Document 1L4KRMp1QVbw4vQ5kYKd22ed

MIDWEST RESEARCH INSTITUTE ADDITIONAL EVALUATION OF THE ENVIRONMENTAL TOXICANTS VINYL.CHLORIDE (VC) AND VINYLIDENE CHLORIDE (VOC): 0) Exposure of VC or VDC Followed by Observation for 12 Months In Rats and Mice (2) The Effect of Dlsulfiram (DS) on VC Exposure in Mice FINAL REPORT January 1976 through December 1978 Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-2-2084) MRI Project No. 3612-B For National Institute of Environmental Health Sciences P.O. Box 12233 Research Triangle Park, NC 27709 I Attn: Or. Robert L. Dixon EPL 00766 MIDWEST RESEARCH INSTITUTE 425 VOLKER S0U..5VARD. KANSAS CITY, SUSSC'jRi 641 -Q a 16 "i.'-TcO.l ADDITIONAL EVALUATION OF THE ENVIRONMENTAL TOXICANTS VINYL CHLORIDE (VC) AND VINYLIDENE CHLORIDE (VDC): (1) Exposure of VC or VDC Followed by Observation for 12 Months in Rats and Mice (2) The Effect of Disulfiram (DS) on VC Exposure in Mice By Chuen-Bin Hong Joseph M. Winston William B. House A. Monaem El-hawari Robert D. Short, Jr. Ellen R. Ellis Ernesto Castillo Jack H. Hagensen j Karen J. Smith ji " Cheng-Chun Lee FINAL REPORT January 1976 through December 1978 Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-2-2084) ] MRI Project No. 3612-B \ ! i PREFACE This report was prepared at Midwest Research Institute, 425 Volker Boulevard, Kansas City, Missouri 64110, under Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-2084) with the National Institutes of Health, Department of Health, Education and Welfare, MRI Project No. 3612-B, "Research Capability for Environmental Toxicants." The research was sponsored by the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709. Drs. Robert L, Dixon and James S. Woods are the con secutive Project Officers. This work was conducted in the Biological Sciences Division, under the directln of Dr. William B. House, between January 1976 and March 1978, and Dr. Harold M. Hubbard, between April 1978 and December 1978. Dr. ChengChun Lee, Assistant/Associate/Deputy Director, Biological Sciences for Pharma cology and Toxicology, was the Principal Investigator. Dr. Joseph M. Winston, Associate Toxicologist, supervised the inhalation chamber operation with the consultation of Dr. William B. House. Necropsy, gross and microscopic examination of tissues were directed and performed by Dr. C. B. Hong, Senior Pathologist, with the assistance of Dr. Larry P. Thornburg, Assistant Pro fessor in Veterinary Pathology, Department of Veterinary Pathology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, for the microscopic examination of tissues. Mrs. Ellen R. Ellis, Histologist, super vised the histology preparation with the technical assistance of Mr. Kerry L. Crabb and Mrs. Janet L. Kliethermes, and Mr. Ernesto Castillo, Assistant Biologist, supervised the necropsies with the technical assistance of Miss Judith Shifrin and Mr. Hung Hoong, under the direction of Dr. C. B. Hong. Dr. A. Monaem El-hawari and Dr. Robert D. Short, Jr., Senior Toxi cologists performed the hepatic drug enzyme studies with the technical assistance of Miss Mary D. Sawyer. Mr. Jack H. Hagensen and Mrs. Karen J. Smith performed the analytical monitoring of chamber concentrations, and chamber and animal care operations. This report was reviewed by the Quality Assurance Unit of Midwest Research Institute (Mr. Garfield K. Liu) and found to be acceptable accord ing to Good Laboratory Practice Regulations (43 FR 60018, 21 CFR 58.185) for reporting of nonclinical laboratory study results. The raw data, tissues, slides and the final report are stored in our Archives. Approved for: MIDWEST RESEARCH INSTITUTE Florence I. Metz, Acting Director Biological Sciences Division January 1979 iii EPL 00768 TABLE OF CONTENTS Page I. Introduction ............................................................................................................... 1 II. Material and Methods............................................................................................. 2 A. Methods of Exposure.................................................................................... 2 1. Chamber Design............................................................................... 2. Chamber Air Supplyand Flow Rates.......................................... 3. Generation of VC andVDCVapor.............................................. 4. Chamber AtmosphereMonitoring................................................. 5. Standards;........................................................................................ 6. - Chamber Sampling................................................... ................... 2 2 2 2 3 3 B. Safety ...... .................................................................................... C. Animals.............................................. ................................................................ 3 4 . 1. Species................................................................................................. 2. Animal Housing ............................................................................... 3. Animal Hygiene............................................................................... 4 4 4 D. Experimental Protocol............................................................................... 5 1. Exposure to VC or VDC and Observed for 12 Months in Rats and Mice...................................................................... 5 a. Experimental Design........................................................ b. Observations ...................................................................... c. Necropsy and Hlstopathology..................................... 5 6 6 2. Exposure to VC With and Without DS Treatment in Mice................................................................................................. 6 a. Experimental Design........................................................ b. Necropsy and Hlstopathology..................................... c. Liver Microsomes............................................................ d. Cytochromes P-450 and b^.......................................... e. Hepatic Mixed-Function Oxidase (MFO) Activities ...................................................................... f. Statistical Analyses ................................................... 6 6 6 7 7 7 v EPL 00769 TABLE OF CONTENTS (Concluded) gage C. The Effects of DS on VC Exposure inMice........................................ 19 1. Survival Time.........................................................................................19 2. Tumor Incidence.................................................................................... 19 3. Hepatic Microsomal Composition and Function. ... 20 IV. Discussion and Conclusion................................................................................... 22 A. Exposure to VC or VDC in Rats.................................................................. 22 B. Exposure to VC or VDC in Mice..................................................................23 C. Protective Effect of DS on VCExposurein Mice..............................25 V. Publications, Presentations and Manuscripts..........................................26 A. Publications................................................................................................. 26 B. Presentations............................................................................. 26 C. Manuscripts........................................................................................................... 26 References................................................................................................................................... 27 Tables 1-32 ............................................................................................................................ 29-60 vii EPL 00770 ADDITIONAL EVALUATION OF THE ENVIRONMENTAL TOXICANTS VINYL CHLORIDE (VC) AND VINYLIDENE CHLORIDE (VDC): (1) Exposure of VC or VDC Followed by Observation for 12 Months in Rats and Mice (2) The Effect of Disulfiram (DS) on VC Exposure in Mice FINAL REPORT ABSTRACT This report summarizes the results of (1) exposure of VC or VDC, 6 hours/day, 5 days/week, up to 10 months in rats and up to 6 months in mice, followed by observation for 12 months, and (2) DS on VC exposure in mice. Exposure of rats of both sexes to 50, 250 or 1,000 ppm VC for 1 or 3 months followed by observation of 12 months did not cause any death during the observation period of 12 months following exposure. Exposure for 6 or 10.months increased the number of deaths and early terminations during the exposure and observation periods when compared with the respective control rats. Observation for 12 months after exposure of various levels of VC did not diminish the development of hemangiosarcoma in the liver and lung. In addition, exposure of 50, 250 or 1,000 ppm followed by the observation period caused neoplastic nodules and hepatocellular carcinoma in the liver and possibly malignant lymphoma involving various organs; and increased the inci dence of mammary gland tumors, predominantly fibroadenoma and adenocarcinoma/ carcinoma, in the females. The increase in deaths and early terminations of rats, and the incidence and severity of tumors, increased with increases in the level of VC and length of exposure. Exposure of rats to 55 ppm VDC for 10 months, but not for 1, 3 or 6 months, increased the deaths and early terminations during the observation period after exposure. Exposure of VDC up to 10 months followed by the observation period did not cause or increase the incidence of any tumors. Exposure of mice of both sexes to 50, 250 or 1,000 ppm VC for 1, 3 or 6 months followed by observation of 12 months increased the number of deaths and early terminations during the exposure and observation periods. Observation for 12 months after exposure of various levels of VC did not diminish the development of hepatic and extrahepatlc hemangiosarcoma, and the increases in incidence of bronchioloalveolar tumor and metastatic adeno carcinoma, originated from the mammary gland, of the lung, and adenocarcinoma/ carcinoma of the mammary gland in the females. As seen in rats, the increase in deaths and early terminations of mice, and the incidence and severity of tumors were proportional to the level of VC and length of exposure. In addition, the mice died or were terminated earlier in groups exposed to higher levels of VC and for longer durations. EPL 00771 ix Exposure of mice to 55 ppm VDC for 3 or 6 months, but not for 1 month. Increased the deaths and early terminations during the observation period after exposure. Exposure of VDC up to 6 months followed by the observation period did not cause or increase the incidence of any tumors. Simultaneous treatment of mice with DS exerted a partial protection against VC-induced carcinogenesis. Treatment of mice with DS during exposure to 250 or 1,000 ppm resulted in an increase in survival time, and appeared to reduce the incidence of VC-induced hepatic hemangiosarcoma in both sexes and VC-induced mammary'gland tumors in females, but had no effect on the inci dence of VC-induced bronchioloalveolar adenoma. Results of the hepatic cytochrome P-450 concentration and MFO activity studies suggest that DS may act by reducing the activation of VC to reactive metabolites via microsomal metabolism. However, it does not rule out that DS may exert its protective action by increasing the availability of free sulfydryl groups to combine with reactive VC metabolites or by a combination of both these two mechanisms. Chu< Sen: M., Ph.D. Lthologist Ct _ Deputy Director Biological Sciences Division EPL 00772 x I. INTRODUCTION Under Contract No. N01-ES-2-2084 (NIH-NIEHS-2-2084) on "Research Capability for Environmental Toxicants," we have performed various studies on the toxic effects of the two commercially available dithiocarbamates, ferbam and thiram.-i/ Following the completion of those studies, the inha lation toxicity of up to 12 months exposure to 50, 250 or 1,000 ppm of vinyl chloride (VC) or 55 ppm of vinylidene chloride (VDC) in mice and rate was studied-- and reported.-- Exposure of rats to 250 or 1,000 ppm of VC re sulted in the occurrence of hemangiosarcomas of the liver, lung and other extrahepatic tissues starting in the 9th month. Exposure of mice to 50, 250 or 1,000 ppm of VC resulted in bronchioloalveolar adenomas, hepatic and extrahepatic hemangiosarcomas, and mammary gland tumors in females starting in the 2nd and 6th month. Exposure of rats and mice to 55 ppm of VDC resulted in a few extrahepatic or hepatic hemangiosarcomas. Additional studies in which rats and mice were exposed to VC or VDC for varying periods of time followed by a 12-month observation phase were carried out to evalu ate the development of tumors during the post-exposure period. This report summarizes the results. Disulfiram (DS) has been used clinically in the treatment of alcoholism because of its ability to sensitize humans to ethanol. This sensitization is a result of the inhibition by DS of aldehyde dehydrogenase, which causes the accumulation of acetaldehyde and a resulting acetaldehyde syndrome.--' Among the other biochemical effects of DS is the inhibition of hepatic drug metabolism in the rat.^?^ Disulfiram has also been demonstrated to inhibit the development of tumors in animals treated with polycyclic hydrocarbons or dimethylhydrazine.-^z^/ In addition, recent work has shown that DS protected mice from the toxic effects of vinylidene chloride, a compound structurally related to VC.--' The present study was also under taken to determine the effect of DS on the carcinogenicity of VC in mice. In addition, hepatic drug metabolism was evaluated in mice exposed chronically to VC with and without DS treatment in order to assess one potential mechanism by which DS might affect VC-induced carcinogenesis. EPL 00773 1 II. MATERIAL AND METHODS Material and methods employed in these studies are described below. A. Methods of Exposure Methods of exposure are essentially the same as described pre viously.-- 1. Chamber Design Five stainless steel cubical type exposure chambers of 3.5 m^ (123 ft3) volume were used. Contaminant entered the air stream' and was then mixed in a plenum at the top of each chamber. Each chamber contained a diffusion plate and two small squirrel-cage fans (100 cfm, 2.85 m3/min) mounted on opposite sides of the top cone above the diffusion plate to ensure complete mixture of the gas with air. 2. Chamber Air Supply and Flow Rates * The air supply to the chambers was drawn from a stack on the roof of the building. Chamber air passed through a coarse filter and then over coils for heating, cooling, and dehumidlfylng. The air then passed through an absolute filter (99.97-99.992 retention of 0.3 /x particles) into the plenum of the chamber. Air flow rates were measured by means of orifice plates installed on the chamber outlet exhause system. Air flow rates were then measured with an air flow transducer (Autotronics 100-SSX). The air flow rate was 26.4 ft /min (0.75 m /min) or approximately 13 air changes per hour. 3. Generation of VC and VDC Vapor Vinyl chloride at a purity of 99.82 was obtained from Matheson Products. It is a gas at room temperature, and was metered with rotameters into the chamber air supply. VDC with a purity of 992 was obtained from the Aldrich Company. Its boiling point is 32C (89.6F) and was heated to 37C (98.6F) to generate the gas. All gas lines and the rotameter were heated to 40C (104F) to prevent condensation. 4. Chamber Atmosphere Monitoring Chamber concentrations were monitored by using a gas chromato graph (Varian-2700) with a flame ionization detector. A 6 ft x 1/8 in. stainless steel column packed with 0.4% Carbowax 1500 on Carbopak A was 2 EPLi 00774 used with a nitrogen carrier flow rate of 80 ml/min. The injection, column, and detector temperatures were 135C, 65C and 1708C, respectively. 5. Standards VC standards at dilutions of 10, 50 and 100 ppm were obtained in lecture bottles from Supelco, Inc., Bellefonte, Pennsylvania. VC standard gas at a concentration of 1,000 ppm was obtained from Matheson Gas Products, Joliet, Illinois. VDC standards were prepared by a serial dilution (weight/volume) of VDC in carbon tetrachloride. The desired final concentrations in a 4 ^1 injection were determined by the following procedure: mgJ$. (ftg/ml) " ppm % mw 24,450 With a molecular weight for VDC of 96.94, the final concentrations of_10, 50 and 100 ppm, the amount of VDC in a 1-ml sample from the chamber would be 0.04, 0.20 or 0.40 (ig, respectively. For 1 til of standard, these same amounts would be obtained from solutions of 40, 200 and 400 mg/liter; but since 4 pi o*f standard were injected, the final dilutions of VDC in carbon tetrachloride were 10, 50 and 100 mg/liter. _ All liquids were injected at a volume of 4 /il and all gases at 1 ml./ Standards at each point on the calibration curve were injected in triplicate. 6. Chamber Sampling The chambers were sampled at least three times daily from a reference point in the center of each chamber; an automatic sampling system was used. The sampling system consisted of 0.25 in. teflon lines that were placed at the reference point in each chamber. The teflon lines ran through a series of sampling valves. The sample lines were purged with chamber atmosphere for several minutes before the anlaytical analysis was initiated. Samples were Injected through twin 1 ml sampling loops which were allowed to reach equilibrium before the sample was injected. The sampling sequence was controlled by a Varian CDS-111 chromatography data system. The con centrations were determined by integration of peak areas by the CDS-111. The integrator was calibrated each day with VC and VDC standards. B. Safety All chambers were operated with a slight negative pressure (0.10.2 in. water) to prevent escape of the test contaminants from the chambers 3 EPL 00775 into the chamber room environment. Before being opened, the chambers were flushed with clean air for a minimum of 30 min. Room monitoring for VC and VDC was performed regularly by obtaining grab samples from the room environ ment with a gas-tight syringe, and the samples were analyzed by gas chroma tography. All air from the chambers traveled under negative pressure to a high-temperature incinerator outside the inhalation building prior to re lease to the surrounding environment. The burner of the incinerator operated at 1700 to 1800F. C. Animals 1. Species Albino CD rats and albino CD-I mice (Charles River Breeding Laboratories, North Wilmington, Massachusetts), were used in these studies. All animals were approximately.2 months of age at the start of the inhala tion studies. The animals were acclimatized for 1 to 2 weeks, before in halation exposures were begun. ' 2. Animal Housing All animals lived in the stainless steel cages during exposure and outside of the chambers. Rats were housed two per cage and micesix to eight per cage. Powdered or block laboratory chow (Wayne Manufacturing Company) was provided at all times except during the 6-hr period of expo sure each day. Water was available ad libitum. The temperature in the chamber and in the room averaged 24 + 1.3C (75 + 5F). The relative humidity in the chambers ranged from 25 to 60%; in the room it also ranged from 25 to 60%, but was regulated at 45 +5% after the 6th month. A 12-hr light cycle was maintained at all times. When out of thg .chambers, the animals were placed in hodling racks as described previously.-- A continuous supply of room air was drawn over the cages while in the holding racks. Provisions were made for covering the front of each compartment in the holding rack in the event of signifi cant off-gassing from the animals. These procedures were found not to be necessary with VC or VDC. 3. Animal Hygiene All cages and feeders were washed and sanitized each week. The floors of the room, the holding racks, and the interior of the chambers were frequently washed with disinfectant. Animal caretakers wore gloves and surgical masks when handling animals. 4 EPL 00776 It was found that when the cages were loaded into the chambers without drop-pans (so not to impair air flow) the animals on the bottom shelves were frequently drenched with urine and water. In order to allev iate this problem, the cages were loaded into the chambers with clean drop pans in place. Sampling studies showed that the vapor distribution was actually improved slightly. Drop pans were washed daily and a clean drop pan without bedding was inserted into each cage before loading into the chamber. D. Experimental Protocol 1. Exposure to VC or TOC and Observed for 12 Months in Rats and Mice a. Experimental Design The experimental design originally called for mice and rats to be exposed to 50, 250 or 1,-000 ppm VC or 55 ppm VDC 6 hr/day, 5 days/ week for periods of 1, 3, 6 or 12 months, followed by 12months of observa tion. However, the results of the initial study indicated that most mice exposed to 250 or 1,j000 ppm VC died between the 7th and 9th months of expo sure, and most rats exposed to these levelsdied between the 10th and 12th months of exposure.JLl2/ Therefore, the experimental design was revised to include the following groups with Indicated number of animals and length of exposure. However, this study was carried out simultaneously with the original study-- whenever the cage and chamber spaces were available and the number in some groups was reduced due to limited space. Exposure Groups Rats Length of Exposure Number of Animals/ Exposure Group Filtered air, 50, 250 or 1,000 ppm VC, or 55 ppm VDC 1 month 3 months 6 months 10 months 4 rats/sex/group 8 rats/sex/group 8 rats/sex/group 16 rats/sex/group Length of Exposure Mice (number/sex/group) VC, ppm Filtered Air 50 250 1.000 VDC, ppm 55 1 Month 3 Months 6 Months 16 16 16 16 16 16 16 16 28 12 12 12 8 8 12 5 OO'H'7 b. Observations Exposure for animals from the various groups was discontinued at the respective time. Thereafter, they were moved to separate animal quarters, placed in plastic cages, and observed for 12 months. Animal housing and hygiene were carefully maintained as previously described. c. Necropsy and Histopathology When moribund or at the end of the observation period, all animals were sacrificed for necropsy. Gross examination of all tissues, especially for the appearance of abnormal growths or other lesions, was per formed. The lung, liver, spleen, kidney, mammary gland and any abnormal tissues were removed, fixed, processed, sectioned, and stained for micro scopic examination. 2. Exposure to VC With and Without PS Treatment in Mice a. Experimental Design Male and female mice were used in these studies. The animals were housed, eight mice per cage, in stainless steel cages, and were allowed free access to feed and water except during the inhalation exposures when the feed was withheld. Mice receiving control feed or feed containing 0.1Z DS were exposed 6 hr/day, 5 days/week for 49 weeks, to filtered atmospheric air, 50, 250 or 1,000 ppm VC. Each exposure group was composed of four to eight mice of each sex. Feeding of the DS diet began 5 days prior to the first inhalation exposure and continued throughout the 49 weeks of exposure. b. Necropsy and Histopathology At the time of death or termination, the tissues of the mice were examined for abnormal growths and lesions. All tumors with adjacent normal tissues and all other tissues without tumors were fixed in 10Z buf fered formalin, processed, sectioned, and stained with hematoxylin and eosin for microscopic examination. c. Liver Microsomes The livers were removed, washed with ice-cold saline, then minced with scissors and homogenized with three volumes of 1.15Z KC1 con taining 0.02 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000 g for 20 min, and the post-mitochondrial fractions were centrifuged further for 60 min at 105,000 g. The microsomal pellets were resuspended, thoroughly mixed with the KCl-phosphate buffer, and recentrifuged for 60 min 6 EPL 00778 at 105,000 g. The washed microsomal pellets were layered with KCl-phosphate buffer and stored in a frozen state. Overnight storage (--14 hr) of microgomes in this manner did not result in significant losses of activity. Before use, the microsomes were suspended in the KCl-phosphate buffer mixture and adjusted to a protein concentration of 3 to 6 mg/ml. Microsomal protein content was determined-i^ using bovine serum albumin as the reference protein. d. Cytochromes P-450 and bs Spectral measurements were performed on the 100,000 g micro somal pellets using an ultraviolet spectrophotometer. Cytochrome P-450 and cytochrome b5 concentrations were measured^/ using extension coefficients of 91 and 164 mM~^ cm"^, respectively. The microsomes were diluted with 0.1 M phosphate buffer (pH 7.4) to yield 1 to 2 mg protein/ml. e. Hepatic Mixed-Function Oxidase (MFO) Activities Enzyme activities were determined under zero-order conditions regarding cof'actor and substrate concentrations and linearity regarding en zyme concentrations and incubation times. Animllne hydrozylase, ethylmorphineN-demethylase and -nitroanisole-0-demethylase were estimated using 3 mg of microsomal protein suspension. The incubation mixtures containing 5 mM MgCl2 0.43 mM NADP, 10 mM glucose-6-phosphate, 2 units of glucose-6-phosphate dehydrogenase, 50 mM Tris-HCl buffer, and 5 mM of each substrate. Incuba tions were carried out at 37C in air for 10 to 15 min in a Dubnoff metabolic incubator. Aniline hydroxylase activity was determined by measuring the formation of -nltrophenol at 420 nm.--' Ethylmorphine-N-demethylase activity was measured by a modification of the procedure of Schenfcman et al. (1967)' formaldehyde was trapped in semicarbozlde (4.1 mM) and estimated according to the method of Nash.--' The activity of -nitroanisole-0-demethylase was determined by a modification of the method of Netter and Sledel.--^ The reactions were initiated by the addition of the microsomal protein. f. Statistical Analyses Survival and tumor incidence data were analyzed using the Fisher Exact Probability Test.A$/ All other data were analyzed by Tukey's Omega Test.--' A significance level of p < 0.05 was used in all cases. EPL 00779 7 III. RESULTS The effects of exposure to VC or VDC followed by observation for 12 months were studied in rats and mice; the effects of DS on VC expo sure were studied in mice. The results are described in the following sections. A. Exposure to VC or VDC and Observation for 12 Months in Rats 1. General Observations and Number of Deaths Rats were exposed to VC or VDC, 6 hr/day, 5 days/week, for periods of 1, 3, 6 or 10 months. During these periods, these rats were exposed for a total of 20, 61, 128, or 213 days, respectively. A number of rats died or were moribund and terminated during the exposure and observation periods. _ The number of deaths and early terminations depended upon the duration of initial exposure and level of VC. More rats died or were moribund and terminated in groups exposed to higher levels of VC and for longer, durations. The clinical signs were rough hair coat, lethargy, and/or the appearance of exF ternal tumor mass.es, .especially mammary tumors in the females. Some deaths ^'occunred at night and autolysis took place in a few rats, especially during the weekends. The number of deaths and early terminations and total number of _ .. rats exposed to VC or VDC followed by observation for 12 months are summar- lzed in Table 1. There was no apparent difference in number of deaths and . early terminations between the males and females of any group. Occasional deaths and early terminations occurred during the observation period in the control rats and rats of the various groups exposed to VC or VDC for 1 or 3 months. The number of deaths and early terminations increased in the rats during the observation period after being exposed for 6 months. Three of 16 control rats (19%) were terminated during the exposure and observation periods. The number of deaths and early terminations for the mice exposed to 50, 250 or 1,000 ppm VC was 4 (251), 4 (25%), and 10 (63%), respectively. Only 1 of 16 rats exposed to 55 ppm VC was moribund and terminated during the observation period. In the rats exposed for 10 months, a total of 13 of 32 controls (41%) died or were terminated during the exposure and observa tion periods. The number of deaths and early terminations in the rats ex posed to 50, 250 and 1,000 ppm VC was 17 of 26 (66%), 17 of 28 (61%), and 30 of 32 (94%), respectively, during the same periods. A total of 20 of 30 rats (67%) exposed to 55 ppm VDC died or were terminated during the exposure and observation periods. The distribution of deaths and early terminations spread throughout the observation period. 00^8 8 2. Tumors The tumor occurrence In rats exposed to VC or VDC for 1, 3 or 6 months followed by observation for 12 months Is summarized in Tables 2, 3 and 4, respectively. The tumor occurrence in rats exposed for 10 months followed by observation for 12 months is summarized in Tables 5 through 8. a. Exposure for 1 Month After exposure for 1 month followed by the observation period, no tumor was found in any of the control rats or rats exposed to 50 ppm VC (Table 2). Two females exposed to 250 ppm VC had fibroadenoma of the mammary gland; one female also had chromophobe-cell adenoma of the pituitary. Hepato cellular carcinoma occurred in one male exposed to 1,000 ppm VC and hepatic hemangiosarcoma occurred in one male exposed to 55 ppm VC. b. Exposure for 3 Months After exposure for 3 months followed by the observation period, epidermoid carcinoma of the lung occurred in one female control; cystadenoma of the ovary and chromophobe-cell adenoma of the pituitary occurred in another female control (Table 3). A few other tumors occurred in rats exposed to various levels of VC. In the lung, there was bronchio loalveolar tumor. In the liver, there were neoplastic nodules, hepato cellular carcinoma, or hemangioma. In the mammary gland, there were fibroadenoma, adenoma, adenocarcinoma, or carcinoma. Other occasional tumors included squamous-cell carcinoma of the skin and chromophobe-cell adenoma of the pituitary. In the rats exposed to 55 ppm VDC, one female had a fibroadenoma of the mammary gland. c. Exposure for 6 Months After exposure for 6 months followed by the observation period, similar types of tumors, seen in the previous group of rats exposed for 3 months, also occurred in these rats of the various groups including the controls (Table 4). Three rats exposed to 250 ppm VC had neoplastic nodules and/or hemangioma of the liver, and five rats exposed to 1,000 ppm VC had hepatocellular tumor, bile duct carcinoma, hepatic hemangioma or hemangiosarcoma. These hepatic tumors were not seen in the control or other treated groups. There was malignant lymphoma involving the lung, liver, spleen, and/or kidney of one male and one female rat exposed to 1,000 ppm VC. Other occasional tumors included papilloma, squamous-cell carcinoma or fibrosarcoma of the skin; bronchioloalveolar tumor; fibrosarcoma of the Intestine; fibroadenoma or adenocarcinoma of the mammary gland; and chromo phobe-cell adenoma of the pituitary. In these groups, a few rats, especially those exposed to 1,000 ppm VC with various tumors, died or were moribund and terminated during the observation period. 9 EPL 00781 d. Exposure for 10 Months After exposure for 10 months followed by the observation period, the tumor Incidence and severity were greatly Increased In various groups (Tables 5 through 8). In the control group (Table 5), 11 rats had tumors including fibroma of the skin in three males, hepatocellular tumor in one male, carcinosarcoma of the kidney in one male, adenoma or fibro adenoma of the mammary gland in five females and chromophobe-cell adenoma of the pituitary in one male and three females. Three of the 11 control rats with various tumors died or were terminated during the observation period. In the group exposed to 50 ppm VC (Table 5), 15 rats had tumors similar to those of the control rats. However, Incidence of the mammary gland tumors in the females, including fibroadenoma, adenocarcinoma, or fibrosarcoma, was higher than that seen in the control group. One female with fibrosarcoma in the mammary gland, also had fibrosarcoma in the lung. Other occasional tumors included squamous-cell carcinoma or fibroma of the skin, hemangioma of the. liver. Islet-cell adenoma of the pancreas, and chromophobe-cell of the pituitary. One male with fibroma and squamous cell carcinoma of the skin died during the 31st week of exposure. In addition, nine of the remaining 15 rats with various tumors died or were moribund and terminated during the observation period. In the group exposed to 250 ppm VC (Table 6), 21 rats had different types of tumors in various tissues. In the liver, the tumor Incidence and severity were considerably increased over that seen in the group exposed to 50 ppm VC. The hepatic tumors included neoplastic nodules in six rats, hepatocellular in one rat, hemangioma in one rat, and hemangiosarcoma in four rats. Two females with hepatic hemangiosarcoma also had hemangiosarcoma of the lung. In the mammary gland, seven females had fibroadenoma. Other occasional tumors included squamous-cell carcinoma r fibroma of the skin, bronchioloalveolar tumor, adenocarcinoma or car cinoma of the kidney, malignant lymphoma of the spleen, and chromophobe cell adenoma of the pituitary. One female rat with fibroadenoma of the mammary gland died during the 40th week of exposure and 10 other rats with various tumors died or were terminated during the observation period. In the group exposed to 1,000 ppm VC (Table 7), 25 rats had different types of tumors in various tissues. In the liver, the tumor incidence and severity were further increased over those seen in the pre ceding group exposed to 250 ppm VC. The tumors Included neoplastic nodules in one rat, hepatocellular carcinoma in six rats, hemangioma in one rat, and hemangiosarcoma in 12 rats. Six of the rats with hepatic hemangiosarcoma also had hemangiosarcoma of the lung. In addition, one other rat without hepatic hemangiosarcoma had hemangiosarcoma of the lung, and two rats had bronchioloalveolar tumor. Malignant lymphoma involving the lung, liver. 10 EPL 00782 spleen, lymph nodes, and/or bone marrow occurred in two rats. Other occasional tumors included squamous-cell carcinoma or sebaceous gland adenocarcinoma of the skin, squamous-cell carcinoma of the nasal cavity, adenocarcinoma of the intestine, metastatic bronchioloalveolar carcinoma of the kidney, granulosal-cell tumor of the ovary, and chromophobe-cell adenoma of the pituitary. One rat with squamous-cell carcinoma of the skin died during the 40th week of exposure, and nearly all the remaining rats with various tumors died or were moribund and terminated during the expo sure period. In the group exposed to 55 ppm VDC (Table 8), the tumor incidence and severity were similar to those seen in the control rats (Table 5). A total of nine rats had tumors including fibroma of the skin in three rats, fibroadenoma of the mammary gland in four rats, and chromophobe-cell adenoma of the pituitary in three rats. One rat with fibroma of the skin died during the 24th week of exposure, and six of the remaining eight rats with various tumors died or were terminated during the observation period. 3. Cumulative Tumor Incidence and Tumorlgenesis The cumulative tumor incidence in control rats and'rats exposed to VC or VDC is summarized in Table 9. These rats were exposed to the filtered air, VC' or VDC for 1, 3, 6 or 10 months followed by observation for 12 months. It must be pointed out that within each treatment group the life span of all rats was not the same. a. In the Liver In the liver, there were three major types of tumors in rats exposed to 250 or 1,000 ppm VC. Neoplastic nodules were seen in seven males and three females of a total of 68 rats (15%) exposed to 250 ppm, and in one male and one female of 72 rats (3%) exposed to 1,000 ppm. Hepato cellular carcinoma occurred in one male of a total of 72 control rats (1%), one male and one female of 68 rats (3%) exposed to 250 ppm, and in four males and three females of 72 rats (10%) exposed to 1,000 ppm. Hemangiosarcoma occurred in one male and four females of a total of 68 rats (7%) exposed to 250 ppm, and in five males and nine females of 72 rats (19%) ex posed to 1,000 ppm. Exposure of 55 ppm VDC did not cause any of these tumors. Nevertheless, hemangiosarcoma was seen in the liver of one male exposed to VDC. Hemangiosarcoma of the liver and lung was similarly reported in rats exposed to 250 or 1,000 ppm VC for 9 to 12 months without the observation period after exposure.The characteristics of hepatic hemangiosarcoma are similar to those seen in mice described in the appro priate section. Classification of hepatocellular tumors in rats described by Squire and Levitt--' was applied in this report. Microscopically, the neoplastic nodules were characterized by ground-glass areas of altered hepatocytes. The size of these nodules were larger than that of one liver lobule; their normal lobular architecture was distorted. They compressed 11 EPL 00783 to the normal surrounding parenchyma, resulting in a sharp demarcating line. The cytoplasma of the altered hepatocytes were eosinophilic and vacuolated, and their nuclei were enlarged. Their sinusoids were dilated or obliterated. In comparing them to neoplastic nodules, hepatocellular carcinomas were larger and the cells were in a great diversity of differentia tion. The tumors included a well-differentiated type and a moderatelydifferentiated type. Other tumors in the liver of rats exposed to VC included hemangioma and bile duct carcinoma. Hemangioma occurred in one male, one and two females, and two males exposed to 50, 250 or 1,000 ppm VC, respect ively. Bile duct carcinoma was seen in one male exposed to 1,000 ppm VC. b. In the Lung About half of the rats with hepatic hemangiosarcoma also had hemangiosarcoma in the lung. Hemangiosarcoma of the lung was seen in two females of a total of 68 rats (3%) exposed to 250 ppm VC, and in three males and four females of 72 rats (lp%) exposed to 1,000 ppm VC. Grossly, there were multiple red patches on the surface of the lung and slightly firm in consistency. Their cut surface was wet and red. Microscopically, they were composed of the basophilic and lmmatured endothelia which encompassed varying amounts of RBC and fluid. In addition, bronchioloalveolar tumor occurred in one male and one female exposed to 250 ppm VC, and three males and one female exposed to 1,000 ppm VC. The microscopic feature of the bronchioloalveolar tumor in most rats was similar to that seen in mice. In a few cases, the tumor was not well delimited and resembled adenomatous change of alveolar epithelium. Fibrosarcoma, probably originated from the mammary gland, was seen in one female exposed to 50 ppm VC. c. In the Mammary Gland Mammary gland tumors observed only in the female rats and ' included fibroadenoma, adenoma, adenocarcinoma, carcinoma, and fibrosarcoma. Fibroadenoma was seen in five of 36 (14%) female controls, 11 of 36 (31%), nine of 32 (28%), and five of 36 (14%) females exposed to 50, 250 or 1,000 ppm VC, respectively, and in five of 36 (14%) females exposed to 55 ppm VDC. Adenoma occurred in one female control, one female exposed to 250 ppm VC, and two females exposed to 1,000 ppm VC. Adenocarcinoma/carcinoma occurred in one female control, four females exposed to 50 ppm VC, and one female exposed to 250 ppm VC. There was fibrosarcoma in one female exposed to 50 ppm VC. Fibroadenoma was characterized by proliferation of both epithelial cells and connective tissues. There was a great variation in structure in terms of amount of connective tissue present. Adenomas were characterized by proliferation of gland epithelium arranged in a uniformly 12 EPL 00784 pattern. The neoplastic cells in adenocarcinoma were arranged in a great variety of ways. Their acinar spaces were varying in size and shape. Papil lary projection and cysts were seen in some cases. Carcinoma of ductular epithelium was most anaplastic and most malignant in this group of tumors. In most cases, the cells were basophilic, and arranged in cords or sheets. The stroma in this tumor was much more than in adenocarcinoma. Squamous differentiation was seen occasionally. A mixture of adenocarcinoma and carcinoma was noted in one rat. d. Malignant Lymphoma Malignant lymphoma was seen in three males and one female of a total of 72 rats (62) exposed to 1,000 ppm VC. It involved the lung, liver, spleen, kidney, lymph nodes and/or bone marrow. Malignant lymphoma also occurred in the spleen of one male exposed to 250 ppm VC. e. Other Organs A number of other tumors occasionally occurred in the con trol and/or treated rats. They were spontaneous and were not related to any group. Chromophobe-cell adenoma of the pituitary occurred in one male and four female controls, two males and three females, two males and three females, and two males and two females exposed to 50, 250 and 1,000 ppm VC, respectively, and in four females exposed to 55 ppm VDC. Other occasional tumors Included papilloma, squamous-cell carcinoma, sebaceous gland adeno carcinoma, fibroma and fibrosarcoma of the skin; squamous-cell carcinoma of the nasal cavity; adenosarcoma and fibrosarcoma of the intestine, Islet-cell adenoma of the pancreas; adenocarcinoma, carcinoma, carcinosarcoma and metastatic bronchioloaveolar carcinoma of the kidney; cystadenoma and granulosal-cell tumor of the ovary, mesothelioma of the epididymis, and pheochromocytoma of the adrenal gland. 4. Other Lesions There were a number of spontaneous or incidental lesions in the tissues of control rats and rats exposed to various levels of VC or VDC followed by observation for 12 months. The lesions for rats exposed to VC or VDC for 1, 3, 6 or 10 months followed by the observation period are summarized in Tables 10, 11 through 13, 14 and 15, and 16 through 20, respectively. The lesions include inflammation or abcess of the skin; inflammatory cyst of the mammary gland; fibroplasia, myocarditis or calcifi cation of the heart; pneumonia; fatty degeneration, focal Inflammation, cystic changes, bile duct hyperplasia, cholangitis, telangiectasis, and/or necrosis of the liver; nephritis, calcification, or fat infiltration of the kidney; mineralization of the testes; prostatitis; cystic or fatty degeneration of the adrenal; and/or inflammation of the mesentery or peritonium. These lesions were inconsistent and occasionally seen in the control as well as in the treated rats. They are not related to VC or VDC. 13 EPL 00785 B. Exposure to VC or VDC and Observation for 12 Months In Mice 1. General Observations and Number of Deaths Mice were exposed to VC and VDC, 6 hr/day, 5 days/week, for periods of 1, 3 or 6 months. During these periods, these mice were exposed for a total of 21, 63, or 128 days, respectively. A number of mice died or were moribund and terminated during the exposure and observation periods. The number and time of deaths and early terminations depended upon the duration of initial exposure and level of VC. The clinical signs Included rough hair coat, lethargy, and/or the appearance of external tumor masses, especially mammary tumors in the females. A number of mice died at night, and autolysis occurred in some mice, especially during the weekends. The number of deaths and early terminations and total number of mice exposed to VC and VDC followed by observation for 12 months are sum marized in Table 21. There was no apparent difference in number of deaths and early terminations between the males and females of any group. After exposure for 1 month, a total .of two of 32 control mice (6%) were termi nated during the observation period. The number of deaths and early terminations for the mice exposed to 50, 250 or 1,000 ppm VC was four (13%), five (16%), and JL0 (31%), respectively. Only one of 16 mice (6%) exposed to 55 ppm VDC for 1 month was terminated during the observation period. After exposure for 3 months, six of 32 control mice (19%) died or'were terminated during the observation period. The number of deaths and early terminations for the mice exposed to 50, 250 or 1,000 ppm VC was eight of 32 (25%), 19 of 32 (59%), and 15 of 20 (75%), respectively. Five of 16 mice (31%) exposed to 55 ppm VDC died or were terminated during the observa tion period. After exposure for 6 months, 11 of 56 control mice (20%) died or were moribund and terminated during the observation period. A total of 15 of 16 mice (94%) exposed to 50 ppm VC died or were terminated during this period. On the other hand, all the mice (100%) exposed to 250 or 1,000 ppm VC died or were terminated. Most of the deaths and early termina tions in the mice exposed to 250 ppm occurred within 9 months after exposure and in the mice exposed to 1,000 ppm within 6 months after exposure. In addition, two of 20 mice (10%) exposed to 250 ppm and five of the 24 mice (21%) exposed to 1,000 ppm were terminated during the exposure period. A total of 11 of 24 mice (46%) exposed to 55 ppm VDC for 6 months died or were terminated during the observation period. EPL 00786 14 2. Tumors The tumor occurrence in mice exposed to VC or VDC for 1 month followed by observation for 12 months is summarized in Tables 22 and 23, for 3 months in Tables 24 and 25, and for 6 months in Tables 26 and 27. a. Exposure for 1 Month After exposure for 1 month followed by the observation period, a few tumors occurred in eight of 32 control mice (Table 22). The tumors included bronchioloalveolar tumor of the lung in three mice and hepato cellular tumor of the liver in four mice. In addition, one mouse had an adenocarcinoma of the mammary gland and a hemangioma of the kidney; another mouse had a malignant lymphoma involving the kidney and lymph node. These tumors were also seen in mice exposed to various levels of VC or VDC (Tables 22 and 23). The incidence and severity of bronchioloalveolar tumor were greatly increased in mice exposed to 250 or 1,000 ppm VC. This tumor occurred in three of 32 mice exposed to 50 ppm, 19 of 32 mice exposed to 250 ppm, and 20 of 32 mice exposed to 1,000 ppm. In addition, adenocarcinoma/ carcinoma of the mammary gland occurred in four of 16 females exposed to 50 ppm VC, two of 16 females exposed to 250 ppm VC, and none of 16 females exposed to 1,000 ppm VC. Hepatic hemangiosarcotna was seen in one male ex posed to 50 ppm VC, and hemangioma in two females exposed to 1,000 ppm VC. Other occasional tumors included metastatic adenocarcinoma of the lung originating from the mammary gland, malignant lymphoma of the kidney and lymph node, or liver and spleen, and carcinoma of the kidney. In the 16 mice exposed to 55 ppm VDC, there were a few tumors including bronchioloalveolar tumor in one mouse, hepatocellular tumor in three mice, and renal carcinoma in one mouse. b. Exposure for 3 Months After exposure for 3 months followed by the observation period, bronchioloalveolar tumor of the lung and/or hepatocellular tumor of the liver were seen in three control mice (Table 24). The incidence and severity of tumors in several organs were greatly increased in mice exposed to all levels of VC (Tables 24 and 25). Bronchioloalveolar tumor of the lung occurred in 12 of 32, 21 of 32, and 16 of 20 mice exposed to 50, 250 or 1,000 ppm VC, respectively. Hepatic hemangiosarcoma was seen in four and five mice exposed to 250 or 1,000 ppm VC, respectively; adenocarcinoma/ carcinoma of the mammary gland was seen in four, six or two females exposed to the respective levels of VC. Extrahepatic hemangiosarcoma also occurred in a few mice exposed to VC involving the mesentery/peritonium or skin. In addition, hepatic and extrahepatic hemangioma was seen In several mice exposed to VC. There were a few metastatic tumors in the lung. The metastatic tumors included adenocarcinoma from the mammary gland in three mice exposed to 50 or 250 ppm VC, and metastatic osteosarcoma in one mouse 15 gpXj 00787 exposed to 1,000 ppm VC. Other occasional tumors in mice exposed to VC for 3 months included squamous-cell carcinoma or fibrosarcoma of the skin, myxoma of the mammary gland, and malignant lymphoma Involving the lung, kidney and lymph node. A number of mice exposed to various levels of VC with various tumors, especially those exposed to 250 or 1,000 ppm, died or were moribund and terminated during the observation period. In the 16 mice exposed to 55 ppm VDC (Table 25), bronchioloalveolar tumor of the lung occurred in three mice and hemanglosarcoma of the mesentery occurred in one mouse. c. Exposure for 6 Months After exposure for 6 months followed by the observation period, a number of tumors occurred in various tissues of 17 of the 56 control mice (Table 26). The percentage of control mice with tumors was not greater than that of the control mice in chamber for 1 or 3 months followed by the observation period. Most tumors occurred in the lung, liver, and mammary gland as seen in the control mica of the previous groups. Occa*sional tumors included fibrosarcoma of the skin,' adenocarcinoma of the Intestine, leiomyoma of the uterus, hemangioma of the spleen, and malignant lymphoma involving the liver, stomach and Intestine. The incidence of the hepatic and extrahepatic hemanglosarcoma, bronchioloalveolar tumor of the lung and adenocarcinoma/carcinoma of the mammary gland in the females were Increased in the mice exposed to 50, 250 or 1,000 ppm VC (Tables 26 and 27). Hepatic hemanglosarcoma occurred in one of 56 control mice, and one of 16, nine of 20, and 13 of 24 mice exposed to 50, 250 or 1,000 ppm VC, respect ively. Hemanglosarcoma was also seen in the mesentery/peritonium or skin of a few mice exposed to VC. Bronchioloalveolar tumor of the lung occurred in 11 of 56 control mice, and 3 of 16, 12 of 20, and 14 of 24 mice exposed to the respective levels of VC. Adenocarcinoma/carcinoma of the mammary gland occurred in 3 of 28 female control mice, and 2 of 8, 5 of 8 and 4 of 12 females exposed to the respective levels of VC. Other occasional lesions in the mice exposed to 250 or 1,000 ppm VC included metastatic adenocarcinoma in the lung originating from the mammary gland, hepatocellular tumor of the liver, adenocarcinoma of the intestine, chromophobe-cell adenoma of the pituitary, and malignant lymphoma Involving several organs. All the mice . exposed to 50, 250 or 1,000 ppm with various tumors died or were terminated during the exposure and observation periods; whereas, only seven of the 17 control mice with tumors died or were terminated before the end of the observation period. A few tumors occurred in six of 24 mice exposed to 55 ppm VDC (Table 26); the incidence and type of tumors were not apparently different from those of the control mice in the chamber for 6 months. EPL, 00T88 16 3. Cumulative Tumor Incidence and Tumorigenesis The cumulative tumor incidence in control mice and mice exposed to VC or VDC is summarized in Table 28. As for the rats, these mice were exposed to the filtered air, VC or VDC for 1, 3 or 6 months followed by observation for 12 months. The life spans of all mice within each treatment group were not the same. a. In the Liver Exposure of various levels of VC caused hemangiosarcoma of the liver. It occurred in two of 80 (2.5%), 13 of 84 (15%), and 18 of 76 (24%) mice exposed to 50, 250 or 1,000 ppm VC, respectively. This tumor was found in only one of 120 control mice and none in 56 mice exposed to 55 ppm VDC. Hepatic hemangiosarcoma was also found in rats of the present study after exposure to VC followed by observation for 12 months, and in mice and rats in a previous study-^i-^/ after exposure to VC without the observation period. This tumor was mostly multiple with random dis tribution among the lobes. Their size ranged from pin-point focus to a big bloody mass of several cm in diameter. Most large tumors ruptured with blood clot and/or bloody fluid filled the abdominal cavity. Many animals with this secondary internal bleeding had markedly enlarged spleen and a few of them also had subcutaneous edema at the lower part of body. Micro scopically, the small tumors consisted of undifferentiated endothella and some RBC which interspersed in the spaces encompassed by the neoplastic cells. The small neoplastic foci were seen in the early stage of tumor or in the peripheral zone of the large, advanced tumor. When the tumor became big, the proliferating immature endothella formed a wide cord, ex panded outwardly, and replaced the hepatocytes. With these cord growth, they anastomosed each other.in an irregular pattern and formed spaces varying from a cleft to a large cavity which was full of RBC. These RBC-filled spaces in the center of the advanced tumor were large and those at the peripheral areas were small. The neoplastic cells were very pleomorphic and anaplastic. They were basophilic and their nuclei were hyperchromatic. Mitosis was not uncommon in this tumor. In some advanced tumors, there was cavernous structure lined by flat endothia mimic of cavernous hemangioma. Extrahepatic hemangiosarcoma and hemangioma of the liver or other tissues were occasionally seen in various groups of mice, including hemangioma of the kidney in one control and of the spleen In another control mouse. There was also a lymphangioma in the liver of one female exposed to 55 ppm VDC. Hemangiosarcomas of the mesentery or subcutaneous tissue were slightly different from those in the liver. Their neoplastic endothelial cells were somewhat uniform, resembled the fibroblast, and arranged in a loose compart ment pattern filled with RBC as seen in the liver. Rupture and hematoma formation from these tumors were seen more often from the mesentery than from the liver. 17 EPL 00789 Hepatocellular tumors were seen in 11 of 120 control mice (9%), 5 of 80 (6Z), 11 of 84 (13%), and 4 of 76 (5%) mice exposed to 50, 250 or 1,000 ppm VC, respectively, and in 4 of 56 mice (7%) exposed to 55 ppm VDC. This tumor appeared to be sex-related and occurred mostly in males. It was seen in only one female control and one female exposed to 50 ppm VC. Grossly, there were solitary or multiple round masses on the surface of the liver. They were paler and softer than the surrounding normal parenchyma. Microscopically, the cells were well-differentiated or slightlyundifferentiated, and arranged in a trabecula or solid pattern. They were mostly spherical nodules without much stroma present in the cell mass, or between neoplastic and normal tissues. The normal hepatic lobular pattern was obliterated. In a few animals, the sizes of neoplastic cells were larger than normal, and the cytoplasm was basophilic and vacuolated. b. In the Lung In the lung, bronchioloalveolar tumor occurred in 16 of 120 control mice (13%). The total incidence was increased in mice exposed to various levels of VC. The tumor occurred in 16 of 80 (20%), 52 of 84 (62%), and 50 of 76 (66%) mice*exposed to 50, 250 or 1,000 ppm VC, respect ively. A total of five of 56 mice (9%) exposed to 55 ppm VDC had this tumor. Bronchioloalveolar tumor-of the lung was reported in mice exposed to VC with out the observation period.2,3/ There were a few occasional metastatic tumors in the lung of mice exposed to VC.' These metastatic tumors were adenocarcinoma originating from the mammary gland. In addition, there was metastatic osteosarcoma in one female'exposed to 1,000 ppm VC for 3 months, and metastatic squamous cell carcinoma originating from the skin in one male exposed to 55 ppm VDC for 6 months. c. In the Mammary Gland Adenocarcinoma/carcinoma of the mammary gland, seen only in females, occurred in 4 of 60 control mice (7%), 10 of 40 (25%), 13 of 40 (33%), and 6 of 38 (16%) mice exposed to 50, 250 or 1,000 ppm VC, and none in 28 mice exposed to 55 ppm VDC. These tumors were also found in rats of the present study after exposure to VC and observed for 12 months, and in mice exposed to VC without the observation period. Adenoma occurred in one female exposed to 250 ppm VC and myxoma in one female exposed to 50 ppm VC. d. Other Organs A number of other tumors occasionally occurred in various organs of these mice. Their occurrence was not related to any group. These tumors included malignant lymphoma involving various organs, chromophobe cell adenoma of the pituitary, squamous--cell carcinoma and fibrosarcoma of the skin, adenocarcinoma of the intestine, carcinoma of the kidney, and lieomyoma of the uterus. 18 EPL 00790 4. Other Lesions Spontaneous and age-related lesions were seen in most mice from the control and treated groups. These lesions included inflammatory and degenerative changes. Amyloidosis occurred in most tissues of the older mice. C. The Effects of PS on VC Exposure in Mice Based on body weight and feed consumption data, it was estimated that the mice fed the 0.1% DS diet consumed approximately 100 to 150 mg/kg of DS daily. The mice receiving DS and exposed to VC appeared to be less lethargic and to have smoother hair coats than their counterparts exposed to VC without the DS diet. 1. Survival Time At the conclusion of'the 49-week study, DS significantly increased the number of survivors of both sexes of mice exposed to 250 ppm VC and in males exposed to 1,000 ppm VC (Table 29). The number of survivors in the control and 50 ppm VG exposed mice was not affected by the DS administra tion. In addition, the DS-treated mice exposed to 250 or 1,000 ppm VC had a longer mean survival time than mice not receiving DS. At 250 ppm VC, females survived 33 weeks compared to 48 weeks for DS-treated females. Males exposed to 1,000 ppm VC survived 27 weeks compared to 43 weeks for those re ceiving DS. Females exposed to 1,000 ppm VC survived 34 weeks compared to 43 weeks for DS-treated females. These differences in survival time would be much greater if the study had not been terminated at 49 weeks. 2. Tumor Incidence The tumors found in the mice exposed to VC with or without DS treatment Included bronchioloalveolar adenomas, hemanglosarcoma of the liver and/or mesentery, and mammary gland tumors In females. The tumor Incidence is summarized In Table 30. DS treatment appeared to have no effect on the incidence of VC-induced bronchioloalveolar adenomas. Bronchioloalveolar adenomas occurred in 80 to 100% of the males and 50 to 100% of the females exposed to 250 or 1,000 ppm VC with or without DS. The incidence of this tumor in controls and 50 ppm VC exposed mice was much lower, ranging from 0 to 25%. At both 250 and 1,000 ppm VC, there appears to be a trend toward a decreased incidence of hemangiosarcomas in both male and female mice treated with DS. At 250 ppm VC, 5 of 6 males and 2 of 5 females had hemanglosarcoma compared to 2 of 8 males and 1 of 8 females in the 250 ppm VC group receiving DS. At 1,000 ppm VC, 4 of 5 males and 3 of 5 females had hemangiosarcomas 19 EPL 00791 compared to 3 of 7 males and none of 4 females exposed to 1,000 ppm VC and treated with DS. However, no statistical differences were demonstrated. One male and one female exposed to SO ppm VC were found to have hemangiosarcomas, compared to none in the 50 ppm VC group treated with DS. No hemangiosarcomas were found in control mice or mice treated with DS alone. There was also a trend toward reduction in the incidence of mam mary gland tumors in female mice at all exposure levels of VC and treated with DS. The mammary gland tumors were composed of ductular adenocarcinoma, squamous-cell carcinoma, anaplastic-cell carcinoma, hemanglosarcoma or a combination of these tumor types. At 50 ppm VC, two of six females had mammary gland tumors compared to none of five in the DS treated females. At 250 ppm VC, two of eight females had tumors compared to one of eight in the females treated with DS. At 1,000 ppm VC, three of five females had mammary gland tumors compared to none of four in the females treated with DS. However, no statistical differences were demonstrated. 3. Hepatic Microsomal Composition and Function * The livers of mice surviving the 49-week study were, removed and the microsomal fractions were examined for cytochrome P-450 and bg concen trations and for MFO activities. Alterations in hepatic microsomal composi tion and function in mice from chronic VC administration with and without DS treatment were compared. The results are summarized in Tables 31 and 32. Treatment with DS alone resulted in a small increase in relative liver weights and a small decrease in microsomal protein concentrations compared to mice fed the control diet. A decrease in cytochrome P-450 con centration in DS treated mice was accompanied by reductions in the MFO activities, although the reductions in the enzyme activities were not parallel. Impairment of -nitroanisole-0-demethylase and ethylmorphine-Ndemethylase activities occurred to a lesser degree than the hydroxylation of aniline. Sex differences in the response to DS treatment were not apparent. Exposure of mice to 50 or 250 ppm VC alone for 49 weeks resulted in a considerable increase in relative liver weights. Microsomal protein concentrations tended to decrease, but this was highly variable. Both cytochrome P-450 and b^ were decreased in mice treated with 50 ppm VC, but these effects were not apparent in the animals exposed to 250 ppm. No significant alterations were observed in MFO activities of VC-treated mice, although ethylmorphine-N-demethylase and -nitroanisole-0-demethylase activities tended to be higher. The increase in relative liver weights of mice exposed to VC and fed DS was not as great as the increase seen in mice exposed to VC alone. A significant decrease in cytochrome P-450 was seen in the male mice fed DS 20 and exposed to 50 ppm VC. No significant changes were seen in cytochrome P-450 in OS-fed females exposed to 50 ppm VC or in males exposed to 250 ppm VC. The lower activity of aniline hydroxylase observed in mice treated with DS alone was also evident in DS-fed mice exposed to VC. The decreases in ethylmorphine-N-demethylase and -nitroanisole-0-demethylase activities seen in DS-fed mice were not seen in DS-fed mice exposed to VC. In fact, ethylmorphine-N-demethylase activities were greater than those of mice fed either the control or DS diets alone. 21 EPL 00793 IV. DISCUSSION AND CONCLUSION A. Exposure to VC or VDC in Rats We reported previously22-1--3/ that most rats exposed to 250 or 1,000 ppm VC died between the 10th and 12th months. These levels caused hemanglosarcoma of both the liver and lung starting the 9th month. Hemangiosarcoma also occurred in the mesenteric lymph node or subcutaneous tissue of two rats exposed to 55 ppm VDC for 12 months without the observation period. In the present study, exposure of 50, 250 or 1,000 ppm VC for 1 or 3 months did not cause any death during the observation period of 12 months following exposure. Exposure for 6 or 10 months increased the number of deaths and early terminations during the exposure and observation periods when compared with the respective control rats. The increase in deaths and early terminations was proportional to the length of exposure and the level of VC. After exposure of 50, 250 or 1,000 ppm VC for 1 or 3 months, the tumor incidence in rats examined at the end of the 12-month observation period including the tumor incidence in the rats terminated earlier was not apparently increased when compared with the spontaneous tumors seen in the respective control rats. Exposure for 6 months, 250 or 1,000 ppm VC caused various tumors in the liver. Exposure for 10 months, all three levels of VC caused a number of tumors in the lung, liver and mammary gland, and probably malignant lymphoma involving several organs. In the liver, exposure of 250 or 1,000 ppm VC for 6 months caused three major types of tumors. Neoplastic nodules occurred in three of 16 rats exposed to 250 ppm (Table 4). The incidence was Increased and occurred in six of 28 rats exposed to 250 ppm VC for 10 months, but not in rats exposed to 1,000 ppm VC (Table 6). Exposure of 1,000 ppm VC for 6 months caused hepatocellular carcinoma in one of 16 rats and hemangiosarcoma in two of 16 rats. The incidence and/or severity of these two tumors were increased in rats exposed to 250 and/or 1,000 ppm VC for 10 months. Exposure of VC might have also caused hemangioma in the liver. Hemangioma was seen in several rats exposed to 50, 250 or 1,000 ppm VC. Bile duct carcinoma occurred in one male exposed to 1,000 ppm VC for 6 months. It is not known whether or not this tumor is related to VC exposure. About half the rats with hepatic hemanglosarcoma also had hemangiosarcoma of the lung. In addition to the hemanglosarcoma seen in the lungs of rats exposed to 250 or 1,000 ppm VC for 10 months, bronchioloalveolar tumors occurred in two rats exposed to 250 ppm VC, and in four rats exposed to 1,000 ppm VC after exposure for 3 months or longer. This tumor was not 22 EPL 00794 seen in control or other treated groups. The increased incidence of this tumor in VC exposed rats might be related to the compound. Exposure of various levels of VC increased the incidence of several tumors of the mammary gland in the female rats, predominantly fibroadenoma and adenocarcinoma/carcinoma. These tumors were seen in seven of 36 control rats (19%) exposed to the filtered air. Their incidence was increased, and occurred in 16 of 36 rats (44%) exposed to 50 ppm VC. The incidence remained high and occurred in 11 of 32 rats (34%) exposed to 250 ppm VC. In rats exposed to 1,000 ppm VC, the incidence of the mammary gland tumors dropped and occurred in seven of 36 rats (19%) , probably due to earlier deaths of this group of rats. Exposure of 1,000 ppm VC caused malignant lymphoma in three male rats and one female rat that involved the lung, liver, spleen, kidney, lymph node, and/or bone marrow. Malignant lymphoma involving the spleen also occurred in one male rat exposed to 250 ppm VC. A number of spontaneous tumors occasionally occurred in various organs of the control rats and the rats exposed to VC. They'are not related to the exposure of VC. Exposure of 55 ppm VDC for 10 months, but not for 1, 3 or 6 months, increased the deaths and early terminations of rats during the observation period after exposure. VDC did not Increase the incidence of any spontan eous tumors or cause any other tumors. B. Exposure to VC or VDC in Mice We reported--2 1--3/ that most mice exposed to 250 or 1,000 ppm VC died starting the 7th month, and that these levels caused bronchioloalveolar tumor of the lung, hepatic hemangiosarcoma with occasional extrahepatic hemangiosarcoma Involving various organs, tumors of the mammary gland in the females and possibly malignant lymphomas involving a number of organs. Exposure of 55 ppm VDC for 9 to 12 months without the observation period caused hepatic hemangiosarcomas in a few mice and probably bronchiolo alveolar tumors. In the present study, exposure of 50, 250 or 1,000 ppm VC for 1, 3 or 6 months increased the number of deaths and early terminations during the exposure and observation periods when compared with the respect ive control mice. The increase in, and time of, deaths and early termina tions were proportional to the length of exposure and the level of VC. After exposure for 6 months, 94% of the mice exposed to 50 ppm and all the mice exposed to 250 or 1,000 ppm VC died or were terminated before the end 23 EPL 00795 of the observation period, whereas only 20% of the control mice died or were terminated during the observation period. In addition, the mice died or were terminated earlier in groups exposed to higher levels of VC and for longer durations. Exposure of VC caused hepatic and extrahepatic hemangiosarcomas. Hepatic hemanglosarcoma occurred in one of 32 mice exposed to 50 ppm VC for 1 month; and in four of 32 and five of 20 mice exposed to 250 or 1,000 ppm VC, respectively, for 3 months. After exposure for 6 months, the incidence was greatly Increased. The tumor was seen in one of 16, nine of 20 and 13 of 24 mice exposed to the respective levels. The severity of this tumor was also increased in some mice exposed for 6 months. One of 56 control mice in the chamber with filtered air for 6 months developed hepatic hemanglosarcoma during the observation period. None of the mice from the other control groups had this tumor. Extrahepatic hemanglosarcoma was seen in several mice exposed to various levels of VC but not in any controls. Hepatic and extrahepatic hemangiomas were occasionally seen in various groups of mice including the controls. Hepatocellular tumors were seen in a number of control mice and mice exposed to VC or VDC. The incidence of these tumors was not apparently altered in the mice exposed to these compounds. Exposure of 250 or 1,000 ppm VC for 1 month or exposure of 50, _250 or 1,000 ppm VC for 3 or 6 months increased the incidence of bronchiolo alveolar tumor of the lung when the mice were kept for observation of 12 months. The cumulative incidence of this tumor In all mice was 16 of 120 control mice (13%), 19 of 80 (24%), 52 of 84 (62%), and 50 of 78 (64%) mice exposed to 50, 250 or 1,000 ppm VC, respectively. In addition, there were a few occasional metastatic adenocarcinoma in the lung originating from the mammary gland in mice exposed to various levels of VC. In the mammary gland of the females, exposure of various levels of VC for 1 month or longer increased the incidence of adenocarcinoma/ carcinoma. These tumors occurred in 4 of 60 female controls (7%), and 10 of 40 (25%), 13 of 40 (33%), and 6 of 38 (16%) females exposed to 50, 250 or 1,000 ppm VC, respectively. The lesser incidence of these tumors In the females exposed to 1,000 ppm was probably due to earlier death of this group of mice. As for the rats, there were a number of spontaneous tumors in various organs of the control mice and mice exposed to VC. These tumors were occasionally seen, and are not related to the exposure of VC. Exposure of 55 ppm VDC for 3 or 6 months, but not for 1 month, increased the deaths and early terminations of mice during the observation period after exposure. VDC did not increase the incidence of any spontan eous tumors or cause any other tumors. 24 epl 00796 C. Protective Effect of PS on VC Exposure in Mice Exposure of mice to 50, 250 or 1,000 ppm VC was reported to cause a high Incidence of bronchioloalveolar adenoma, mammary gland tumors and hemangiosarcoma.-=JLi' In the present study, treatment with DS appeared to reduce the incidence of hemangiosarcoma of the liver in both sexes and mam mary gland tumors in females. However, DS treatment had no apparent effect on the incidence of VC-induced bronchioloalveolar adenoma. Also, treatment of mice with DS during exposure to 250 or 1,000 ppm VC resulted in an in crease in the survival time. These data suggest that DS exerts at least a partial protection against VC-induced carcinogenesis. Whether this pro tective effect of DS is an actual reduction in the occurrence of the VCinduced neoplasms or simply an Increase in the latency period to tumor formation is not clear from the present study. Treatment of mice with DS alone for a period of 49 weeks decreased the hepatic cytochrome P-450 concentration and reduced the MFO activities. These data are similar to previously reported studies in rats after single doses of D&5/ or 12 daily doses of DS.-- Exposure to 50 ppm VC alone for 49 weeks increased the relative liver weight and decreased the cytochrome P-450 and b^ concentrations without any apparent effect on the MFO activities. The effects on liver,weight and cytochrome levels were not apparent in mice ' exposed to 250 ppm. Watanabe et al.i2/ reported that exposure to 5,000 ppm VC for 7 weeks did not result in any changes in microsomal enzyme activity. Combination treatment of DS plus VC, in the present study, decreased the effects of VC on liver weight and cytochrome levels. The activity of aniline hydroxylase was decreased whereas the activities of ethylmorphine-Ndemethylase and -nitroanisole-0-demethylase were unaffected or slightly increased. It has been reported that VC is metabolized, at least in part, by the hepatic MFO system to a reactive metabolite(s) which may covalently bind to cellular macromolecules and initiate the carcinogenic response to VC.-- Repeated exposure to VC was also reported to increase the binding of reactive VC metabolites to hepatic macromolecules ^ Our data show that DS inhibits the MFO system. This suggests that DS may act by reducing the activation of VC to reactive metabolites via microsomal metabolism. It has been also suggested that a potential pathway of VC detoxification may be the conjugation of reactive VC metabolites with nonprotein sulfhydryl groups.jQJ Since DS is rapidly metabolized to compounds bearing free sulfhydryl groups,--^ it may exert its protective action by increasing the availability of free sulfhydryl groups to combine with reactive VC metabo lites. Conceivably, DS may act by a combination of these two mechanisms. EPL 00797 25 V. PUBLICATIONS. PRESENTATIONS AND MANUSCRIPTS A. Publications Lee, C. C., J. C. Bhandari, J. M. Winston, W. B. House, P. J. Peters, R. L. Dixon, and J. S. Woods: Inhalation Toxicity of Vinyl Chloride and Vinylidene Chloride. Environ. Health Persp., 21:25-32 (1977). Lee, C. C., J. C. Bhandari, J. M. Winston, W. B. House, R. L. Dixon, and J. S. Woods: Carcinogenicity of Vinyl Chloride and Vinylidene Chloride. Toxicol, and Environ. Health. 4:15-30 (1978). B. Presentations Lee, C. C., J. C. Bhandari, W. B. House, P. J. Peters, J. S. Woods, and R. L. Dixon: Inhalation Toxicity of Vinyl Chloride (VC) or Vinylidene Chloride (VDC) in Rats and Mice. The Pharmacologist, 1J:245 (1976). - -'u Winston, J. M., C. C. Lee, J. C. Bhandari, R. L. Dixon, and J. S. Woods: A Study of the Carcinogenicity of Inhaled Vinyl Chloride (VC) and Vinylidene Chloride (VDC) in Rats and Mice. First International Congress on Toxicology, March 30-Aprll 2, 1977, Toronto, Canada. Lee, C. C.: Toxicity and Carcinogenicity of Vinyl Chloride Compared to Vinylidene Chloride. Conference on Comparative Metabolism and Toxicity of Vinyl Chloride Related Compounds. May 2-4, 1977, NIH, Bethesda, Maryland. Winston, J. M., J. C. Bhandari, A. M. El-hawari, R. D. Short, and C. C. Lee: The Effect of Disulfiram on Vinyl Chloride-Induced Carcinogenesis in Mice. Society of Toxicol., Abstract (17th Annual Meeting), 211, 1978. C. Manuscripts Winston, J. M., A. M. El-hawari, J. C. Bhandari, R. D. Short, J. S. Woods, and C. C. Lee: Vinyl Chloride-Induced Carcinogenesis: The Effect of Treat ment with Disulfiram. Submitted 1979. Hong, C. B., J. M. Winston, L. P. Thornburg, C. C. Lee, J. S. Woods, and R. L. Dixon: Additional Study on the Carcinogenicity of Vinyl Chloride. In preparation. EPL 00798 26 REFERENCES 1. Lee, C. C. et al.: Toxicological Evaluation of Ferric Dimethyldithiocarbamate (Ferbam) and Dithlocarbamate (Thiram) with Acute Toxicity of Manganese and Zinc Ethylenebisdithiocarbamates (Maneb and Zlneb). NIH Contract No. N01-ES-2-2084 (Continuation of NIH-NIEHS-72-2084), 1-356, 1975. 2. Winston, J. M. et al.: Evaluation of the Environmental Toxicants Vinyl Chloride (VC) and Vinylidene Chloride (VDC). NTH Contract No. N01-ES2-2084 (Continuation of NIH-NIEHS-72-2-2084), 1-129, 1977. 3. Lee, C. C., Bhandarl, J. C., Winston, J. M., House, W. B., Dixon,-R. L. and Woods, J. S.: Carcinogenicity of Vinyl Chloride and Vinylidene Chloride. J. Toxicol. Environ. Health, 4, 15-30, 1978. 4. Hald, J., Jacobsen, E. and Larsen, V.: The Sensitizing Effect of Tetra ethylthiuram Disulphide-(Antabuse) to Ethyl Alcohol. Acta Pharmacol. Toxicol.. 4, 285-297, 1948. 5. Stripp, B., .Green, F. E. and Gillette, J. R.: Dlsulfiram Impairment of Drug Metabolism by Rat Liver Microsomes. J. Pharmacol. Exp. Ther., 170, 347-354, 1969. 6. Zemaitis, M. H. and Greene, F. E.: Impairment of Hepatic Microsomal Drug Metabolism in the Rat During Daily Dlsulfiram Administration. Biochem. Pharmacol., 25, 1355-1360, 1976. 7. Wattenberg, L. W.: Inhibition of Carcinogenic and Toxic Effects of Polycyclic Hydrocarbons by Several Sulfur-Containing Compounds. J. Natl. Cancer Inst.. 52, 1583-1587, 1974. 8. Wattenberg, L. W.: Inhibition of Dimethylhydrazine-Induced Neoplasia of the Large Intestine by Dlsulfiram. J. Natl. Cancer Inst.. 54, 1005-1006, 1975. 9. Short, R. D., Winston, J. M., Minor, J. L., Hong, C. B., Seifter, J. and Lee, C. C.: Toxicity of Vinylidene Chloride In Mice and Rats and Its Alteration by Various Treatments. J, Toxicol. Environ. Health, 3, 913-921, 1977. 10. Lowry, 0. H., Rosebraugh, N. J., Farr, A. L. and Randall, R. J.: Protein Measurements with Folin Phenol Reagent. J. Biol. Chem., 193, 265-275, 1951. EPL 00799 27 11. Ornura, T. and Sato, R.: The Carbon Monoxide-Binding Pigment of Liver Microsomes. J. Biol. Chem., 239, 2370-2378, 1964. 12. Imai, Y., Xto, A. and Sato, R.: Evidence for Biochemically Different Types of Vesicles in the Hepatic Microsomal Fraction. J. Biochem. (Japan). 60, 417-428, 1966. 13. Schenkman, J. B., Remmer, H. and Eastabrook, R. W.: Spectral Studies on Drug Interaction With Hepatic Microsomal Cytochrome. Mol. Pharmacol., 3, 113-123, 1967. 14. Nash, T.: The Colorimetric Estimation of Formaldehyde by Means of Hantzch Reaction. Blochem. J., 55, 416-421, 1953. 15. Netter, K. J. and Siedel,. G.: An Adaptively Stimulated O-demethylatlng System in Rat Liver Microsomes and Its Binetic Properties. J. Pharmacol. Exp. Ther., 146, 61-65, 1964. 16. .Goldstein, A.: Biostatistd.es; An Introductory Text, New York: Macmillan, 1964. 17. :-' Steel, R. G.. and Torrie, J. H., Principles and Procedures of Statistics. , New York: McGraw-Hill, 1960. - 18. Squire, R. A. and Levitt, M. H.: Report of a Workshop on Classification of Specific Hepatocellular Lesions in Rats. Cancer Research, 35, 3214- 3223, 1975. 19. Watanabe, P. G., Zempel, J. A. and Gehrlng, P. J.: Comparison of the Fate of Vinyl Chloride Following Single and Repeated Exposure in Rats. Toxicol. Appl. Pharmacol., 44, 391-399, 1978. 20. Hefner, R. E., Jr., Watanabe, P. G. and Gehrlng, P. J.: Preliminary Studies of the Fate of Inhaled Vinyl Chloride Monomer in Rats. Ann. N. Y. Acad. Sci.. 246, 135-148, 1975. 21. Watanabe, P. G. and Gehrlng, P. J.: Dose-Dependent Fate of Vinyl Chloride and Its Possible Relationship to Oncogenicity in Rats. Environ. Health Perspec.. 17, 145-152, 1976. 22. Stromme, J. H,: Metabolism of Dlsulfiram and Diethyldithiocarbamate in Rats With Demonstration of an In Vivo Ethanol-Induced Inhibition of the Glucuronic Acid Conjugation of the Thiol. Blochem. Pharmacol., 14, 393410, 1965. BPL 00800 28 TABLE 1 THE NUMBER OF DEATHS AMD EARLY TERMINATIONS AND TOTAL NUMBER OF RATS EXPOSED TO FILTERED AIR (CONTROL). VC OR VDC FOR 1. 3, 6 OR 10 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS Month During Observation Period Control MF 50 MF VC, ppm 250 MF 1, 000 MF VDC, ppm 55 MF After exposure for 1 month 6-9 Total 1 0/4^ 0/4 0/4 0/4 0/4 0/4 0/4 0/4 1/4 0/4 After exposure for '3 months .3-6 lJ>/ . . . 1 6-9 - - - - - - - - - 9-12 32 Total 0/8 4/8 oTs 2/8 o7? m T7a o7? o7? o7? After exposure for 6 months During exposure 1' i 3-6 1 i - - - l - - ... 6-9 1---i 3 3 - 9-12 Total ** -- 3 2i 2i T/8 1/8 4/8 "oTi" 2/8 2/8 J/8 5/8 1/8 - -_ After exposure for 10 months During exposure 2 0-3 1 3-6 1 6-9 3 1 9-10 23 Total 6/16 7/16 3 -i3 1i1 145 1 5 2 6/10 11/16 11/16 _1 1 1 -4 3 2433 2537 1256/12 15/16 15/16 11/14 - 3 1 5 9/16 a/ Number of deaths and early termlnations/total number of rats studied, b/ Number of deaths and early terminations during the period. 29 EPL 00801 TABLE 2 SUMMARY OF TUMORS IN RATS EXPOSED TO VC OR VDC FOR 1 MONTH FOLLOWED BY OBSERVATION FOR 12 MONTHS^/ Treatment: Sex: Rat No.: Weeks After Exposure: 250 ppm VC Female 546 549 52 52 TumorsbR/ Liver Hepatocellular carcinoma _ He^ngiosarcoma__________ Mammary Gland _ Flbradenomja_______________ Pituitary _ ChromDhb-ell_adenoma 11 1 1,000 ppm VC Male 596 52 55 ppm VDC Male 656 52 3 4 a/ Includes only rats with tumors. b/ Tumor classification: 1 - benign; 2 - malignant, well-differentiated; r;\ 3 - malignant, moderately differentiated; 4 - malignant, poorly differentiated. 00802 30 TABl.K 1 tuhokm in rats, exposkh tu mrateo ajii (cowruni). vi: ok voc for i hohtiis follow; i> by observation for j 2 howtos" / I^ Tlt!"r" Treatment: Sex: Rut No.: Week After Exposure: Control Fcaaie AW 418 47_ 49_ 50 ppw VC__ 250 ppi> VC___________ 1,000 |i|> VC_____ 55 ppm VOC _ Finale____ ____ Hale __ Peaale____ Hale______ ____Fceala Female 475 479 409 492 524 ~ 525 527 559 560 561 602 604 606 614 640 641 704 41 45 52_ 52 52 52 52 52 52^ 52. 52 52 52 52 52 52 52 Skin Sqijauoub-ra) I curdiiiun Conn Rrunrli Jolualveolar tumur Epiileroid_curi:lnumn _ l.lver HooplustIc nod u lea lieputuce 11 ular carcinoma jlcmongloau ___ ________ Epididymis Mesothelioma^___________ Hiuaory Claud Fibroudeiuima Adenoma Adenocarcjnumu Cicdnuaui______ _ _ (Ivory Cystadonoma________ _ Pimlc.i ry CtiroKijihobe-eej[ I _adenoma a/ Inc I ode it only rota with tumors. Ii/ Tumor c lues ii lent Uni: 1 - benign; 2 - mallgnunt, well-differentiated; 1 - Malignant, Moderately differentiated; 4 - malignant, poorly differentiated. 00803 TAttl.K A |j/ Tuwora- SUHHAKf OR TUMORS IN RATS EXPOSED TO FII.TENR.il AIR (CONTROI.). VC OR VIIC FOR 6 NONIUS FOI.LOWED BV OBSERVATION RH 12 MOHTIIS"/ Treatment: Sex: Hat Ho.: Week After Exposure: Control Renale 399 421 31 52 50 J*l vc Hale Peaule 436 454 494 497 504 19 52 52 52. 52 250 ppn VC Halo Rennie 513 518 555 568 52 52 14 51 612 592 U-f 21 1,000 ppn VC Hale 589 599 60S 611 28_ 34 52 52 Fondle 632 611 641 34 18 52 55 ppw VPC Hjila Rennie 674 711 52 57 Skin Papilloma Si|tiaiOU(i-cull carclnoaa KJbroBurcona 2 2 1 #2 Lung Broni'titu loulveolar iuw>r liver 2 Neoplastic nodules 1 .1 1 Itup.uocellulsr carcinoma Bile duet carcinoma IksBuingioiiu llemangiuttarcuaa 1 * 2 1 2 44 InusUnfi Fibrosarcoma HuMury Gland fuot Rlbrundenona AilenocarcliiuM 1 2 11l ' 1 Pituitary Clirunupltobe-cell adwiuM l Halignant l.ynphuna Lung, liver, spleen and/or kidney 42 a/ Includes only rats with tuaora. I*/ Tumor claaalfIcatlon: 1 - benign; 2 - malignant, well-differentiated; 1 - Malignant, moderately different In ted; 4 - Malignant, poorly differentiated, c/ During exposure. EpL 00804 TAIMLE S gIPgWRY OP TIWOHS IN HATS gXPOSKO TU KltTKftKP AIK (COHTKOI.) OH VC POM 10 HrWTtlS HUl-OUKII BY OBSKRVATIOH 0R 12 MONTHS'^ Trent MuiiL: Sex: Hul Ho; Mk After kx|ioaur Tumore-^ ___ Control____________ _____ ___________ __________ Hole ____________________ Female 162 170 372 174 169 401 402 408 409 412 413 11 32 32 32 52 46 46 52 52 52 52_ 466 H-* Hale ____________________ 431 444 456 470 499 15 52 52 11 29 50 pom VC___________________________________________ Female 474 472 476 477T47B 498 484 486 *500 34 17 47 y 47^ 50_ 52_ 52 52 Skin Squummiu-cell cureIDOOM Fibroma _________ 1____]____________ _1_ Long FI l>rosarcoma_ ________________________ 1.1 ver l1e|>atocellulor 2 carcinoma _ Hemangioma______________________________ Pancreas! Inlet-cell W _ adenoma____ _ OJ Kidney Carcinosarcoma ______________________ J Hinty Gland fib roodemma 1 Adenoma Ad unoca re1noma 1'lit roaur coma______ __________________ Adrenal _ Flicoclicomicytome_________ _ I'llnJtury Citrnmu|>liobe-cell .1 adenoma 2 2 111i11 I1 a/ lncludou only rata ultli tumors. * b/ Tumor classification; 1 - luiilto; 2 - aullgnunt, we] l-dlffcrtsntlated; 1 - malignant, moderately differentiated; 4 - malignant, poorly differentiated, c / During ex|iouuru. Sos TAHI.K 6 SUHHARY OE TUHIIHS IN HATS KXTOSKII TO VC 1QR ID HOHTltS WJI.LOHttl) HY OHS KKVATI OH HI Hi 12 MOHTHS^ T_uworsh- / Yreataenti Sax: Rat Ho.i Week After Expoaure 25(1 ppe VC Hale KmIb 506 502 514 508 517 5)8 519 520 521 517 576 567 542 541 544 545 55(1 551 552 551 554 12 18 29 11 49 52 52 52 52 52 4()/ 25 26 17 19 47 52 52 52 52 52_ Skin Si(|iiiiwiuH'CeH curciiMU 2 Flhruaui _ ___ _______ _ ________________ _____________ __ Lung Bruiuliloloolveular tuaur _ {jeiu(i^lomcMa___ _ _______ _______________________ __________ Liver Neupludtlc iMidile XX IlcpetoceJ lular carcliMHu llM;mgloaa IleauiugUntarcoM___________ __ ____________ _______ __ _______ 2 Kidney Adenoca rc I tiuM CarcjnuM ________ _________ __________ _______________ ___________ Haltgnmit lymphoma U1 Spleen____________ __ _ ____ _________ _____ _______ __________ Kiiuwacy 01 and [IbiUiiJeouM ______ ________________ ____ ____________ rtiuliary Chr<jiiu)|a|iobe-CfeJ 1 _adencMM___________ ___________ _____ _____ 1l 2 I 2 2 1 2 I 1 1 1 1111 a/ Included only rms with tuaora. b / Tuiuur eliiBttlf Icntloni ) - benign; 2 - nallgnant, well-different la ted; 1 - malignant, moderately differentiated; 4 - malignant, poorly differentiated, c/ during capoeure. p ' ('U { * EPL 00806 TAUJ.E 7 SUHHAKY Or THHOKS IH RATS tXltWtll It) VO FOR 10 HOHTItS FOI.IJIWKU IIV (IHSHKVAT10H rOR 12 HOWTOS9/ Treat Muiut; Sum: Rat Ho.; Heuk After Rxpoaure; _______________________ 1,000 pimm Vf.____ ________ Hale_________ 577 579 5HO 563 578 562 585 567 590 591 607 598 647 615 613 616 617 2 J0_ 10 II 17 22 27 33 IS 36 67 52 40/ 9 12 20 25 Fh le 588 616 619 26 31 34 621 42 622 623 624 631 42 46 46 52 Tiuatiru!>./ Skin S`|<mimjiiu 11 card luma Selmcoouu ulaiul ojeno- carclnuM lains Siuni'hluluttlveoUr ttiaor HcMumgUsu|^cunui_______ Mosul Cavity SquamoiiiJ-ceH circlnoM _ Liver Neoplastic nodules Hepatocellular carcinoma Hemangioma lleuonj^lodaruoan________ u> intentIne In Adenocarcinoma _ Kidney Ciirdnou Metastatic bronchiolo alveolar carclnona Ovary franu loau I-cel 1 tuoi Hiiwaary HI and f I {) road ciamui _ I'ltulUry Chromophobe-cel I adenoma___________ Malignant Lymphoma lams, liver, spleen, lymph tunica and/or hone marrow 11 24 124 44 44 22 a/ Includes only rata with tumors. h/ Timor c1osalEIcation: I - benign; 2 - malignant, well-dlfferentlated; ] - malignant, moderately differentiated; 4 - malignant, poorly differentiated, t/ During exposure. epl 080 7 TABLE 8 SUMMARY OF TUMORS IN RATS EXPOSED TO VDC FOR 10 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS^/ Treatment: 55 ppm 7DC Sex: Male Female Rat No.: 684 676 677 687 688 689 690 691 692 Week After Exposure: 24^ 52 52 23 43 45 46 46 46 Skin Fibroma Mammary Gland Fibroadenoma Pituitary _ Chromohbe_-cell_ad_enoma 111 1 111 111 a/ Includes only rats vlth tumors. b/ Tumor classification: 1 - benign; 2 - malignant, well-differentiated; 3 - malignant, moderately differentiated; 4 - malignant, poorly 1 differentiated. cj During exposure. ~ - -........... 36 EPL 00808 TABLE 9 CSMPIATTVE TPMOR INCIDESC2 IN RATS CTOSZD TO FILTERED AIR (CONTROL). VC OR VDC FOLLOWED BY OBSERVATION FOR 12 MONTHS Tumors Livar Neoplastic nodule* Hepatocellular carcinoma 811a duct carcinoma Hemangioma Hamangioaarcoma Lung Bronchioloalveolar cumor Fibrosarcoma Hamanglo sarcoma Mammary Gland Flbroadanoma Adanoma Adanocarclcooa/carcinoma Fibrosarcoma Malignant Lymphoma Lung, livar, spleen, kidney, lymph noda and/or bona marrow Pituitary Chromophobn-call adanoma Skin Papilloma Squamous-call carcinoma Sabacaous gland adanocarcinoma Fibroma Fibrosarcoma Masai Cavity Squamous-call carcinoma Intastioa Adanocarcinoma . Fibrosarcoma Pancraas Islac-call adanoma Kidney Adanocarcinoma Carcinoma Carcinosarcoma Matastatlc bronchioloalvaolar carcinoma Ovary Cystadanoma Granuloaal-call tumor Epididymis Masothslioma Adranal Gland Phaochromocytama Control MF 30 ppm VC Nf F 250 opm VC MF 1.000 oom vc VF 55 oom VDC MF 0/361/0/36 1/36 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/30 0/30 0/30 1/30 0/30 0/36 0/36 0/36 0/36 0/36 7/36 1/36 0/36 1/36 1/36 3/32 1/32 0/32 2/32 4/32 1/36 4/36 1/36 2/36 i/36 1/36 3/36 0/36 0/36 9/36 0/34 0/34 0/34 0/34 1/34 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/30 0/30 0/30 0/36 1/36 0/36 1/36 0/36 0/36 1/32 0/32 2/32 3/36 0/36 3/36 1/36 0/36 4/36 0/34 0/34 0/34 0/36 0/36 0/36 0/36 0/36 0/36 0/36 3/36 1/36 1/36 0/36 0/30 0/30 0/30 0/30 11/36 0/36 4/36 1/36 0/36 0/36 0/36 0/36 9/32 1/32 1/32 0/32 0/36 0/36 0/36 0/36 3/36 2/36 0/36 0/36 0/34 0/34 0/34 0/34 5/36 0/36 0/36 0/36 0/36 0/36 0/30 0/36 1/36 0/32 3/36 1/36 0/34 0/36 1/36 4/36 2/30 3/36 2/36 3/32 2/36 2/36 0/34 4/36 0/36 0/36 0/36 3/36 0/36 0/36 0/36 0/36 0/36 0/36 0/30 2/30 0/30 1/31 1/30 0/36 1/36 0/36 0/36 0/36 0/36 1/36 0/36 2/36 0/36 1/32 0/32 0/32 0/32 3/32 0/36 3/36 1/36 0/36 0/36 0/36 1/36 0/36 0/36 0/36 0/34 0/34 0/34 3/34 0/34 .0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/30 0/36 0/36 0/32 0/36 1/36 0/34 0/36 0/36 0/36 0/36 0/36 0/30 0/30 0/36 0/36 0/36 0/36 0/32 0/32 0/36 0/36 1/36 0/36 0/34 0/36 1/34 0/36 0/36 0/36 1/30 0/36 0/36 0/32 0/36 0/36 0/34 0/36 0/36 0/36 1/36 0/36 0/36 0/36 0/36 0/36 0/30 0/30 0/30 0/30 0/36 0/36 0/36 0/36 0/36 0/36 0/36 0/36 1/32 1/32 0/3 2 0/32 0/36 0/36 0/36 1/36 0/36 1/36 0/36 0/36 0/34 0/34 0/34 0/34 0/36 0/36 0/36 0/36 0/36 1/36 0/36 0/36 0/30 0/30 0/36 0/36 0/36 0/36 0/32 0/32 0/36 0/36 0/36 1/36 0/34 0/36 0/34 0/36 0/36 0/36 0/30 0/36 0/36 0/32 1/36 0/36 0/34 0/36 0/36 1/36 0/36 0/36 0/36 0/32 0/36 0/36 0/34 0/36 a/ Sumbar of cats with tunors/tocal number of rats studied. EPL 0 0809 TABLE 10 SUMMARY OP LESIONS OTHER THAN TUMORS IN.RATS EXPOSED TO FILTERED AIR (CONTROL), VC OR VDC FOR 1 MONTH FOLLOWED BY OBSERVATION FOR 12 MONTHS!*/ Lesions^ Trea tment: Control 50 ppm VC 250 ppm VC Sex: Male Male F Male Rat No.: 365 366 367 368 435 439 440 481 512 513 514 515 Week After Exposure: 52 52 52 52 34 52 52 52 52 52 52 52 Skin _ InJ^lamat^on________ Lung _ Liver ________ Fatty degeneration _ _Fca_l j^flanmtlon_ Kidney u> _ Nephritis^__________ oo _2_ 2 12 2 3 a/ Includes only rats with lesions. b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - ve^y severe. EPL 00810 TABLE 11 SUMMARY OF LESIONS OTHER THAN TUMORS IN RATS EXPOSED TO FILTERED AIR (CONTROL) OR VC FOR 3 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS**/ Treatment: Control a/ Includes only rats with lesions. b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. EPl TABLE 12 SUMMARY OF LESIONS OTHER THAN TUMORS IH RATS EXPOSED TO VC FOR 3 MONTHS FOLLOWED BY OBSERVATION POR 12 MONTHS*?/ a/ Includes only rats with lesions. b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. EPL 00812 TABLE 13 SUMMARY OF LESIONS OTHER THAN TUMORS IN RATS EXPOSED TO VDC FOR 3 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS^ Lesions--^ Liver Fatty degeneration Kidney Nephritis Treatment: 55 ppm VDC Sex: Male F Rat No.: 666 667 668 670 671 707 Week After Exposure: 52 52 52 52 52 52 1 S t| 1 223 31 1 <N| f a/ Includes only rats with lesions. b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. EPL 00813 41 TABLE 14 SUMMARY OP LESIONS OTHER THAN TUMORS IN RATS EXPOSED TO FILTERED AIR (CONTROL) OR VC FOR 6 MOUTHS FOLLOWED BY OBSERVATION FOR 12 MONTHSg/ Treatment: Control *> to a/ Includes only rats with lesions. hi/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe, c/ During exposure. EPLi 00814 TABLE 15 SUMARY OP LESIONS OTHER THAN TUMORS IN RATS EXPOSED TO VC OR VDC FOR 6 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHSg/ a/ Includes only rats with lesions. bf Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. cf During exposure. EPL 00815 TAM.K I 6 SUMMARY (IK LKSjONS tlTJIKH 11IAN TUMORS IN RATS RXTIKijW TO fll/rKRKII AIM (COHTHOI.) PIHt 10 HtMIOS FOLMIUKO BY DBSKRVAT MIN TOR 12 MONTHS'?/ l.cainna b/ Rut No.: Ucek After Rx (insure ............. ... ... ...................... Hales ___ If. I 142 187 iiV 18J" 170 171 172 171 It II 14 4147 52 52 52 52 174 18B 1119 190 52 52 52 52 Heart f I bropj aB la LI ver Patty degenernt(on Focal InflaautIon Lliolangltln Telnni'lectJislB K Idney Nephritis Frostale I it [ 1 nnmnt I on t> Hesettr nry htf liiMuit Ion leafons4/ Rat No.: Meek After Kxpostire Fei*nle_ 197" 400 401 402 416 408 409 4 Ml 41 12 15 44 44 52 52 52 52 52 412 411 52 52 Heart FI hropineIa Liver Fatty tie Kent rat Ion PocuJ III! luiam.it loll Kidney Nephritis Adrenal Fatty degenerntIon Peritoneum Inf ItiuMtlffli____ I2I 2I 1 a/ Includes only rnln wllli lea Ions. Jt/ Severity of loulntin: I - telolM.il, 2 - moderate, 1 - nevere, and 4 - very nevere. EPL 00816 TABLE 17 - `,<r* f J ' ; - V' SUMMARY OF LESIONS OTHER THAN TUMORS IN RATS EXPOSED TO 50 PPM VC FOR 10 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS**/ Males af Includes only rats with lesions. b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. c( During exposure. EPL 00&17 TABLE 18 SUMMARY OP LESIONS OTHER THAN TUMORS IN RATO EXPOSED TO 250 PPM VC FOR 10 MONTHS FOLLOWED BY OBSERVATION FOR 12 MOHTHSg/ Males a/ Includes only rats with lesions. b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. EPL 00818 TABLE 19 SUMMARY OF LESIONS OTHER THAN TUMORS IN RATS EXPOSED TO 1,000 PPH VC FOR 10 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS**/ Males C' b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. EPL 00819 TABLE 20 SUMMARY OP LESIONS OTHER THAN TUMORS IH RATS EXPOSED TO 55 PPM VPC FOR 10 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS*/ Males b/ Severity of lesions: 1 - minimal, 2 - moderate, 3 - severe, and 4 - very severe. During exposure. EpL 00820 TABLE 21 THE NUMBER OF DEATHS AND EARLY TERMINATIONS AND TOTAL NUMBER OF MICE EXPOSED TO FILTERED AIR (CONTROL). VC OR VDC FOR 1. 3, OR 6 MONTHS FOLLOWED BY OBSERVATION FOR 12 MONTHS Month During Observation Period Control MF 50 MF vc. PPm__ 250 MF 1,000 MF VDC, pom 55 MF After exposure for 1 month 3-6 - - 6-9 9-12 01 l*/ 0 Total 1/16VT7T6 1 1 1 3/16 1 0 0 1/16 1 0 1 2/16 1 0 2 3/16 1 2 5 8/16 0 1 1 2/16 0 0 0 0/8 0 1 0 1/8 After exposure for 3 months During exposure 0 0 . 0-3 2 0 3-6 0 0 6-9 1 1 9-12 11 Total 4/16 2/16 0 _0 0 1 2 3/16 0 0 1 3 1 5/16 02 00 14 32 43 8/16 11/16 01 1 .o 60 04 03 7/10 8/10 0 1 0 2 1 4/8 0 0 0 1 0 1/8 After exposure for 6 months During exposure 0 0 0-3 2 0 3-6 1 1 6-9 2 0 9-12 14 Total 6/28 5/28 0 1 0 1 5 7/8 0111400 2 3336 11 2347211 4400010 010103 3 8/8 12/12 8/8 12/12 12/12 6/12 5/12 a/ Number of deaths and early terminations during the period, b/ Number of deaths and early terminations/total number of mice studied. EPL 00821 49 TABU 22 ' SUHHAH* OK TOHtWS IN HICK EXPOSED TO FH.TKRBI1 AIM (COHTHOI.) I HI VC BOB I HOHTIt FIH.I.UWKD BY OBSEBVATHW FOB II HOHTIIS^ Tini~^ Hinaatiiry Clund Adeiiucurc ! nuM/citrclntiMa lung bronchioloalveolar tiiaur HuitistaiJc utleaocrclnoM Liver tlti|iAioolhilar tutter lleaumgluaarcuiu Klduey IWHMlIglOIW Malignant Lymphuma Kidney and lyMpti node Treettae nt: Control 50 p rat VC Sea: ' Hale Kenale Hole Female House Ho.: 37) 377 378 379 182 186 748 419 441 442 441 451 452 475 484 492 491 496 Week Alter Exposure: 45 52 52 52 52_ 52 12 52 52 52 52 52 52.. 24 52 52 4 '2 2 11 1 I ' 11 12 12 4 *1 3 32 22 X 1 OLn Treatment: So: Hale ___250 ppm VC Fuata tu Tiiiuai a I/ House Hu.: 520 521 522 522 524 525 526 527 528 529 530 531 532 546 554 556 557 558 560 562 563 564 567 799 Muck Alter Exposure: 52 52 52 52 52.. 52 52 52 52 52 52 52 52 26 44 50 52 52 52 52 52 52 52 52 Heawtary Gland Muniicartliiiiaa ___________ 1 til's Hiontblojo.ilyeninrtumor___________ |_____ l t.lvur Hcp.<LoreMolar luaur H.il IgnanL lymplioam _______ l_ 1 J.lyer anj up]cun ______________ _ 222 11 1 u/ liu IuJen unly mice wllli tumors. b/ Tumor classification; 1 - bunion; 2 - mallgaunt, well-til f ferentfatcil; 3 - autlgnsut, moderutely tlif fcrentlated; 4 - malignant, poorly illfferentlnteil; X - pre-aunt. EPL 00822 TABLE 23 SIMHAkY Of THHOkli IH MICE EXPOSED TO VC 08 VDC POH l HOHTU POLUIVIEIt MV OBSEKVATltffl TOM 12 HOHTHS--^ TiiMora-^ lung bronrhlujou 1 vunlnr tuuur Liver llupetocul lular tuaor lleMung^owi ____ ___ Treatment: 1,000 ppm VO Sex: Hale Eenale House Ho.t 587 590 595 596 600 '601 601 604 605 606 607 622 624 625 626 627 628 629 6)0 6)1 Meek After Exposure: 27 31. 47 48 51_ 52 52_ 52 52 52 52 52 52 52 52 52 52 52 52 52 --------------------------- 1 2 _1- 2 1 _- _ 2 _ _1_ 2 2_ 1 1 _1_ - i _ 2 _ 1 _ _1_ 1 -- 11 2_ _ 1 2 -- L/ lunora- Trent will: i Sex: House Ho.; Meek After Exposure: 55 ppm VDC Male SSI 664 666 52 52 52 Lung Hrunclijoluatveular tuwir____ Liver llejinloeel 111 1 r Itieor Klilney LurclnoMn _ _ _ 1 I _ JL . I 3 j/ Includes only nice with tuners. 6/ Tuuor clues IfI cutInn: l - benign; 2 - anlignunt, uel1-dlfferentlated; 3 - malignant, Moderately differentiated; 4 - malignant, poorly differentiated. gpL 00823 TAUJ.tt 24 vtsiiHHAHif tty tuhoks in hii:b kxi-osko ri> Hi.Tmm aik (ammoi.) <ik vc. turn i mwrus yoi.uwnit ohsekvatiuh row 12 mouths^ bf Tumors Treatiieiii: Ctiiii ro I SO yc S<txs ____Mule_ _________________ Hale Penalc House Hu.: 710 712 733 440 460 462 464 465 475 477 478 km 4B1 497 501 779 780 781 783 Week After KNpnuuru; 52 52 52 10 52 52 52 52 52 52 28 33 36 41 52 52 52 52 52 Skin SqiiaKitid-i eH ____ HdiMury Cliiiiil Adenucardtiow/caictniMui Hyxoju ______ _____ __________ laing ftronchiuloujvoolsr tiwur llutastatlc aJej>ocarc]nou __ Uver Hepatocellular tuaur Hemangioma______________ _______ Spleen Huuuglcwa___ _________________ t.ympli Node Hetaang I u*a______ __________ __ _ Mesentet y-Perltontum UK)i lluunijltiaa i}uMityloearcuaa___________ HjHgnuiit l.ynphona Unk Idney and_1y^>Inode 1111 1 l 1 42 1 lI1 XX I1 Tumors-^ Treataantt Sea; House Ho. j Ueek After Eapoaure: _______ ______ 516 32 32 250 ppm VC Kale * Penale 519 531 534 535 786 787 788 791 792 793 547 552 550 551 553 BOO 802 803 804 805 806 38 40 41 49 51 52 52 52 52 ' 52 14 22 26 26 31 43 52 52 52 52 52 Skin fibrosarcoma __ liuiai^ludarcoMi ___ _ _ ______ _______ Mammary ClAOtl AJeiiucur^looma/carclnofU________ ___ iUDB ttronchiuluulvuular tumor 2 Metastatic adenocarcinoma_____________________ l.iver llepatocel lular tumor IJemangioear coma ___ _ ___ Mcseiii ery-Teri LutiJum {lumiHi^uBarcnM__________________ _ I 22 4 24 3 22 21 232 332 2 1 1 1 12 11 2 1 1 X 22%2 2 2 J2 22 a/ Inclmleu only mice with tumors. !/ Tumor classifJculIon: 1 - benign; 2 - malignant, wetl-JlffereutJated; 3 - malignant, moderately differentiated; 4 - X - present. ilignant, poorly differentiated; EPL 00824 TAttt.E 25 SUWART HR TUMORS JN MICK EKWtSKI) TO VC HR VOC FllR XMOMTIIS RIH.IjIIMKP BY OmKRVATItM FOR 12 MOUTHS^ Tumor Skin llcmanRluwi ijemi ng 1 ojiiircoma Hammary Claud AdenocercI noma/core Inoaa Ijmg Bronchioloalveolar tiawr HetaatatJc oateosarcoma l.lver Utt|tatnculltthr tumor Hemangioma 1 lemang1ouorcoma Trua latent: i.oon PPft VC Sex: Hale Female House Ho.: 608 592 596 597 598 599 609 811 812 555 620 621 631 636 635 821 823 826 Week After Exposure: 19 31 33 3X. 35 IX. 52 52 52 36 .38 38 39 61 61 63 52 52 --- -- -- -- ---- -- I . -- ---- 1 -- 22 3221 1 2223 1 1 2 21 11 21 17 1 OUlr Intorah / Treatment: Sex: Mouse Ho.: Week After Exposure: 55 ppm VDC_____ Ma la_____ _f_ 828 670 831 706 16 63 52 52 l.ung Si oiiLlilolua Iveulur tumor________________ Hcbcutury tlcmingI oanrcoma ___ _____ _______ 2l1 2 il/ Ificlmlfctf only mice with tunort, h/ Tumor cluaalfJcatlon; 1 - benign; 2 - ntllgoant, wel 1-ilifferentloted; 3 - uU|Huntj aoJerately differentiated; 4 - malignant, poorly dlfferentlauil) X - preboot SPL 0825 TABLE lb SUfMAKY OK 11MONS.IN NICE l-.Kl-iSJilI TO J'jJ.l'KKKn AJK jlSINTKOIJ, VC OKVI1C FI* Jt HOKTHS Fm.I.OUEII BY OIISKgWATlOH POM 12 HOHTIIS-^ * bf Tiiiiiu ru Treatment: Sex: ________________ ___________ CuntroI Hale____________ ___ Female Huuuu Hu.i 170 164 " 174 166 192 * 721 724 H17 405 406 421 421 401 424 426 764 768 Week After Ex|>usuru: 18 2| li 52 52 52 52 52 45 45 49 51 52 52 52 52 52 sk In Flbruenreuma Niiwury Clued Ailunucm 1 owi/u re t {wu Lung biunclylu loa l yen l ar turner liver llepatocel lular lluianngl uaurceaul ________ IncestInc AdenocareInows Uterus kuluKytesa .............. .................... Spleen [leaning luma ____ ____ Malignant lymphoma Liver, alumueliuml Intestine 1 is 1IIMI Id*/ Treetaunt: Sex: House Hu.: Week After Expuaure SO ppm VC Hale________________________F_e_m__a_l_e_ 55 ppm VPC Hale____ _____ ___ F 418 419 771 414 435 474 471 477 500 649 657 825 811 814 701 44 45 47 48 49 21 22 10 12 11 19 41 52 52 51 Skin Iteaungluau S<|nunouu-i:el I cnrclnomu____ Kausury III anil Ailenncsrc I noma /cureinome _ _________ _________________ Lung Bronchioloalveolar tumor HulusLutlc tiwur __ Liver llepatocel lular tumor Meaning 1 us:: r coma lymphangioma___ ____ Kidney Parr I nuaw ____ _ Mcuculery-IVrltonIurn llcmangluau Mcaningl nan maun Ha 11 gnnnt Lymph,uu Jteait, Ijvur, kidneyj spleen, lymph nude, ond/ornkln 1 1 111 2 2 4 4 2 4 1* 1 2 2 1 2 / Includes unly mice wltli tumors. {/ luaiur classification; 1 - benign; 2 - malignant, wel 1-dl ffereut I a ted ( 1 - mallgnaut, mnderutely dlffereiitluted; 4 X - pr e*.* nt . auil ignant, pour ly differ eu tinted; EPL, 00826 TAW.K i) suhhahy op thhuhs ih^hu-l mmsitii to w. hh> hhhtiis h.mwwd m oswatioh mi 12 huhtms^' Tuauru-^ Skin Hunungluiinretiaa .......................... Hnnuury 01 and Adunonut A<lenui:iiruljH>w.a/riirclnoa _ _ ______ Uni Bronchioloalveolar tunror Hetastntlc adciiircarcliiiHnu _____ liver {kweilearrcoM _ _____ _______ ___ _ IntentIne AdenoeareInoaa ___ ______ Hceenlery-Per1tunInu Nemang loan reoeur___ ^ Pituitary t:|iruieo;iltobu~cu j I adetruntn lUllun.ulL l.yatphonu mtn rung, liver, kidney, eyleen end jjrnyli node _Tenure-I*/ Skin Hearing leu llenungI ae arenas__ _____ Hommery (ilejid AJuoocnrc Inum/cnrelniim l.nitK Hronchloliratveul nr tuner fletagi.it 1c adcriucarc hionr l.lvet lle|intueul lnl.tr tnairr l{enuin|;J uun reaMd Treatnuntt Sum Hchim Ha. 1 Weak After Kxpaaure 250 pun VC Hula Tenuile 505 506 507 SOS 509 512 511 514 515 516 566 541 601 542 541 545 548 12 12 11 16 20 26 28 12 12 40 n' 5 9 10 14 20 25 'Is" 1 1.21 l t 2 2_ _ 4 _ _4 _ _4_ 2_ 2 1 1 12 1 I .2___ 1 _ _ 1_ 2 11 21 2 12 l 4 Treetaunt t 1.000 epa VC Sum Hale Peule Houm Mo. i 560 581 562 584 586 588 581 645 641 611 614 615 616 617 618 619 622 Week After Trentunit 14 14 17 21_ 22 21 40 24-^ 25|/ 569 9 1(1 11 14^ JB __________________ _4_________ l ______ - __________ ___ ________ _____________ 2_______________ _2_ 2_ _ 2 I121 121 2 111 12 1 I_ 8 1 2 2 _ _2 .. 1'. _ 2_ __________ 2 _ _ 1 _ 2_________2 2_ 2 2 2 n/ I m l mien on I y mice wllli tuaure. b/ I nnrti r tleenl 1 lent Iini: X - preeent. !/ Ihjl Ing expoiilire. I - benign; 2 - nu 11 gueiil. uel 1-dl ( ferunt latud; 1,- anllgnnnL, auderatuly differentiated; 4 - malignant, poorly different luted 00827 TABLE 28 CUMULATIVE TCMOft IMCIDEHCE Of MICE EXPOSED TO FILTERED AlK (COWTEOL) VC OR VDC FOLLOWED BT OBSEKVATIOB FOE 12 MONTHS Twr Llvar Hepatocellular tumor Hemangioma Hamangioaarcoaa Lymphangioma Control XF 50 ppm VC MF 250 oum VC 1.000 PDS VC 55 ppm VDC MTMFMF 10/S04/ 1/60 0/60 0/60 0/60 1/60 0/60 0/60 4/40 1/40 1/40 0/40 1/40 2/40 1/40 0/40 11/44 0/44 8/44 0/44 0/40 0/40 5/40 0/40 4/38 0/38 6/38 0/38 0/38 3/38 12/38 0/38 4/28 0/28 0/28 0/28 0/28 0/28 0/28 1/28 Lung Bronchioloalveolar tumor Mata*tails tunorj Manurv Cland Adenoma Adanoearcinoma/eareinoma Myxoma Malignant Lymphoma Haart, lung, llvar, aplaan, kidney. lymph noda, atonach. lntaatlna and/or kin Pituitary Chromophobe-call adasosa Skin Spuanoua-call sarelsona Fibroaarcona Hemangioma Hanangloaarcoma 8/60 0/60 8/60 11/40 0/60 0/40 6/40 29/44 23/40 27/38 23/38 4/40 0/44 2/40 0/38 2/38 0/60 0/60 0/60 0/60 A/60 0/60 7 2/60 0/60 0/40 0/40 0/40 0/40 10/40 1/40 2/40 0/40 0/44 0/44 0/44 1/40 13/40 0/40 1/44 1/40 0/38 0/38 0/38 0/38 6/38 0/38 0/38 *0/38 ' w` 0/60 - .% 0/60 1/60 0/60 0/60 0/60 0/60 0/60 0/60 0/60 0/40 0/40 0/40 1/40 0/40 0/40 1/40 0/40 0/40 0/40 0/44 0/44 1/44 0/44 0/44 1/40 0/40 0/40 0/40 3/40 0/38 0/38 0/38 0/38 0/38 0/38 0/38 0/38 0/38 2/38 4/28 1/28 0/28 0/28 0/28 0/28 0/28 1/28 0/28 0/28 0/28 1/28 0/28 0/38 0/28 0/28 1/28 0/28 0/28 0/28 0/28 0/28 lntaatlna Adanocarcinoma 1/60 0/60 0/40 0/40 1/44 0/40 0/38 0/38 0/28 0/28 Kidney Carclnona Baaanglona 0/60 0/60 0/40 0/40 0/44 0/40 0/38 0/38 2/28 0/28 0/60 ' 1/60 0/40 0/40 0/44 0/40 0/38 0/38 0/28 0/28 Utaruc Lleomyoma 0/60 2/60 0/40 0/40 0/44 0/40 0/38 0/38 0/28 0/28 Splaan Baaanglona 0/60 1/60 1/40 0/40 0/44 0/40 0/38 0/38 0/28 0/28 Lymph Noda Haaangloaa 0/60 0/60 0/40 1/40 0/44 0/40 0/38 0/38 0/28 0/28 Maaentary-Parltoniua Hanangiona Hanangloaarcoma 0/60 0/60 0/60 0/60 0/40 1/40 2/40 1/40 0/44 2/44 0/40 2/40 0/38 0/38 0/38 0/38 0/28 1/28 o/;s 0/28 a/ Sumbar of mica with tunor*/total number of mice atudled. EPL 00828 TABLE 29 SURVIVAL AND MORTALITY OF MICE EXPOSED TO VINYL CHLORIDE WITH OR WITHOUT DISULFIRAM FOR 49 WEEKS VC (EPg) 0 50 250 1,000 Diet -DS +DS -DS +DS -DS +DS -DS +DS Survival After 49 Weeks Male Female 6/8 (75)--^ 8/8 (100) 8/8 (100) 6/8 (75) 6/8 (75) 3/7 (43) 5/8 (62) 5/8 (62) 3/8 (38) 8/8 (100)c/ 0/8 (0) 7/8 (88)^ 0/8 (0) */8 (75)/ 0/8 (0) 3/7 (43) Mean Week of Death or Termination^/ Male Female 47 49 49 45 43 46 44 43 42 33 49 48 27 34 43 43 / Study terminated after 49 weeks. Number of survivors/ number of exposed (% survivors). Significantly different from corresponding VC exposure without DS. |n |oJ|p EPL 00829 57 TABLE 30 SUMMARY OP TUMORS IN MICE EXPOSED TO VINYL CHLORIDE WITH OR WITHOUT PISULFIRAH Tumor Bronchioloalveolar adenoma Hemangiosarcoma (liver and/or mesentery) Mammary gland--^ Diet -ns +DS 0 Male Female 0/4 (oW 0/8 (0) 1/8 (12) 0/6 (0) Vinyl Chloride (ppn) '50 250 Male Female Male Female 1.000 Hale Female 1/6 <17) 0/6 (0) 6/6 (100) 4/5 (80) 4/5 (80) 5/5 (100) 1/4 (25) 1/5 (20) 7/8 (88) 4/8 (50) 7/7 (100) 4/4 (100) -DS 0/4 (0) 0/8 (0) 1/6 (17) 1/6 (17) 5/6 (83) 2/5 (40) 4/5 (80) 3/5 (60) +DS 0/8 (0) 0/6 (0) 0/4 (0) 0/5 (0) 2/8 (25) 0/8 (0) 3/7 (43) 1/4 (25) * -DS 0/4 (0) 0/8 (0) 0/6 (0) 2/6 (33) 0/6 (0) 2/5 (40) 0/5 (0) 3/5 (60) +DS 0/8 (0) 0/6 (0) 0/4 (0) 0/5 (0) 0/8 (0) 1/8 (12) 0/7 (0) 0/4 (0) a/ Number of nice with tumor/number of mice examined (Z with tumor). b/ Tumors composed of ductular adenocarcinoma, aquamous-cell carcinoma, anaplastic-cell carcinoma and/or hema nglosarcoma. 1 ';,TAW.K Hi ' ' - : .-'r f * ' KKKKCT Of IHSUI.KIKAH (JUS) ANII VINYI. Cjll/MHlt>K (VC)OHTOK CUHtHKlThM AHI) KN/.VMK AtnlVm (IK TIIK IIKPATIC SBlXrril KNMIPI jlSHIlf AKTliaiUlM IN HA1.K. MICK l.lvcr Vli-lj-lil (Z Ixuly w<-l);lil ) I'cnlriil Diet____ _________ vc (pi*).......... ........... >U 250 i.7 I O.zV 6.6 ( 0.2ll/ 6.4 I II.l*'i 4.4 + ((.!! l*S lllr-l___ VC (p|) 111 4. *1 t II. 6,/ 250 4.4 I 0.7 HIcromiKil prut cl ii (nift/p liver) Cytiiclirnac 8-440 (iMMilu/ng pruletn) 19.11 + 11.9 14.?.+ 1.6 11.8 + 1.9 1.17 + 0.01 11.9(1 + 0.06^ 1.14 + 11.07 14.4 + Cl. 7 II. I I II. 1 12.9 + 1.6 11.87 + O.liz,!1*^./ II.Uh I O.ltt1'^ 1.02 + 0.01 Cfliirliriiw (nwile/aK pruteln) 0.41 + 0.1)1 11.14 + 11.010.40 + 0.04 0.44 + 0.01 0. 18 + 0.02 0.48 + 0.02 Anllliic liyili oxy Inac (ole I'lll'/ni; prote In/aiin) 1.118 + 0.01 0.94 + 0.02 0.97 + 0.10 0.29 + U.04l,/ I). 17 + (l.llfcl'-W 0.49 + 0. lO*1'!^ Cn VO KlbyImnrplilnc-N-dcnctliylane 8.6 1 0.2 9.1 I 0.8 9.6 + 0.6 5. I + II. O1^ 11.6 ( O. 1I'iKiJ^ 10.6 + O.?1-''1^ (nwiile IICII0/M|t prolelii/olii) p-NItroiifllHtilc-IK-dcinelliylUHC (mile PNI'/np prot eln/ialii) 4.1 + 0.1 4.6 I (I. I 4.7 + 0.I 1.4 + O. lhl 4.4 I 0. l'1^ 4.9 + 0.4d/ / All values Indicate Mean I- S,K. f two td nix dote ml nut limn, 6/ Significantly different frn alee rui'ulvlnx roniml dint alone* p * 0.00. c / SiftnlIIruntly different frnp leu nut fed OS mid ex|Hnud to nmw funiontnitlon of VC, P * </ SI Knl Hi.nit ly different from mUv reel!IvIi>k IMS dirt nlnm | * 0,OS. o83l TABLE 32 EFFECT OF DISULFIRAM (PS) AND VINYL CHLORIDE (VC) OH THE COMPOSITION AND ENZYME ACTIVITY OP THE HEPATIC SMOOTH ENDOPLASMIC RETICULUM IH FEMALE MICE t Liver weight (% body weight) Microsomal protein (mg/g liver) Cytochrome P-450 (nmole/mg protein) Cytochrome b^ (nmole/mg protein) Aniline hydroxylase (nmole PHP/mg proteln/min) Ethylmorphlne-N-demethylase (nmole HCHO/mg proteln/min) jv-Nitroanlsole-O-demethylase (nmole PNP/mg proteln/min) Control Diet ___________vc <PP) ' 0 50 4.8 + 0.`1-/ r , .; 5.8 + O.lk/ 16.0 + 0.5 15.6 + 1.7 1.07 + 0.03 0.44 + 0.01 0.90 + 0.11 0.36 + 0.01-^ 1.15 + 0.05 1.09 + 0.05 8.4 + 0.2 11.8 + 2.0 5.4 + 0.2 6.5 + 0.6 DS Diet VC (ppm) 0 50 5.0 + 0.2 5.3 + 0.2 14.9 + 0.5 0.81 + 0.02--^ 13.8 + 0.5 0.94 + 0.07 0.38 + 0.01 0.44 + 0.02--/ 0138 + 0.02k/ 3.9 + 0.1^/ 0.66 + 0.05-*--*" 11.9 + o.3k*k/ 3.5 + 0.3k/ 6.1 + 0.3k/ a/ All values Indicate mean + S.E. of two to six determinations, b/ Significantly different from mice receiving control diet alone, p < 0.05. cj Significantly different from mice not fed DS and exposed to same concentration of VC, p < 0.05. d/ Significantly different from mice receiving DS diet alone, p < 0.05. EPL 00832