Document 9JNGqx5py50zGbqo55E6jRBrp

RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) By: J. D. Burek, W. J. Potts, A. A. Crawford J. S. Murray, J. A. John, T. J. Bell, and F. J. Murray Reviewed By: K. S. Rao February 7, 1980 Toxicology Research Laboratory Health and Environmental Sciences, USA Dow Chemical Company, U.S.A. 1803 Building 00 1309^7 C-ONF TDFNT TAl LIST OF TABLES Table Page 1. Analysis of the Test Material.................................................................. 18 2. Routine Tissues Collected During the Necropsy Examination . . 19 3. Probe Study No. 1: Number of Tissues and Organs Examined Histologically from Rabbits, Rats and Dogs Exposed to 50 ppm DBCP.................................................................................................... 20 4. Probe Study No. 1: Body Weights (kg) of Male Rabbits Exposed to 50 ppm of DBCP....................................................................................... 21 5. Probe Study No. 1: Summary of the Gross and Histopathologic Findings in Male Rabbits Exposed to 50 ppm DBCP by Inhalation.....................................................................................................22 6. Probe Study No. 1: Body Weights (g) of Male Rats Exposed to 50 ppm of DBCP............................................................................................23 7. Probe Study No. 1: Summary of the Gross and Histopathologic Findings in Male Rats Exposed to 50 ppm DBCP by Inhala tion ............................................................................................. .... 24 8. Probe Study No. 1: Body Weights (kg) of Male Dogs Exposed to 50 ppm of DBCP....................................................................................... 26 9. Probe Study No. 1: Semen Evaluation of Dogs Exposed to 50 ppm of DBCP.....................................................................................................27 10. Probe Study No. 1: Summary of the Gross and Histopathologic Findings in Male Dogs Exposed to 50 ppm DBCP by Inhala tion .................................................................................................................. 28 11. Probe Study No. 2: Body Weights (kg) of Male Rabbits Exposed to 10 ppm of DBCP....................................................................................... 29 12. Probe Study No. 2: Semen Evaluation of Rabbits Exposed to 10 ppm of DBCP............................................................................................30 DO 1 3 O 9 f-) 8 CONF TDFN7 T At INTRODUCTION DBCP (l,2-dibromo-3-chloropropane) Is a soil fumigant and an agricultural nematocide. It has been shown to cause testicular atrophy in laboratory animals (Torkelson, e. al.., 1961), and has been associated with reduced sperm counts and sterility in exposed male workers (Whorton, et^. al., 1977). A study was designed to further assess the effects of inhaled DBCP on spermatogenesis and fertility in laboratory animals, to determine the maximum level of exposure to DBCP that does not have an effect on reproduction, and to evaluate the reversibility of these effects. Prior to starting the larger study on spermatogenesis and fertility, two probe studies were conducted. The first probe study consisted of exposing male rabbits, male dogs and male rats to 50 ppm of DBCP by inhalation. The purpose was to confirm that DBCP did have an effect on spermato genesis, and to determine the species of choice for the larger reproduction and fertility study. The second probe study was conducted to further evaluate the appropriateness of the rabbit for the larger reproduction and fertility study. The purpose of this report is to summarize the results of the two probe studies. 00 130969 OONFTDFNT T Al -4- Semen Evaluation. Semen evaluation was performed on dogs and rabbits. Semen collections were performed prior to placing the animals into the chambers. Semen was collected in a graduated test tube from each animal by means of an artificial vagina. After collection, the volume of semen and its color were recorded. The semen was then placed in a constant temperature bath at 37C for 15 minutes to allow the semen to liquify. The motility and ratio of live and dead sperm were determined after the semen had liquified. To assess motility, a large drop of semen diluted with warm saline was placed on a glass slide under a cover slip and examined at 400 X magnification. The first 100 sperm observed were ranked as follows: 1) Progressively motile (moving from place to place), 2) nonprogressively motile (twitching tails but staying in one place), and 3) non-motile (no movement). The ratio of live and dead sperm was determined by staining a drop of semen with eosin-aniline blue stain^ and counting 100 sperm per specimen (Seager and Fletcher, 1972). The sperm count was determined using the hemocytometer method described by Miller (1955) after the method of Gordon, et. al. (1965). The actual count was determined using a Coulter Counter, Model ZBI. 3 Chambers. The first probe study at 50 ppm DBCP was conducted using a 1-m Rochester-type exposure chamber. The main airflow in the chamber was 3-182 1/min, for an air turnover rate of ^11 air changes/hour. The air was pulled through the chamber by an exhaust pump, such that the chamber '"Aniline blue, 5 g; eosin B, 0.75 g; sodium citrate, 2.9 g; water, 100 ml. DO 130970 CONFTDFNT TAl -6- used as in the previous probe. However, certain modifications were ; made, in order to provide the ability to run 3 chambers from the same apparatus (see below). The air effluent direct from the vapor generator I was diluted ^3:1 with compressed air, and this mixture led to a 3-neck 1-liter plenum flask, through one neck. A second neck was capped off. I The third neck joined the main chamber air intake line, but through a slight throttle, such that the pressure in the 1-liter flask was 'vl" ^0 positive with respect to ambient room air. Once target concentration (10 ppm) was reached, it was found to be a simple matter to hold the * concentration to within 3% of target by making small adjustments in airflow as required. I Design of Probe Study No. 1. In this probe study, 2 male rabbits, 5 male rats and 2 male dogs were exposed by inhalation to 50 ppm of DBCF for 6 hours/day, 5 days/week. The duration of exposure was two weeks for rabbits and rats. Exposure of dogs did not stop after two weeks, but was continued in order to maximize the potential effect in that species. A control group was not included in the probe study. Rabbits were weighed on days 1, 6, and 14 of the study (the first day of exposure was considered to be day 1 of the study). Rats were weighed on days 1, 3, 6, 8, 10, 13, and 14. Dogs were weighed on days 1, 6, 13, and 20. On days when the animals were exposed to DBCF, body weights were recorded prior to placing the animals in the exposure chambers. Semen from dogs was collected and analyzed (See section on semen evalua tion) on days -1, 6, 13, and 20 of the study. Attempts were made to collect semen from rabbits during the study, but no samples could be DO 130371 CONFTDFNTTA 8- - technique with fluorescent light Illumination Immediately following the decapitation. Prior to formalin fixation, the weights of the testicles were obtained and recorded from all five rats. The exposure of the dogs did not stop on the 14th day of the study* but was continued on to maximize the potential effect in the dogs. Both dogs died on October 29, 1977 (25th day of the study) and were presented for necropsy examination. Both dogs were examined externally and inter nally and any observations were recorded. Representative tissues collected during the necropsy examination were preserved in phosphate buffered 10% formalin. The routine tissues collected during the necropsy examination are listed in Table 2. Prior to the formalin fixation, the weights of the testicles were obtained and recorded from both dogs. Representative sections from most of the tissues shown in Table 2 were processed from all animals from this probe study. Tissues were processed using conventional methods, embedded in paraffin, sectioned (5-6 u) and stained with hematoxylin and eosin. A complete list of the organs and tissues that were examined microscopically are presented in Table 3. It should be noted that the list of tissues evaluated included animals that died as well as animals that were killed during the study. In a few cases, a tissue specimen was not obtained at necropsy or was lost during the processing. The parathyroid glands were evaluated only to the extent that they were included in the routine sections obtained from the thyroid gland. DO 13097? OONFTDFNTTAl -10- RESULTS Probe Study No. 1. Two male rabbits were exposed to 50 ppm of DBCP. One rabbit (77-6712) died on the 10th day of the study; the other rabbit was sacrificed on the 14th day of the study. Significant weight loss was noted in both of these rabbits (Table 4). Repeated attempts to collect semen from these rabbits on days 6 and 7 of the study were unsuccessful. Although both bucks were able to mount a teaser doe, they were unable to maintain an erection and ejaculate. No problems were encountered in collecting semen from either of these bucks one -day prior to the initial exposure. Also, before being placed on this study, both of these bucks were used on numerous occasions in this laboratory as semen doners for artificial insemination; difficulties in obtaining semen did not occur during that period. The gross and histopathologic findings and the testicular weights of the two rabbits exposed to 50 ppm DBCP are summarized in Table 5. Both rabbits had testicles that were decreased in size based on organ weight, morphological appearance or both at the time of gross necropsy. Histo logically, the testicles of both rabbits had bilateral germ cell degen eration, individual cell necrosis, focal necrotic debris, apparent decrease in the number of mature spermatids and spermatocytes, and one rabbit, (77-6713), also had necrotic debris, atypical germ cells and a few multinucleated spermatids free in the tubular lumina of one epididy mis. These morphological alterations in the testicles and epididymides were considered to be treatment-related alterations. 1 rF/N/T -12- alterations that were observed in the testicles in all five rats. These changes consisted of focal or multiple tubular degenerative changes, apparent decrease in the number of germ cell layers within the tubules, occasional multinucleated spermatids, and an apparent accentuation or increased prominence of Sertoli cells in a few seminiferous tubules. These changes were rather subtle, but they appeared to be treatmentrelated. In addition to the testicular alterations, the kidneys from 4 of the 5 rats had epithelial cell changes at the corticomedullary junction. These changes consisted of enlarged pleomorphic epithelial cells within the tubules that were suggestive for a regenerative process. Such changes are unusual in rats of this strain and age and are, there fore, interpreted as probably treatment-related. There were a few other tissue changes observed, but these were consistent with normal or spon taneously occurring changes that are commonly seen in rats of this strain, sex and age. Two dogs were exposed to 50 ppm of DBCP. As in the other two species, dogs exposed to DBCP had significant weight loss (Table 8). Vomiting was observed on the 3rd and 4th days of exposure. One dog was emaciated and had a dull coat and excessive dandruff; this dog died on the morning of the 25th day of the study. The other dog died during the afternoon of the 25th day; it had been unable to stand when taken out of the exposure chamber that afternoon. Evaluation of the semen of the dogs revealed no changes attributable to DBCP with respect to sperm count, volume of semen, color of semen, or motility of sperm (Table 9). DO 130974 OONFTDFNTTAl -14- Evaluation of semen collected on the morning of the 5th day of exposure revealed no remarkable changes compared to pre-exposure values (Table 12). Only 1 of the 4 rabbits (77-7210) exposed to 10 ppm of DBCP for 14 days had a gross lesions that consisted of a thickend, hyperemic and hemor rhagic appearance to the wall and mucosa of the urinary bladder. This finding was suggestive for cystitis, but was not considered treatmentrelated since no other gross lesions were observed in these 4 animals. Only the testicles and epididymides were evaluated histologically (from these rabbits). All showed slight histologic alterations within the testicles that consisted of focal individual germ cell necrosis, focal tubules with multinucleated spermatids, and focal tubules with decreased numbers of germinal cells lining the tubular lumen. These changes were considered to represent slight treatment-related effects. HO 100975 CONF T DFNT T A[ -16- QUALITY ASSURANCE STATEMENT This report represents data generated prior to the enactment of the V FDA Good Laboratory Practice Regulations. The study was conducted according to standards used in this laboratory at that time. The report accurately reflects all of the data generated. All data and reports are located at the submitting laboratory. i Study Started: Nov. 28. 1977 Protocol Audited: 1977 Data Audited: Jan. 23, 1980 Final Report Audited: Jan. 21^ 1980 Report Issued: Feb. 7, 1980 Reported: 1977 Reported: Jan. 24, 1980 Reported: Jan. 24, 1980 W. E. Hoover (Date) ^ Quality Assurance Toxicology Research Laboratory Health and Environmental Sciences, USA 1803 Building Dow Chemical U.S.A. Midland, MI 48640 DO 130976 OONFTDFNT TA( -18- TABLE 1 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) Analysis of the Test Material Pre-exposure Post-exposure X by weight DBCP 97.3 96.610.4 Allyl Chloride 0.4 0.5910.06 1,2,5,6-Tetrabromohexane 0.92 1.2010.1 2-Chloropropane 0.35 0.3810.04 1-Chloropropane 0.03 0.0410.004 Epichlorohydrin 0.81 1.0010.1 l-bromo-3-chloropropane 0.11 0.1010.01 00 130977 CONf T DF'M7 1 A -20- TABLE 3 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) PROBE STUDY NO. 1; Number of Tissues and Organs Examined Histologically From Rabbits, Rats and Dogs Exposed to 50 ppm DBCP Species_______________________________ Number Examined_____________________ _ Liver Gallbladder Kidneys Urinary Bladder Heart Anterior Mediastinal Blood Vessles (or aorta) Mesenteric Blood Vessels Trachea Lungs Tongue Esophagus Stomach Small Intestine Cecum Colon Salivary Glands Pancreas Testicles Epididymis Prostate Seminal Vesicles Coagulating Glands Brain Spinal Cord Peripheral Nerve Pituitary Gland Adrenal Glands Thyroid Gland Parathyroid Gland(s) Spleen Thymus Anterior Mediastinal Lymph Node(s) Mesenteric Lymph Node(s) Subcutaneous Lymph Node(s) Skin Mammary Gland Skeletal Muscle Eyes Rabbit 2 2 1 2 2 2 1 2 2 2 0 2 2 2 2 2 2 2 2 2 2 0 0 2 1 2 2 2 1 1 1 0 0 0 0 1 0 1 2 Rat 5 5 0 5 5 5 5 3 5 5 5 5 3 3 3 3 5 5 5 4 5 5 4 5 5 1 5 5 5 1 5 5 5 4 2 4 4 4 4 CM CM CM CM CSI Dog 2 2 1 1 2 2 0 2 2 1 2 2 2 2 2 2 0 0 2 0 1 2 2 2 1 2 2 1 2 0 1 0 2 2 HO 130978 oonftdfnttai 00 130979 oonftdfntt Pa thology Number 77-6712 77-6713 TABLE 5 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) PROBE STUDY NO. 1: Summary of the Gross and Histopathologic Findings In Hale Rabbit3 Exposed to 50 ppm DBCP by Inhalation Summary of Gross and Histopathologic Observations Gross: Soiled hair around external nares from nasal discharge. Petechial hemorrhage and hyperemia of small intestine mucosa. Decreased lngesta In gastrointestinal tract. Purulent material in nasal turbinates. Pale liver. Debris in right outer ear canal. Testicles weighed 3.17 gms together. Histopathology: Testicles - bilateral multifocal gernf cell degeneration, individual germ K cell necrosis and increased prominence of Sertoli cells. Epididymis - unilateral 1 decreased sperm in tubular lumlna. Lungs - severe necrotizing bronchopneumonia. Trachea - mild suppurative tracheitis. Liver - slight fatty change and slight tinctorial variation to hepatocytes. Kidneys - generalized congestion and focal interstitial aggregates of mononuclear lymphoid cells. Gross: Hemorrhagic foci in lungs. Decreased testicular size and weighed 2.86 gms together. Histopathology: Testicles - bilateral multifocal germ cell degeneration, individual germ cell necrosis, focal necrotic debris, an apparent decrease in the number of spermatids and spermatocytes and occasional multinucleated spermatids. Epididymis unilateral decreased sperm. Increased necrotic cellular debris, atypical germ cells and a few multinucleated spermatids in the tubular lumina. Lung - mild acute bronchitis and bronchiolitis and peribronchial aggregates of mononuclear lymphoid cells. Heart - few arterioles with necrotic debris and mineral deposits in vessel wall. Pathology Number 77-6714 77-6715 TABLE 7 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) PROBE STUDY NO. 1: Summary of the Gross and Histopathologic Findings in Male Rats Exposed to 50 ppm DBCP by Inhalation Summary of Gross and Histopathologic Observations Gross: Apparent decreased thymic size. Hlstopathology: Testicles - localized tubules with decreased germ cells, occasional multi- nucleated spermatids and increased prominence of Sertoli cells. Liver - altered tinctorial properties and vacuolation of hepatocytes. Kidneys - enlarged and i pleomorphic tubular epithelial cells at the corticomedullary junction. Lungs - $1 slight perivascular, peribronchial and interstitial aggregates of mononuclear 1 lymphoid cells. Gross: No significant lesions. Hlstopathology: Testicles - localized tubules with decreased germ cells, occasional multinucleated spermatids and increased prominence of Sertoli cells. Liver - altered tinctorial properties and vacuolation of hepatocytes. Kidneys - enlarged and pleomorphic tubular epithelial cells at the corticomedullary junction. Lungs slight perivascular and peribronchial aggregates of mononuclear lymphoid cells. DO 1 3 0 9 8 0 CONFTDFNTT > -26- TABLE 8 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) PROBE STUDY NO. li Body Weights (kg) of Male Dogs Exposed to 50 ppm of DBCP Animal No, NZ16 AE26 1 13.4 10.0 Day of Study 6 13 20 12.0 11.0 10.1 9.0 8.2 7.6 25 Dead Dead r>0 1 30981 CONF TDFNTTA! no 13098? CONFTDFNTT Pathology Number 77-6889 77-6890 T> TABLE 10 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOCS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) PROBE STUDY NO. 1: Summary of the Gross and Histopathologic Findings in Hale Dogs Exposed to 50 ppm DBCP by Inhalation _______________ Summary of Gross and Histopathologic Observations______________________ Gross: Cachectic and dehydrated. Severe edema, hemorrhage and/or congestion of the left hind foot, subcutaneous tissue, muscles, testicles, urinary bladder, urethra, gastroin testinal tract, kidneys, anterior mediastinum, and pericardium of heart and lungs. Mottled liver. Decreased size of prostate gland. Testicles weighed 7.1 gms together and were tan in color. lllstopathology: Testicles - degeneration, necrosis and loss of germ cells with increased prominence of Sertoli cells. Liver - slight fatty change and macrophages with brown granular cytoplasmic pigment. Lungs - suppurative pneumonia and focal nematodlasls (Filaroides hirthi). Kidneys - multifocal degeneration and necrosis of tubular i epithelial cells. Multiple organs - hemorrhage, congestion and/or edema. oo Gross: Cachectic and dehydrated. Severe edema, hemorrhage and/or congestion of the tongue, subcutaneous tissue, testicles, lymph nodes, urinary bladder, prostate, gastroin testinal tract, adrenal gland, and lungs. Mottled, enlarged and friable liver. Ascites. Perforated gastric ulcer. Decreased ingesta in gastrointestinal tract. Testicles weighed 10.8 gms together. Questionable decreased size of thyroid glands. Decreased size of prostate gland. , Histopathology: Testicles - degeneration, necrosis and loss of germ cells. Liver - slight fatty change, macrophages with brown*granular cytoplasmic pigment and focal necrosis with suppurative inflammation. Kidneys - multifocal degeneration and necrosis of tubular epithelial cells. Lungs - suppurative bronchopneumonia. Colon - submucosal granulomata. Lymph nodes - lymphoid cell depletion. Multiple organs - hemorrhage, congestion and/or edema. TABLE 12 RESULTS OF TWO INHALATION PROBE STUDIES IN RABBITS, RATS AND DOGS THAT WERE EXPOSED TO 1,2-DIBROMO-3-CHLOROPROPANE (DBCP) j I; PROBE STUDY NO. 2: I Semen Evaluation of Rabbits Exposed to 10 ppm of DBCP i Animal Day of Number Study 819 -5 5 Sperm Count, millions/ml Vol. of Semen, ml Color Motility of Sperm (%) Non- Non- Progressive Progressive Motile 128 0.5 Milky 66 87 1.0 Milky 79 9 25 8 13 1130 -4 5 58 0.5 Milky 83 130 0.25 Milky 78 6 ;u 8 14 931 -4 5 51 0.5 Milky 70 47 0.5 Milky 70 13 17 15 15 930 -4 5 48 0.4 Milky 76 168 0.5 Milky 75 7 17 10 16 DO 1309B3 GONFTDFNTTAl TOXICOLOGY RESEARCH LABORATORY HEALTH AND ENVIRONMENTAL RESEARCH DOW CHEMICAL U.S.A. LOCATER SHEET FILE NO. ftET-fo -/7/T-C/s) DEAD STORAGE NO, /5?3 TITLE OF REPORT: pah of ttlutfahoit jpy'M*'*- /* *,4 DATE REPORT ISSUED: 7-/^-77 AUTHOR(s): UUihyutu ZT. &.(/,** M-M- SMUdihr, ' J* ' /e.J, Notebook(s) - (Number and Page(s): n AJik. -tZ-f 4) 2) --/3-// 5) 3) SPECIMENS: 1) 2) 3) 4) 6) PATHOLOGY NUMBERS: ADDITIONAL DOCUMENTS: 1) 2) 3) 4) LOCATION: **/ RESTRICTED FOR USE WITHIN THE DOW CHEMICAL COMPANY ONLY. THIS PAGE SHOULD FOLLOW THE DOW SUMMARY PAGE AND BE ATTACHED TO COPIES MAINTAINED ONLY IN THE TOXICOLOGY RESEARCH ARCHIVES AND QUALITY ASSURANCE UNIT FILES. 1 80984 OONFTOFNTTAl J THE DOW CHEMICAL COMPANY IIOCHEMICAL RESEARCH LAE. MIDLAND, MICHIGAN 41*40 REQUEST FOR TOXICOLOGICAL STUDIES wo? bldg. TOX. NO. filTnro_T /7/5^-ijp/5). Hr-A t<~ m^-Ciit) AME OF MATERIAL TO BE TESTED ZZhdetu. iUBUITTTEEOO BBYY A*. /W4 SUPERVISOR (SifrituraT j, N C I, ~ of) g * '*'**' cn^t.,,uy 1-n-it, T(Daf and Dutldm|) j(ph*Mr (ftp PROJECT SAFETY COORDINATOR <D*'' ESTIMATED cost 2- S. cTL'V ACCOUNT NUMSER LEDGER LOCATION GROUP SECTION FliHCTIOIT^' SUaFUNg SUFFIX PROBLEM NO. TO BE USED BY TOXICOLOGY La*. .S' / PROBLEM INDEX .HQ, PROBLEM 0 o\o\o,/ j* f DO NOT WRITE ROUTE OF ADMINISTRATION S,,h LENGTH Of treatment BELOW THIS LINE COMPLETE ALL INFORMATION CN REVERSE NOTES, CHANGES. ADVERSE EFFECTS NOTED SPEOES: oat Of dog Dmf OTHER DOSES braarbrbiitt OgFm monkey DDmf GUIpNiEgA DpFm hamster ODmf CHECK POINT MPLE RECEIVED TOXICOLOGY estimated starting time ESTIMATED REPORTED DATE REVISED ESTIMATE REPORTED DATE &-7C, /0-7C> 2-77 REPORT DRAFTED REPORT ISSUED FATE OF SAMPLE 1 1 RETURNED 1 iDESTROYED DATE | | REFERENCE SAMPLE STORED | | REFERENCE SAMPLE TO K STORAGE NOTES DATE PROJECT COMPLETED ' DO 130985 COHF'TOFNTTAl si- -Y R & D REPORT DOW CHEMICAL U.S.A. RESTRICTED: for use within The Dow Chemical Company only. iifartmknt Toxicology Research Laboratory HET K-1715-(15) 7-18-77 LAB. Ha.-'- PaoailM NO. 5 i 1.6 0 i 0 10 i 011 |2 | 8 Pate of Ethylene Dibromlde in Rats Following Inhalation Exposure 31 UTHO*|l) P. G. Watanabe, J. D. Young, M. M. Echlachter, J. A. Zerapel, R. J. Rarbowski tUTHDn(l) 10NATU*() PAGES IN FULL REPORT CRI NUMBER nviiaiii'a am DATA REFERENCES (book aid paga): This report is: I INTERIM m FINAL and mainly: 0 NEW review (Rafar alio to aarliar ralatad raportj and publication!.) LATENT STATUS: I J ditdoaura lubmittad I I caaa filad I I no patant action required DESCRIPTIVE SUMMARY WITH CONCLUSIONS: The fate of ethylene dibromide (EDB) was studied in rats following inhalation exposure to determine if a change in the disposition of EDB in the body occurred with Increasing exposure concentrations. Male rats were exposed to 7, 25, or 75 ppm ^ u-labeled EDB for 6 hours and the routes and rates of elimination of Re activity were followed for 48 hours after termination of exposure. Total metabolism of EDB and hepatic glutathione (GSH) levels were also determined. The percentage of recovered radioactivity excreted by the various routes were similar for all exposure levels. The urinary excretion of radioactivity was the major route of elimination accounting for 80% of the recovered radioactivity. The rates of excretion of urinary Re-activity were similar for all exposure levels with a half-life (t-i^) 5.1-5.6 hours. Urinary metabolites of EDB following inhalation exposure appear to be derived from GSH conjugation. Hepatic GSH levels were 94, 77, and 59% of control following 6 hour exposures to 7, 25, and 75 ppm EDB respectively. The metabolism of EDB appears to constitute a detoxification mechanism and the data suggest that EDB is detoxified more efficiently at a low exposure (6-7 ppm) compared to higher exposures (25, 75 ppm). Thus, the fate of inhaled EDB in rats is dependent on the exposure concentration and toxicity may result in high exposures because the ability to detoxify EDB has been impaired. Furthermore, it may not be appropriate to extrapolate linearly the same toxicity incurred at levels of exposure greater than 6-7 ppm EDB to lower levels of exposure in rats. &UV /Isita- DISTRIBUTION: Saa Back Pag* / v- ML Dtstd. sttnatte. ** ISC 3 DO 130986 OONFTDFNTTAl FATE OF ETHYLENE DIBROMIDE IN RATS FOLLOWING INHALATION EXPOSURE P. G. Watanabe, J. D. Young, M. M. Schlachter, J. A. Zempel and R. J. Karbowski June 18, 1977 Toxicology Research Laboratory Health and Environmental Research Dow Chemical U.S.A. Midland, Michigan 48640 DO 130987 CONFTDFNTTAI, ABSTRACT The fate of ethylene dibromide (EDB) was studied in rats following inhalation exposure to determine if a change in the disposition of EDB in the body occurred with increasing exposure concentrations. Male rats were exposed to 7, 25, or 75 ppm *C-labeled EDB for 6 hours and the routes and rates of elimination of 14C-activity were followed for 48 hours after termination of exposure. Total metabolism of EDB and hepatic glutathione (GSH) levels were also determined. The percentage of recovered radioactivity excreted by the various routes were similar for all exposure levels. The urinary excretion of radioactivity was the major route of elimination accounting for 80% of the recovered radioactivity. The rates of excretion of urinary 14 C-activity were similar for all exposure levels with a half-life (tjy2) of 5.1-5.6 hours. Urinary metabolites of EDB following inhalation exposure appear to be derived from GSH conjugation. Hepatic GSH levels were 94, 77, and 59% of control following 6 hour exposures to 7, 25, and 75 ppm EDB respectively. The metabolism of EDB appears to consti tute a detoxification mechanism and the data suggest that EDB is detoxified more efficiently at a low exposure (6-7 ppm) compared to higher exposures (25, 75 ppm). Thus the fate of inhaled EDB in rats is dependent on the exposure concentration and toxicity may result at high exposures DO 130988 CONFIDENT TAl -iibecause the ability to detoxify EDB has been impaired. Furthermore, it may not be appropriate to extrapolate linearly the same toxicity incurred at levels of exposure greater than 6-7 ppm EDB to lower levels of exposure in rats. rni? 1 '?0989 C0Nf"TDFNT TA -1- INTRODUCTION Ethylene dibromide (1,2-dibromoethane, EDB) is used extensively as a gasoline additive, fumigant and chemical intermediate. Exposure to EDB results primarily from industrial applications where inhaled EDB vapor constitutes the common route of entry into the body. Acute and subchronic studies on the toxicity of inhaled EDB in experimental animals (50-3000 ppm) showed that EDB caused dose-related damage to the liver, lung and kidney (Rowe, et al., 1952). Toxicity was not observed in rats exposed repeatedly to 25 ppm EDB (7 hours/day, 5 days/ week) for 6 months. The current threshold limit value for industrial exposure to EDB is 20 ppm. The metabolism of EDB has been the subject of numerous investigations. There appears to be a general consensus that the toxicity of EDB is due to the parent molecule and that the metabolism constitutes a detoxification process. Detoxi fication of EDB by conjugation with hepatic glutathione (GSH) mediated by GSH-S-alkyl transferase has been shown to occur in vitro (Nachtomi, 1970). Consistent with this observation was the identification of 5-hydroxyethyl cysteine, its Nacetylated analogue and its respective sulfoxide as major urinary metabolites in rats following oral intubation of EDB (Nachtomi, 1966; 1970). DO 130990 OONFTDFNTTAl -2- Of particular significance is the finding that EDB is detoxi fied by conjugation with GSH in vivo. This detoxification process can be saturated if doses sufficient to deplete GSH are given. This has been observed following administration of large doses of acetaminophen and bromobenzene (Gillette, 1974). If exposure to high levels of EDB similarly deplete GSH levels in the body, this may lead to increased concentra tions of EDB and an increased reaction with intracellular macromolecules resulting in toxicity. In addition to morpho logical and functional damage to the cell, chronically induced oncogenesis may be mediated by such reactions (Miller and Miller, 1971) . Recent concern over the potential hazard of exposure to EDB was prompted by the report of induction of stomach cancer (squamous cell carcinoma) in rats and mice orally intubated with EDB at 40-200 mg/kg/day (Olson, et al., 1973). Assessing the hazard of exposure to EDB from these results is limited severely because stomach cancer was produced only at very high dose levels which caused overt toxicity and increased mortality in all the treatment groups. Furthermore, adminis tration of a reactive chemical such as EDB in a concentrated solution via oral gavage is an unrealistic route of adminis tration to assess the toxicity of a material such as EDB for which inhalation is the predominant route of exposure. DO 130991 CONFTDFNT T Al -3- Studies on the fate of EDB following inhalation exposure are needed to determine if the fate of inhaled EDB changes with increasing exposure concentration. Thus the objective of this study was to characterize the fate of EDB, including reaction with intracellular macromolecules, following exposure to various atmospheric concentrations of 14 C-EDB in rats. DO 13099? OONFTDFNTTAl -4- METHOD Material. ^C-labeled EDB (1,2-^C-dibromoethane) was purchased from New England Nuclear (Lot #884-249, specific activity 60 mCi/ mmole). Radiochemical purity was 94-95% as determined by gas-liquid chromatography on a Hewlett-Packard, Model 5750, gas chromatograph. EDB (0.2 ul) was chromatographed on a column of 10% SP-1000 on Chromosorb W (Supelco, Inc.), 100/ 120 mesh (6' x 1/8"). The helium carrier gas flow rate was 20 ml/min and the oven temperature was 125C. Following passage through a thermal conductivity detector the effluent gases were trapped by bubbling directly in scintillation vials containing 20 ml of scintillant containing Concifluor (Mallinkrodt Chemical Works). Fractions were collected every 30 seconds and the radioactivity was determined by counting in a liquid scintillation spectrometer. The non-labeled EDB used was 99% chemically pure. The non-labeled and 14 C-labeled EDB were mixed as vapors in a Saran bag (Anspec) to the desired specific activity and concentration. Animals. Male Sprague-Dawley rats (Spartan Research Laboratory) weighing 195-250 g were used in the study. All animals were housed in rooms in which a constant humidity, temperature, and 12-hour light-dark cycle (7 AM - 7 PM EST) were maintained. DO 130990 C0NFTDFNT7 Al -5- Food and water were provided ad libitum except during exposure. Rats used for determination of blood EDB levels were surgically prepared by implanting a cannula in the right jugular vein (Harms and Ojeda, 1974). These rats were allowed to recover 2-3 days prior to use. Exposure. All rats were exposed under dynamic condition's to room air (controls) or to various concentrations of EDB. The exposures were conducted in a 1 liter plexiglass inhalation chamber which is designed to expose 4 rats simultaneously. In this apparatus the rats head protrudes into the chamber allowing exposure of the head only to the test atmosphere. The EDB atmosphere was generated by pumping an appropriate volume of concentrated EDB vapor from a Saran bag (Anspec) at a controlled rate into the chamber airflow (approximately 6 1/min). The concentrated EDB vapor was metered with a Masterflex peri staltic pump (Cole-Parmer Instrument Company) or a precision dual syringe pump. The concentration of EDB was monitored by passing the input airflow into the chamber through a gas cell of an infrared spectrophotometer (Wilks) calibrated at a wavelength of 8.5 ym. The chamber atmosphere was also analyzed at hourly intervals by gas chromatography. The column used for analysis was similar to the one described for determining the radiochemical purity of EDB. Samples for ^C-activity no i 30994 -6- determinations were taken hourly by bubbling 1 ml aliquots 1 of the chamber atmosphere into a scintillation solution containing Concifluor (Mallinckrodt Chemical). Radioactivity was determined by liquid scintillation spectro- j metry. The inhalation chamber was operated in a laboratory 1 fume hood to prevent contamination of the working environment. Fate of Radioactivity Following Exposure to Inhaled 14 C-EDB Groups of 4 rats were exposed to 7, 25 or 75 ppm 14 C-EDB for 6 hours. The mean analytical concentrations measured by gas chromatography were 7.20.2 (SD), 24.8+0.4 and 74.7+1.2 ppm EDB. The respective specific activities were 582, 211 and 280 DPM/pg EDB. Control animals used for non protein sulfhydryl determinations (4/group) were exposed concomitantly to room air. Following the exposure one rat per exposure level was placed in a glass Roth-type metabolism cage for the collection of urine, feces and expired air. Room air was drawn through the cages at 400-500 ml/min. The air leaving the chamber was passed first through a tube containing 1 g of activated charcoal to adsorb expired EDB. Subsequent transit through a trap containing 120 ml of 5 M ethanolamine in 2-methoxyethanol enabled the collection of 14 C02* The trap for 14 C02 was maintained at room temperature. DO 1 3099.S CONF IDFNT T Al -7- Samples of excreta were collected for 48 hours after termination of exposure and analyzed for 14 C activity. Expired EDB was collected at 0.5 hr intervals for 4 hr; the C02 trap and urine receptable (immersed in a dry ice bath) were changed at 12 hr intervals for 48 hr; and feces were collected every 24 hr. At the termination of the study (48 hr) the animals were killed by a blow to the head. Samples of liver, kidney, lung, fat, stomach, skin, testes, muscle and brain were collected for analysis of 14 C act.iv.ity. The remaining carcass was homogenized (50% w/v) in distilled water and analyzed for 14 C activity. Samples of excreta and tissue were prepared for scintillation counting as described previously (Watanabe, et al., 1976). Radioactivity was determined by counting in a Mark II or Mark III (Searle Analytic, Inc.) liquid scintillation spectro meter. External standard channel ratios were used to determine the counting efficiency. Counts per minute were converted to disintegrations per minute using a standard quench curve. The remaining nine rats (3/exposure level) were killed by a blow to the head immediately following exposure. The liver was removed and a sample (1 g) was taken for determination of DO 130998 CONFT OF NT T A -8- non-protein sulfhydryl content (primarily GSH) by the method of Sedlak and Lindsay (1968). The remaining liver was frozen immediately on dry ice and stored at -20C until analyzed. The carcass, liver, lung and skin were analyzed for total radioactivity as described previously. The radioactivity determined in the tissues and carcass was non-volatile, therefore this radioactivity represented the total amount of metabolized EDB. Macromolecular binding of radioactivity to liver and lung tissue was determined by the method of Jollow, et al., (1973). Separation of Uri. nary 14 C-Activity. Urinary 14 C-activity was separated by high pressure liquid chromatography. The column (stainless steel, 5 mm x 0.5 m) was packed with Zipax SAX (duPont) ion exchange resin. Fifty yl of urine from the 0-12 hour collection interval from each rat exposed to 7, 25, or 75 ppm 14 C-EDB was injected on the column and eluted with a linear gradient from distilled water to 10% acetic acid, 1% pyridine over a 25 minute period (flow rate 1 ml/min). Fractions were collected at 1/2 or 1/4 minute intervals. Radioactivity in the fractions was determined by liquid scintillation spectrometry. 00 130997 conftdfnttai -9- Blood Levels of EDB During and Following Inhalation Exposure. Two groups of 3 rats/group were exposed to 6 or 78 ppm EDB for 6 and 7 hours respectively. The mean analytical concen trations of EDB measured by gas chromatography were 5.80.2 (SD) and 78.21.1 ppm EDB. Blood samples were taken at hour intervals during the exposure, and following exposure blood samples were obtained at 15 minute intervals for 1 hour. The blood was analyzed for EDB by gas liquid chromatography on a Hewlett-Packard Model 5700 or Varian Series 2400 gas chromato graphs equipped with flame ionization detectors. Whole blood (1 yl) was chromatographed on a column of 6.5% SP-1000 on Carbopack A (Supelco). The He carrier gas flow rate was 24.5 ml/min and the column temperature was 100C. DO 13 0 9 9 R CONF TDFNTTAl -10- RESULTS The routes of excretion following inhalation exposure to various concentrations of EDB are shown in Table 1. The percentage of the recovered radioactivity excreted by the various routes was similar for all exposure levels suggesting that the routes of excretion were independent of the exposure concentration. Urinary excretion of radio activity was the major route of excretion accounting for 80% of the total radioactivity recovered. There was signi ficant excretion of radioactivity as expired COj representing 7-8%. Only about 1% of the recovered radioactivity was eliminated by the lungs as EDB. The rates of excretion of urinary radioactivity over a 48-hour period are shown in Figure 1. The excretion curves were fit by linear regression analysis of the logarithmically trans formed data and the elimination was described by a mono exponential function. The apparent first order rate constant for urinary excretion following 7, 25, and 75 ppm exposures were 0.136+0.019 (SD), 0.12710.016 and 0.12410.013 hr"1 respectively. These corresponded to respective half-lives of 5.1, 5.5, and 5.6 hr. The distribution of radioactivity in various tissues immediately following exposure is shown in Table 2. Highest concentrations of radioactivity were observed in the liver and kidney. All OO 130999 OONFTDFNTTAl -11- other tissues analyzed had equivalent or lower levels of radioactivity than that in the carcass suggesting that the metabolites of EDB are generally distributed throughout all tissues with no preferential accumulation in any one tissue other than liver and kidney. The total amount of EDB metabolized, macromolecular binding to hepatic and lung tissue and hepatic non-protein sulfhydryl levels following exposure to EDB are given in Table 3. The total amount of EDB metabolized and macromolecular binding to liver and lung tissue all increased proportionately (3-fold) to the increase in exposure concentration between 25 and 75 ppm. However, the 3.7 fold increase in exposure between 7 and 25 ppm resulted in only a 2-fold increase in total metabolism and liver binding and slightly over 1-fold increase in lung binding. Thus, the binding in the liver and lung correlated reasonably well with the total amount of EDB metabolized but rats metabolized proportionately more at 7 ppm than at 25 or 75 ppm. Hepatic non-protein sulfhydryl content (primarily glutathione, GSH) was depressed significantly from controls only following 6 hour exposures to 25 and 75 ppm. No statistically significant (Student t-test, p < 0.05) depression in hepatic GSH was observed following the 6-hour, 7 ppm exposure. no i31ooo CONP TDFNT 1 Al -12- A typical profile of urinary radioactivity separated by high pressure liquid chromatography is shown in Figure 2. The 14 C-activity was separated into four radioactive peaks labeled A, B, C, and D. Peaks B and C co-eluted with j authentic radiolabeled standards of N-acetyl-S^-(2-hydroxy- 1 ethyl) cysteine, and thiodiglycolic acid respectively. However, when the same urine samples were rechromatographed one month later, peak A was not observed and peak B was increased quantitatively equalling the sum of the two previous peaks A and B. This led to the postulate that peak A may be a cyclized form of N-acetyl-S-(2-hydroxyethvl)j cysteine which hydrolyzed on storage. Thus both A and B appear to represent cysteine conjugates of EDB. Blood levels of EDB during and following exposure to 6 and 78 ppm for exposure times of 6 and 7 hours respectively are shown in Figure 3. Upon exposure to 78 ppm EDB the blood levels approached a steady state of approximately 0.78 yg EDB/ml blood within the first hour. Following exposure the clearance of EDB over the next hour was described by a monoexponential process with an apparent first-order rate constant of 0.0370.003 (SD) min-^ which corresponds to a half-life of 18.7 minutes. Upon exposure to 6 ppm the time required to reach a steady state appeared to take longer. At termination of the 6 hour exposure the concentration was 0.067 yg EDB/ml blood. no 131001 OONF TDFNT1 Al -13- Clearance of EDB from the blood after the 6 ppm 6 hour exposure was not determined because no EDB was detected in the samples taken 15 minutes post-exposure. If it is assumed that the pulmonary uptake of EDB is an analogous process to a constant rate intravenous infusion, then the approach to steady state blood levels can be predicted from the following relationship: C= (1-e Kt) t Va.K 1 (1) Ct is the concentration of EDB in the blood at a given time (t); Vd is the volume of distribution; K is the elimination rate constant from blood; and kort is the constant rate of pulmonary uptake which is analogous to a constant infusion rate. Parameters were obtained by using the observed data for the uptake and elimination of blood EDB during exposure to 78 ppm. Subsequently those derived parameters were used to predict blood level data for both the 78 and 6 ppm exposure. 13100? OONF IDFNTTAl -14- Pulmonary uptake (kQ) was calculated by dividing the total amount of EDB recovered in the body at 6 hours (determined in rats given 14 C-labeled EDB) by the exposure time (6 hr). The volume of distribution was then calculated at the 6 hr point by substituting the measured mean blood concentration of EDB at that time (C^) into equation (1). Once estimates were obtained for all parameters then the blood concentra tion of EDB at any time (C^.) could be predicted. The uptake curve showing the predicted approach to steady state blood levels of EDB are shown along with the observed data in Figure 3. While the predicted uptake of EDB in blood closely approximated the observed data on exposure to 78 ppm, the uptake on exposure to 6 ppm was slower than that predicted during the first 2 hours of exposure. The blood concentration of EDB immediately following 6 and 78 opm exposures increased proportionately to the increase in exposure levels. DO 13100.3 rONF TDFNT TAI 15- DISCUSSION The results indicate that rats rapidly metabolize and excrete metabolites of EDB upon inhalation exposure to atmospheric concentrations ranging from 7-75 ppm 14 C-EDB. The percentage of radioactivity eliminated by the various routes of excre tion are similar for all exposure concentrations indicating that the routes of elimination were independent of the exposure level. Likewise, the elimination rate of 14 Cactivity in the urine, accounting for 80% of the total amount of metabolized EDB, was independent of the exposure concen tration from 7-75 ppm EDB. The distribution of radioactivity in various tissues showed nothing remarkable. The highest concentrations of radio activity when expressed on a per g tissue basis were found in liver and kidney which are the predominant organs involved in metabolism and excretion. It is noteworthy that the concentration in the stomach, which was the target organ following oral intubation (Olson, et al. , 1973), was not greater than other non-target tissues. Furthermore, the concentration of radioactivity in the stomach did not increase disproportionately with the increase in the exposure levels. This is additional evidence that stomach cancer observed in rats following massive doses by oral gavage may be unrelated to the hazard of exposure to EDB via inhalation. no 101004 CONFTDFNTTAl -16- Chromatographic analyses strongly suggest that two of the major urinary metabolites of EDB are N-acetyl-S-(2-hydroxyethyl) cysteine and thiodiglycolic acid. These same metabolites were reported by Nachtomi (1970) to result from oral administration of EDB to rats. It appears that following inhalation exposure, rats rapidly conjugate EDB with glutathione (GSH) and the GSH conjugates are subse quently converted to cysteine conjugates and further oxida tion products of cysteine prior to urinary excretion. Only a very small quantity of EDB per se is eliminated in the expired air; thus metabolism of EDB via conjugation consti tutes the primary means for detoxification. Reaction of chemicals with intracellular macromolecules is believed to be one mechanism for toxicity including carcino genicity. The elegant works of Mitchell, et al. , (1973); Potter, at al. , (1974); and Jollow, at al^, (1974) showed that large doses of bromobenzene and acetaminophen overwhelm their GSH-dependent detoxification through depletion of hepatic GSH. As GSH is depleted the electrophilic inter mediates formed by biotransformation of these compounds react with macromolecules rather than GSH. Toxicity is a function of the magnitude of those reactions with macro molecules. The target organs for toxicity in rats following DO 131005 CONFIDFNTTAI -17" inhalation exposure to EDB are the liver, lung, and kidney (Rowe, et al., 1952). However, macromolecular binding of radioactivity to liver and lung tissue following exposure to EDB did not increase disproportionately with the increase in exposure concentration. Instead, the binding was directly related to the total amount of EDB metabolized. Since the metabolism of EDB is primarily a detoxification process it appears that for EDB total binding in these tissues may not correlate with toxicity. This is understandable since the methodology used measures reaction with total macromolecules, and toxicity and/or carcinogenicity is likely a function of interaction with specific macromolecular sites including nucleic acids. An aspect of the findings in the research reported herein worthy of discussion is that the amount of EDB biotransformed increased only 2-fold when the exposure concentration was increased from 7 to 25 ppm, a 3.4 fold increase. However, when the concentration was increased from 25 to 75 ppm, the amount of EDB biotransformed increased proportionately. The less than proportionate increase in the amount of EDB biotransformed in going from an exposure of 7 ppm to 25 ppm suggests that at the higher concentration the biotransforma tion process is less efficient. It is unlikely that the DO r.ONF 1 31 00f' TDFNTTM -18- difference in efficiency is related to biotransformation occurring in organs subsequent to the absorption of EDB into the blood. This is concluded because the percent of the total body burden of ^C-activity expired as EDB per se subsequent to a 6 hr exposure was only 0.9 and 1.2% in rats exposed to 7 and 25 ppm EDB respectively. This small, indeed insignificant, difference is insufficient to explain why the total amount of EDB biotransformed increased only two-fold in rats exposed to a 3.4-fold higher concentration. To explain the apparent discrepancy it is speculated that in rats EDB may be cleared from the lungs by not only blood flow but by biotransformation of EDB in pulmonary tissue. Further, this speculated pulmonary biotransformation of EDB is conceived to be saturated by exposure to high concentration of EDB and exhausted as exposure to 7 ppm EDB continues. This mechanism provides for a more efficient uptake of EDB from alveolar air when the concentration is low because pulmonary biotransformation in addition to blood flow will maintain a greater concentration gradient. At high concentrations the pulmonary biotransformation process will be overwhelmed leading to a smaller concentration gradient and consequently less efficient uptake. no 131007 CONF TDFNT TAt -19- Another observation supports the foregoing speculation. The time required to attain a steady state level of EDB in the blood was longer for rats exposed to 6 ppm, 5 to 6 hours, than in rats exposed to 78 ppm, 1 hr. This suggests that early in the exposure to 6 ppm EDB is efficiently taken up and biotrans formed by pulmonary tissue limiting the uptake of EDB per se by blood. As exposure continues, the pulmonary biotrans formation process is exhausted and consequently more of the EDB per se reaches the blood, thus accounting for the greater duration required to reach a steady state concentration in the plasma of rats exposed to 6 ppm than rats exposed to 78 ppm. In rats exposed to 78 ppm EDB pulmonary biotransformation is conceived to be quickly overwhelmed and hence steady state levels of EDB in plasma are quickly attained. In summary, the chemical process governing the fate of inhaled EDB is conceived to occur in accordance with the following model. Expired EDB T Atmospheric____ ^ Central EDB * .g* Compartment ii Metabolism nn Metabolism J Excretion of Urinary Meta bolites HO 13100R CONFTDFNTIAl -20- In this model the central compartment is composed of blood and those tissues with a large blood flow, i.e., liver and kidney. The essential aspect of this model is that biotrans formation by lung must be a saturable process. This specu lation is not inconsistent with previous reports that glutathione transferases are active in the lung (Grover and Sims, 1964; Bend, et al^ 1976) but that their maximum activity is considerably less than that in liver. One of the objectives of this study was to obtain pharmaco kinetic information on the fate of inhaled EDB in rats to lend perspective to results on the chronic toxicity of inhaled EDB. One such study is currently in progress (Powers, M., personal communication). Rats are being exposed daily (6 hours/day, 5 days/week) to 10 and 40 ppm EDB. Based on the evidence for increased biotransforma tion of EDB by the lung at exposure levels of 6-7 ppm observed in this pharmacokinetic study, proportionately more EDB may be absorbed into the blood and distributed to the tissues of rats exposed to 40 ppm than those exposed to 10 ppm. This may result in toxicity and furthermore, it may not be appropriate to extrapolate linearly the toxicity at this level to 10 ppm where pulmonary biotransformation of EDB may be more efficient. no 131009 GONF TDFNT TAl -21ACKNOWLEDGEMENTS The authors wish to express their appreciation to B. Isenbarger for analytical assistance and D. Gransden for synthesis of radiolabeled standards for metabolic identification. io -22REFERENCES Bend, J. R., Ben-Zvi, Z., Van Anda, J, Dansette, P. M., and Jerina, D. M. (1976). Hepatic and extrahepatic glutathione transferase activity toward several arenej oxides and epoxides in the rat. Carcinogenesis, Vol.j I, R. I. Freudenthal and Jones, P. W., ed., pp. 63-75, Raven Press, New York, NY. I Gillette, J. R. (1974). A perspective on the role of chemically reactive metabolites of foreign compounds in toxicity.-I Biochem. Pharmacol., 23, 2785-2794. Grover, P. L. and Sims, P. (1964). Conjugations with gluta thione. Biochem. J., 90, 603-606. I Harms, P. G. and Ojeda, E. R. (1974). A rapid and simple procedure for chronic cannulation of the rat jugular vein. J. Appl. Physiol., 36, 371. Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J, R. and Brodie, B. B. (1973) . Acetaminopheninduced hepatic necrosis. II. J. Pharmacol. Exp. Therap., 187, 195-202. Jollow, D. J., Mitchell, J. R., Zampaglione, N. and Gillette, J. R. (1974). Bromobenzene induced liver necrosis. Pharmacol., 11, 151-169. Miller, J. A. and Miller, E. C. (1971). Chemical carcino genesis: mechanisms and approaches to its control. J. Nat. Cancer Inst., 47, 5-14. no 131011 CONFTDFNTTAt -23- Mitchell, J. R., Jo1low, D. J., Potter, w. Z., Gillette, J. R. and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis. IV. J. Pharmacol. Exp. Therap., 187, 211-217. Nachtomi, E., Alumot, E., Bondi, A. (1966). The metabolism of ethylene dibromide in the rat. I. Identification of detoxification products in urine. Israel J. Chem., (5-6), 239-46. Nachtomi, E. (1970). The metabolism of ethylene dibromide in the rat. The enzymic reaction with glutathione in vitro and in vivo, Biochero. Pharmacol., 19, 2853-60. Olson, W. A., Habermann, R. T. Weisburger, E. K., Ward, J. M., Weisburger, J. H., (1973). Brief Communication: Induction of stomach cancer in rats and mice by halogenated aliphatic fumigants, J. Nat'l. Cancer Inst., 51 (6), 1993-5. Potter, W. Z., Thorgeirsson, S. S., Jollow, D. J., and Mitchell, J. R. (1974). Acetaminophen induced hepatic necrosis. V. Pharmacology, 12, 129-143. Powers, M., Personal communication, Hazelton Laboratories American Inc., 9200 Leesburg Turnpike, Vienna, Virginia 22180. Rowe, V. K., Spencer, H. C., McCollister, D. D., Hollingsworth, R. L., Adams, E. M. (1952). Toxicity of ethylene dibromide determined on experimental animals, A.M.A, Arch. Ind. Hyg. and Occup. Med., , 158-73. Sedlak, J. and Lindsay, R. M. (1968). Estimation of total protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's Reagent. Analyt. Biochem., 25, 192-205. DO lOIOlD OONF TDFNT TAI -24- Watanabe, P. G., McGowan, G. R., and Gehring, P. J. (1976). Fate of ^^C-vinyl chloride after single oral administra tion in rats, Toxicol. Appl. Pharmacol., 36, 339-352. Zbinden, G. (1976). Glutathione, in Progress in Toxicology, Vol. 2, pp. 60-68, Springer-Verlag, New York, NY. 1^0 131013 CONF TDFNTI A TABLE 1 Percentage 14 C-activity Eliminated By Male Rats During 48 Hours Following a 6 Hour Inhalation Exposure to EDBa Expired as: EDB C02 Urine Feces Tissues Total 7 ppm % yg equiv. EDB Exposure Levels 25 ppm % yq equiv. EDB 75 ppm % yg equiv. EDB 0.9 7.3 81 .3 2. 7 7.8 14 110 1225 40 117 1506 1.2 8.9 79.9 2.5 7.5 61 294 2633 83 246 3317 1.7 8.1 80.4 2.6 7.2 181 849 8442 270 760 10502 irnio i aExpressed as percentage of the total radioactivity recovered. one rat per exposure level. no io io i4 CONF TDFNTT i> -26- TABLE 2 Tissue Distribution of 14 C-Activity in Male Rats Following Inhalation Exposure to EDBa For 6 Hours Tissue Liver Kidney Carcass Lung Fat Stomach Skin Testes Muscle Plasma Brain 7 ppm 45.815.5 48.2110.9 6.110.7 6.9 + 0.7 7.310.9 8.311.1 3.7 + 0.3 4.5 + 0.4 3.110.4 3.310.8 2.7 + 1.4 Exposure Level 25 ppm 112.7+19.2 86.618.1 13.312.7 10.412.1 10.111.7 9.8+1.5 8.0+1.7 7.812.5 6.011.6 7.711.5 1.311.3 75 ppm 255.7110.6 228.816.9 58.2115.4 34.211.4 62.4121.4 38.9113.3 19.2+2.8 24.713.7 23.5110.1 22.215.1 17.712.2 Expressed as ug equiv. EDB/g tissue; animals were killed immediately after exposure; mean S.D., 3 rats/exposure level. DO 13T01C' GONFTDFNT TAl TABLE 3 Total Metabolism, Macromolecular Binding and Hepatic Non-Protein Sulfhydryl Levels in Male Rats Following Inhalation Exposure to EDBa Exposure Cone. <PPm> 7 Mg Equivalents EDB Metabolized 1554+173 Liver ug Equivalents EDB Bound per g Protein Lung Mg Equivalents EDB Bound per g Protein Hepatic Non-Protein Sulfhydryl Content (GSH, % Control) 7.710.7 2.2+1.5 94 25 3129+401 14.413.0 . 2.710.3 75 1176612218 47.216.4 8.211.6 77 Mi I 59 aRats were exposed for 6 hours to EDB and killed immediately following exposure. ^Control rats (4) were exposed to room air and killed simultaneously with the EDB exposed group. cMean 1 S. D., 3 rats/exposure level. ^Statistically different from controls. Student t-test, p < 0.05. o U) o CO NFIDFNTTA! -28- LEGENDS Figure 1 Urinary excretion of 3,4C-activity (yg equivalents j EDB) versus time (hr) following inhalation exposurje to 7, 25, and 75 ppm. Lines were fit by linear regression analysis and the apparent first order rate constants (k) and half-lives elimination were determined. fr Figure 2 Typical separation of urinary metabolites (0-12 hour sample) by high pressure liquid chromato graphy (hplc) on a SAX (duPont) ion exchange column. The profile shows the separation of radioL activity representing metabolites A, B, C, and D by hplc versus time (min, flow rate = 1 ml/min). Figure 3 Blood concentration of EDB (yg/ml) versus time (hr) during and directly following exposure to atmospheric concentrations of 6 and 78 ppm EDB (solid line). The arrow designates termination of exposure. The elimination curve of EDB following exposure to 78 ppm was fit by linear regression analysis. The dashed lines are the predicted uptake profiles using parameters derived from the 78 ppm exposure. DO 131017 CONFTDFNT TAl I*g Equiv. EDB Excreted Per 12 Hour Intervals Figure 1 00 131018 OONFIOFNTTA Fiqure 2 Radioactivity, DPM Oz VO -T\ T! -jj zo i> 28 Blood EDB, /ttg/ml Figure 3 DO 131000 C0NFTDFNT1 Al