Document LpgvxnLbDRNMgB4qNJ41wpZyb

TOXICOLOGY CARCINOGENICITY, AML REPF. ACTIVE EFFECTS OF SINGLE AND MULTIPLE EXPOSURES TO VINYL CHLoAIDE IN RATS AND MICE CA 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 I, Aberdeen Proving Ground, Maryland. B.P. McNAMARA, Ph.D. Chemical Systems Laboratory PROJECT OFFICERS: JOSEPH MCLAUGHLIN, JR., Ph.D. Consumer Products Safety Commission AUTHORS OF RELEASE: ROBERT M. HEHIR, Ph.D., Consumer Products Safety Commission GEORGE BIERBOWER, D.V.M., Consumer Products Safety Commission DONALD A. WILLIGAN, D.V.M., Ph.D., Donald A. Willigan, Inc., GERALD KOLAJA, 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 RICIIAF.D L. DIMM1CK, Chemical Systems Laboratory JOSEPH S. WILES, Chemical Systems Laboratory DISCUSSION A, . Factors tn Carcinogenicity B. Vinyl Chloride Carcinogenicity CONCLUSIONS Appendix GGC 003384 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. 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 GGC 003385 osmium tetroxide, dehydrated in graded alcohol solutions, cleared in propylene 3 oxide and embedded in Epon (O.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/Dawley/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 F-j > Fg ancf ^3 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, m. 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 GGC 003386 4 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 c examined at 8,16 and 20 months are as follows (Appendix, Tables VII-X, inclusive 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% GGC 00.33S7 6 TABLE 1. VINYL Cm-ORIDE STUDIES Experimental Group* PPM DAYS SUMMARY (1 hr/day) (ppm-hrs) SPECIES/NUMBER EXPOSED. 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 D 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 Sprague-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 like controls Fischer Rats 180 negative TOTAL EXPOSED TOTATTcDNTRQL COMBINED TOTAL m2263 3039 *A - 170 ICR Mice (82 male and 88 female) and 171' 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. tKi*L) .<Tl ' V-; .? V V' f. Extruded areas of hepatocyte cytoplasm (in bleb formation)(Fig. XI 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 (50, 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 (500 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. 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^ and chemical carcinogenesis7'^5 by numerous authors. The Food Protection Committee, Food and Nutrition Board of National o 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. This relationship has been reported for 1,2,5,6,-dibenzoanthracene (DBA)4, 12 12 20-methylcholanthrone (MC) , and DMBA plus croton il , p-dimethylaminoazo- 13 *| A benzene , and carbon tetrachloride. Dose response curves have been given for ultraviolet light^5 and ionizing radiation.1*6^7. 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.^ 12 GGC 003390 v-':i^M&r Vi'r':. 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 Lee et al_. Viola et al_. ,2^ 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. Caputo23 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. Keplinger et al_., 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. 1A OGC OOS?si TABLE 3 X m OTHER VINYL CHLORIDE STUDIES AUTHORS VIOLA, BIGOTTI23' CAPUTO CAPUTO, VIOLA,2^ * BIGOTTI KEPLINGER et al. 25 LEE 21 CONSUMER PRODUCTS SAFETY COMMISSION SPECIES RATS RATS RABBITS MICE (RATS St HAMSTERS) MICE ICR MICE A/J MICE FISCHER RATS RESULTS (CARCINOGENESIS) - PPM-HRS POSITIVE AT 28, 800K NEGATIVE AT 48K POSITIVE AT 320K, 1280K, 3200K 6400K, 12,800K POSITIVE AT 720OK POSITIVE AT 5GK . ALL TESTS ESSENTIALLY NEGATIVE BELOW AND POSITIVE ABOVE 48K 50 AND 500 - NEGATIVE 5000 - BORDERLINE POSITIVE 5O,OG0 - POSITIVE 5CG0 - POSITIVE 50, 500, 5000 AND 50,000 - NEGATIVE ` 24,500 - EOSINOPHILIC. LOCI, NO CANCERS \ 16 i GGC 00339: tumors in rats in the C?SC 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 (3-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.^ The Environmental Protection Agency`S has established 1 8 GGC 003393 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 concent ration and exposure time, Ct.^'5,6 2. There were VCM doses which were not carcinogenic and there appeared to be a total accumulated dose above which tumors were produced1. 3 ' 15 1 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 (Cts) of 5,000 ppm-hrs and above. *t 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. '.c!8. The Federal Radiation Council (Report No. 1, Background Material for the Development of Radiation Protection Standards, 1960, Government Printing Office, Washington, DC). . 19. C. Mai torn*. The Value of Predictive Experimental Bioassay in 1 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 O.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 gaskampes, 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 GO: 1&Q3: 55 rtrrtiVUl Table II . Exposure Schedule for Animals Exposed Repeatedly to VCM Species Fischer Rat A/J Mouse Fischer Rat A/J Mouse Sex U 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 7/1S/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 15 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 k~ - r 50 PPM CONTROL 50.000 PPM 5.000 PPM 500 PPM % IW .iT A U T Y (CUMULATIVE) 26 Guc on: :97 O AJivinw no) A in v ." r'/j % (^7 _ . 80-t Figure III 'SPONTANEOUS MORTALITY MALE FISCHER RATS 28 OGc fin'z-r - % MORTALITY (CUMULATIVE) ^^ Figure V SPONTANEOUS MORTALITY 30 GGC OL' m Figure VII SPONTANEOUS MORTALITY 60 AJ MICE 100 DAY STUDY VINYL CHLORIDE MONOMER 50 40 30 20 10 1-12 13 32 GGC 003400 7. MORTALITY (CUMULATIVE) 70 -i 60 60 40 30 20 10 - OQQ OcoOAm 1-9 10 11 Figure IX SPONTANEOUS MORTALITY FISCHER RATS 100 DAY STUDY VINYL CHLORIDE MONOMER yC----- "C------- 1> O'" AGE OF PATS (MONTHS) ' ' 3k EXPOSED \i CONTROL X <T fiH i MEAN ANIMAL. WEIGHTS I N CRM Figure XI GROWTH CURVES OF FEMALE ICR MICE EXPOSED ONCE TO VCM r. .zt.;r V-'w Ti ^CTA'S,C* j? I *0 36 MEAN A N IM A L WEIGHTS I N GRAMS Figure XIII GROWTH CURVES OF FEMALE FISCHER RATS EXPOSED ONCE-TO VCM oo& CooN (oN 38 MEAN A N IM A L WEIGHTS I N GRAMS Figure XV GROWTH CURVES OF A/J MICE EXPOSED TO VCM IN 100 DAY STUDY ho t'Ot' 200 DOS Figure XVII GROWTH CURVES OF FISCHER RATS EXPOSED TO VCM IN 100 DAY STUDY 500- 4*75-5 cn 450-5 < vc 425-f o 2 400-5 H 375- m h- X 350-53 ID UHJ 325- >V^ rf if c/ -J 300 -51 t < X H 275- X 250-3 X< 225-| LtJ 200-5 yj>M. ** '"I >> A* , /V ya 5a ~A "A -A A* A" A 175-f 150 J A'* +-MRLE EXP --MALE jTfTTrmfflTrnLTrjTnmmyrrrrrrrffjT^ CAT a=FEM?LE EXP -FEMALE CNT -10 10 ' 30 50 70 SO 110 20 40 b'0 80 100 WEEKS POST EXPOSURE 42 OID OOt% ooo I 1 Table V. Summary of Incidence of Histo1ogicalJflfcProvcn Neoplasms Within Tissues ICR Mice Exposed to Vinyl Chloride Monomer Inhalation Exposure) 1 -hr Inhalation Exposure in ppm 50,000 5,000 500 50 0 (Control) TOTALS VCM Spontaneous Deaths 0-15 Months 7- 12 Months M F Total M F Total M 0 0 0 5 11 16 33 022A2 6 22 0 5 5 6 8 14 19 0 0 0 4 15 19 9 20 07 i 20 1 1 14 *! "J7 J.L ............ 5 ... P Schedule Sacrifice 8 Months }' ...... 8 Months IB Months | Total 1' FM "1 32 11 '....J Total . _tt._ F Total 1 5' 7 17 24 Totals Male 49 Female 61 27 2 1 3 ... . 1 14 33 !l ! 1 J H1 ! 18 , 27 If 0 0 0 _ L_ 1r 41 55 is0 ii 1r 91 j 174 f 7 0 i0 L______ _____ i ; 2 J9 1 ii I 5 5 2 21 19 | 13 24 14 12 63 18 33 29 31 16 15 14 18 !37 43 .__... 'TT f 'l**n^ ks 51 j 1\ 47 : 54 i ------------------- -i i:, f> V oo oo *o4T*' 44 f Table VII . Over-all Summary Incidence of Non-neop'lastic Changes and Histological ly-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 n 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 4 * 1 2 2 1 6 6 13 1 2 1 1 1 1 1 1 - hepatic cell adenoma 1 ii - cholangiocarcinoma 1 TT 6 13 T6" Lung Number Evaluated : 43 47 26 90 - edema - pneumonitis - bronchio-alveolar hyperplasia 2 21 1 2 - bronchio-alveolar adenoma - bronchio-alveolar carcinoma 15 16 56 68 3 12 10 17 22 68 81 * Total includes animals from scheduled sacrificed (8, 16, 20 month periods) ancT-spont3'iTeous~deaths' Table IX. 9Summary of incidence of Histologi -oven Neoplasms Within Tissues From AJ Mice Exposed to Vinyl Chloride Monomer vMultiple Inhalation Exposures) Sacrifice Pe riod Spontaneous Deaths 0-6 Months 1-hr inhaleition Exposures to Vinyl Chloride Monomer 50 p>pm x 00 C 0NTR0L 500 ppm x IOC { CONTROL M F TOTAL M F TOTAL M F TOTAL M F TOTAL 0 00000000000 T 0 T A 1 cl V CM MF 02 CONTROLS M JT 00 7"12 Months 8 3 11 0 0 0 2 1 3 0 0 0 10 13-20 Months 23 20 43 3 5 8 36 27 63 2 3 5 59 Scheduled Sacrifice 8 Months 3 1 4 0 1 1 6 6 12 0 0 0 9 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 40 0 ) 1 47 5 8 70 1 14 3 4 85 33 32 Totals 63 74 137 21 20 41 87 85 172 20 25 45 150 159 41 ix 45 x ooo oo CM o CO 48 ( 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 Lung .Number Evaluated - bronchopneumonia : 85 26 67 10 Incidence of Response 50, 000 Vinyl chloride 5,000 500 MF 86 87 MF 83 93 MF 87 100 50 M 90 91 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. Oo oft* o<0 50 i Table XIII. Incidence of H i s roiorj i cj l '"ven Keoplasms Within Tissues H rom J-ischer Rats Exposed to Vinyl Chl^K .Gnome r (Single Inhalation Exposure) Sacr.i f tci Organ Period Tissue 50,000 ppm M F Tota I Scheduled Lung 8 months Liver Kidney Stomach Other* Total 16 months Lung Liver 0 0 0 0 2 2 2 0 00 00 00 00 02 02 02 00 Kidney 0 00 Stomach Other* Total 2*1 months Lunq Liver Kidney S tomach Other* 0 2 4 0 1 0 0 20 00 24 26 22 34 00 I1 28 48 Total 21 34 ;55 5 ,000 ppm M F Total - 500 ppm 1 F Total M 0 00 C 00 0 0 00 0 00 0 0 00 0 0 00 0 00 00 0 0 0 00 0 00 0 0 00 0 0o o 0 00 1 0 00 0 0 0 l 01 00 00 1 0 0 0 00 0 00 0 51 6 :i2 0 12 6 5 1 6 31 3 0 13 7 0 0 o 13 Is 3 2 __ 5_ _ 0 0 o \1 36 4 12 0i l 9 0 01 <o 0 01 34 49 83 !70 30 100 54 37 52 i!i_ 85 37 119 64 50 ppm K F Total 00 00 00 00 00 0 0 01 00 00 00 0 0 2 .... 3 .. 0 6 7 12 0 00 22 76 27 91 0 ppm (control) M F Total 00 00 0 0 00 0 00 0 00 00 00 0 0 0 00 0 00 0 00 0 11 -3 _ 14 11 3 14 32 5 91 10 10 1 01 1 47 23 60 27 70 87 oaa o0 01ii o 52 Table XV. Summary of Incidence of Histok. cally-Proven Neoplasms Within Tissues From Fischer Rats Exposed to Vinyl Chloride Monomer (Multiple Inhalation Exposures) 1-hr inhalation Exposures to Vinyl Chlorine Monomer Sacrific^ Period i __ SQ...Ippm x 100 M F Total Control M jj| F Total 500 ppm x 100 i Control M F -Total i H If Total Spontaneous IDeaths 0 0 0 0 0 0 0 0 0 0 0 0` 0-6 Months ! Totals VCM M F 00 Control MF 00 7-12 Months 0 0 0 3 0 3 0 0 0 0 0 0 . ... 0 0 30 13"IS Month 14 6 20 5 0 5 10 6 16 3 0 3 24 12 80 19-24 Month 111 51 162 52 29 Scheduled Sacrifice 0 0 0 0 0 8 Months 16 Months 8 10 18 4 1 * ............ -...... 24 Months 43 43 86 32 29 Totals 176 110 166 96 59 81 52 29 81 33 34 67 00 I I 00 0 5 10 1 11 00 ' 0 61 i2 71 113 69 27 96 145 ?i jn 4 j108 1 !f .222...... J.1..0..5..... * -:6-1...... 1166 163 0 18 85 290 80 85 10 1) 4 114 101 218 .. 201 63 0 1 -cv: J 56 > 120 ooo -Wooo. 54 Table XVII Incidence of Histological! n Ikoplesms Within Tissues From Fischer Rats Exposed to Vinyl Chloride utcmer (Multiple Inhalation Exposures) Scheduled Sacrifice Sacrifice Period 8 Months Organ Tissue Lunq Liver ........ Kidney 50 ppm x 100 M F Tota 1 M 0 0 00 00 0 0 0 00 Control 1 F 1Total o io i ! 0 nI 0 0! 500 ppm x 100 M F Total 000 .0 0... __ 0oo Con t ro1 MF 00 00 00 Total 0 0 0 jStomach 0 0 0 0 0 0 0 0 0 0 0 0 Other* Total 16 Months , Lunq Liver Kidney I0 0 0 0 o 0 0 1 1 0 0 0 0 0 0 0 0 0 1a 0 1 1 0 0 0 0 0 00 0 ` 0 i n . 0 I. 0 n .. ...... .0. . ... 0. 0 0 .. 0 .. n 0 .1 0 Lul_ 0 0 0. 0 0 0 \ 2 0 ! 2 0 0 0 Stomach 0 0 : 0 o 0 \ 0 7 0 0 0 0 0 0 Other* 8 10 ! 13 4 , 5 l a 1 !5 0 j Total i 8 10 18 4 : 5 10 1 11 0 24 Months ] Lunq \ ' Liver 1 ..... 0 ' 7__ r\ 1 J_ i 2i 4 2 6 3 \7 i 11 5 Kidney 0 U K/ J f c - j Stomach i 0 rtv- r* i Total j0 i 35 j 43 01 0 41 | 76 43 j 86 u *1-Cj 32 0 0 ;| 0 0 0 1 --/vl, ~ J. Jl llw 68 . 5^ C(j 25 61 11 42 71 113 1 69 0 oo I 0 0 3| 4 LaJ c 0i 0m _ f3 j 27 LA ooo oow to 56 Table XIX. Summary of Matings and Percentage Pregnancy Observed in the VCM Three-Generation Study. Here and in all similar tables. the exposure level refers to that of the F0 generation. ooG"i 4owoS. W 58 Table XX, Average Litter Size for the Three-Generation VCM Study, Total Pups Born Fl Generat ion 195 F2 Genera tion 239 F3 Genera tion 225 Control Total Number Li tters 22 Average Li tter S i ze STD Dev 8.9 0.63 Total Pups Born 202 50 ppm Total Number Litters l 1 Average j Litter \ Size a, 1 | Total STD Pups Dev Born 0.64 184 19 12.6 0.67 234' 18 13.0 0.69 255 19 11.8 0.67 204 18 j 11.3 j 0.69 285 ________3 500 ppm Total Number Li tters Average Litter Size STD Dev 23 8.0 0.61 21 12.1 0.64 21 12.6 0.64 o0o tooAn A 60 Table XXIII. Numbers, Sex, and Weights of F-j Generation Age of Pups Days Total Number of Pups Control Male Female 50 ppm Male Female 1 102 92 105 93 ' 500 ppm Male Female 93 88 ^ Average Weight (Grams) Control Male Female 50 ppm Male Female 6.88 6.60 6.98 6.69 500 ppm Male . ' Female1-------------------------------a 7.05 6.76 4 102 7 102 92 105 89 105 93 93 14 102 .. . 21 102 er,,. 89 105 92 89 105 1 92 i -----a. 1 j 92 88 92 88 92 j 92 j, 88 f * j 88 t 1 10.50 10.03 10.50 15.29 14.71 15.30 27.8 5 44.5 t :rr 26.8 ! \ 42.4 i *i 28.6 * i f 44.3 i A 10.15 14.87 10.72 10.37 15.76 15.41 27.2 42.8 28.6 45.4 23.4 44.1 i'J eo v .* OO *1 62 I i f Table XXV. .t Numbers, Sex, and Weights iff ^ Generation Age of Pups Days Total Number of Pups Control Male Female 50 ppm Male Female 1 112 111 91 108 4 112 111 91 108 500 ppm Male Female Average Weight (Grams) | Control j Male Female t 50 ppm Male Female 139 123 6.82 6.38 6,84 6.61 139 123 10.18 9.47 10.35 9.76 500 ppm Male Female 6.66 6.30 9.87 9.07 7 111 111 91 108 139 123 14.63 13.90 14.81 14.18 13.89 13.25 14 111 111 90 108 135 121 25.41 25.39 27.46 25.81 25.53 23.95 21 111 Ill t ____ ______ 5 1( 90 108 134 121 j 34.77 33.85 36.75 ' 35.14 35.11 33.40 ooo woo fn eh Figure XVIII Control - 8 months FIGURE LEGEND Control hepatocyte morphology with normal appearing endothelium adjacent. X5,C00. Negative 198,033 Figure XIX Single Exposure - 8 months post-exposure FIGUil'J LEGEND llepatocytes show lipid droplet accumulation. At center of field, cytoplasm devoid of recognizable organelles is in shape of bleb. Adjacent belb is apparently free in space of Dis.se. This may represent a means whereby cytoplasmic material may be shed. 1-15,000. Negative A52S91 GGC 00341 V:' Figure XX Single Exposure - 8 months post-exposure f FIGURE LEGET Figure shows portion of two b..:`:U;ocyt^ small dense bodies with one or more elector are lipofuchsin granules. x-i.OOO. Note numbers of lucent regions. These Negative 7JrA54,0-lG GGC 00 68 hi' Figure XXI Multiple Exposure - 8 months post-exposure FIGURE LEGEND Kupffer cell shows evidence of active phagocytic function. Large residual bod$' suggests prior engulfuient of entire cell (perlr-ps a white blood cell). X5,000. Negative #193,06S OGC CCS*zo 69 Figure XXII VIKYIi a:iminE/MULTIPIE DOSE ig post exposure controls Ferres VP76 136-138 C; lCiigeS V. ere c^O;-;in tills control group. Tn hepatocytes, c.left-1 ? ko spue ns v, :;ro sea", in. dense bodies. These wore suggestive of a crystalline i"r.ixr.'.rl ex'.rcr.ted during processing. Kupffer cells were large and ccrtr.ined large residual bodies. Vesicular endoplnsniic reticulv and bizarre sh;: :d riitochondria were occasional findings. 4 70 Gc^, Figure XXIII Multiple Exposure - 16 months post exposure FIGURE LEGBd) Indented, nucleus at center of field consistent with adenocarcinoma. Cell at upper left is ncotic nod shows mitochondria with high amplitude swelling uw:' ftxculout densities. X5,000. Negative l" GGc 003422 71 Figure XXIV Controls - 24 months { FIGURE LEGEND Normal appearance oT hepatocyte in this control group. X5,000. Negative 203,298 GGC 00542.? 72 Figure XXV VP77 67-71 MALES 10 WEEKLY EXPOSURES TO PPM VIdYL QILORIDE POST EXPOSURE 106 WEEKS General Summary ot Findings: Changes within hepatocytes included increased lipofuchsin granules and endoplasmic reticulum dilatation with a material of law electron density filling endoplasmic reticulum cisten io. J:i addition, bile canaliculi frequently shaved distended lumen; with t;:_ s.implication of the plasma membrane at this site. Non--hepatc ;ytio changes included enlarged Kupffer cells with abundant lysoso.-.r. mvi bodies and a mononuclear infiltrate into hectic parenehy>- GGC 003424 73