Document 8oMNOmGvM9Z1nz139rkm7Ye

TOXICOLOGY, CAP.CIMOGENICITY, AMI REPRODUCTIVE EFFECTS OF SINGLE AND MULTIPLE EXPOSURES TO VINYL CHLORIDE IN RATS AND MICE (A pre-publication release as of 1 April 1979) This pre-publication release reports a research project sponsored by the Consumer Products Safety Commission under an interagency agreement (1974, 1975-1979) with Chemical Systems Laboratory (formerly Edgewood Arsenal), Aberdeen Proving Ground, Maryland. B.P. McNAMARA, Ph.D. Chemical Systems Laboratory PROJECT OFFICERS: JOSEPH MCLAUGHLIN, JR., Ph.D. Consumer Products Safety Commissi on AUTHORS OF RELEASE; ROBERT M. HEHIR, Ph.D., Consumer Products Safetv Commission GEORGE BIERBOW'ER, D.V.M., Consumer Products Safety Commission DONALD A. WILLIGAN, D.V.M., Ph.D., Donald A. Willigan, Inc., GERALD IfOLAJA, MAJ., D.V.M., Ph.D., Chemical Systems Laboratory GLEN E. MARRS, JR., MAJ., D.V.M., Chemical Systems Laboratory DAVID E. HINTON, Ph.D., Univ. of West Virginia Medical School RICHARD L. DIMM1CK, Chemical Systems Laboratory JOSEPH S. WILES, Chemical Systems Laboratory SL 096599 SUMMARY REPORT I, INTRODUCTION II, EXPERIMENTAL PROCEDURE A, Exposure Procedures for Toxicology and Carcinogenesis 1. Doses 2, Exposure and Sampling Technique 3, Observation 4. Pathology a. Gross and Light Microscopy b. Electron Microscopy B, Reproduction Study and Carcinogenicity III, RESULTS A, Toxicology (single and multiple exposures) 1. Observations During Exposure 2, Observations After Exposure B, Gross Pathology C, Light Microscopic Pathology 1. Mice (single exposures) 2. Mice (10 exposures) 3. Mice (100 exposures\ 4. Rats (single exposure) 5. Rats (multiple exposures) 6. Rats (49 exposures from the reproduction study) 7. Summary of Light Microscopic Studies D, Electron Microscopy Study E, Multigeneration Study in Rats 1 SL 096600 IV. DISCUSSION A* Factors in rcinogentcity B. Vinyl Chloride Carcinogenicity V, CONCLUSIONS VI. Appendix SL 096601 Toxicology, Carcinogenicity ^nd Reproductive Effects of Single and Multiple Exposures to Vinyl Chloride in Rats and Mice * * I. INTRODUCTION. The background information on vinyl chloride monomer (_VCM)_ with technical details is reviewed in "Scientific and Technical Assess ment Report on Vinyl Chloride and Polyvinyl Chloride", US Environmental 1 Protection Agency, EPA-600/6-76t004, June 1975, VCM studies in rats and mice reported herein were conducted at the Chemical Systems Laboratory (CSL) under interagency agreement With Consumer Products Safety Commission (CPSC). II, EXPERIMENTAL PROCEDURES. A, Exposure Procedures for Toxicology and Carcinogenicity, 1, Doses. Male and female rats (Fischer 344) and mice (A/J or ICR) were? totally exposed in one of the following ways: t a. One single one-hour exposure at 0, 50, 500, 5,000 or 50,000 ppm. (Fischer 344 rats, ICR Mice). (Appendix, Table I) b. Ten one-hour exposures at 500 ppm (one-hour per day, five days per week for two weeks). (Fischer 344, A/J Mice) (Appendix, Table II) c. One-hundred one-hour exposures at 50 ppm (one-hour per day, five days per week for 20 weeks). (Fischer 344 rats, A/J Mice) 2. Exposure and Sampling Techniques. The exposures were conducted in 1000 liter, stainless steel dynamic flow chambers of the Rochester type. Construction insured laminar flow and uniform exposure of the test animals. During one-hour exposure periods four 0.5 cc gas samples were collected at selected times for concentration analysis, A Hewlett-Packard series 5830A Reporting Gas Chromatograph equipped with a dual-flame ionization detector was used for VCM analysis. 1 SL 096602 The system is sensitive to 60 parts per billion of vinyl chloride. Following exposure the animals were air-washed in the chamber until less than 1 ppm of VCM was detectable. They were then placed in the animal holding area for the remainder of the observation period (up to 24 months). 3. Observation. Animals were observed twice daily for indications of their general health. The following signs were noted: mortality, health, external sores, subcutaneous masses, alertness, and activity. All test groups were weighed weekly for the first eight weeks post exposure and monthly thereafter. No blood chemistry or hematology studies were performed. ,n 4. Pathology. a. Gross and Light Microscopy. A complete gross pathological examination was performed on most control and exposed animals which died or were sacrificed. Autolysis precluded such examination in a few cases. Light microscopic examination was made of the following tissues: lung, trachea, heart, liver, stomach, small intestine, large intestine, spleen, kidneys, bladder, bone marrow (sternum), adrenals, pancreas, duodenum, brain (2 sections), pituitary, spinal cord (cervical), skin, thyroid, uterus-ovaries, eye, ear, nose, muscle and bone (femur). Particular emphasis was placed on examination of brain, lung, liver and the Zymbal gland in the rodent ear. All rodents were to be serially sacrificed at 8, 16, 24 months post exposure. However, the life span of the mice forced some changes in the later times of sacrifice and termination of mouse experiments. For the single exposure the planned 16 and 24 month sacrifices were replaced by 2 SL 096603 Xl an 18-month sacrifice. The latter is minimum suggested by the National Cancer Institute for cancer bioassays in small rodents. For the multiple dose studies (500 ppm x 10 exposures; 50 ppm x 100 exposures) in mice the final sacrifice was at 20 months rather than 24 months. The change was made in consideration of the risk of animal loss through death and possible cannabolism. b. Electron Microscopical Studies. For the electron microscopical studies the following animals were selected at random: Groups consisting of five males and five females Fischer 344 rats from each of the single one-hour (50, 500, 5000 and 50,000 ppm) ex posures with equal numbers of their corresponding control group. The groups were sacrificed at 8, 16 and 24 months, respectively, after the exposure. Groups consisting of five males and five female Fischer 344 rats from each of the multiple one-hour exposures (10 x 500 oom, 100 x 50 ppm) with equal numbers of their control group. The groups were sacrificed at the end of 16 and 24 months, respectively, after the final exposure. One group of five male and five female Sprague/Dawley/Wistar rats from the parenteral generation of the reproduction group (49 x 50 ppm, 49 x 500 ppm) with equal numbers of their control group. This group was sacri ficed 24 months after the final exposure. The rats were anesthetized with pentobarbital and their livers were perfused via the vena cava with chilled electron microscopical fixative 2 consisting of 4% formaldehyde, \% glutaraldehyde in 0.1 M phosphate buffer (E.M. McDowell & B.F. Trump, Histological Fixatives Suitable for Diagnostic Light and Electron Microscopy, Arch, Path. Lab. Med. 100:405-415, 1976). After mincing, the tissues were post-fixed in 1% phosphate buffered 3 SL 096604 osmium tetroxide, dehydrated in graded alcohol solutions, cleared in propylene 3 oxide and embedded in Epon (J.H. Luft, Improvements in Epoxy Resin Embedding Methods, J. Biophys. Biochem. Cytol, 9:409-414, 1961). B. Reproduction Study and Carcinogenicity. Fq male and female parents Sprague/Oawley/Wistar Rats were exposed to 50 ppm or 500 ppm of VCM one-hour per day, five days per week for 10 weeks (49 exposures) before they were mated. (Appendix, Table III) This assured exposure of all forms of male germ cells. The females were exposed during all phases of the oogenic cycle. The parents were evaluated for numbers of matings, percentages of pregnancies, fertility and lactation indices. The and F^ off-spring were evaluated for litter-size, percent of stillborn pups, post-natal growth, viability, survivability and reproduction anomalies. The parenteral generation of Sprague/Dawley/Wistar Rats were maintained for 24 months post exposure for carcinogenic evaluation. HI. RESULTS. A. Toxicology (single and multiple exposures). 1. Observation During Exposure. Exposures of rats and mice for one-hour to concentrations of 50, 500, 5,000, and 50,000 ppm produced no remarkable signs of toxicity 'except for mice exposed to the highest level. Fifty percent of the males were hyperventilating after 45 minutes of exposure; twitching and possible 4 SL 096605 .t i ataxia were noted. At 59 minutes tremors were seen. Females showed some hyperactivity at 40 minutes and 25% showed respiratory difficulty and ataxia after 55 minutes. No other effects were noted. There were no remarkable signs of toxicity during the ten repeated exposures of mice and rats at 500 ppm of VCM, nor in rats during the 49 exposures at 500 ppm, nor in either species during the 100 exposures at 50 ppm. 2. Observations After Exposure There were no consistent or dose-related differences between control and exposed (single or multiple) mice or rats in: death rate, (Appendix, Fig I IX, inclusive) toxic signs, gain in body weight. (Appendix, Figs X-XVIII inclu: B. Gross Pathology There was a suggestion of a higher frequency of masses in the lungs and livers of mice and rats exposed once or repeatedly at the higher dose levels. (500, 5,000, 50,000 ppm) C. Light Microscopic Pathology 1. Mice (single exposure) Histological examination at 8 and 18 months in ICR mice exposed \ once to concentrations of 50, 500, 5000 or 50000 ppm showed the following chanc attributable to VCM. (Appendix, Table IV-VI, inclusive) The development of 'lunt adenomas increased with exposure to higher dose levels of vinyl chloride: control - 12/120 or 10% 50.000 ppm - 45/137 or 32.8% 5.000 ppm - 24/143 or 16.3% 500 ppm - 18/139 or 12.9% 50 ppm - 14/139 or 10.1% Progression to carcinoma was minimal: 5 SL 096606 t1 control - 0/120 or 0% 50.000 ppm - 3/137 or 2.2% 5.000 ppm - 1/143 or 0.7% 500 ppm - 1/139 or 0.7% 50 ppm - 0/139 or 0% Pneumonitis was evident in all animal groups which were exposed to VCM in doses of 500 ppm or more. 2. Mice (10 exposures) Changes attributable to VCM in A/J mice exposed ten times at 500 ppm examined at 8,16 and 20 months are as follows (Appendix, Tables VII-X, inciusi-. The induction of pulmonary adenomas: controls - 31/90 or 34.4% 500 ppm - 124/166 or 74.7% Progression to malignancy (carcinoma) in the test group was greater than in the controls: controls - 3/90 or 3.3% 500 ppm - 22/166 or 13.3% 3. Mice (100 exposures) Marginal increases in the occurrence of adenomas of the lungs were attributable to VCM in A/J mice exposed 100 times at 50 ppm and evaluated at 8, 16 and 20 months. (Appendix, Tables VII-X, inclusive) controls - 29/84 or 34.5% 50 ppm - 65/158 or 44.1% Progress to malignancy (carcinoma) was apparent: controls - 2/84 or 2.4% 50 ppm - 7/158 or 4.4% 6 SL 096607 4. Rats (single exposures) 1 Except for aggravation of latent pulmonary changes, particularly bronchopneumonia, changes attributable to VCM were not apparent in any of the tissues evaluated microscopically at 8, 16 or 24 months from Fischer 344 rats exposed to concentrations of 50, 500, 5000 or 50,000 ppm. (Appendix , Tables XI-XIV, inclusive) 5. Rats (multiple exposures) No changes attributable to VCM were apparent in any of the tissues evaluated microscopically at 8, 16 or 24 months from Fischer 344 rats exposed ten times at 500 ppm or 100 times at 50 ppm. (Appendix, Tables XV-XVII, inclusive) 6. Rats (49 exposures from the reproductive study) The following histological observations were made 24 months post exposure in Sprague-Dawley/Wistar rats which had been exposed 49 times at 50 ppm or 500 ppm of VCM. (Appendix, Table XVII) Neoplastic and non-neoplastic lesions were observed in approxi mately ; ;al frequency in control and test animals. The only lesions that occurred in higher frequency in ,:s VCM exposed animal than in control rats were eosinophilic cell alterations presented as foci and/or areas. The appearance of these foci was related to dosage. The nature of these lesions is controversial. Some pathologists feel that basophilic lesions have greater significance with respect to neoplasm development than do other cellular alterations. Others feel that all of these cellular alterations may be part of a spectrum capable of progressing to the formation of neoplastic nodules. 7. Summary of Light Microscopy Studies The carcinogenic, or possible related changes (eosinophilic foci) attributable to exposure of VCM in rats and mice are summarized in Table 1. 7 SL 096608 TABLE 1. VINYL L.,uORIDE STUDIES Experimental Group* PPM DAYS SUMMARY ' SPECirs/NUMBER" EXPOSED (1 hr/day) (ppm-hrs) RESULTS A 50,000 1 50,000 ICR Mice 180 33% adenomas Fischer Rats 178 negative C 500 49 24,500 Sprague-Dawley/ 49 eosinophilic Wistar Rats changes A 5,000 1 5,000 ICR Mice 180 16.8% adenomas Fischer Rats 180 negative B 500 10 5,000 AJ Mice 180 74% adenomas Fischer Rats 180 negative B 50 100 5,000 AJ Mice 179 41% adenomas Fischer Rats 180 negative C 50 49 2,450 Sorague-Dawley/ 47 Eosinophilic Wistar changes A 500 1 500 ICR Mice 180 like controls Fischer Rats 190 negative A 50 1 50 ICR Mice 180 1 ike controls Fischer Rats 180 negative TOTAL EXPOSED TOTAL "CONTROL COMBINED TOTAL 2263 7% 3039 *A - 170 ICR Mice (82 male and 88 female) and 17T Fischer Rats (92 male and 79 female) served as control animals for the single exposure (50,000; 5,000; 500; 50 PPM) studies. B - 190 AJ Mice (89 male and 101 female) and 200 Fischer Rats (100 male and 100 female) served as control animals for the multiple exposure (10 X 500 PPM and 100 X 50 PPM) studies. C - 45 Sprague-Dawley/Wistar (20 male and 25 female) served as control animals for the multigeneration reproduction study. SL 096609 fX 0. Electron Microscopical Results. 1. General. In general, these studies indicate that exposure to vinyl chloride increased organelle turnover (Fig. XIX) as well as loss of volume control (bleb formation) and increased lysosomal activity in the liver of rats (Figure XIX). These alterations progressively decreased as recovery time after exposure increased. Hepatocellular carcinoma was seen in one male Fischer rat which had received ten exposures of 500 ppm. Lymphosarcoma was noted in one female Fischer rat which had received a single exposure at 500 ppm. Since these were individual cases, and since no cancers were seen at 50,000 ppm, the lymphosarcoma and the hepatocellular carcinoma are not likely related to the vinyl chloride exposure. Thus, it appears that exposure to vinyl chloride did not produce cancer in rats in any of these single or multiple exposures. General conclusions concerning these various segments of the electron microscopical results follow. "\ 2. General Conclusion of 8-Month Recovery to Single Exposure 50, 500, 5000, 50,000 ppm-hr) of Vinyl Chloride in Fischer Rats. a. Alterations from control (Fiq. XVIII) occurred in all treated animals at each concentration. b. Changes were less severe in female animals. c. Hepatocytic alteration included lipid accumulation (Fig. XIX), increased dense bodies (Fig. XX), lipofuschin granules (Fig. XX) and residual bodies. These are an indication of cytoplasmic sublethal injury. d. Alteration was incremental with increasing exposure concentra tion. e. Most severe changes involved cellular necrosis with subsequent phagocytosis by Kupffer cells.- (Fig XXI) seen in single and multiple exposures. 9 SL 096610 f. Extruded areas of hepatocyte cytoplasm (in bleb formation)(Fig. x may indicate a means of removal of altered portions of cells. 3. General Conclusion of 8-Month Recovery to Multiple Exposures (500 ppm X 10 exposures; 50 ppm X 100 exposures) to Vinyl' Chloride in Fischer Rats. a. Changes were seen in male and female treated animals. (Fig. XXI) b. Less alteration was encountered in female rats. c. Cellular changes was greater than that seen in single dose animals. d. Alterations involving hepatocyte nuclei, not seen in single dose animals, were encountered in males of this group. 4. General Conclusion of 16-Month Recovery After Single Exposure T50, 500, 5000, 50,000 ppm x 1 hr) to Vinyl Chloride in Fischer Rats. a. Normal morphology (Fig. XXII) was noted after 16 months in the lower exposure concentrations. When compared to the above alterations at 8 months the 16 months recovery period appeared to be sufficient for return to control morphology. b. When changes in controls were subtracted from those in treated animals, the most significant finding was hepatocyte necrosis in high dose males. 5. General Conclusion of 16-Month Recovery to Multiple Exposures (50Q ppm X 10 exposures; 50 ppm X 100 exposures in Fischer Rats. a. Electron microscopic evidence supportive of a diagnosis of hepatocellular carcinoma was seen in one male given 10 doses of 500 ppm vinyl chloride. (Fig. XXIII) b. Other changes included cleft-like spaces in hepatocytes and Kupffer cells. These were interpreted as age-related changes in hemoglobin/ hemosiderin metabolism. c. Changes were more severe in males. 10 SL 096611 6. General Conclusion to 24-Month Recovery from Single Dose ISO, 500, 5000, 50,000 ppm)`Vinyl Chloride in Fischer Rats, a, . Age-related changes including formation of cleft-like spaces^^ (possibly related to hemoglobin/hemosiderin), inflammatory infiltrate (pericholan gitis) and some collagen accumulation were seen in controls (Fig. XXIV) and treated animals in this group. b. In this group hepatocytic injury - not encountered in controls - was observed. At 5000 ppm one female showed evidence of lymphosarcoma. 7. General Conclusion to 24-Month Recovery After Multiple Doses (50 ppm X 10) of Vinyl Chloride in Fischer Rats. a. Controls and treated animals showed changes similar to those seen after 24 months recovery to single exposure. b. These changes were regarded as age-related and non-spe<$ificlFig. 8. General Conclusion to 49 Exposures for 1 Hr at Daily Intervals Followed by 24 Month of Recovery (50 and~500 ppmTTSPrague/ .Pawley/Vlistar Rats). a. Age-related non-specific change was encountered in all grcupj b. Most advanced changes were related to 500 ppm group of malei rats in which cell swelling and platelet aggregation were seen. E. Multigeneration Study in Rats (Sprague/Dawle.y/Wistar). No consistent changes attributable to VCM were found in Fq parents which were exposed to 50 ppm or 500 ppm of the vinyl chloride monomer one hour per day, five days per week for ten weeks before mating and evaluated for numbers of matings, percentages of pregnancies, fertility and lactation indices. The F.j, F^, and F^ offspring were evaluated for litter size, percent of stillborn pups, post-natal growth, viability, survivability and reproduction anomalies. (Appendix, Table XIX - XXV, inclusive) Electron microscopical examination of the parent rats which were he for 24 months after the 49th exposure revealed age related changes, cell swelling and platelet aggregation but no tumors,(Fig. XXV).' n SL 096612 IV. DISCUSSION. A. Factors in Carcinogenicity. Theoretically, a single molecule of a carcinogenic substance may produce a cancer-if it is not destroyed in the body before it reaches a suscept ible body cell, if it makes a carcinogenic biochemical combination ("hit") with the cell, if this combination is not repaired, if the cancer cells are not de stroyed by the immune system, and if other host factors are favorable to carcinogenicity. On the other hand it may be assumed that not every molecule will make a carcinogenic "hit", that some "hits" will be repaired, and some "hits" may not develop into tumors because of unfavorable host factors, and in some situations the cancer cells may be destroyed by the bodies immune system. The higher the dose rate the greater the number of hits and the greater the frequency of tumor production. The above considerations suggest the existence of "no effect" doses, threshold doses, and dose response curves which have the hockey stick shape 4 described by Bryan and Shimkin. These considerations have been discussed for radiation 5 ' 6 and chemical carcinogenesis 7 ' 8 by numerous authors. The Food Protection Committee, Food and Nutrition Board of National 9 Academy of Sciences - National Research Council noted that dose-response re lationship applied to carcinogens. The higher the dose, the greater is the incidence of response and the shorter the time required to elicit the response. 4 This relationship has been reported for 1,2,5,6,-dibenzoanthracene (DBA) , 20-methylcholanthrone (MC) 12 , and DMBA plus croton oil 12 , p-dimethylaminoazobenzene 13 , and carbon tetrachloride. 14 Dose response curves have been given for ultraviolet light^ and ionizing radiation.^,17. There are non-tumorigenic levels of exposure to carcinogens for given experimental conditions. Carcinogens do not produce cancers in all exposed animals. In bioassays, the lower dose levels sometimes do not produce tumors while higher dose do produce tumors. 11 ' 17 ' 12 SL 096613 r The possibility is recognized that "no effect" doses may exert carcinogenic effects which are too weak to be detected with the numbers of animals used in routine testing. The concept of lifetime accumulative, non-tumorigenic and tumorigenic doses of radiation has been adopted. The Federal Radiation Council 13 stated that for occupationally exposed personnel the accumulated dose of radiation to the whole body, head, trunk, active blood forming organs, gonads or lens of the eye shall not exceed: 1. In any calendar quarter, 1.25 roentgen equivalent mammal; (rem) 2. Total lifetime dose of 5 (N-18) rem where N equals the present age in years. B. Vinyl Chloride and Carcinogenicity. Maltoni described a dose-response relationship for the carcinogenic effect of vinyl chloride in animals. The neoplastic response was related to the length of exposure. 19 ' 20 Lee et aK, 21 noted that the incidence and severity of tumors increased with the concentration of VC and the length of exposure. The above statments indicated that the total dose (concentration X exposure time) may be of importance in the carcinogenicity of vinyl chloride. Total inhaled dose can be approximated by the Haber concept. 22 In its simplest form this concept states that the total inhaled dose, Ct (mg min/cu m) is the product of C (concentration in mg/cu m) X t (time in minutes). 13 SL 096614 The concentration can be expressed also in part per million (ppm) and the time can be expressed in hours producing Ct in ppm-hr. Factors for breathing rate and detoxication can be included when these data are available. However, the simplified Ct approximation of total inhaled dosage is sometimes useful. A rough calculation of total dosages has been made for some of the data of Maltoni ^'20; Lee et al_. Viola et al_. Caputo et al. and Keplinger et al. 25 These Ct calculations are summarized in Tables 2 and 3. In the studies of P.L. Viola, A. Bigotti and A. Caputo 23 tumors were seen in rats which had been exposed to 30,000 ppm of vinyl chloride four hours per day, five days per week for 12 months. Positive effects were obtained at the total dose (CT) of 28,800,000 ppm-hrs. A. Caputo, P.L. Viola and A. Bigotti 24 exposed rats to vinyl chloride four hours per day, five days per week for 12 months. The con centrations were 20,000; 10,000; 5,000; 2,000; 500; or 50 ppm. The total dose (Ct) for the 50 ppm was 48,000 ppm-hrs. No tumors were produced at this level. The total dose for 500 ppm was 480,000 ppm-hrs. Tumors did occur at the latter dose appearing as early as eight months was 320,000 ppm-hrs. Tumors appeared in the rabbits after nine months at 10,000 ppm. Thus the lowest total dose was 7,200,000 ppm-hrs. M.l. Kepi inger et , 25 exposed rats, hamsters and mice to vinyl chloride. Only the data on mice was sufficiently complete for examination of total dose effects. The animals were exposed seven hours per day, five days per week for eight months. The lowest Ct was 56,000 ppm-hrs. This Ct and all higher ones did produce tumors in mice. 14 SL 096615 TEST BT1 BT3 BT6 BT7 BT4 TABLE 2 MALTONI VINYL CHLORIDE STUDIES Calculations based on reference 19 SPECIES RESULTS (CARCINOGENESIS) - PPM-HRS RATS RATS RATS RATS MICE QUESTIONABLE AT 52K* NEGATIVE AT 17K QUESTIONABLE AT 85K POSITIVE AT 170K, 850K, 2000K, 3000K POSITIVE AT 24.600K NEGATIVE AT 52K, 260K QUESTIONABLE AT 520K POSITIVE AT 2600K, 6200K, and 10.400K POSITIVE AT 30K, 150K, 300K, 600K, 1500K and 3600K K* = thousand (000) 9T9960 TS 15 TABLE 3 OTHER VINYL CHLORIDE STUDIES t AUTHORS VIOLA, BIGOTT1,23 CAPUTO CAPUTO, VIOLA,24 ' BIGOTTI KEPLINGER et al.,25 LCE ^ CONSUMER PRODUCTS SAFETY COMMISSION SPECIES RATS RATS RABBITS MICE (RATS ft HAMSTERS) MICE ICR MICE A/J MICE FISCHER RATS RESULTS (CARCINOGENESIS) - PPM-HRS POSITIVE AT 28, 800K NEGATIVE AT 48K POSITIVE AT 32GK, 1280K, 3200K 0400!(, 12.8G0K POSITIVE AT 7200K POSITIVE AT 5GK * .-.Li. TESTS ESSENTIALLY NEGATIVE BELOW AND POT: AVE ABOVE 50 AND 5Q0 - NEGATIVE 5000 - BORDERLINE POSITIVE 50,000 - POSITIVE 5CG0 - POSITIVE 50, 500, 5000 AND 50,000 - NEGATIVE 24,500 - EOSINOPHILIC. LOCI, NO CANCERS , 1 'j i i ; ; SL 096617 16 t 1 Calculations (Tables 1 and 2 Appendix) on the Consumer Products Safety Commission studies indicate that strongly positive carcinogenic effects may not appear in rats until the total dose (Ct) of VCM reaches or exceeds 50,000 ppm-hrs. The maximum response in the rats was the ap pearance of eosinophilic cells which might suggest a pre-cancerous change. The CPSC VCM tests in mice indicate that increased frequencies of adenoma may appear in this species at Ct1s of 5,000 ppm-hr and above. This total dose for carcinogenicity is in general agreement with Lee et al., ?l (48,000 ppm-hrs) and Maltoni IQ ' ?n (between 30,000 ppm-hrs and 150, 000 ppm-hrs), and the other investigators. ' ' In general, in orders of magnitude, carcinogenic tendencies are seen in some species at Ct's of 5.000 to 50,000 ppm-hrs. Definite carcinogenicity appears in both rats and mice at Ct's of 50,000 to 500,000 ppm-hrs, and high incidences of carcinogensis are noted in mice and rats at Ct's of greater than 500,000 ppm-hrs. The previous studies^'^0'^ present CPSC study are in agreement as to the dose-time relationship for carcinogenesis related to vinyl chloride exposure. All of these studies considered collectively may indicate that there may be a life-time total dose for vinyl chloride below which carcinogenicity is not likely to occur. This "no cancer" Ct seems to be below 5000 ppm-hr for mice. For histological confirmed carcinogenesis the "no cancer" Ct in rats appears to be greater than 50.000 ppm/hrs. The results of the light or the electron microscopical study did not reveal carcinogenic responses in rats at Cts from 50-50,000 ppm-hrs. However, Ct's of 2450 ppm-hr produced eosinophilic loci but no 17 SL 0966X8 tumors in rats in the CPSC tests. As an additional consideration in the total dose concept, Ct's of 5000 ppm-hr produced increased incidence of adenomas in mice when the exposures were at 5000 ppm for one day, 500 ppm for 10 days or 50 ppm for 100 days. The increased incidence of adenomas produced by the single exposure of mice at 5000 ppm is of borderline significance. How ever, there is the indication that even single exposure of sufficient magnitude may be carcinogenic in sensitive species. The dose-response relationship and the "no effect" dose con cept have been described for other carcinogens. It is difficult to relate these animal studies to man. Data on vinyl chloride exposure in plants have been limited. However, acute dizzyness, headache, nausea and chronic liver damage have been seen in vinyl chloride workers. It is assumed that peak exposure levels of several thousand parts per million were experienced at times. Air monitoring of one group of plants during 1950-59 indicate that timeweighted (8-hr) average exposure were 120-385 ppm. (This would give daily Cts of 960-3080 ppm-hrs). Peak exposures possibly exceeded 1000 ppm. This may not have been typical of all polyvinyl chloride plants.^ Data on vinyl chloride in ambient air are limited also. Atmospheric measurements in the vicinity of production plants indicate that concentrations are below 1 ppm. One peak grab sample of 33 ppm has been reported at 0.5 kilometer from the center of one plant.^ The time-weighted threshold limit value of the American Conference of Government Industrial Hygienest for 1977 was 200 ppm. There is a notice of intended change. 26 The Environmental Protection Agency27 has established 18 SL 096619 the following emission limits for vinyl chloride: (1) formation and purification processes is 10 pmm, (2) emissions from equipment preceding and including the stripper in the plant process flow is 10 ppm, (3) emissio^J^ from equipment following the stripper are to be controlled by stripping dispersion resins to 2000 ppm and other resins to 400 ppm. Assuming that man is as sensitive as the mouse or rat the carcinogenic effects of vinyl chloride might be expected after an accumulated Ct of about 5000 ppm-hrs or 'approximately 150,000 ppm-hr;(Maitoni BT3). res; :-cti. Based on the high pollution values given above (1000 ppm-hr per day) car cinogenic doses could have been accrued in one week (mouse) or 30 weeks (rat). At the TLV of 200 ppm (daily Ct of 1600 ppm-hr) the time to accumulate a carcinogenic dose would be 3 days (mouse) or about 19 weejts $ (rat). Comparable accumulation times at 10 ppm (80 ppm-hrs per day) f would be about 12 weeks (mouse) or 375 weeks (rats). The calculations are based upon a five day working week. IV. CONCLUSIONS. A. Except at the highest concentration, 50,000 ppm, where possibly anesthetic-type effects were seen vinyl chloride produced no pharmacotoxic (excluding pathology) signs in mice or rats during or after exposure. B. No reproductive or teratogenic changes attributable to vinyl chloride were found. Mating; % pregnancies; fertility, lactation indices, % still-borns; litter size; post-natal growth; viability, survivability, anomalies. C. Vinyl chloride seemed to produce pneumonitis in mice and to aggravate bronchopneumonic in rats. D. Vinyl chloride produced eosinophilic changes in rats but no 19 SL 096620 frank (light microscopy) carcinogenesis. E. Vinyl chloride produced increased in frequency of tumors in mice. F. Electron microscopical studies revealed some hepatocellular changes but no carcinogenesis related to the vinyl chloride exposures at Ct's of 50,000 ppm-hrs or less. 1. The carcinogenic effectiveness of VCM depends upon concentration and exposure time, Ct. 4 ' 5 ' 6 2. There were VCM doses which were not carcinogenic and there appeared to be a total accumulated dose above which tumors were product, .13 J 5 Similar effects have been noted with other chemicals. 3. Tumors were not seen in mice at Ct's of 500 ppm-hrs nor in rats at 50,000 ppm-hrs or less.^'^''^'^'^ 4. Carcinogenic effects of VCM were seen in mice at total doses iI (Cts) of 5,000 ppm-hrs and above. ' 20 SL 096621 i 1. Scientific and TechnJcal Assessment Report on Vinyl Chloride and Polyvinyl Chloride, US Environmental Protection Agency, EPA-600/6-75-004, June 1975. 2. E.M. McDowell & B.F. Trump. Histological Fixatures Suitable for Dignostic Light and Electron Microscopy. Arch. Path. Lab. Med. 100:405-414, 1976. 3. J.H. Luft, Improvements in Epoxy Resin Embedding Methods, 0. Biophys. Biochem. Cytol. 9:409-414, 1961. 4. Bryan, W.R., and M.B. Shimkin. Quantitative Analysis of Dose-Response Data Obtained with Three Carcinogenic Hydrocarbons in Strain C3H Male Mice. 0. Natl. Cancer Inst., 3:503-531, 1943. 5. J.B. Storer. Radiation Carcinogenesis, Chapter 16, pp 453-483 in Cancer 1. Etiology: A Comprehensive Treatise. Editor: F.F. Becker. Plenum Press, N.Y., London. 1975 6. A.C. Upton, J.L. Randolph and J.W. Conklin. Late Effects of Fast Neutrons and Gamma-Rays in Mice as Influenced by the Dose Rate of Irradiation: Induction of Neoplasia, Radiation Res. 41:457, 197G. * jr 7. U. Saffiotti. Identifying and Defining Chemical Carcinogens, pp 13111362. Origins of Human Cancer, Book C. Human Risk Assess.'r-cc. Editors: H.H. Hiatt, J.D. Norton, J.A. Winsten. Cold Spring Cnarte" Conferences in Cell Proliferation. Vol. 4. 1977. Cold Spring Charter Laboratory. 8. T.H. Maugh II, Chemical Carcinogens: How Dangerous ere Low Doses? Science, 202:37-41. Oct 1978. ^ Problems in the Evaluation of Carcinogenic Hazara frcm the Use of Food Additives. Publication 749. The Food Protection Committee, Food and Nutrition Board of National Academy of Sciences - National Research Council. December 1959. 10. Horton, A.W., and Dorothy T. Denman. Carcinogenesis of tne Skin. A Re-examination of Methods for Quantitative Measurement the Potencies of Complex Materials. Cancer Research 15:701-709, 1565. 11. Poel, W.C. Effect of Carcinogenic Dosage and Duration of Exposure on Skin-Tumor Induction in Mice. J. Natl. Cancer Inst., 22: 19-44, 1959. 12. Graffi, A. Untersuchungen uber den Mechanismus der Carcerogenese und die Wirkungsweise cancerogener Reize. Abhandl. deut. Akad, Wiss. Berlin, 53:1-27, 1953. 13. Druckery, H. Pharmacological Approach to Carcinogenesis, in Ciba foundation Symposium on Carcinogenesis: Mechanisms of Action. Boston: Little, Brown and Company, 1959. pp 110-130. 21 SL 096622 14. Eschenbrenner, A.B., and Eliza Miller. Studies on Hepatomas. I. Size and Spacing of Multiple Doses in the Induction of Carbon Tetra chloride Hepatomas. J. Natl. Cancer Inst., 4:385-388, 1944. 15. Blum, H.F., On the Mechanism of Cancer Induction by Ultraviolet Radiation. J. Natl. Cancer Inst., 11:463-495, 1950. 15. Finkel, Miriam P. Mice, Men and Fallout. (The potential danger of strontium 90 is appraised on the basis of data from animal experi ments). Science 128:637-641, 1958. "17. Mole, R.H. The Dose-Response Relationship in Radiation Carcinogenesis. Brit. Med. Bull. 14:184-189, 1958. J8. The Federal Radiation Council (Report No. 1, Background Material for the Development of Radiation Protection Standards, 1960, Government Printing Office, Washington, DC). 19. C. Maltoni. The Value of Predictive Experimental Bioassay in Occupational and Environmental Carcinogenesis. An Example: Vinyl Chloride. Ambio. 4:18-23, 1975. 20. C. Maltoni and G. Lefemine. Carcinogenicity Assay of Vinyl Chloride. Ann. NY Acad. Sci. 246:195-218, 1975. 21. C.C. Lee, J.C. Bhandari, J.M. Winston, W.B. House, R.L. Dixon and J.S. Woods. Carcinogenicity of Vinyl Chloride and Vinylidene Chloride, J. Tox. Environ. Health 4:15-30, 1978. 22. F. Haber, "Funf Vortrage aus den Yahren 1920-23": No. 3. Die chemie im Kriege: No. 5. Zur geschichte des gaskarnpes, Juluis Springer, Berlin, 1924 - reference through Prentiss: Chemicals in War, McGraw-Hill Book Company, Inc., New York, 1937. 23. P.L. Viola, A. Bigotti and A. Caputo. Oncogenic Response of Rat Skin, Lungs and Bones to Vinyl Chloride, Cancer Research 31:516-522, 1971. 24. A. Caputo, P.L. Viola and A. Bigotti. Oncogenicity of Vinyl Chloride at Low Concentrations in Rats and Rabbits. J. Int. Res. Commun. 21:1582, 1974. 25. M.L. Keplinger, J.W. Goode, D.E. Gordon, and J.C. Colandra, Interim Results of Exposure of Rats, Hamsters and Mice to Vinyl Chloride. Ann. NY Acad. Sci. 246:219-224, 1975. 26. Threshold Limit Values for Chemical Substances and Physical Agents in The Workroom Environment with Intended Changes for 1977, American Conference of Governmental Industrial Hygienist. 27. Title 40-Protection of the Environment. Environmental Protection Agency, Part 61-National Environmental Standards for Hazardous Air Pollutants, Stand ard for Vinyl Chloride, Federal Register, Vol 41, No. 205, Thursday, Oct 21, 1976. 22 SL 096623 SL 096624 APPENDIX Table I. Sinyle Exposure Schedule of Animals To VCM Species Sex Dose ppm Exposure date AM Pi-l M 50 3/4/75 500 3/11/75 5000 3/18/75 Fischer 50000 3/25/75 Rat F 50 3/4/75 500 3/11/75 5000 3/18/75 50000 3/25/75 M ICR Mouse F 50 500 5000 50000 3/4/75 3/11/75 3/18/75 3/25/75 50 500 5000 50000 3/4/75 3/11/75 3/18/75 3/25/75 Rat M Neg/Cont. F Neg/Cont/ Mouse M Neg/Cont. F Ney/Cont. Exposure group si/-': (s) 90 90 85 90 90 100 95 88 90 90 90 90 90 90 90 90 92 79 82 88 At;tj at time of exposure weeks 15 16 17 18 15 16 17 18 15 16 17 18 15 16 17 18 15-18 15-18 15-18 15-18 2 i APPENDIX Table II. Exposure Schedule for Animals Exposed Repeatedly to VCM Species Fischer Rat A/J Mouse Fischer net l A/ J Moua Sex M F M F M F M F Dose ppm 50 500 50 500 50 500 50 500 Neg . control Neg. control Neg. control Neg . control Exposure periods days 100 10 100 10 100 10 100 10 100(c) 10(C) 100(c) 10(c) 100(c) 10(c) 100(c) 10(c) Exposure dates From To 8/27/75 7/7/75 8/27/75 7/7/75 7/7/75 8/27/75 7/7/75 8/27/75 1/26/76 7/18/75 1/26/76 7/18/75 7/18/75 1/26/76 7/18/75 1/26/76 -- _ - - NOTE: (c) control for corresponding dose above. Exposure Group size(s) Start End 90 86 90 90 90 87 90 90 90 87 90 90 90 88 90 90 50 50 50 50 50 47 50 50 40 39 50 50 50 50 50 50 Age Start 21 14 21 14 I5 8 15 8 21 14 21 14 15 8 15 8 End wks 41 16 41 16 35 10 35 10 41 16 41 16 35 10 35 10 SL 096625 24 APPENDIX Table III. VCM Multigeneration Study Fg Parents Group I II III Compound Air VCM VCM Dose Control Low Dose (50 ppm) High Dose (500 ppm) Number of Males 25 25 Number of Females 25 25 25 25 SL 096626 11 100- 90 - 80 - 70 - R TALITY (CUMULATIVE; _ 60 LU > P <_) D 3 co - !> < Jir. ' 40 * 30 - Figure I SPONTANEOUS AVO TOTALITY MALE i C fi /A 1 C 2 S1 N G L E D O S E! STUDY VINYL CHLOPJDE i<\ CM O/A E R m 50 PPM y k control /ft/ 50.000 PPM P 5'000 ppr'1 ' jf/ ^ ^ n} ' ) // j//l / M7 / / / 1' ! / /I / / j 20 ~ 10 - yf / y/ /n^-n I o4 i | , r | i i ! i i i--------1--------,-------1-------- j----------, 4 C 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 AGE OF MICE (MONTHS) 26 SL 096627 100 Figure II SPONTANEOUS MORTALITY 90 - FEMALE ICR MICE SINGLE DOSE STUDY 80 - VI K'/L C I-; LO Rl D E MONOME R 70 - 60 - 50,000 ppr, -5,000 PPM 50 PPM CONTROl 500 PPi % M O RTALITY (CUMULATIVE) AGE OF MICE (MONTHS) 27 SL 096628 50,000 P[ G.ooo ppr COu'TROl 50 PPM 500 ppm 28 SL 096629 Figure IV CONTROL 5,OOC PPM 500 PPM 50 PPM 50,000 I'Fi1 29 SL 096630 % MORTALITY (CUMULATIVE) Figure V SPONTANEOUS MORTALITY 30 SL 096631 /o i/'LM .ALirV (CUr.'lULATIVE) Figure VI SPONTANEOUS M O RTA L IT'V w to 31 60 -i 50 - 40 UJ > H <.j !D sn o ~ 30 > i_j o1^ 20 Figure VII SPONTANEOUS MORTALITY AJ MICE 100 DAY STUDY VINYL CHLORIDE MONOMER p c/exposed // // t /J// n Q CONTROL Pr // o exposed // T ^ J// // AO CONTROL OAilViniAir.O) A1T !0!A! 10 " / -... _ - ^i 1-12 13 I 14 i---------- 1---------- 1---------- 1---------- 1------------ 1------- 1--------- 1-------------i 15 16 17 18 19 20 21 22 23 AGE OF MICE (MONTHS) 32 SL 096633 Figure VIII SPONTANEOUS MORTALITY 70 FISCHER RATS 100 DAY STUDY VINYL CHLORIDE MONOMER L0 - 40 - dlEXPOSED C^ONTROL EXPOSED Q COMTHGL T % M ORTALITY (CUS'ULATIY tn r1 ok(oTOri it* 33 ' P 70 60 50 40 30 20 TO CO f OuoooCOJrir '/ MORTALITY (CUMULATIVE) Figure IX SPONTANEOUS MORTALITY FISCHER RATS 100 DAY STUDY VINYL CHLORIDE MONOMER AGE OF RATS (MONTHS) 31* (^EXPOSED Il Figure X GROWTH CURVES OF HALE ICR MICE EXPOSED ONCE TO VCM cn O' tD H 10 *r LD tf'J4 -J H < < LU s: tFn o couCr>T\\i 35 MEAN ANIMAL WEIGHTS I N CRAi Figure XI GROWTH CURVES OF FEMALE ICR MICE EXPOSED ONCE TO VCM w> f OT EXPOSURE tC-O1 o U(C'JTFJii MEAN ANIM AL WEIGHTS I N GRAMS Figure XII GROWTH CURVES OF MALE FISCHER RATS EXPOSED ONCE TO VCM cn F ov0oucoo\\t 37 MEAN ANIM AL WEIGHTS I N GRAMS Figure XIII GROWTH CURVES OF FEMALE FISCHER RATS EXPOSED ONCE TO VCM WEEKS POST EXPOSURE CO t1 oID cr> w VO 38 mean a n im a l w eig hts i n grans Figure XIV GROWTH CURVES OF A/J MICE EXPOSED TO VCM IN 10 DAY STUDY to c* oKD C(TT\\ O 39 MEAN ANIMAL WEIGHTS IN GRAMS Figure XV GROWTH CURVES OF A/J MICE EXPOSED TO VCM IN 100 DAY STUDY okO cn CTi *i0 Figure XVI GROWTH CURVES OF FISCHER RATS EXPOSED TC VCM IN 10 DAY STUDY 500- MEAN ANIMAL WEIGHTS I N GRAN 450 H CO 400- 350--1 300250200- jr JT ..TV* if Si. / ,^A' *=7C A nv, if--A .A /A' 150- Ji* t---MRLE EXP *-MRLE CNT a=FEMRLE EXP =FEhflLE CNT 100' rmmirpnnm TpinmiqiiiurmpTimrujimuiiijiiiiiinijiir.iinijiiiiiiiiijrrrniiiijnumTTj 10 10 30 50 70 90 110 0 20 40 60 80 100 WEEKS POST EXPOSURE C/3 Ir1 o 0CCO3\ i * ft tf' tfc>. to 41 Figure XVII GROWTH CURVES OF FISCHER RATS EXPOSED TO VCM IN 100 DAY STUDY 500 475 MEAN ANIMAL WEIGHTS I N GRAMS 450-5 425-5 400-5 375 350-5 325-5 300 275-3 250 225-5 200-5 r-j- -A A 175 +=HRLE EXP s;=HRLE CNT a=FEM?LE EXP =FEI1flLE CNT 150 mmrri|mmn rjTnrrfnij rniiTrcrjnurrmyi *m iirrjTrrrrrraTiTi ;mn jTirrnrmiTirTTiHmrnTrrfmrrr^ -10 10 30 SO ?0 SO 110 0 20 Xu SO 80 100 fCO WEEKS POST EXPOSURE o ua&<CT>O\i Table IV. Over-all Summary Incidence of Non-neoplastic Changes and Histologically-Proven Neoplasms Within The Liver and Lungs of ICR Swiss Mice Exposed to Vinyl Chloride. Single Inhalation Exposure Tissue/Response Exposure : Dose Level (PPM) : Sex of Animals Animals Per Group* : Control 0 MF 62 77 Liver :Number Evaluated : 50 75 - hepatic cell necrosis - hepatic cell vacuolation (lipidosis) - hepatic cell hypertrophy - hepatic cell hyperplasia - angiectasis - sinusoidal reticulosis 2 10 2 11 1 Incidence of Respons e Vinyl chloride 5 Moo 5,000 500 M FM FM F /4 82 76 82 72 75 63 78 Dcj 76 67 72 3 b5 74 2 4 on 2 12 3 84 44 i4 5 6 5 13 50 M 81 64 2 1 1 F 80 68 3 4 - hepatic cell adenoma - hepatic cell carcinoma - hemangioma - heinangiosarcoma 21 12 2 416 19 2 21 11 Lung - pneumonitis :Number Evaluated : 50 1 - bronchio-alveolar adenoma - bronchio-alveolar carcinoma 4 70 6 8 61 76 65 21 10 13 31 14 14 121 78 66 17 19 10 8 11 71 15 4 10 8 1 68 7 6 * Total includes animals from scheduled sacrificed (8 and 18 month periods) and spontaneous deaths. SE^)96644 43 Table V. Summary of Incidence of Histologica1iy-Proven Neoplasms Within Tissues ICR Mice Exposed to Vinyl Chloride Monomer (Si 'e Inhalation Exposure) t 1 -hr 1 nhalation Exposure i n ppm 50,000 Spontaneous Deaths 0-6 Months 7-12 Months M F Total M F Total 0 0 0 5 11 16 Schedule Sacrifice 13- 8 Months _____8 Months | 18 Months M F Tota1f Ih T F Total --tt_. F Telal 33 32 65 4 1 5' 7 17 24 Tota1s Male 49 female_____ 61 5,000 500 022 42 6 22 27 49 j 2 33 ! 0 5 5 68 14 19 14 1 | 13 01 i 5 13 18 33 j 45 1 5 24 29 31 5 ] 51 50 0 0 0 4 15 0 (Control) TOTALS VCM 2 0 020 7 7 19 ------- _____ _ 1 36 19 9 1 14 55 83 18 0 0 0 -4\ * ! 41 551 00 *_____ ______ ... f1 91 j 174 f :: 7 j ;; ! J 25 ;1 d 2 14 2 12 i 1 ' 19 I 68 ; .. 16 15 14 18 !37 _ ! 47 i 54 1 tIto1 0 U0CifT11*i 44 Table VI. Incidence of Hi stologically-Proven ileopiasms Within Tissues From ICR Mice Exposed to Vinyl Chloride Monomer (Single Inhalation Exposure) sacrifice Organ Period Ti ssue 50,000 ppm 5,000 ppm M F Tota l M F rota! 500 ppm M F Total M 50 ppm 0 ppm (Control) F rotal M F Total ichedulec Lung 41 51 12 0 0 00 00 0 0 . Q .... 8 months Liver Kidney 0 0 0 0 01 00 01 1 t 00 0 0 10 0 0, -- 0 00 00 0 0 0 0 ... 00 Stomach 0 0 00 00 0 0 0P 0 0... n nn 0 the r* 0 0 00 00 0 0 00 0 . 0 ._ 0 . 0 0 Totals It 1 ?2 13 1 0 10 00 0 00 18 months Lunq 5 6 11 3 7 10 4 12 16 1 4 ____ 1 _______2__ 8 Liver ? 3 52 02 1 1 20 11 1 2 - 1-- Kidney 0 0 00 11 0 3 30 22 0 00 S tomach 0 0 00 00 0 1 11 01 0 00 Other-' 0 8 80 55 0 7 70 77 0 33 Totals 7 14 24 5 13 13 . .. 5 ......... 2.4 _2L_ 2--____L4,, J-6___ 2 12 14 Spontan eous Deaths 0-6 man Lung Liver Kidney S tomach 0 0 0 0 0 0 0 0 00 00 00 00 11 00 00 00 0 0 0 0 I 1 0 _____2_ 0 _______2__ 0 i 10 00 d 00 1 10 0 00 00 00 0 0 00 00 7-12 mon Other-Totals Lunq _JL _JL Liver___ _JL Kidnev 1 0 ___ 00 36 22 2 ___ 01 02 31 00 1 .... 0 1 2 4 0 1 0 0 1 2 l 2 20 5 50 2 32 1 31 1 20 00 66 24 01 22 20 2 -----------tr~ 00 00 00 2 2 0 0 0 Stomach Other* Totals JL 1 5 0 4 1) 00 50 16 4 00 11 26 0 2 6 0 00 4 6f 8 14 4 11 10 11 15 19 0 0 0 00 11 11 13-18 mon Lunq L i ver j Kidney Stomach im Other* Total 22 5 It 0 2 33 16 5 0 0 1] 32 38 12 10 ? 41 00 13 4 65 22 5 '7 5 10 34 00 5 8 1 0 14 18 A 927 49 1 ) 2 4 2 0 6 14 7 5 .......5 10 4 9 13 . 12 4 0 00 -2 6 5 7 12 1 0 1 0 I 11 1 0 -------- g5-- 6 --3 n 33 JL____J.Q__ 10 9 18 27 1 Li._____ | 14 41 -id! 9^9960 7S Table VII . Over-all Summary Incidence of Non-neoplastic Ghanges and Histologically-Proven Neoplasms Within The Liver and Lungs of AJ Mice Exposed to Vinyl Chloride (Multiple Inhalation Exposure) Tissue/Response Incidence of Response Exposure : Control Vinyl chloride Dose Level (PPM) : 0 500 Hours Exposure : 10 x 1 10 x 1 Sex of Animal :M F M F Animals Per Group* : 46 48 78 92 Liver : Number Evaluated : 45 48 78 89 - hepatic cell necrosis - lymphoid cell infiltrate - hepatic cell lipidosis - neutrophil infiltrate - bile duct hyperplasia - granulomatous foci - sinusoidal reticulosis - hepatocyst - amyloidosis - angiectasis 46 1 2 2 1 6 13 1 2 1 1 1 1 1 1 - hepatic cell adenoma - cholangiocarcinoma Lung : Number Evaluated : 1 11 43 6 47 1 13 76 - edema - pneumonitis bronchio-alveolar hyperplasia 2 21 bronchio-alveolar adenoma bronchio-alveolar carcinoma 15 16 3 17 22 56 12 63 fatal includes animals from scheduled sacrificed (S, 16, 20 iiionTfiT perioiasT^f'ST'poritaneaiiy-de'arfrs T6~ 90 1 2 68 10 81 SL 09664 Table VIII. Over-all Summary Incidence of Non-neoplastic Changes and Histologically-Proven Neoplasms Within The Lungs of AJ Mice Exposed to Vinyl Chloride. Multiple Inhalation Exposure Tissue/Response Lung - edema - congestion - focal hemorrhage - pneumonitis - bronchio-alveolar hyperplasia - osseous metaplasia - bronchio-al veolar adenoma - bronchio-alveolar carcinoma - reticulum cell sarcoma LXpOS'.Ii'l: dose Level (PH'1) Hours Exposure Sex of Animal Animals Per Group* Con lcoI " "0 ~ 100 x 1 M 39 Incidence of Response V i ny1 chloride 50 100 x 1 F MF 47 81 83 : Number Evaluated : 39 2 4 2 5 45 1 3 3 77 81 1 2 45 3 1 11 18 2 13 18 27 38 34 1 30 43 * Total includes animals from scheduled sacrifice (8, 16, 20 month periods) and spontaneous deaths. SL 096648 47 Table IX. Summary of Incidence of Histo logi --`11y-Proven Neoplasms Within Tissues From AJ Mice Exposed to Vinyl Chloride Me .er (Multiple Inhalation Exposures) Sac rifice Period Spontaneous Deaths 0-6 Months 50 p pm x 100 CONTROL M F T0TA1 M F TOTAL 0 00000 500 ppm x 10 j M F TOTAL CONTROL ------- -------- -i-v < n iVCM M F TOTAL M F 0000000 2 7-12 Months 8 3 1 i 0 0 0 2 1 3 0 0 0 to 4 CONTROLS MF 00 00 1 3-20 Months 23 20 43 3 5 8 36 27 63 2 3 5 59 Scheduled Sacrifice 8 Months 3 1 40 1 1 66 0009 16 Months 10 6 16 2 3 5 14 8 22 1 1 2 24 20 Months 19 44 63 16 11 27 29 41 70 17 21 38 48 Totals 63 74 137 21 20 41 87 85 172 20 25 45 150 .. ______ 47 5 70 14 3 85 33 --------- __-- 159 41 8 l 4 32 45 SL 096649 48 6 SL 096650 Table XI. Over-all Summary Incidence of Non-neoplastic Changes Within The Lungs of Fischer Rats Exposed To Vinyl Chloride. Single Inhalation Exposure Tissue/Response Exposure Dose Level (PPM) Sex of Animal Animals Per Group* : Control :0 :M F : 89 74 Incidence of Response Vinyl chloride 50,000 5,000 500 MF MF MF 86 87 83 93 87 100 50 MF 90 91 Lunq :Number Evaluated - bronchopneumonia : 85 26 67 10 80 77 45 13 80 87 13 10 85 95 80 15 3 28 74 3 % incidence : 30.6 14.9 56.2 16.9 16.3 11.5 17.6 3 35.0 4.1 * Total includes animals from scheduled sacrifice (8, 16, and 24 month periods) and spontaneous deaths. SL 096651 50 Table Xl1. Summary of Incidence of H i stoloyica11y-Proven Neoplasms Within Tissues From Fischer Rats Exposed to Vinyl Chloride Monomer (Sinlge Inhalation Exposure) I-hr 1nhalation Exposure i n ppm 50,000 Spontaneous Deaths 5 0-6 Months MF T* 7-12 Months M F T* 13"18 Months 19-2*1 Months ] 8 Months M F T* M F 1 ! T* | M JF T* i 00 0 ! 0 4 4 16 20 36 86 47 133 2. \ 0 2 1 \ <1 1 Schedule Sacrifice 16 Mon t h s 2*1 Months M F T* M F T-1 4 2 6 21 3** 55 5,000 0 0 0 2 2 4 20 9 29 71 37 108 0 0 0 5 1 6 37 52 89 Totals Male Female 129 107 135 101 500 0 0 0 0 2 2 18 18 36 57 64 121 0 0 0 13 0 13 82 37 119 170 / 50 0 0 0 0 2 2 18 15 33 89 28 117 0 0 0 7 0 7 64 27 91 178 0 (Control) 0 0 0 0 0 0 13 3 16 102 56 158 ! 0 0 0 11 3 14 60 27 87 186 Tota1s VCM 0 0 0 2 10 12 72 62 134 303 176 *79 2 0 2 29 3 32 204 150 354 121 72 89 , cfo oco cn ctnn to Table XI t I . Incidence of rl i s t o loj i ca ! I y-P r.j ;en ['ooplasms Within Tissues From Fischer Rats Exposed to Vin/1 Cfi1 \de hc-nomei (Single Inhalation Exposure) Sacr i f i cc Organ Period Tissue 50,000 ppm M F Total ichedu1ed Lung 8 months Liver Kidney Stomach Other* Total 16 months Lung Liver Kidney Stomach Other* Total 24 months Lunq Liver Kidney S tomach Other* Total 0 0 0 0 2 2 2 0 0 0 2 4 0 1 0 0 20 2) 00 00 00 00 02 02 02 00 00 00 24 26 22 34 00 11 28 48 34 55 5 ,000 ppm j M F Tota1j rt 0 00 c 0 00 o 0 00 0 0 00 0 0 00 0 0 00 0 0 00 i 0 00 0 0 00 0 0 00 0 5 1 6 12 5 1 6 f 13 0 0 o It 3 2 58 0 0 01 0 1 10 34 49 83 70 37 52 89 82 500 ppm a F Totalj M 0 0 S0 0 o 10 0 ___! 0 0 o i0 00 0 00 0 1 01 1 00 0 00 0 00 0 0 12 6 0 13 7 36 4 12 01 1 9 0 000 30 100 54 37 119 64 50 ppm 0 ppm (control) F Total M 00 0 00 0 00 0 00 0 00 0 00 0 0 10 00 0 00 0 00 0 0 6 11 0 7 11 2 3....... } 3_. 12 9 00 1 000 22 76 47 27 91 60 F Total 00 00 00 00 00 00 00 00 00 00 3 14 3 14 25 1 10 01 11 23 70 27 87 SL 096653 52 1; 0 ;E Table XIV. Incidence of H i s lo 1 < -g i 1 1 y-F ovl-h H.-opiaiins Within Tissues From Fischer Rats Exposed to Vinyl Chloride Monomer (Single Inhalation Exposure) i Sacrifice Organ Period Tissue Spontaneous Deaths 0-6 Months Lung Liver Kidney Stomach 50, 000 ppm M F Total 00 0 00 0 00 0 00 0 5, 000 ppm M F Total M 0 0 0 10 0 0 00 0 0 0 f0 0 0 00 Other* 0 0 0 0 0 00 Totals 0 0 0 0 0 0' 0 7-12 Lung 00 Months Liver 0 0 0* 0 0 0 0 0 0 0 00 Kideny 0 0 0 0 0 00 Stomach Other* 0 0 0 4 0 0 0 00 4 2'' 2 4 0 Iota 1s 0 4 4 2 2 40 13-18 Months Lung. Liver K i dney S tomach 1 2 0 0 4 3 1 0 5 5 1 63 5 4 2 i 6 12 !0 . i0 0 0 0 0 '0 19-24 Months Other* 13 Totals 16 Lung 4 Liver 12 Kidney 2 Stomach j 0 Other* ; 63 12 25 1 1 5 16 t 13 20 36 I'0 9 29 ha * 8 ! 11 5 16 1 4 |JL_! 3 4 13 ) 6 Ul. J., 2 !0 1 I0 7 0 ;0 0 34 102 43 27 { 7:. (; "1, -/ Totals j 86 ?7 ul'33 - 71 37 [108 57 600 ppm i 50 ppm 0 ppm (control) M F Fota 1 M 00 0j 0 0 0 000 000 0 00 0 00 0 00 0 00 0 00 000 000 0 0- 0 0 00 0 00 1 10 000 0 00 0 00 0 00 0 00 1 10 220 1 10 220 5 83 1 20 4 62 0 40 0 02 0 20 0 00 1 10 9 22 13 24 13 18 j36 jis 15 33 13 8 12 11 0 11 8 10 16 9 ! ' 1 3...... 0 9 14 j Lj 3 31 3 V' h 0 f' 0|0 2 8 64 3 74 fSlP* |89 28 f 117 i 1 102 F Total 0 0. 00 0. 0 00 00 00 00 00 00 00 00 00 00 00 00 00 3 16 3 16 08 8 22 I4 14 46 120 56 | 158 SL 096654 53 / Table XV. Summary of Incidence of tt olo-jic;>liy-Proven Neoplasms Within Tissues From Fischer Kats Exposed to Vinyl Chloiide Monomer {Multiple Inhalation Exposures) _sa.Sacrif ic^ 1ppm x 100 Period t M F Total Spontaneous Dea ths 000 0-6 Months M 0 Control F Total 00 500 ppm x 1 Of) ] M i F -Total 1 0 l0 0 Control M tF 00 ^ota 1 0 _1 7-12 Months 0 0 0 3 0 3 0 0 0 0 0 0 VCM M 0 F 0 00 1 Control MF 00 30 13-18 Month 1*4 6 20 5 0 5 10 6 16 3 0 3 .. 19-2*4 Month 111 51 162 52 29 81 52 29 81 33 3*4 67 Scheduled Sacrifice 0 0 0 0 0 0 0 l 1 0 0 0 8 Months 8 1816 Months 10 * 1 5 (0 1 M 0 0 0 : 2*4 163 0 18 24 Months **3 **3 86 -Totals 176 110 166 32 23 61 42 7) 1)3 69 27 96 59 1145 11*4 108 222 Jl05 |61 t-- . , --, ,, 1 96 85 166 '290 12 8 0 80 85 63 1 00 ?1 k 1 1 1*4 101 218 . 201 56 120 SL 096655 / 5** IdOit; AVI. ^ ^ - , MjjUe,ii Rais Exposed to Vinyl Chloride Mog^fc'r vilu'tiple Inhalation Exposure) SL 096656 j7 Table XVJ1. Incidence of H i s to l og i ca i 1 v-P rovc:i lloirUsiri'j Within Tissues From Fischer Rats Exposed to Vinyl Chic j Monomer (Multiple Inhalation Exposures) Sacrifice Organ Period Tissue 50 ppm x 100 Schedu1 ed Sacrifice Control I 9 M F Total M F Total 500 ppm x too M F Iota 1 Control MF Total 8 Months Lunq Liver Kidney S tomach Other* Total 00 0 .. . 0 00 00 00 00 00 00 00 00 00 00 16 Months , Lunq L i ver Kidney 0 0 0 S tomach 0 24 Months Other* _3____ Total 8 | _Lurui ___ 1 _J _ 0 00 0 00 0 0 Cl 0 Q io s 10 18 ? h 10 18 4 , _ 1_ _ 1 0 0 0 o o 00 n1 0 01 0 b 0J 0 01 0 00o Q..._ _0. ... 000 00 0 110 00 .0 _0 . 00 00 00 o 0i 0e iS p. 0 0s 0 n ,, 2 1 |0 00 0 i_ o_ _ n ____IL- ____Q___ !0 o ___Q__________Q_ ____0___ 0 !2 0 0! o 00 0 0 io 0 16 0 190 1 ! ! 1 11 0 01 0 0 0 . 0 1 1 _ ' - ; 1 4__ * _3 01 3 4j 9 K i J.n. . 0 j Stomach 0 j QUiSX*_______ 35 j Total 43 u 0!0 7hi i . 43 j 86 u 25 .32 - 0 0 :i 0 0 f 0 f 1 25 68 i ' ^ 71 i23 61 ? bl j 69 113 - 0i 0 i( 23 83 27 96 SL 096657 56 Table XVIII. Incidence of Eosinophilic Changes Within Liters of EWA Colony (Sprague-Dawley) Rats-MuItipie Inhalation Exposures to Vinyl Chloride (VC) Exposure Dose Level (PPM) Hours Exposure Group+ Control 0 49 A wL ,T,, . VC _J[ __ j ,,6 Males: Animals per group Eosinophilic Focus/Foci % Eosinophilic Area/Areas % 3 1/9 11.1 0/9 0.0 16 ITU 7-7 1/13 7.7 25 2/22 9-1 1/22 4.5 13 [i 0 /13 j76.9 { 1/13 j 7.7 a 12 3/1 1 27.3 i/1 1 9-1 Eosinophilic AIterations++ / Females: Animal per group Eosinophi1ic Focus/Foci % Eosinophi1ic Area/Areas % Eosiniphitic Al terations++ % 1/9 11.1 18 0/18 0.0 4/18 22.2 4/18 22.2 1/13 7.7 7 0/7 0.0 0/7 0.0 0/7 0.0 2/22 9-1 25 0/25 0.0 4/25 16.0 4/25 16.0 ji0/13 576.9 i f 11 1 4/11 36.4 2/11 13.2 5/11 45.4 I 1 4/11 36.4 14 1/13 7.7 0/13 0.0 1/13 7.7 + Group: A = Scheduled sacrifice, B = Spontaneous deaths, T = Total ++ Eosinophilic fee: and/or areas in the same animal are tabulated one time as an eosinophilic alteration * = Different from control at 5-0 - >2.5% level (P = .05 - .025) ** = Different from control at 2.5 - >0.5% level {f* = 0.25 - .005) *** = Different from control at 0.5 - >0.05% level (P = .005 - .0005) aaaa = Different from control at0.05% level (P = .0005) _JL -A-- 25 13/24*** 54.2 2/24 8.3 11 4/io 40.0 0/10 0.0 14/24**** 4/10 58.3 40.0 25 5/24** 20.8 2/24 8.3 6/24 25-0 19 2/19 10.5 1/9 5-3 3/19 15-8 VC 50 _____ 14 4/11* 36.4 3/11 27.3 5/11** 45.4 6 1/6 16.7 1/6 16.7 1/6 16.7 25 8/21** 38.1 3/21 14.3 9/21** 42.8 25 3/25* 12.0 2/25 8.0 4/25 16.0 57 SL 096658 Ta a XIX. Summary of Matings and Percentage P re< i fo ?Gon. i ri G^n. F2 Gen. Control* Tota 1 Fema1es Mated Total Females Pregnant j | | % Pregnant | Females 25 22 53 Tola! FemaIts Mati.d 1 25 j j 1 1 1 20 19 19 19 Ji t' " i 19 I 1* fi i 100 |^ l Here and in all similar tables the exposure level refers to tha of the Fg gene ration. SL 096659 58 I Table XXL Summary of Average Litter Size for Each Treatment Group and Generation. Control 50 ppm VCM 500 ppm VCM F.j Generation Fg Generation Fj Generation * Significantly different, p = 0.05 Average Litter SizeSTD DEV 11.10 11.32 10.92 3. S3* 12.57 11.93 0.38 0.39 0.36 0.36 0.39 0.39 i 59 Table XX. Average Litter Size for the Three-Gene rat ion VCM Study. Control 50 ppm 1 500 ppm Total Pups Born F1 Generat ion 195 r2 Generat ion 239 F3 Generat ion 225 Tota 1 Number L i tters 22 19 19 Average L i tter S i ze STD Dev 8.9 0.63 12.6 0.67 11.8 0.67 Tota 1 Pups Born Total ! Number ( Litters 1 Average j Litter V* cS.ize 202 , 9.6 23* 18 13.0 i204 11.3 18 Total STD Pups Dev Born 0.64 184 0.69 255 0.69 285 Tota 1 Numbe r Lit ters 23 21 21 Average Lit ter S i ze STD Dev 8.0 0.61 12.1 0.64 12.6 0.64 SL 096661 60 Table XXII. Percentage of Stillborn Upus in the VCM Three-Generation Study F1 Generation F2 Generation F3 Generation Control Total Pups Born Total Stillborn % Stillborn 195 1 0.51 Total Pups Born 202 239 0 0.00 234 225 2 0.89 204 ______________l 50 npm Total % Stillborn Stillborn 5 2.48 2 0.85 5 ----------------- i 2.45 | 500 ppm Total Pups Born Total Stillborn 184 3 255 2 265 3 % Stillborn 1.63 0.78 1.13 co 0 cn 01 cr\ to Table XXIII. Numbers, Sex, and Weights o. F-| Generation Age of Pups Days Total Number of Pups Control 50 ppm Male Female ----M---a--l-e-----_tf Female j 500 ppm Male Female 1 102 92 105 93 93 88 --' 4 " " .7 102 102 92 105 93 92 88 89 105 93 92 88 Average Weight (Grams) Control Male Female 6.88 6.60 50 ppm Male Female 6.98 6.69 10.50 15.29 10.03 14.71 10.50 15.30 10.15 14.87 ; 14 102 89 21 102 fc. --^ 89 \ 105 105 j92 92 88 27.8 26.8 28.6 t fi *; : 92 92 i 83 j 44.5 | 42.4 ! 44.3 !i ........._ f 27.2 42.8 500 ppm Male . Female q 7.05 6.76 10.72 10.37 15.76 15.41 28.6 45.4 23.4 44.1 SL 096663 62 Table XXIV. Numbers, Sex, and Heights of Generation of Pups Days Total Number of Pups Control Ma 1 e Feina 1 e 50 ppm Ma1e Fema1e 1 120 119 114 118 4 118 119 114 7 118 119 113 117 116 14 118 119 113 116 21 118 : 119 113 116 500 ppm Male Female 123 130 122 130 j Average Weight (Grams) | Control } Male 11 1 | 6.38 Female 6.13 50 ppm Male .female 6.55 6.16 j 9.70 9.13 9.82 9.21 500 ppm Male Female 6.52 6.18 10.00 9.27 122 130 14.38 13.85 14.57 13.45 14.72 13.97 122 130 27.77 27.26 28.10 26.15 28.80 27.44 122 130 40.89 40.57 41.54 38.67 42.93 41.20 SL 096664 - fcfl* 63 Table XXV. Numbers, Sex, and We join f Generation Age of Pups Days Total Number of Pups Control Male Female 50 ppm Male Female 1 112 111 91 108 4 112 111 91 108 7 111 111 91 108 14 111 111 . 90 108 21 111 Ill 90 108 500 ppm Male Female Average Weight (Grams) \ Control I Male Female 50 ppm Male Female 500 ppm Male Female 139 123 139 j 123 .J 139 123 135 121 134 121 6.82 10.18 6.38 9.47 14.63 r 13.90 25.41 25.39 t _________ 1 1 | 34.77 33.85 6.84 10.35 6.61 9.76 6.66 6.30 9.87 9.07 14.81 14.18 13.89 13.25 27.46 25.81 25.53 23.95 36.75 35.14 35.11 33.40 Table XXVI. Viability, Survival, and Lactation Indexes in a Three- Gener\.tT,n Study of Reproductive Performance After Expose & of the FgU Parents to VCM Gas. Low Dose High Dose Generate; Control 50 ppm 500 ppm F1 100 100 99.4 Viability Index F2 99.2 99.7 99.6 F3 100 100 100 Survival In. ex Day 21 F1 f2 F3 93.5 99.2 99.6 99.5 98.7 99.5 Lactation Index F1 . 98.5 F2 100 F3 39.6 99.5 99.1 99.5 Viability Index = Day 21 Survival Index = Lactation Index = No. c r or ns alive at Day ^ x 100 No. or pups born alive No. oh rues alive at Day 21 x No. o.' pups bo it, alive No. of nuns alive at Day 21 lnn No. c-r pups alive et Day 4 Banerjee, B.N., Course Director,, Terotol cgy-Princi pi es and Procedures Related to Fetal Development. The Center for Professional Advancement, Sommerville, New Jersey. September 1974. Course Notes. 9*. 4 99.6 97.3 99.4 100 97.3 65 SL 096666 Figure XVIII Control - 8 months FIGURE LEGEM) Control hepatocyte morphology with normal appearing endothelium adj acent. X5,COO. Negative #193,033 66 SL 096667 Figure xviii ' Control - 8 months f;*'. ' j, ' ' - 7' , I. V-x '< ~ ?v ' \ :./ -it-- '** . .f1, 'V v : ' '< \ ' -4 J, 'J .<V ` t 1 , - !l/ A- ' V ; .. ' ' ?>*. ' -- \x .-vv A-' I*.", ' tj.j'-"'1;' ` r -- '*'?,-> 1'/ ' S........ /' ' : V vS ' ' \ " ?- \+-< . :v %. . , 1 ' j , v-" ** 4; ' *. S k *4, ^ ' rtf, ` ^ ''^S.*r^SS*'-, .*,*.*, * "* tr* ' 2 + . . ',*< < '* ff '' / - j*S # . ^ , y i.'a - ^ .*/.* ' ' i4 j '4 ^ -1 . -t . ] . ".*,>? . > * ^ v - .'< 'l;1 *l , ; ^ i ' . > " 1S' y' . * * * ",'** `'S5 ' *'-A' . ^ m'S4'' '> - . *SS-' . ' ,' , * .V 7 7 , . I`.1 `* S . ^ ^* . * * *' J.'4 . >* : I.I S '7 .VI'' r , . ' 1 -0r; > ,4..,, ?*s, ..'CJ v ' , . C I . .* ' - t ' ? ` 4.', '' * * r-' ' I-. ,,U, 4^4 , ^- 4 " ' - f .s, , r , . " ' S* * : ` S'*- - *>. . * ^. 4 * o - : ^ 1 ; s* s;. V'->1 . T *4 - .:r.: / * - '4 - J - . *\s*. *f* 'V- v; S'C~:S ' C ^ -4 A! ' i ' 1 i%*'* IL . 4- J 't\ ' , 4_ ' -' . , M i-V..'.fc '. ' .t,',*C* r '3' . S ' '* s . r ' ,**:< '- ;S IS"r ' . .<7^ f V <7 ' 'r-4 ' >! t'.' .* fT . -v ' ' 'V :_V 1 '\ , V ''/ s'/ f *' A'* .A ' \`S' -C* >7 .*- 1 ^ VS'-, ", ,/-' ' ^ '"yji v - ' V, 'A Sv/.- .' . s _* 4' ' i Jt". I IV S"..-:; ' 4 ?'* ,'7' "Ijr -x ) ^:S 7/ '// - / : ' `r' s/ .4 s;..... -v 4v . S; , . v `C 4 *<l; ", A V . 4\\ { '! ` .4' ' f. ^ *. ' .S -V . ' - U4 - - V-. t- rv..' ' . 'Sv ' - *.. 4..* S'1 ' `* ' 4 <; , /' S*,4 t^, . 4 . * ``v,4 t r,^ ' 4#* .* 7 f* v\*v; ' v , ,. I' ' 4 ` * fi'tV f \i t- ,f, ,I .... * * . 's'S-Sii:. .-'VyV* ,,>< -.S' t SL 096668 Figure XIX Single Exposure - 8 months post-exposure ' , TjiGETi Ilepatccytcs show lipid droplet accumulation. At center of cytoplasm devoid of recognizable oi'ganelles is in shape oo. Adjacent belb is apparently free in space of Disse. nay represent a moans whereby cytoplasmic material may be XI,000. 67 SL 096669 Figure XIX* Single Exposure - 8 rnonths post-exposure .* ' * - Y,;.Y-.:- ` , ) ' ,1 v. y , ,Y. ; V ' ' # >> ' r*'- ' -J ! J ' ^ < *- ^ , v. * , % * y r-:=1'',>A :> " " v c' ** v* **.'1,, \ t `m ' >1 * t.tj. -*< < -V /*\ : ^*~y *r - :>/'' , "V ,m1 y.\ V- . / 'Vi { r: j ' ; ,, * -' ' 1 -i' '1. r.(- ;'' ' 3 ' .'' *.* ':- , ' > ,r. j v v i ,^ ' .* /; .*r' \*'``-t; 7"*- f-, `i,'v ` ' ) .r y* .; A<"s.-' ; i*'- :i fir* * /;YA ,t ; *y) 'F<s C'9'^V '.:) 1/ , * - ^ 7V'/ * iV - V'v / ' . ' C '', -T* '* sv r- .* V ' 7i . '.('?.,<, v\ v'-' ',\ " J . \ ./- -J` .' ' ' ^ ' s 'r' '>' ,s:' 'V,-; ' tv . *" -\ *?* ' * ` ^ T *'. *L * ' > . -^ .- * v .... .*. / 'V '- -'v.``ts/ .' s `\ - ' i- ' * ' ^* j-%v * .V *... '.. .-.`v v h. ' , s ^ '.V * * *" ' v ` ! >: r-V ,,.^v }/ ;- :v ,1'.- . '.;w . , .... \ - .. Y- ;V,"V* -S .-;v< : . ' - V ^ T' ' r-:\ . . rv.*-v^ vLr : ; .:'V'Y /.-/ \Y;,; > . V'* Y.Y. i\!}*'*,* <# {! 4 *\ vv \ Y\ 'V'Y/' ` , J. J.. -, - *-! v.t ;* , - "* : ,'V*v-.-^- ___ -. -o Y '-'YvY"i *V* t -' ;'-, tJ-'J.V Y )'./:J " 7' , ' /.*. Yt.TY ; -7 ');.T !v'Vr";: ' ` yV; . ,-v^. Y'YY'yY.V , w7su`- 1 (--r s `V VlU -- 1. ,7 `j A SL 096670 Figure XX Single Exposure 8 months post-exposure FIGURE LEGEND Figure shows portion of to.' !. ana.ll dense bodies with one or 1 : ^ are lipofuchsin granules. Xi.C'CL Note numbers of -lucent regions. These Negative Oojj. \ 7?11 54,01 68 SL 096671 Figure XX Single Exposure - 8 months post-exposure . /* .-A* -i A ) <iv' </ Hj % * ':i -;tr. SL 096672 Figure XXI Multiple Exposure - 8 months post-exposure .FIGTBTl LCGE-D Kupffer cell shows evidence of active plrigouytic function. Large residual tody suepests prior c-ngulf:/ -nt of entire cell (perhaps a white blood cell). X5,000. t f ' vtive r?'19S,0oG 69 SL 096673 figure XXI Multiple Exposure - 8 months post-exposure T Ps- '%*, * ** -*v J\. i\/-'st- .;.i:v.J* . V, , lr r-*i. f` V-', V f i Ir F i ''jt'e X/ i i vieyg g : 'G/GOLTiriE dose 10 ::g;og i-;ft exdosehe controls Fe.Ge: VP7G 13G-13S in tills control group. in hepntccytes, clcfL-1 Oi'O tv---'it' . in dense bodies. These v.eic suggestive of a cryot.lline e. ..' 1 e.,' " 1 cha'ing processing, Kupffer cells were large nod ce: ` . in E'rge r'eidual bodies, \csiculn r ondopl ascnic reticular, tind biz,.r son. d r,itochenclrl a v.ere occas*ional findings. * 70 SL 096675 Figure XXII Controls - Multiple Exposure - 16 months post-exposure FiGunr: Hopxtxrp; o ,, .r . - . "v mmr-rous dense bodies and lipofuchsin granules. CIc^l-F../ r.ire spaces in dense bodies may represent crystalline .- " i ..racked during processing. X5,000. 4 SL 096676 tr Ftgyre xxil Cjwovrn--trol - Multiple Exposure - 1_S mo-'nTt'h',_prro^s.t,-^erxrposfur re r" ` . , > ~*rt- SL 096677 Figure XXIII Multiple Exposure - 16 months post exposure FIGURE LEGE:ID Indented nucl'. ^ ; rt .-.zj' of field consistent with adenexmreinoma. Cell at upper left v n. ;o s ,d stows mitochondria with high amplitude swelling '! . 'cesir.:e-~. X5.000. Mi'egai.iv e 71 .* SL 096678 SL 096679 Figure XXIV Controls - 24 months to f FIGURE LEGEND Normal appearance of hcpatocyte in this control group. X5.000. Negative ^209,293 72 SL 096680 . Figure XXV VP77 67-71 mLES 10 WEEKLY EXPOSURES TO PPG VL'.YL GlLORlDE POST EXPOSURE 106 ddS k* General Summary o Findings: Changes within hepatocytes included increased Ihoiuchsin granules and endoplasmic reticulum dilatation witn a r.o-L .rL.j. of low electron density tilling endoplasmic reticulur, cistern,:..-. Jo adiition, bile canalicuii frequently snowed distended lumens wio: oh. sii .plication of the plasma membrane at this site. N-on-hapntcp tic o' ngos induced enlarged I'upffer cells v.'ith abundant lysoscw.- . vd ' . bodies and a iif.nonucloar infiltrate into hectic parenchy . SL 096682 73 `* SL 096683 i