Allen. 1972. Chlorinated aromatic hydrocarbon induced modifications of the hepatic endoplasmic reticulum: concentric membrane arrays. Environ. Health Per spec t. 1:137-143. 29. Norback, D.H. and J.R. Allen. 1973. Biological responses of the nonhutnan primate, chicken, and rat to chlorinated dibenzo-pdioxin ingestion. Environ. Health Perspect. 5:233-240. 30. Pate, B.D., R.C. Voight, P. J. Lehn, and J.H. Hunter. 1972. Animal survey of Test Area C-52A, Eglin AFB Reservation, Florida. AFATL-TR-72-72, Air Force Armament Laboratory, Eglin AFB, Florida, April 1972. 31. Poland, A.P. and E. Glover. 1974. Genetic expression of aryl hyrdocarbon hydroxylase activity. J. Biol. Chem. 249(17):5599-5606. 32. Poland, A.P. and E. Glover. 1975. Genetic expression of aryl hydrocarbon hydroxylase by 2,3,7/8-tetxachlciirodibenzo-p--dioxin: evidence for a receptor mutation in genetically non-responsive mice. Mol. Pharmacol. 11C4):389-398. 33. Poland, A.P. and E. Glover. 1976. Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. Biol. Chem. 251(16):4936-4946. 34. Rawls, R.L. and D.A. O'Sullivan. 1976. Italy seeks answers following toxic release. Chem. Eng. News 54(35):27-35. 35. Schwetz, B.A., J.M. Norris, G.L, Sparschu, V.K. Rowe, P.J. Gehring, J.L. Emerson, and C.G. gerbig. 1973. Toxicology of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5:87-99. 50 �36. Shirley, E. 1977. The analysis of organ weight data. Toxicology 8:13-22. 37. Vos, J.G. 1972. Toxicology of PCBs for mammals and for birds. Environ. Health Perspect. 1:105-117. 38. Vos, J.G., J.A. Moore, and J.G. Zinkl. 1974. Toxicity of 2,3,7,8tetraciilorodibenzo-p-dioxin (TODD) in C57B1/6 mice. Toxicol. Appl. Pharmacol. 29(1):229-241. 39. Weibel, E.R., G.S. Kistler, and W.P. Scherle. 1966. Practical stereologicai methods for mcrphometric cytology. J. Cell Biol. 30:23-38. 40. Weibel, E.R. 1973. Stereologicai techniques for electron microscopic morphanetry. Pages 237-296 in M.A. Hayat, Ed., Principles and Techniques of Electron Microscopy, Biological Applications, Vol. 3, Van Nostrand Reinhold Co., New York. 41. Wilson, J.W. and E.H. Leduc. 1948. The occurrence and formation of binucleate and multinucleate cells and polyploid nuclei in the mouse liver. Am. J. Anat. 82:353-392. 42. Young. A.L. 1974. Ecological studies on a herbicide equipment test area (TA C-52A), Eglin AFB Reservation, Florida. AFATL-TR-7412, Air Force Armament Laboratory, Eglin AFB, Florida, January 1974. 43. Young, A.L., C.E. Thallcen, W.E. Ward. 1975. Studies of the ecological impact of herbicides on the ecosystem of test area C-52A, Eglin AFB, Florida. AFATL-TR-75-142, Air Force Armament Laboratory, Eglin AFB, Florida, October 1975. 51 �� 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. 0106 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 Cockerham, Lorris G. Alvin L. Young Charles E. Thalken Description An account of the resource <strong>Corporate Author: </strong>U.S. Air Force, Air Force Systems Command, Frank J. Seiler Research Laboratory Date A point or period of time associated with an event in the lifecycle of the resource 1980-03-01 Title A name given to the resource Histopathological and Ultrastructural Studies of Liver Tissue from TCDD-Exposed Beach Mice (Permyscus Polonotus) Subject The topic of the resource dioxin animal testing 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. 097 Folder The folder containing the original item. 2499 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 Cockerham, Lorris G. Alvin L. Young Source A related resource from which the described resource is derived Environmental Toxicology and Chemistry Date A point or period of time associated with an event in the lifecycle of the resource 1982 Title A name given to the resource The Absence of Hepatic Cellular Anomalies in TCDD-Exposed Beach Mice--a Field Study 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. 097 Folder The folder containing the original item. 2500 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 Cockerham, Lorris G. Alvin L. Young Source A related resource from which the described resource is derived Human and Environmental Risk of Chlorinated Dioxins and Related Dioxins Date A point or period of time associated with an event in the lifecycle of the resource 1983 Title A name given to the resource Ultrastructural Comparison of Liver Tissues From Field and Laboratory TCDD-Exposed Beach Mice 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. 112 Folder The folder containing the original item. 3088 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 Cockerham, Lorris G. A. L. Young Source A related resource from which the described resource is derived Environmental Toxicology and Chemistry Date A point or period of time associated with an event in the lifecycle of the resource 1982 Title A name given to the resource The Absence of Hepatic Cellular Anomalies in TCDD-Exposed Beach Mice - A Field Study https://www.nal.usda.gov/exhibits/speccoll/files/original/58a8f5e4d971563acebe4d0020cdfe16.pdf a8cff01934b888cbee89f46cbe2c9d12 PDF Text Text item D Number °4852 Author Cockerham, Lorris G. D Not Scanned Corporate Author RBDOrt/ArtiGlB TitlB Typescript: A Review of the Teratogenicity of TCDD Journal/Book Title Year 1976 Month/Day J ne 25 Color Number of Images " D 14 Descriptor! Notes Thursday, February 14, 2002 Page 4852 of 4919 �A REVIEW OE* THE fHfMOGENICIW OP TCDD 25 June 1976 Major Lorris G* Cockarham, M.S. Animal Miyatologiat Professor of Biological Sciences Department of Chanistry and Biological Sciences (DK33S) United States Air Force Academy, Colorado 80340 NCTCEi IMs is a report prepared in support of the ABIC Research Project on the Disposition of Herbicide Orange. The views expressed by the author are his own and do not represent the policies nor ooratdt support from the United States Air Faroe Academy Command, �A REVIEW OF OTE TERA3XX3ENICHY OF TCDD INTRODUCTION During the manufacture of the herbicide 2, add ( , , - ) a highly toxic iapurity, 2,3,7,8Ha9trachloeodiberizc>~p245T dioxin (TODD) , may be formed. TODD was named as the culprit in cases of drijoracne and chtcdc edema disease In the 1940 's and the 1950' . s Attention waa focused cm the possible taratogenlc effects of TOO) in the late 1960's whim 2,4,5-T was used as a defoliant in certain areas of South Vietnam* Recent descriptions of the teratogenic effects of TODD began when Courtney et al. (5) released a study using mice and rats treated with 2,4,5-T containing approximately 30 parts per million (ppm) of TOD. Using two strains of mice, Courtney et ajU (5) reported an increase in the incidence of deft palate in both the C57BL/6 and the AKR strain of mice when pregnant females were subjected to 2,4,5-T either subcutanaoualy or orally on days 6 through 14 or days 9 through 17 of gestation for the C57BI/6 strain and days € through 16 for the AKR strain. Only the C57HL/6 strain showed an increased incidence of cystic kidney. Doses varied from 21.5 mg to 113*0 mb 2,4,5-T per Kg of body weight with the 2,4,5-T containing approodmately 30 ppm of TCDD, Subsequently, other taratological studies using mice have shown that varying dose levels could affect prenatal development in mice. �Subsequently, other fceratological studies using mice have shown that varying dose levels could affect prenatal development in mice. Specifically, the only taratogenic affects observed were an increased frequency of cleft palate and cystic kidneys, a special type of kidney abnormality. Other toxic signs were noised but cannot be referred to as taratogenic since they can also be demonstrated in the adult after treatment with TCDD ( 5 . 1) Courtney and Moore ( ) by using varying dose levels and mixtures 6, of 2,4,5-T and. TCDD, attempted to determine if the previously reported teratogenic results (Oourtney et al. (5) ware due to 2,4,5-T, TCDD or the combination of the two. Cleft palates were produced in all strains of mice tested using 2,4,5-T, TCDD and the combination with no potentiation of the teratogenic effect using the ccnbination. Interestingly, the less pure grade of 2,4,5-T (Technical) produced less kidney anomalies than did the purest grade of 2,4,5-T (analytical) while treatment with TCDD produced a narked increase in kidney anomalies. An additional finding of the study was that the C57B1/6J strain of mice are the most susceptible to TCDD-induced effects. A later study by Hart and Valerio (10) was able to show only a marginally significant increase in incidence of cleft palate and no causal relationship between other observed abnormalities and treatment with 2,4,5-T. In JiMition to using 2,4,5-T instead of pure TOD, the investigators used the CD-I strain of mice rather than the C57B1/6J strain which is more sensitive to the teratogen. �Naubert and Dillraan ( 4 performed an exhaustive study of the 1) ernbryotojdc effects of both 2,4,5-T and TCDD In mice. In this study with NMRI mice only the appearance of cleft palate was considered as evidence of teratogenicity. it was found that both 2,4,5-T and TCEO produced cleft palate in mice, even with a single oral dose* The maximum teratogenic effect was seen with 2,4,5-T if the single oral dose was given on day 12 or 13 of gestation while with TCDD the maximal effect was obtained on day 11 of gestation* A potantiation of the effects of 2,4,5-T and TCDD was obtained when teratogenic doses of one were combined with threshold doses of the other. It was shown that TCDD was capable of producing cleft palate in this strain of mice at doses exceeding 2yg/kg which is well above that expected as a contamination of 2,4,5-T preparations. It was also suggested that other impurities than TCDD may be responsible for the teratogenic effects seen with preparations of 2,4,5-T. Using 2,4,5-T and 2,4-^lLchlorc^henoxyacertdc acid (2,4-D) separately and combined (TCDD content was less than 1 ppm) in varying dose levels, Bage, Cekanova and Larsson (1) found that high dosages were teratogenic in NMDRI mice. The most ccramon finding was isolated cleft palate followed by malformations of ribs and vertebra. Cystic kidneys, reported by Courtney at al. (5) after 2,4,5-T treatment, were not observed. As stated by the investigators, the dose resulting in teratogenic effects was extremely high as compared with the dose a pregnant wotan could normally be exposed to. They also noted that the results of their study and other studies did not substantiate any special risk to the human erabryo from the regular use of phonoxy herbicides. �the findings of Moore et at. (13) supported the cfcservations made Jay Courtney and Moore (6) of the teratogenic effects of TCDD on mice of the C57B1/6 strain, deft palate was reported with an incidence of 55.4 percent while kidney anomalies were found with an incidence of 95.1 percent when the dose was 3yg TCDD/kg given on gestation days 10 through 13* With a dose level of lyg/kg on the sane days, the incidence levels dropped to zero and 58.9 percent for cleft palate and kidney anomalies, respectively. Starting with Courtney et al. (5) the rat has also been used to test the teratogeniciiy of TCDD. The only teratogenic evidence seen was that of cystic kidneys. These investigators failed to say what strain of rats were used or to describe the renal anomalies other than as cystic kidneys. The 2,4,5-T used in the treatment contained approximately 30 ppm of TCDD. The results of a study by Emerson et al. (8) failed to substantiate the finding of Courtney et al. (5)« Examination of fetuses failed to reveal any serious teratogenic effects when the dams were given daily doses of 2,4,5-T containing approximately 27 ppm of TCDD on days 6 through 15 of gestation. In this study Spragxie-Dawley rats were used as expertoantal animals. Sparschu, Dunn and Howe (17) gave TCDD orally to pregnant female Sprague-Dawley rats on days 6 through 15 of gestation. Mo gross teratogenic effects were noted in the fetuses but both maternal and fetal toxicity was associated with TCDD at high doses (8.0ttg/kg/day). �Using a CD strain of rats, Courtney and Moore (6) found that neither 2,4,5-T nor TCDD produced cleft palate in rat fetuses when given on days 6 through 15 of gestation. However, it was found that 2,4,5-T produced only a minimal response in kidney malformation while TCDD produced a 34 percent incidence of malformation. Since many of the conclusions concerning the teratogeaiicity of TCDD were drawn from results of studies using TCDD contaminated 2,4,5-T, Emerson et al. (9) attempted to evaluate the teratogenic effects of a routine production lot of 2,4,5-T containing 0.5 ppm TCDD. Groups of Sprague-Dawlay female rats ware dosed once daily on days 6 through 15 of pregnancy with 1,3,6,12 or 24 mg 2,4,5-TAg. Examination of all fetuses following Caesarean section on day 20 of gestation revealed no teratogenic effects. Therefore, in this experiment, 2,4,5-T was not teratogenic to rats nor was TCDD at the ccaiocaitration of 0.5 ppn. Khera, Huston and MoKinley ( 1 , using 2,4,5-T herbicide containing 1) less than 0.5 ppat TCDD, found no apparent teratogenic effects using 25 rag 2,4,5-T/kg/day on Wistar rats during days 6 through 15 of gestation. Using a dose level of 50 mg/kg/day, the significance of the teratogenic effects observed was inconclusive. To determine if TCDD actually caused the teratogenic effects attributed to 2,4,5-T by Courtney et al. ( ) Sparschu, Dunn and Bowe (18) 5, subjected Sprague-Dawlay rats to orally administered TCDD on days € through 15 of gestation at levels of 0, 0.03, 0.125, 0.5, 2,0 and 8.0 yg/kg/day. Cleft palate and cystic kidneys were not observed in this study although both maternal toxicity and artoryotoxicity were �evident at the higher doses. It is believed, however, that the 1CDD present as a cjontamtoant in the 2,4,5-T tested earlier may hove aooounted for the reported observations of teratogenic effects which 'were attributed to 2,4,5-tf. Following the prcxsedures used by Bnsrsoti et al. ( ) bat increasing 9, the dose levels to 50 and 100 reg 2,4,5-<PAg/day> Sparschu et al. (19) showed no teratogenic effects with ooimercdal-grade 2,4,5-7 containing 0.5 ppn TCDD. The higher dose showed severe mternal toxicity and seme fetal toxicity. Thompson, Bnerson and Sparschu ( 0 , again using ooranercially 2) produced 2,4,5-T, treated Sprague-Dawley cats orally with doses of 1, 3, 6, 12, 24 or 50rag/kg/dayon days 6 through 15 of gestation* A dose of 100 lag/kg/day of 2,4,5-T was given to rats on days 6 through 10 of gestation. Bats treated with tp to 50 mg/kg/day did not reveal teratogenio effects* The 100 ing/kg/day dose produced maternal toxicity and death and some fetuses which showed toxic effects, but no teratogenic effects. Khera and Roddick (12) reported no teratogenic effects by treating female Wistar rats orally with 0.125*16 yg/tag/day of TCDD during days 6 through 15 of gestation. However, postnatal survival of the pups was severely decreased with increased prenatal doses* The investigators reasoned that this was due to entaryonic damage induced by the TCDD. Another test animal used to study the teratogenic effects of TCDD was the golden Syrian hamster. Collins and Williams (4) administered 2,4,5-T containing varying amounts of TCDD to groups of hamsters on days 6 �6 through 10 of gestation. Fetotoxicity and abnormalities such as the absence of eyelid and delayed bead ossification increased with TCDD content in the 2,4,5-T. cleft palates and cystic kidneys were not observed in hamsters. Snarson et al, (9) also evaluated the teratogenic effects of a routine production lot of 2,4,5-T containing 0.5 pps TCDD using groups of female New Zealand White rabbits. Each group received 0, 10, 20 or 40 rag 2,4,5-T/kg daily on days 6 through 18 of pregnancy. No anatomical malformations were observed and it was concluded that under the conditions of this experiment,, 2,4,5-T was not teratogenic to rabbits. •nwmpaon, Bnerson and Sparschu (20) also used groups of New Zealand White rabbits to study the taratogenic effects of 2,4,5-T. Visceral and skeletal examinations did not reveal teratogenic effects in any of the groups of rabbits given daily oral doses of 10, 20 or 40 rag 2,4,5-T/kg on days 6 through IS of gestation. Sheep were used by Binns and Balls (2) to test the teratogenioity of 2,4,5-T containing 1 ppn of 1X3DD. Teratogenic effects were not induced in any of the lanfcs from ewes fed as much as 113 ng 2,4,5-T/kg body weight for various periods during gestation. Dougherty, Coulston and Golberg (7) subjected mature female Rhesus monkeys (Macaca roulatta) to 2,4,5-T containing less than 0.05 ppn TCDD. CJancentrations of 0*05, 1*0 or 10.0 mgr 2,4,5-TAg body weight ware given to the test groups from day 22 through 28 of pregnancy. Detailed examination of the live infants and one stillborn fetus revealed no gross develcpnantal abnorrtality in any of the groups. Therefore, within �the oanditions of this experiment, there was no evidence that 2,4,5-T containing less than 0.05 ppn TCDD is teratogenic in the Rhesus monkey. Neubert et al. ( 5 , in reviewing the data on the teratogenic 1) effects obtained using pure TCDD, noted that doses as low as 1-10 yg/kg could produce nalformations of certain types. The two major types of malformations were cleft palate, reported only in mice, and kidney abnormalities, observed in both mice and rats, No mention was made of using pure TCDD in teratological experiments performed with any other species, even those that are highly sensitive to TCDD. The investigators found that potentiation can occur after treatment with a single dose of a combination of two or more known teratogenes* This was the case with the TCED~2,3,5-T combination, but not when nonteratogenic doses of 2,4,5-T are used with less than 10-20 ppm TCDD. DISCUSSION One Pood and Drug Aarainistration expects three studies to be completed in order for a drug to be acceptable for use ( 6 . These 1) three tests are: (1) study of fertility and general reproductive performance; (2) teratological study; and (3) perinatal and postnatal study. The United Kingdom's Ctmnittee on Safety of Drugs differs from the FDA in that it will accept a teratology study alone, but the drug must be administered from day one of gestation until just before term rather than during the period of embryogenesis as accepted by the FDA. All of the articles reviewed above reported teratogenic studies with the addition of postnatal studies being reported by Khera, Houston and MsKinley ( 1 , Khera and Ruddick ( 2 , and Moore et al. ( 3 . 1) 1) 1) 8 �According to Baboon ( 6 , when a teratology study is done, the 1) drug should be given during the period of organogenesis, which would be about days 6 through 15 for mice and rats, and days € through 18 for rabbits, With the exception of Bag©, Cekanova and Larsson (1) (days 6 through 14 for ndoa) and Moore et al. (13) (days 10 through 13 for mice) all investigators administered the test doses over the prescribed time period. In addition, Moore at al. ( 3 , Neubert and Dillman (14) 1) and Noubert et al. ( 5 tested single doses during the period of 1) organogenesis. All the investigators in the reviewed articles have satisfied the HIA recommendation of two dose levels. With ti» esxceptions of references 1, 2, 10, 13 and 19 the reports also satisfied the British recommendation of at least three dose levels. Some teams such as Neubert and Dillman (14) employed as many as 20 dose levels. It has been reccranended (16) that offspring be removed by Caesaraan section one or two days before term* For the most part all offspring were collected in this manner. Birms and Balls (2) allowed the ewes to go to term (160 days) as did Oourtney and Moore (€), Thompson, Emerson and Sparschu ( ) and Khera and Ruddiok (12) with rats. Dougherty, 2, Ooulston and Qolberg (7) also allowed the monkeys to go to term (164 days). With few exceptions, fetuses have been examined for both visceral anomalies and skeletal abnormalities. For some reason Binns and Balls (2) did not report what examinations were made, but only that the offspring were normal after birth* .fin. examination for skeletal abnormalities was not mentioned by Courtney and Moore ( ) Moore et al. ( 3 , and 6, 1) �Nsubert gt al. ( 5 . For this reason these four reports nay be considered 1) somewhat inocwplete. To properly test a drug &xr tetrabogenic effects at least two species of test animals should be used (16) * Many criteria are to be considered when selecting test animals to be used in teratological investigations. According to Clegg (3) scma of these criteria are availability, ease of maintenance, economics, gestation period/ estrus cycle, fertility in captivity, litter size, etc. All criteria are probably best met by the hamster, mouse, rabbit and rat. Extrapolation of the experimental data to man would necessitate the selection of test animals whose embryonic development and metabolic parameters are similar to those in man. Manhattan primates may be the animals of choice when metabolic data on the drug is not known* Only four of the investigating teams reviewed used two species of test animals (references 5, 6, 9 and 20). 3be rat was selected by all four, the rabbit by two, and the mouse by two* One team of investigators used sheep (2) which is not one of the recxamvended species, but was probably readily available. Collins and Williams (4) selected hamsters while Dougherty, Ooulston and Golberg (7) used the Rhesus monkey which would seem to be an animal of choice if one were trying to extrapolate the results of the teratological investigations to man. Strangely enough, the highly TCCO-sensitive guinea pig was never selected as a test animal. It would be interesting to compare the high toxicity seen in the adult of this species with any possible teratogenic effects on the fetus and with data obtained from other animals such as rats and mice* 10 �Very faw taratologloal studies have been done with TCDD alone aa the agent. Usually TCDD is studied in confeination with another agent such aa 2,4,5-7. Many of the early studies were done with very high concentrations of TCDD. indeed, one ouch study was completed before the concentration of the TOB in the 2,4,5-T was even known ( ) Later 5. investigations also used TCDD in ccwbination with 2,4,5-T, but at a much lower and more realistic concentration ( ) In addition, in an attempt 7. to more closely approach the embryonic development and metabolic parameters found in man, Rhesus monkeys have been employed as test animals. The highly cmtroversial question as to the potential hazard to man resulting from exposure to 2,4,5-T containing even a minimal amount of TCDD still remains to be settled* However, it appears from these studies that the teratogenic effects of TCDD in 2,4,5-T will not be seen at concentrations below approximately 10 ppiu 11 �REETSKENCES 1. Bage, G., E. Ctefeanova, and K.S. Larsson. 1973. Teratogenic and ettoL-yotoxic effects of the herbicides di and tricdilorophenoxyacetic acids (2,4-D and 2,4,5-T). Acta Pharmacol. Toxiaol. 32:408-416, 2. Binns, W. , and L. Balls. 1971. NOnteratogenic effects of 2,4,5tricdilxjrcplienaxyacetic acid and 2,4,5-T propylene glyool butyl esters herbicides in sheep. Teratology 4:245. (Abstr.) 3. Clegg, D.J. 1971. Teratology, Ann. Rev. Pharmaool. 11:409-424. 4. Collins, T.F.X., and C.H. Williams. 1971. Teratogenic studies vdth 2,4,5-T and 2,4-D In the haraster. Bull. Environ. Oontara. 6:599-567. 5. CSourtney, K.D., D.W. Gaylor, M.D. Hogan, and H.L. Falk. 1970. Teratogenic evaluation of 2,4,5-T. Science 168)864-366. 6. Courtney, K.D., and J.A* Moore. 1971. Teratology studies with 2,4,5-txichloropheiiaxyacetlc acid and 2,3,7,8-tetraohlorodibenzop-dioxin. Toxicol. Appl. Pharmacol. 20:396-403. 7. Dougherty, W.H., F. Ooulston, and L. Golberg. 1973. Nonteratxsgeaiicity o£ 2,4,5-teichloroplieiitaxyacetic acid in monkeys (Macaca mulatta) . Toxicol. Appl. Phannacol. 25:442. (Abstr.) 8. Etorson, J.L. , D.J. Thottpson, C.G. Gerbig, and V.3. Bobinson. 1970. Teratogenic study of 2,4,5-tri<±lorophenoxyacetic acid in the rat. Toxicol. Appl. Phamacol. 17:317. (Abstr.) 9. Bnerson, J.L., D.J. Thompson, R.J. Strebing, C.G, Gerbig, and V.B. Bobinson, 1971. Teratogenic studies on 2,4,5-trichloroplienox7acetic acid in the rat and rabbit. Fd. Oosmet. Toxicol. 9:395-404. 10. Hart, E.R., and M.G. Valeria, 1972. Teratogenic effects of 2,4,5-T in mice. Toxicol. Appl. Pharmacol* 22:317. (Abstr.) 11. Khara, K.S. , B.L. Huston, and W.P. ftoKinley, 1971. Pre- and postnatal studies on 2,4,5-T, 2,4-D, and derivatives in wister rats. Tooticol. Appl. Phannacol. 19:369. (Abstr.) 12. Khera, K.S., and J.A. Roddick. 1973. Polydilorodibenzo-p-dioxiiiis: Perinatal effects and the dominant lethal tests in vdstar rats. Pages 70-84 in E.H. Blair, ed. CMorodio«ins--Qrlgin and fate, Advances in chemistry series 120, American Chemical Society, Washington, DC, �13. Moore, J.A., B.N. Gupta, J.G. Zirikl, and J.G. Vos. 1973. Postnatal effects of maternal exposure to 2,3,7,8-tetrachlorocllbenzop-dioxin (TCDD), Envlxon. Health Perspect, No. 5s 81-85. 14. Neubert, D., and X. Dillmann. 1972. Bribryotoadc effects in mice treated with 2,4,5-trlchlorophiewwyaoetic acid and 2,3,7,8tetxachlorodiberazo-rfp-<2ia>£in» Naunyft-Schtaiedeberg'B Arch. Pharmacol. 272:243-264. 15. Neubert, D., p. Zens, A. RotJienwallner, and H.J. Mother* 1973. A survey of the embryotoxic effects of TCEO in mammalian species. Environ. Health Perspect* No. 5: 67-79. 16. Baboon, J.M. 1970. Testing drugs for teratogenicity and their effects on fertility* Brit. Mod. Bull. 26(3);212-216. 17* Sparscnu, G.L., F.L. Dunn, and V,K. Howe. 1 7 . Teratogenic study 90 of 2,3,7,8H»tracMjorodibenz&-p-dloxin in the rat* Toxiool. Appl. Pharmacol. 1 : 1 - 1 . (Abstr.) 73738 18. Sparscnu, G.L., F.L* Dunn, and V.K. Acme. 1 7 * Study of the 91 teratogenicity of 2,3,7,8-t»traicMorcx3iben2»-p-dlxxx^ in the rat* Fd. CJosmet. Toxicol. 9:405-412, 19. Sparscnu, G.L., P.L. Dunn, R.W. Lisowe, and V.K. Rowe. 1 7 . 91 Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic add on foetal development in the rat. Fd. Goemet. Tcxicol. 9»527-530» 20* lhampson, D.J., J.L. Emerson, and G.L. Sparschu. 1 7 . Study of 91 the effects of 2,4,5-tric^oropheno«!^acetic acid (2,4,5-T) on rat and rabbit fetal development. Iteratology 4:243. (Abstr.) � 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. 162 Folder The folder containing the original item. 4852 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 Cockerham, Lorris G. Date A point or period of time associated with an event in the lifecycle of the resource June 25 1976 Title A name given to the resource Typescript: A Review of the Teratogenicity of TCDD