Document 7RE8OG1voqaeam0BBDV1LMBko

4. V / Yh ^ C fr- -r DOW CHEMICAL L^.S.A. 6ot^L February 11, 19B0 RECEIVED MIDLAND. MICHIGAN 4*640 FEB 19 I960 b o-L^*4 fbcSS<^~_ nARR Z. G. Bell, PPG Industries T. J. Benya, Ethyl Corporation W. D. Harris, Unixoyal, Inc. G. K. Hatfield, Diamond Shamrock M. N. Johnson, M.D., The B. F. Goodrich Company C. D. Kary, Shell Chemical Company J. V. Lawrence, SPI J. T. Seavell, CMA / W. M. Smith. Air Products & Chemic aIs,(JLnc./ J. Stafford, ICI R. JSL. Wheeler, Union Carbide Corporat RECEIVED RESEARCH A DEVELOPMENT, FEB 14 1980 W* M. SMITH cpscTyintl chloride studt in rats and mice Inresponse to an informal request for information Dr. Robert Hehir ^nfcpsc has sent the enclosed manuscript. While considerably improved over previous drafts, the authors relate the lung adenomas and car cinomas in mice to vinyl chloride, choosing to minimize the possible relationship to pneumonia. It is my understanding that the manuscript will be submitted to Toxicology & Applied Pharmacology. I'd appreciate any comments you might have. Attachment dm AN OPERATING UNIT OP 1 HE LOW CHIMICM COMPANY l \Yl' AP00009258 U.S. CONSUMER PRODUCT SAFETY COMMISSION WASHINGTON. D.C. S0S07 February 6, 1980 Dr. Theodore R. Torkelson Health 6 Environmental Sciences Building #1603 Dow Chemical U.S.A. Midland, Michigan 48640 Dear Ted; In response to your telephone request of 2/5/80, I am enclosing for your information a copy of the paper entitled* "Toxicology, Carcinogenicity and Reproductive Effects of Single and Multiple Exposures to Vinyl Chloride in Rats and Mice." This paper is now being submitted for publication. If you or your colleagues have comments or questions, please contact me directly at (301) 492-6504. Technical Director of Laboratories cc:Dr. J. McLaughlin, CPSC Dr. B. McNamara, CSL-Edgewood Arsenal Don R. Clay, DAED/ES, CPSC AP00009259 1 TOXICOLOGY, CARCINOGENICITY AND REPRODUCTIVE EFFECTS OF SINGLE AND MULTIPLE EXPOSURES TO VINYL CHLORIDE ZN RATS AND MICE by R.M. HEHIR,1 B.P. MCNAMARA,2 J. McIAUGHLIN, JR.,3 D.A. WILLIGAN,4 G. BIER30WER,5 J.F. HARDISTY,6 *pd Proofs tosR.M. Hehir, Ph.D. Consumer Product Safety Commission 5401 Wes'tbard Avenue Bethesda, Maryland 20207 Robert M. Hehir, Ph.D., Consumer Product Safety Commission* B.P. McNamara, Ph.D., US Army Chemical Systems Laboratory2 Joseph McLaughlin, Jr., Ph.D., Consumer Product Safety Commission^ Donald A. willigan, D.V.M. , Fh.D_, Donald A. VJilligan, Inc.4 George Bierbower, D.V.M., Consumer Product Safety Commission^ Jerry F. Hardisty, D.V.M., Experimental Pathology Laboratories, Inc. DISCLAIMER The views, opinions and conclusions expressed in this paper-are those of the authors and do not necessarily represent the views of the Consumer Product Safety Commission. \s AP00009260 HEHIR et. al. 2 ABSTRACT From a regulatory point of view there is serious concern for the safety of humans exposed to single and intermittent low level concen trations of known carcinogens. Vinyl chioride monomer (VCM), already identified as a human and animal carcinogen, was selected as a model agent to explore this area of interest for two reasons - one, expo sure to consumers and industrial workers was wide-spread and secondly, some basic information on its mode of action was available and more was being developed, in the traditional cancer bioassay, animals are repeatedly exposed over their normal life span to small doses of suspect chemical. In the current studies rats and mice were exposed in an inhalation chamber to single one-hour doses of vinyl chloride ranging from -50 to 50,000 ppm. Zn addition; other groups were given -10 onehour exposures to 500 .ppm or 100 one-hour exposures to 50 ppm of the same chemical. All animals were kept in cages for the remainder of their lives, generally 18-24 months. Moribund animals were euthanized and survivors were sacrificed on schedule and their tissues examined for pathological changes. A multi-generation reproduction study in Sprague- Dawley/Wistar rats, carried out simultaneously, did not demonstrate adverse effects on reproductive performance. Moreover, the original parents from the vinyl chloride reproduction study were also maintained for two years and subsequent examination failed to disclose any carcino genic effect. Specifically, the oncogenic study demonstrated dose rel effects for single one-hour exposure of vinyl chloride at high levels 5,000 and 50,000 ppm. These concentrations increased the incidence of pulmonary adenomas and carcinomas in mice. Repeated exposures of A/J mice AP0000926I HEHIR et. al. 3 to the same chemical at 500 ppm x 10 one-hour exposures also increased the incidence of pulmonary ademonas and carcinomas which are considered \ highly significant (P-.001) when compared to matched controls. However, at a lower repeated dose of 50 ppm x 100 one-hour exposure no significant increase in tumors was observed. Rats exposed to identical concentrations of vinyl chloride failed'-to elicit a tumorigenie response. The liver as a target organ received special attention. Electron microscopic (EM) studies of the Fischer rats exposed.for one hour to vinyl chloride showed sublethal cytoplasmic injury which subsequently recovered. Examinations of the Sprague-Dawley/Wiatar rats which were held for 24 months after the 49th exposure to 2,450 or 24,950 ppm vinyl chloride revealed only age related., changes. Running Title: Biological Effects of Short-Term Exposures to VCM AP00009262 KEHIR et.al 4 INTRODUCTION The responsibility for regulating vinyl chloride fell between the overlapping jurisdiction of several federal agencies. Federal proposals to control vinyl chloride*s use as an aerosol propellant, to limit occupational exposure and emission from chemical and fabricating plants as well as to regulate its shipment were initiated by the Food and Drug Administration, the Environmental Protection Agency, the Consumer Product Safety Commission, the Department of Transportation and the Occupational Health and Safety Administration in (1974). These actions followed reports of long-term chronic toxicity studies by Viola et.al., (1971) and Maltoni(1974), which demonstrated vinyl chloride's carcinogenic activity in experimental animals. .The need for immediate action was further heightened by the epidemiological studies of Creech and Johnson (1974). Their report uncovered four cases of hepatic angiosarcoma in chemical plant workers associated with polyvinyl chloride production. Further industrial investigations by Tabershaw et al., (1974) identified additional cases. The relationship between vinyl chloride and angio* sarcoma in man and animal has been substantiated by a number of American and European scientists. For the most part, the government's primary attention was focused on reducing occupational exposures, in addition, basic research projects were also being initiated by other federal agencies. Considering the scope of the federal research programsand proposed rulemaking which would eliminate unnecessary exposure to vinyl chloride it appeared that the potential problem was well under control. However, vinyl chloride has been referred to by medical AP00009263 HEHIR et.al. 5 researchers as a "potent carcinogen." What did ye know about the potential hazards to consumers dealing with a host of everyday products containing vinyl chloride in such items as aerosol hairsprays, pestiides, paints, etc.? Vinyl chloride is a base ingredient in one of the most commonly used plastics, polyvinyl chloride (PVC), which was omni present in their everyday life i.e., food wrappings, pipes, home and auto upholstery and hundreds of other sundry products. The fact was ve could find no relevant information on the long-term biological effects of acute or intermittent inhalation exposures no any known carcinogen. Preliminary studies by FDA and EPA demonstrated that the use of certain aerosol hairsprays or pesticides could result in exposures of vinyl chloride of 200 and 400 ppm respectively for short-duration. Bearing in mind that Maltoni and Lefemine (1974) and Keplinger el: al., (1975), implicated even low level repeated doses of vinyl chloride i.e., 50 ppm as being capable of inducing angiosarcoma in laboratory rodents there was genuine concern for single high level and intermittent lower level exposures of vinyl chloride in humans. Therefore, the CPSC had two primary objectives in mind in sponsoring this research: (1) to study the long term effects of graded single and short-term intermittent ex posures to vinyl chloride and (2) to study the effects of vinyl chloride on reproduction. Furthermore, we felt that this program would complement other on going research efforts to determine the pharmacokinetics and mechanism of action for this chemical. Quite apart from the objectives just mentioned, there was a secondary objective, to look at the question of a "threshold effect" or "safety factor" for low dose exposures for a well defined carcinogenic substance like vinyl chloride. There is a AP00009264 HEHIR at.at. 6 natural scientific curiosity about the question of a "threshold effect" and while we did not hope to resolve the debate on this highly controversial issue we did expect to develop sufficient scientific data to help formulate regulatory policy and to sustain future decisions to control low level human exposures to chemical carcinogens. The experimental protocols were jointly developed by the staff of Chemical Systems Laboratory (formerly Sdgewood Arsenal) and by the Health Science staff at the Consumer Product Safety Commission. The protocols were reviewed and critiqued by both governmental and private sector experts and their suggestions were considered and in corporated into the final experimental design. MATERIALS AND METHODS Vinyl Chloride Monomer The vinyl chloride monomer gas (99.9% minimum purity), used in these inhalation studies, was obtained from Matheson Gas Products in size IF cylinders containing 200 lbs. of the monomer under 34 psi at 70F. Animals Rats: Fischer 344, male and female (Charles River Laboratory, Wilmington, Mass.). Sprague-Dawley/Wistax, male and female (Chemical Systems Laboratory Colony, Edgewood, Maryland). Mice: AJ Strain, male and female (Jackson Laboratory, Bar Harbor, Maine). AP00009265 1 hehir et.al. ICR Strain/ male and female (Charles River Laboratory, Wilmington, Mass*) Exposure and Sampling Techniques The exposures were conducted in 100 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 ml 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. 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). 7 AP00009266 HEHIR et. al 8 Exposure Procedures for Toxicology and Carcinogenicity Male and Female rats (Fischer 344) and mice (A/J or ICR) were totally exposed in one of the following ways: a. One single one-hour exposure at 0, 50, 500, 5,000 or 50,000 ppm. Fischer 344 rats, ICR mice, exposure shown in Table 1. b. Tien one-hour exposures at 500 ppm (one-hour per day, five days per week for two weeks). Fischer 344, A/J mice, exposure schedule shown in Table 2. 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, exposure schedule shown in Table 2. 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. Cf No blood chemistry of hematology studies were performed. Pathology 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. AP06609267 HEH1R et. al. 9 All rodents were to be serially sacrificed at 8, 18, 24 months pest 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 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 cannibalism. Electron Microscopic Studies For the electron microscopic studies the following animals were selected at, random. Groups consisting of five male and five female Fischer 344 rats from each of the single one-hour (50-500-5,000 and 50,000 ppm) exposures with equal numbers of their corresponding control group were sacrificed at 8, 16, and 24 months. Groups consisting of five male and five female Fischer rats from each of the multiple one-hour exposures (10 x 500 ppm, 100 x 50 ppm) with equal numbers of their control group were sacrificed at the end of 16 and 24 months, after the final VCM exposure. One group of five males and five female Sprague-Dawley/Wictar rats from the parental generation of the reproduction group (49 x 50 ppm, 49 x 500 ppm) along with equal numbers of their control group were sacrificed 24 months after the final exposure. AP00009268 HEHIR et. al. 10 The rats were anesthetized with pentobarbital and their livers were perfused via the vena cava with chilled electron microscopic fixative consisting of 4% formaldehyde, 1% glutaraldehyde in 0.1 H phosphate buffer as described by .M. McDowell and B.F. Trump (1976). After mincing, the tissues were post-fixed in 1% phosphate buffered osmium tetroxide, dehydrated in graded alcohol solutions, cleared in propylene oxide and embedded in Epon by the modified techniques of J.H. Luft (1961). Reproduction Study and carcinogenicity , Fe male and female parents or Sprague-Dawley/Wistar rats were exposed as outlined in Table 3 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. This assured exposure of all forms of male germ cells. The females were exposed during all phases of the oogenic cycle. Each parental generation was evaluated for fertility, litter size, number stillborn and ability to lactate. The F^, and F^, *"<3 off-spring were evaluated for post-natal growth, viability, survivability and reproduction anomalies. The parental generation of Sprague-Dawley/Wistar rats were maintained for 24 months post exposure for carcinogenic evaluation. RESULTS Toxicology (single and multiple exposures) 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 AP00009269 HEHIR et. al. 11 males were hyperventilating after 45 minutes of exposure; twitching and ataxia were noted. At 59 minutes tremors were seen. Females showed some hyperactivity at 50 minutes and 25 percent showed respi ratory difficulty and ataxia after 55 minutes. Mo other effects were noted. There were no remarkable signs of toxicity during the ten re peated 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. Observation After Exposure There were no significant differences in mortalities at P=.05 levelbetween exposed and control*animals using Student - T or ChiSquared analysis at any level. However, when the KOLMOGOROV - SMIRNOV two sample test is used to analyze the VCM mortality data there is a significant difference at P".05 level between 50,000 ppm and control male single exposure ICR mice and between 50,000 ppm and 50 ppm single dose ICR mice. No other dose level or species showed significance. Gross Pathology There was a suggestion of higher frequency of masses in the lungs and livers of mice and rats exposed once or repeatedly at the higher dose levels, i.e., 500, 5,000 and 50,000 ppm. Light Microscopic Pathology Mice (single exposure) Histological examination at 8 and 16 months in ICR mice exposed once to concentrations of 50, 500, 5000 or 50,000 ppm showed the following changes attributable to VCM. Summary of histo logically identified changes in the liver and lung are shown in Table 4. AP00009270' HEHIR et. al. 12 The development of lung adenomas Increased with exposure to higher dose levels of vinyl chloride but the progression to carcinoma was minimal as summarized in Table 5. Pneumonitis was evident in all animal groups which were exposed to VCM in doses of 500 ppm or more. Mice(10 x Ihr x 500 ppm and 100 x Ihr x 50 ppm exposures) A summary of the histopathologic changes observed in the liver and/or lung of A/J mice at 16 and 20 months following their multiple exposures to VCM are presented in Tables 6 and 7. The Increased incidence of pulmonary adenomas and carcinomas was attributed to the VCM exposure. The pulmonary find ings are presented for comparative purposes in Table 8. The effects of multiple exposures.of VCM on A/J mice in Table 8 were compared by the z test for difference of means and proportions. Accordingly, there is a highly significant difference in the number of pulmonary adenomas (P< .001) and carcinomas (P-- .001) in the A/J mice exposed to 500 ppm VCM for 10 x one hour exposures when compared to their matched controls. By the same token, there is no significant difference in pulmonary adenomas and carcinomas in test versus control animals at the 50 ppm x 100 x one hour exposure level. Rats (single exposures) Except for aggravation of latent pulmonary changes, particularly broncho pneumonia as shown in Table 9, 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. Rats (multiple exposures) No changes attributable to VCM were apparent in any of the tissues evaluated microscopically at 6, 16, or 24 months from Fischer 344 rats ex-' posed 10 times at 500 ppm or 100 times at 50 ppm as shown in Table 10. Rats (49 exposures from the reproductive study) The following histological observations were made 24 months post exposure AP00009271 t HEHIR et. al. 13 in Sprague-Dawiey/Wistax rats which had been exposed 49 times at 50 ppm or 500 ppm of VCM. Neoplastic and non-neoplastic lesions were observed in approximately equal frequency in control and test animals. The only lesions that, occurred in higher frequency in the VCM exposed animal than in control rats were eosinophilic cell alterations presented as foci and/or areas as shown in Table 10. The appearance of these foci was related to dosage. The nature of these lesions is controversial. Summary of Light Microscopy Studies The carcinogenic/ or possible related changes (eosinophilic foci) attrib-. ut&ble to exposure of VCM in rats and mice are summarized in Table 11. The significance of these histologic changes will be discussed later in the paper. Electron Microscopic,Results In general, these studies indicate that exposure to vinyl chloride in creased organelle turnover as well as loss of volume control (bleb formation) and increased lysosomal activity in the liver of rats. These alterations progressively decreased as recovery after exposure increased. Hepatocellular carcinoma was seen in one male Fischer rat which had re ceived 10 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 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. AP00009272 HEHIR et. al. 14 General Conclusions of S-Mont'h Recovery to Single Exposure to 50/ 500, 5000, and 50,000 pcm-hr of Vinyl Chloride In Fischer Rats * a. Alterations from control occurred in all treated animals at each concentration. b. Changes were less severe in female animals. c. Hepatocytic alteration included lipid accumulation, increased dense bodies, lipofuschin granules and residual bodies. These are an indication of cytoplasmic sublethal injury. d. Alteration was incremental with increasing exposure concentration. e. Most severe changes involved cellular necrosis with subsequent phagocytosis by Kupffer cells seen in single and multiple exposures. . Extruded areas.of hepatocyte cytoplasm (in bleb formation) may indicate a means of removal of altered portions of cells. General Conclusions of 16-Month Recovery After Single Exposures to 50, 500, 5000, and 50,000 ppm x 1 hr to Vinyl Chloride in Fischer Rats a. Normal morphology 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. General Conclusions to 24-Month Recovery from Single Dose Exposures of 50, 500, 5000, and 50,000 ppm Vinyl Chloride in Fischer Rats a. Age-related changes including formation of cleft-like spaces AP06609273 HEHIR t. al. 15 (possible related-to hemoglobin/hesnosiderin) , inflammatory infiltrate (pericholangitis) and some collagen accumulation were seen in controls and treated animals in this group. b. In this group hepatocytic injury - not encountered in controlswas observed. At 5000 ppm one female showed evidence of lymphosarcoma. General Conclusions of 8-Month Recovery to Multiple Exposures to 500 ppm x 10 exposures; 50 ppm x 100 exposures to Vinyl Chloride in Fischer Rats a. Changes were 6een in male and female treated animals. b. Less alteration was encountered in female rats. c. Cellular changes were greater than that seen in single dose animals. d. Alterations involving bepatocyte nuclei, not seen in single dose animals, were encountered in males of this group. General Conclusions of 16-Month Recovery to Multiple Exposures of 500 ppm x 10 exposures and 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. b. Other changes included cleft-like spaces in hepatocytes and Kupffex cells. These were interpreted as age-related changes in hemo globin/hemosiderin metabolism. c. Changes were more severe in males General Conclusions to 24-Month Recovery After Multiple Doses of 500 pom x 10 and 50 ppm x 100 of Vinyl Chloride in Fischer Rats a. Controls and treated animals showed changes similar to those AP00009274 HEHXft et. al 16 seen after 24 months recovery to single exposure. b. These changes were regarded as age-related and non-specific. General Conclusions to 49 Exposures for 1 Hr at Daily Intervals Followed by 24 Months of Recovery (SO and SOD ppm) (Sprague-pawley/wistar Rats) a. Age-related non-specific change was encountered in all groups. b. Most advanced.changes were related to.300 ppm group of male rats in which cell swelling and platelet aggregation were seen. Multi-generation Study in Rats (Sprague-Dawley/Wistar) No consistent changes attributable to VCM were found in F0 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 effect cn fertility, viability and survival of offspring and ability to lactate. The F^, F^, and F^ offspring were evaluated for litter size, percent of stillborn pups, post-natal growth, viability, survivability and reproduction anomalies as shown in Tables 12-18. Electron microscopic examination of the parent rats which were held for 24 months after the 49th exposure revealed age related changes, cell swelling, and platelet but no tumors. DISCUSSION AND CONCLUSIONS 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 susceptible body cell; if it makes a carcinogenic biochemical combin ation Chit") with the cell; if this combination is not repaired? if the AP00009275 REHIR et. al 17 cancer cells are not destroyed by the immune system, and if other host factors are favorable to carcinogenicity. On the other hand it may also 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 second set of considerations above suggest the existence of "no effect" doses, threshold doses, and dose response curves which have the hockey stick shape described by Bryan and Shimkin (1943). These consider ations have been discussed for radiation by Storer (1975) and Lipton et al., (1970) and for chemical carcinogenesis by Saffiotti and Maugh (1976). The Food Protection Committee, Food and Nutrition Board of National Academy of sciences - National Research Council in (1959) noted that a dose response relationship could be applied to carcinogens. The higher the dose, the greater the response and the shorter the time required to elicit that response. This relationship has been reported for 1, 2, 5, 6dibenzoanthracene (DBA) by Bryan and Shimkin (1943) for 20-methylcholanthror>e (MC) and for 9, 10-dimethyl-l, 2-benzanthracene (DMBA) plus croton oil by Graffi (1953) for p-dimethylamino&zobenzene by truckery (1959) and for carbon tetrachloride by Eschenbrenner and Miller (1944). .Dose response curves have been given for ultraviolet light by Blum (1950) and for ionizing radiation by Finkel (1958) and Mole (1958). There are non-turnorigenic levels of exposure to carcinogens for given experimental conditions. Carcinogens do not produce cancers in all AP6bb09276 HEHIR et. al. 16 exposed animals, in bioassays, the lower dose levels sometimes do not produce tumors while higher dose levels sometimes do produce tumors. This remark is supported by experimental observations of such.researchers as Poel (1959), Graffi (1953) and Druckery (1959), The possibility that a "no effect" dose may exert a carcinogenic effect which is too weak to be detected with the numbers of animals used in routine testing is recognized. The concept of lifetime accumulative, non-tumorigenic and tumorigenic doses of radiation has been adopted. The Federal Radiation Council (i960) 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. Vinyl Chloride and Carcinogenicity Maltoni (1975) and Maltoni and Lefemine (1975), described a doseresponse relationship for the carcinogenic effect of vinyl chloride in animals. The neoplastic response was related to the length of exposure. Lee et al., (1976) noted that the incidence and severity to tumors increased with the concentration of VCM and the length of exposure. The statements above indicate that the total dose (concentration X exposure time) may be of importance in the carcinogenicity of vinyl chloride. The total inhaled dose can be approximated utilizing concepts developed by Haber .in (1924). 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). AP00009277 t HERIR at. al. 19 The concentration can be expressed also in parts per million (ppm) and the time can be expressed in hours producing Ct in ppn-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 (1975) and Maltoni and Lefemine (1975)j Lee ct al., (1978); Viola et al., (1971); Caput et al.,(1974) and Keplinger et al., (1975). These Ct calculations are summarized in Tables 19 and 20. In the studies f P. L. Viola, A. Bigotti and A. Caput (1971) 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. xl. Viola and A. Bigotti (1974) exposed, rats and rabbits to vinyl chloride four hours per day, five days per week for 12 months. The concentrations 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. Ho tumors were produced in rats at this dose 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 or at 320,000 ppm-hrs. Tumors appeared in the rabbits after nine months following similar treatment at a concentration of 10,000 ppm VCM. Thus the lowest total dose demonstrating earcinogenosis in rabbits was 7,200,000 ppm-hrs. M. L. Keplinger et al., (1975) 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 AP00009278 HEHIR et. al. 20 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. * Calculations from the data of the Consumer Product Safety Commission studies (Tables 5 and 11) indicate that strongly positive carcinogenic effects may not appear in rats until the total dose (Ct) of VGK reaches or exceeds 50,000 ppm-hrs. The maximum response in the rats was the appearance of eosinophilic cells which might suggest a pre-eaneerous change. The CPSC VCM tests in mice indicate that increased frequencies of adenoma may appear in this species at Ct's of 5,000 ppm-hr and above. This total dose for carcinogenicity is in general agreement with Lee et^ al., (1978) 78,000 ppsn-hrs and Maltoni (1975) and Maltoni and Lefemine (1975) between 30,000 ppm-hrs and 150,000 pptn-hrs, and other investigators, in general, in orders of magnitude, carcinogenic tendencies axe seen in some species at Ct's of 5,000 to 50,000 ppm-hrs. Definite carcinogencity appears in both mice and rats at Ctjs of 50,000 to 500,000 pptn-hrs, and high incidences of carcinogenesis are noted in mice and rats at Ct*s of greater than 500,000 ppm-hxs. The previous studies of Maltoni (1975), Maltoni and Lefemine (1975), Lee et al., (1978), Viola et al., (1971), Caputo et al,, (1974), Keplinger et al.,(1975) and the present CpSC study are in agreement as to the dosetime 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-hrs 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 S'' AP00009279 HEHIR et. al. 21 electron microscopic study did not reveal carcinogenic responses in rats at Cts from 50 to 50,000 ppm-hrs. However, Ct's of 2450 ppm-hrs produced eosinophilic foci but no overt tumors in rats in the CPSC tests. As an additional consideration in the total dose concept, CT's of 5000 ppm-hrs 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 a'denomas produced by the single exposure of mice at 5000 ppm is of borderline significance. However, there is the indication that even a single exposure of suffi cient magnitude may be carcinogenic in sensitive species. Under the conditions of exposure, VCM produced pneumonitis in all mice abov^ 500 ppm. The high level of pneumonitis among test mice was a result of test exposure, not a prior condition of spontaneous disease. Furthermore, the pneumonitis was recognized and evaluated as an inherent response, not as an undesirable variable of exposure. The idea that viral infection of the lungs (i.e., influenza) might lead to cancer is not new. For example, Steiner and Loosli (1950) studied the effects of human influenza virus (Type A) on the incidence of lung tumors in mice . The supposition was that infection might, under certain conditions, cause cancers but more specifically from the proliferative changes that occurred in the lung following recovery. The Steiner-Loosli (1950) experiment was a test of that supposition. In their experiment the hyperplasia which developed as a result of infection did not go on to tumors. On the contrary these investigators noted possible inhibitory or anticarcinogenic effects which they felt deserved further study. Under AP00009280 HEHIR et. al. 22 the conditions of this toxicologic study, pneumonitis and puljnonary adenomas were induced in mice. This is a conclusion strictly limited by the available data. Relationships, synergistieally or otherwise, between pneumonitis and pulmonary adenomas or carcinomas are neither explicit nor ascribable. There is a real possibility that vinyl chloride could induce pneumonitis in man. However, this does not mean that pulmonary cancer may or may not be the inevitable consequences.. It is, therefore, 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 (1950-595 indicate that time-weighed (8-hr) .average exposure were 120-385 ppm. This would give daily Cts of 960-3080 ppsj-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 concentra tions are frequently below 1 ppm. One peak grab sample of 33 ppm has been reported in an EPA Scientific and Technical Assessment Report (1975) at 0.5 kilometer from the center of one plant. AP00009281 HEHIR et. al. 23 The time-weighted threshold limit value of the American Conference of Government Industrial Hygienist for (1977) was 200 ppm. There is a notice of intended change. The Environmental Protection Agency in (1976) established the following emission limits for vinyl chloride; (1)formation and purification processes is 10 pf*&, (2) emissions from equipment pre ceding and including the stripper in the plant process flow is 10 ppm, and (3) emissions from equipment following the stripper are to be controlled by stripping dispersion resins to 2000 ppm and other resins to 400 ppm. The Code of Federal Regulation 40, Protection of Environment, (1976) states that fugitive emissions from loading and unloading of lines, rotating pumps, reciprocating pumps, rotating compressors, agitators, manual venting of gases, and opening of equipment must be ducted through control systems so that the concentration of vinyl chloride in the exhaust does not exceed 10 ppm. Assuming that man is as sensitive as the mouse or rat the carcino genic effects of vinyl chloride might be expected after an accumulated Ct of about S000 ppm-hrs or approximately 190,000 ppm-hr (Maltoni BT3, Table 19) , respectively. Based on the high pollution values given above (1000 ppm-hr per day) carcinogenic doses could have been accrued in one week (mouse) or 30 weeks (rats). At the TLV of 200 ppm (daily Ct of 1600 ppm-hr ) as postulated in Table 21 the time to accumulate a carcinogenic would be three days (mouse) or about 19 weeks (rat). Comparable accumulation times at 10 ppm (80 ppm-hrs per day) would be about 12 weeks (mouse) or 375weeks (rats). The calculations are based upon a five day working week. There are a number of methods which could be used to obtain risk estimates for low doses of this carcinogenic agent. Aside from the fact AP00009282 HEHIR at. al. 24 that none of the current extra-polation procedures ean be justified on a biological basis, the exercise permits one to compare theoretical risk levels for various chemicals. Whereas the Arroitage and Doll (1961) multi stage model assumes the risk is approximately linear even in the low dose range; the probit model of Mantel and Bryan (1961), has a hockey stick appearance. That is to say, as the dose approaches zero, the response function in this region is extremely flat (zero slope). By comparison, the log probit model for high levels of risk and high tumor rates gives a lower predicted dose than the linear extrapolation model. Realizing that any low dose extrapolation method is arbitrary and subject to biological and mathematical criticism, we never-the-less selected a model developed by Guess and Crump (1976) to analyze our data. Instead of assuming the shape of the dose response function we allowed the program to fit. the experimental-data<to the best model by using a Monte-Carlo goodness - of-fit test. We used a computer program (GLOBAL) which has been described in a paper by Crump, Guess, and Deal (1977). The procedure is based upon a dose response function of the form: -Q(d) P(d) *1- exp OOj 0(d) - i=0 .. (1) (2) where Q(d) is a polynomial with non-negative coefficients of unknown de gree, where for all i events and ** o for all but finitely many i. P(d) is the probability that an animal will develop a particular response which is linear in d dose rate for small d if q^>o. Moreover, p(d) can be arbitrarily flattened by taking enough of the low order coefficients equal to zero, since at very low doses P(d) - P(0)'v,exp {-q^ q^d* where 1 is the smallest positive integer for which > 0. Accordingly, Crump, Guess and Deal (1977), interpret P(d) as the probability that an animal will AP00009283 HEHIR et. al 25 develop a particular type of tumor during the duration of the experiment while under continuous exposure to a dose rate d of the chemical being tested. By using the experimental values in table 5, comparing the single VCM inhalation exposures of ICR mice and the progression of pulmonary lung lesions to carcinoma over an 18 month period in the GLOBAL computer program one can theoretically determine an approprate (dose-response) mathematical model and also the risk given the dose or conversely the dose given excess risk. These values along with the upper and lower 95 and 99 percent confidence limits are presented in Table 22 for the multi stage polynomial model. By interpolation of the data presented in Table 22 we estimate an approximate two-fold increase in the risk of developing a pulmonary carcinoma in the experimental animal when exposed to 5000 ppm VCM compared to the spontaneous background level. At the highest exposure level, i.e.r 50,000 ppm, the theoretical risk of developing the same pulmonarystumor-is increased about nine fold. Examining the dose given excess risk, the model predicts that a continuous exposure to 2.4 ppm VCM would induce one excess cancer case in a million. CONCLUSIONS An overall summary of the biological effects of VCM on the laboratory animals studies is presented in Table 25. - A. Except at the highest concentration, 50,000 ppm, where possibly anesthetic-type effects were seen, VCM produced no pharmacotoxic (excluding pathology) signs in mice or rats during or after exposure. AP00009284 HEHIR et. al 26 B. No reproductive changes attributable to VCM vere found. These include mating, percent pregnancies, fertility, lactation, percent still-boras, litter size, post-natal growth, neonatal viability, neonatal survivability, and fetal anomalies. C. VCM seemed to produce pneumonitis in mice and to aggravate bron chopneumonia in rats. D. VCM produced eosinophilic changes in rats but no frank (light microscopy) carcinogenesis. E. VCM produced an increase in frequency of lung tumors in mice. F. Electron microscopic studies in rats revealed some hepatocellular changes but no carcinogenesis related to the VCM exposures at Ct's of SO,000 ppm-hrs or less. G. The carcinogenic effects of VCM in mice seem to depend upon concentration and exposure time, Ct. There were VCM doses which were not carcinogenic and there appeared to be a total accumulated dose above which tumors were produced. Tumors were not seen in mice at Ct's of SOD ppm-hrs, however, carcinogenic effects of VCM were seen in mice at total doses (Cts) of 5*000 ppm-hrs and above. 0 AP00009285 HEKIR t. ftl. REFERENCES American Conference of Governmental Industrial Hygienist. 1977. Threshold limit Values for Chemical Substances and Physical Agents in the Workroom Environment with Intended Changes for 1977. Aunitage, P. and Doll, R. 1961, Stochastic Models for Carcinogenesis. In Proceedings of Fourth Berkeley Symposium on Mathematical Statistics and Probability, Berkeley California June 20 - July 30, I960 (Neyman, J. Editor) Vol. 4, Berkeley California; University of California Press. Pages 19-38 Blum, H.F, 1950. On the Mechanism of Cancer Induction by Ultraviolet Radiation. J. Nat. Cancer Inst. 11: *163-*195 Bryan, W.R., and Shimkin, M.B. 19**3. Quantitative Analysis of DoseResDonse Data Obtained with Three Carcinogenic Hydrocarbons in Strain C3H Kale Mice. J. Nat. Cancer Inst. 3:503-531. Caputo, A., Viola, P.L., end Bigotti, A. 197*1. Oncogenicity of Vinyl Chloride at Low Concentrations in Rats and Rabbits. J. Int. Res. Ccsmun. 21:1582 Creech, J.L., Jr. and Johnson, M.N. 197*1. Angiosarcoma of Liver in the Manufacture of Polyvinyl Chloride. J. Occip. Med- 16:150-151. Crump, K.S., Guess, H.A., and Deal, K.L. 1977. Confidence Intervals and Test Hypotheses Concerning Dose Response Relations Inferred from Animal Carcinogenicity Data. Biometrics 33: *1370*151. Druckery, H. 1959. Pharmacological Approach to Carcinogenesis, in Ciba Foundation Symposium on Carcinogenesis: Mechanisms of Action, pp 110-130. Boston: Little, Brown and Co. Environmental Protection Agency. 1975. Scientific and Technical Assessment Report on Vinyl Chloride and Polyvinyl Chloride, EPA600/6-75-00*1. Environmental Protection Agency. 1976. Federal Register. Title *10 Protection of the Environment. Part 6l - National Environmental Standards for Hazardous Air Pollutants, Standard for Vinyl Chloride. Vol. *11, No. 205. Eschenbrenner, A.B., and Miller, E. 19*1*1. Studies on Hepatomas. I. Size and Spacing of Multiple Doses in the Induction of Carbon Tetrachloride Hepatomas. J. Nat. Cancer Inst. *1:385-388. AP00009286 HEHIR et. al. Federal Radiation Council. I960. Report No. 1, Background Material for the Development of Radiation Protection Standards-. Government Printing Office, Washington, DC. Finkel, Miriam P. 1958. Mice, Men and Fallout. (The potential danger of strontium 90 is appraised on the basis of data fran animal experiments) Science 128:631-641. Pood Protection Ccnrnittee, Food and Nutrition Board of National Academy of Sciences - National Research Council. 1959. Problems in the Evaluation of Carcinogenic Hazard fran the Use of Food Additives. Publication 749- Guess, H.A., and Crunp, K.S. 1976. Low-Dose-Rate Extrapolation of Data from Animal* Carcinogenicity Experiments - Analysis of a New Statistical Technique. Mathematical Bioscience 32:15-36. Graffi, A. 1953. Untersuchungen uber den Mechanismus der Carcerogenese und die Wirkungsweise cancerogener Reize. Abhandl. deut. Akad. Wess. Berlin. 53:1-27. Haber, F. 1924. "Funf Vortrage aus den Yahren 1920-23"; No. 3. Die chemie im Kriege: no. 5- Zur geschiechte des gaskanpes. Julius Springer, Berlin.-reference through Prentiss: Chemicals in War. McGraw-Hill Book Company, Inc., New York, 1937. Horton, A.W., and Deman, Dorothy T. 1965. Carcinogenesis of the Skin. . A Re-examination of Methods for Quantitative Measurement of the Potencies of Complex Materials. Cancer Research 15:701-709. Keplinger, M.L., Goode, J.W., Gordon, E.E., and Calendra, J.C. 1975* Interim Results of Exoosure of Rats, Hamsters and Mice to Vinyl Chloride. Ann. N.Y. Acad. Sci. 246:219-224. Lee, C.C., Bhandari, J.C., Winston, J.M., House, W.B., Dixon, R.L., and Woods, J.S. 1978. Carcinogenicity of Vinyl Chloride and Vinylidene Chloride. J. Tox, Ehviron. Health 4:15-30. Luft, J.H. 1961. Brorcrvements in Epoxy Resin Bibedding Methods. J. Bicphys. Biochem. Cytol. .9:409-414. Maltoni, C. 1974. Conmunication sent to the Proceedings on the Pro posed Permanent Standards for Occupational Exposure to Vinyl Chloride. Occupational Safety and Health Administration, US Department of Labor, Washington, DC. Maltoni, C. 1975* The Value of Predictive Experimental Bioassay in Occupational and Environmental Carcinogenesis. An Example: Vinyl Chloride. Ambio. 4:18-23. AP00009287 HEHIR et. al. Kaltoni, C. and LeFemine* G. 1974. Carcinogenicity Bioassay of Vinyl Chloride: 1. Research Plan and Early Results. Environ. Res. 7:387-405. Maltoni, C., and LeFemine, G. 1975. Carcinogenicity Assay of Vinyl Chloride. Ann. NY Acad. Sci. 246:195-218. Mantel. N. and Bryan, W.R. 1961. "Safety" Testing of Carcinogenic Agents. J. Nat. Cancer Inst. 27:455-470. Maugh II. T.H. 1978. Chemical carcinogens: How Dangerous Are Low Doses? Science 202:37-41. McDowell, E.M.,and'Trump, B.E. 1976. Histological Fixatives Suitable for Diagnostic Light and Electron Microscopy. Arch. Path. Lab. Med. 100:405-414. - Mole, R.H. 1958. The Dose-Response Relationship in Radiation Carcino genesis. Brit. Med. Bull. 14:184-189. Poel, W.C. 1959. Effect of Carcinogenic Dosage and Duration of Exposure on Skin-Tumor Induction in Mice. J. Nat. Cancer Inst. 22:19-44. Saffiotti, U. 1977. Identifying and Defining Chemical Carcinogens, pp. 1311-1362. Origins of Human Cancer, Book C. Hunan Risk Assess ment. Editors: Hiatt, H.H., Norton, J.D., Winsten, J.A., Cold Spring Charter of Conferences in Cell Proliferation. Vol. 4. Cold Spring Charter Laboratory. Spiegel, M.R. 1961. Theory and Problems of Statistics McGraw-Hill Book Company. Steiner, P.E. and Loosli, C.G. (1950) The Effect of Human Influenza Virus (Type A) on the Incidence of Lung Tumors in Mice, pancer Research 10:6, 385-392. Storer, J.B. 1975. Radiation Carcinogenesis, Chapter 16, pp 453-483 in Cancer 1. Etiology: A Comprehensive Treatise. Editor: Becker, F.F. Plenum Press, NY., London. Tabershaw, I.R., and Gaffey, W.R, 1974. Mortality Study of Workers in the Manufacture of Vinyl Chloride and Its Polymers. J. Occupa. Med. 16:509-516. Upton, A.C., Randolph, J.L., and Conklin, J.W. 1970. Late Effects of Fast Neutrons and Gamma-Rays in Mice as Influenced by the Dose Rate of Irradiation: Induction of Neoplasia. Radiation Res. 41:467. Viola, P.L., Bigotti, A., and Caputo, A. 1971. Oncogenic Response of Rat Skin, Lungs and Bones to Vinyl Chloride Cancer Res. 31:516-522. AP00009288 HEHIR et. al. fiOQTOWLEDGMEHT The authors would like to thank Drs. Gerald Kolaja and Glen E. Marts for performing the gross pathology; Dr. David E. Hinton for preparing the electron micrographs, and Mr. Richard h. Dimmiek and Mr. Joseph E. Wiles for their technical assistance -throughout these experiments. We also v?ish to acknowledge Mr. Milton X. Christensen, Mr. Edmund J."'Owens, Mr. Michael A. Lochner, and Mr. Ronald Pellerin for their help and cooperation. Finally, the authors would like to thank Mr. Steve Ganocy for his help with the statistical analysis and risk assessment model. -------------------------- ) AP00db9289 AP00009290 Table 1. Single Exposure Schedule of Animals to VCM Species Sex Fischer 3^ Rat M P Dose ppm 50 500 5000 50000 50 500 5000 50000 Exposure date AM PM Exposure group size(s) 3/'l/75 3/11/75 3/18/75 3/25/75 3/4/75 3A1/75 3/18/75 3/25/75 90 90' 85 90 90 100 95 83 ICR Mouse M 50 500 5000 50000 P 50 500 t 5000 50000 3/V75 3/11/75 3/18/75 3/25/75 3/4/75 3/11/75 3/18/75 3/25/75 .90 90 90 90 90 90 90 90 Rat Mouse ' M Neg/Cont. F Neg/Cont. M Neg/Cont. F Neg/Cont. 92 79 82 88 Age 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 Table 2. Exposure Schedule for Animals Exposed Repeatedly to VO! Species Sex Dose ppm Fischer Rat M F 50 500 50 500 M 50 A/J House 500 F 50 500 Fischer Rat M Neg. control F Neg. control A/J Mouse 'M Neg. control F 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 -- r _ -- __ 1/26/76 7/10/75 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 slze(s > Start End 90 86 90 90 90 87 90 90 90 87 90 90 90 88 90 90 50 50 50 50 50 97 50 50 .... *10 39 50 50 50 50 50 50 Age Start wks 21 1*1 21 19 15 8 15 8 21 19 21 19 15 8 15 8 aid 91 16 91 16 35 10 .35 10. . 91 16 91 16 35 10 35 10 > -Q O ooO ro Grouo X II III Table 3* VCM Multigeneration Study Fq Parents 5/ Compound Air Dose-^ Control Number of Males 25 VCM Low Dose (50 ppm) 25 VCM High Dose 25 (500 ppm) Number of Females 25 25 25 a/ Sprague-Dawley/Wistfir rats. b/ Exposed to 50 or 500 PPM of VCM one-hour per day, five days per week for 10 weeks (*I9 exposures) before mating. Table 4. Over-all Slumary Incidence of Non-neoplastic Changes and HistologicaUy-Proven Neoplasms Within the Liver and Lungs of ICR Swiss Mice Exposed to Vinyl chloride. Single Inhalation Exposure Tissue/Response Exposure : Control Dose Level (ppm): 0 Sex of Animals : M P Animals Per GrouEft : 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 - hepatic cell adenoma - hepatic cell carcinoma - hemangioma - hemangiosarccma 2 2 1 Incidence of Response Vinyl chloride 50,000 5,000 500 M PM FM F 7*1 82 76 82 72 75 63 78 68 12 67 72 3 55 74 6 it 2 12 3 5 28 84 13 fl i| 14 5 1 it 1 6 l 1 19 50 MF 81 80 64 68 23 14 1 2 2 Lung - pneumonitis :Number Evaluated: 50 1 70 6 61 12 65 21 10 13 12 66 17 19 n 11 15 4 - bronchio-alveolar adenoma - bronchio-alveolar carcinoma it 8 31 lit 14 1 21 10 8 . 10 8 1 ___________________________________________________________________________________.______________________________________________________________j__ a/ Total includes animals from scheduled sacrificed (8 and 18 month periods) and spontaneous deaths. 68 7 6 i ) * Table 5: HISTOLOGICAL EXAMINATION OF ICR MICE AT 8 AND 18 MONTHS FOLLOWING A SINGLE (1-HOUR) EXPOSURE TO VINYL CHLORIDE MONOMER VINYL CHLORIDE CONCENTRATION 50.000 PPM* 6.000 PPM* 500 PPM* 60 PPM 0 (CONTROL) HISTOLOGICAL CHANGES ATTRIBUTABLE TO VINYL CHLORIDE INDUCTION OF PULMONARY ADENOMAS 45/137 OR 33.3% 24/143 OR 16.0% 10/139 OR 12.9% 14/139 OR 10.1% 12/120 OR 10.0% PROGRESSION TO CARCINOMA 3/137 OR 2.2% 1/143 OR 0.7% 1/139 OR 0.7% 0/139 OR 0% o/i2a on 0% PNEUMONITIS WAS EVIDENT IN ALL ANIMAL GROWS WHICH WERE EXPOSED TO VCM AT DOSES OF 600 PPM OR MORE. AP00009294 Table 6. Over-all Summary Incidence of Non-neoplastic Ghanges and Histologically-Proventifeoplasns within the Liver and Lungs of AJ Mice Exposed to Vinyl Chloride (Multiple Inhalation Exposure) Tissue/Response Exposure Dose Level (PPM) House Exposure Sex of Animal Animals Per Group3 :M : 46 Incidence of Response Control Vinyl Chloride 0 500 10 x 1 10 x 1 P MF 48 78 92 Liver : Number Evaluated : 45 48 78 89 - hepatic cell necrosis - lumphoid cell infiltrate - hepatic cell lipidosis - neutrophil infiltrate - bile duct hyperplasia - granulomatous foci - sinusoidal reticulosis - hepatocyst - amyloidosis - anglectasis * 46 1 2 2 1 6 13 1 2 1 1 1 1 1 1 - hepatic cell adenoma - cholangiocarclnoma 1 1 11 13 TS Lung Number Evaluated : ii 47 76 90 - edema i. - pneumonitis - bronchio-alveolar hyperplasia 2 21 1 2 - bronchio-alveolar adenoma - bronchio-alveolar carcinoma 15 16 56 68 3 12 10 IT 22 81 a/ Total includes animals from scheduled sacrified (87 l67 20 month periods) and spontaneous deaths. AP00009295 Table 7. 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 Exposure : Dose Level (PPM) : Hours Exposure Sex of Animal ; Animals Per Group a :' Lung : Number Evaluated : - edema - congestion - focal hemorrhage - pneumonitis - bronchio-alveolar hyperplasia - osseous metaplasia Control 0 100 x 1 M 3? 39 2 4 2 5 - bronchio-alveolar adenoma - bronchio-alveolar carcinoma - reticulum cell sarcoma 11 2 IJ Incidence of Response Vinyl chloride $0 Ito x 1 FM 47 81 45 11 8l 11 2 3 45 33 1 18 27 38 34 1 IB" 30 *3 CO /ibtal Includes animals from scheduled sacrifice (8, 16, 20 month periods) and spontaneous deaths. Table 8s HISTOLOGICAL EXAMINATION OF A/I MICE AT 0, 10, AND 20 MONTHS FOLLOWING MULTIPLE (1-IIOUR) EXPOSURES TO VINYL CHLORIDE MONOMER NUMBER OF EXPOSURES ft CONCENTRATION OF VCM 0 (CONTROLS) 10 X 500 PPM 2/ O (CONTROLS 100 X SO PPM */ HISTOLOGICAL CHANGES ATTRIBUTABLE TO VINYL CHLORIDE MONOMER INDUCTION OF , PROGRESSION TO PULMONARY- ADENOMAS CARCINOMA 31/90 OR 34.4% 3/00 OR 3.3% 124/16$ On 74.7% 22/166 On 13.3% 29/64 OR 34.5% 2/84 OR 2.4% 65/158 OR 44.1% 7/158 OR 4.4% / Highly significant difference in the mnber of pulmnary adenonas (P< .001) and carcinomas (Par .001) observed at tho 500 ppa X 10 one hr exposure level versus control by 2 test (Spiegel L961). b/ No significant difference for pulmonary adenomas (P* 0.14) and carcinomas (Pi 0,19 observed at the lover multiple exposure level. AP00009297 Table 9. Over-all Surtmary 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 Group3 : Control 0 MF 89 74 Incidence of Response Vinyl Chloride 50,000 5.000 500 M F M FM F 86 87 83 93 87 100 50 MF 90 91 huniS - bronchopneiiijonia :Number Evaluated : 85 26 67 10 B0 11 80 87 05 95 80 Zi 45 13 13 10 15 3 28 3 % incidence : 30.6 14.9 56.2 16.9 16.3 11.5 17.6 3 35*0 4.1 a/Total includes aniirtals from scheduled sacrifice (8,16, and 24 month periods) and spontaneous deaths. AP00009298 liable 10. Incidence of Eosinophilic Changes Within Livers of EWA Colony (Sprague-Dawley) Rats Multiple Inhalation Exposures to Vinyl Chloride (VC) Exposure Dose Level (PPM) Hours Exposure Group a/ Control 0 49 A0 T VC 500 49 A BT A Males: Animals per group Eosinophilic Focus/Foci % Eosinophilic Area/Areas % 9 1/9 11.1 0/9 0.0 16 1/13 7.7 1/13 7.7 Eosinophilic Alterations b/ 1/9 1/13 % 11.1 7-7 Females: Animals per group 18 7 Eosinophilic Focus/Foci % 0/18 0/7 0.0 0.0 Eosinophilic Area/Areas % 1715"~o77 22.2 0.0 Eosiniphilic Alterations b/ 4/iB 0/7 % 22.2 0.0 25 2/22 9.1 1/22 4.5 2/22 9.1 25 0/25 0.0 1725 16.0 172? 16.0 13 12 10/13*** 3/11 76.9 27.3 1/13 1/11 7.7 9-1 10/13 4/11 76.9 36.4 11 14 4/11 36.4 Tfil 18.2 1/13 7.7 0/13 0.0 5/11 1/13 45-4 7.7 25 13/24*** 54.2 2/24 8.3 14/24**** 58.3 25 5/24** 20.8 2/24 8.3 6/24 25.0 11 4/10 40.0 0/10 0.0 4/10 40.0 19 2/19 10.5 1/9 5.3 3/19 15.8 a/ Group: A Scheduled sacrifice, B * Spontaneous deaths, T = Total b/ Eosinophilic foci and/or areas In the same animal are tabulated one time as an eosinophilic alteration * * Different from controlat5.0 ->2.5? level (P " .05 - .025) ** Different from controlat 2.5 ->0.5? level (P - 0.25 - .005) *** = Different from controlat0.5 - >0.05? level (P = .005 - .0005) **** = Different from controlat0.05? level (P = .0005) VC 50 49 BT 14 25 4A1* 8/21** 36.4 38.1 3/11 3/21 27.3 14.3 5/11** 9/21** 45.4 42.8 6 25 1/6 3/25* 16.7 12.0 1/6 16.7 1/6 16.7 2/25 8.0 4/25 16.0 Table 11. SUMMARY - VINYL CHLORIDE LIFETIME CANCER STUDIES Experimental Group - PPM A 50,000 DAYS (1 hr/dav) 1 (ppm-hrs) SPECIES 50,000 ICR Mice Fischer Rats NUMBER EXPOSED RESVJLTS 100 178 2.2% carcinoma/33% adenoma negative A 5,000 1 5,000 ICR Mice Fischer Rats 100 180 0.7% Carcinoma/16.8%adenocna negative A 500 1 500 ICR Mice Fischer Rats 180 190 0.7% Carcinoma negative A 50 1 50 ICR Mice Fischer Rats 180 180 negative negative B 500 49 24,500 Sprague-Dawley/ Eosinophilic 49 Wistar Rats chanqes B 50 49 2,450 Sprague-Dawley/ 47 Eosinophilic Wistar Rats changes C 500 10 5,000 AJ Mice 160 74% Adenoma Fischer Rats 160 negative C 50 '100 5,000 AJ Mice 179 41% Adenoma Fischer Rats 180 negative TOTAL EXPOSED TOTAL CONTROL COMBINED TOTAL 2263 776 3039 A - 170 ICR Mice (62 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 - 45 Sprague-Dawley/Wistar (20 male and 25 female) served as control animals for inultigeneration reproduction study* C - 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. AP00009300 l Table 12. Surrmary of Matings and Percentage Pregnancy Observed In the VCM Ihree-Generation Study. \ 1 Control3 50 DPT?- > 500 DOT3 Total Gen. W Females Mated Total Females Pregnant Total ' Total % Pregnant Females Females Females Mated Pregnant Total % Pregiant Females Females Mated Total Females Pregnant % Pregiant Females po 25 22 88 25 ' 21 8*1 25 23 92 *i 20 19 95 19 18 9** .7 21 21 100 P2 19 19 100 18 18 100 21 21 a/Here and in all similar tables, the exposure level refers to that of the FQ generation. h/ Generation 100 AP00009301 Table 13. Average Litter Size for the Three-Generation VCM Study. Total GEN. Pups Bom Fi 195 Control Total Average Number Litter Litters Size STD Dev 22 8.9 0.63 50 ppm Total Total Pups Number Bom Litters Average Litter Size 202 21 9.6 STD Dev 0.6*1 Total Pups Born l8*t 500 pnra Total Average Nurrber Litter Litters Size 23 8.0 SID Dev 0.61 f2 * 239 19 12.6 0.67 23*1 18 13*0 0.69 255 21 12.1 0.6*1 F3 225 19 11.8 , 0.67 20*1 18 11.3 0.69 285 21 12.6 0.6*1 Table 14. Percentage of Stillborn Pups in the VCM Three-Generation Study GEN. ?! Total Pups Bom 195 Control Total Stillborn 1 % Stillborn 0.51 Total Pups Bom 202 50 ppm Total Stillborn 5 % Stillborn 2.48 Total Pups Bom 184 500 ppm Total Stillborn 3 % Stillborn 1.63 F2 239 0 0.00 234 2 0.85 255 2 0.78 225 2 ' 0.89 204 5 2.45 265 3 1.13 Table 15. Nuribers, Sex, and Weights of F^ Generation Ajre of Pups Days 1 Total Number of Pups Control 50 ppm Hale Female Male Female 102 92 105 93 500 ppm Male Female- 93 88 Average Weight (Grams) Control 50 ppm 500 ppm Male Female Male Female Male Female 6.88 6.60 6.98 6.69 7.05 6.76 . 4 102 92 105 93 92 88 10.50 10.03 10.50 10.15 10.72 10.37 7 102 89 105 93 92 88 15.29 14.71 15.30 14.87 15.76 15-41 14 102 89 105 $2 92 88 27.8 26.8 28.6 27.2 28.6 23.4 21 102 89 105 92 92 88 44.5 42.4 44.3 42.8 45.4 44.1 AP00009304 Table 16. Numbers, Sex, and Weights of F^ Generation Age of Pups Days 1 Total Number of Puds Control 50 ppm 500 ppm Male Female Male Female Male Female 120 119 114 118 123 130 Average Weight (Grams) Control 50 ppm 500 ppm Male Female Male Female Male Female '6,38 6,13 6.55 6.16 6.52 6.18 4 118 119 114 117 122 130 9-70 9.13 9-82 9.21 10.00 9-27 7 118 119 113 116 122 130 14.38 13.85 in. 57 i3.n5 14.72 13.97 14 118 119 113 116 122 130 27,77 27.26 28.10 26.15 28.80 27.44 21 118 119 113 116 122 130 1(0.89 '10.57 41.5*1 38.67 42.93 41.20 AP00009305 i i | Tkble 17 . Nuntoers, Sex, and Weights of Generation Age of Pups Days 1 Total Number of Pups Control 50 ppm 500 ppm Male Female Male Female Male Female 112 111 91 108 139 123 Average Weight (Grams) Control 50 ppm 500 ppm Male Female Male Female Male Female 6.82 6.38 6.84 6.61 6.66 6.30 4 112 111 91 108 139 123 10.18 9.<17 10.35 9-76 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 26.41 25.39 27.46 25.81 25-53 23-95 21 111 111 90 108 135 ' 121 34.77 33.85 36.75 35.11 35.11 33.40 AP00009306 Table is . Viability, Survival, and Lactation Indexes in a ThreeGeneration Study of Reproductive Performance After Exposure of the F0 Parents to VCM Gas. Generation Control Low Dose 50 pern High Dose 500 ppm pi 100 100 Viability F2 99-2 99.7 F3 100 . 100 99.4 99.6 100 Survival Index F1 f2 F3 98.5 99.2 99.6 99.5 98.7 99.5 99.4 99.6 97.3 Lactation Index F1 P2 F3 98.5 100 99.6 99.5 99-1 99.5 Viability Index Day 21 Survival Index = Lactation Index = No. of ouos alive at Day x 100 fro." of pups bom alive No. of pups alive at Day 21 No. of puds bom alive x xuu of pups alive at Day 21 10 No. of pups alive at Day 4 A 99.4 100 97.3 Eanerjee, B.N., Course Director, Teratology-Principles and Procedures Related to Fetal Development. The Center for Professional Advancement, Sorrmerville, New Jersey, September 197*1. Personal Cornmication. AP00009307 Table 19, MALTONI VINYL CHLORIDE STUDIES^ pi Calculations based on reference TEST Em BT3 BT6 ET7 SPECIES RATS RATS RATS RATS ET^ MICE K* = thousand (000) a/ C. Kaltoni 1975 b/ C. Maltoni and 0. Lefemine 1975 RESULTS (CARCINOGENESIS) - PPM-HRS 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 lO^OOK POSITIVE AT 30K, 150K, 300K, 600K, 1500K and 3600K AP00009308 20.tabus one VINYL CHLORIDE STUDIES AUTHORS VIOLA, BIGOITI, CAPUJX) 1971 CAPtnO, VIOLA Biccrrn 197*1 KEPLINGER et al.. 1975 lee 1978 CONSUMER PRODUCTS SAFETY COMMISSION 1979 * SPECIES RATS RATS RABBITS MICE (RATS & 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 6*1OOK, 12,800K POSITIVE AT 7200K POSITIVE AT 56K ALL TESTS ESSENTIALLY NEGATIVE BELOW' AND POSITIVE ABOVE 78K. 50 AND 500 - NEGATIVE 5000 - BORDERLINE POSITIVE 50,000 - POSITIVE 5000 - POSITIVE 50, 500, 5000, AND 50,000 - NEGATIVE 2*1,500 - EOSINOPHILIC LOCI, NO CANCERS s AP00009309 i AP00009310 Table 21: TLY YS ACCUMULATED PPM/HRS TIV 200 PPM 10 PPM FIVE DAY WORK WEEK TIME* TO ACCUMULATE CARCINOGENIC POSE MOUSE RAT 5000 PPM/HR 150,000 PPM/HR 3 DAYS 10 WEEKS 12 WEEKS 375 WEEKS f s I J iH Table 22: VINYL CHLORIDE INHALATION - PUIX-10NARY RISK </ 0 50 500 5000 50000 Carcinoma--^ 0/120 0/139 1/139 1/143 3/137 Single Exposure (ICR mice) Risk Given Dose 95 Percent P(d)-/" u 99 Percent L U .002723 .002744 .002931 .004803 .023329 -.002118 -.002090 -.001842 -.000001 -.001435 .007563 .002744 .007703 .009604 . 0*18093 -.003639 -.003609 -.003341 -.001507 -.009218 .009085 .008096 .009203 .001113 .055876 (d) - P (o)/ .1 .01 .001 .0001 .00001 .000001 .0000001 .00000001 a/ 50,000 24,134 2,403 240 24 2.4 .24 .024 Dose Given Excess Risk 95 Percent Lu 22063 10675 1063 106 11 1.1 .11 .011 113312 54561 5431 543 54 5 .54 .054 99 Percent L 18767 9084 904 90 9 1 .09 .009 u 133209 64122 6383 638 64 6 .64 .064 a/ Vinyl chloride exposure In ppm. W Number of mice with carcinoma/number of mice total. c/ Risk or probability that animal will develop a particular tumor (Method: d/ Upper and lower confidence limits of 95 and 99 percent. e/ Risk of excess cancers at various dose levels. Crimp, Guess, Deal 1977). , AP00009311