Document 7R85q69Z8q2eab8bdQJ6qnpre

1 Environmental Health Perspectives VoL 21, pp, 25-32, 1977 ihalation Toxicity of Vinyl Chloride nd Vinylidene Chloride* ' C. C. Lee,t J. C. Bhandari,f J. M. Winston,! . B. House,! P. J. Peters,! R. L. Dixon,! id J. S. Woods! Exposure of mice to 1000 ppm of vinyl chloride (VC), 6 hr/day, 5 dayVwtck, caused some acute deaths with toxk hepatitis and marked tubular necrosis of the renal cortex. Starting the sixth month, mice exposed to 1000, 250, or 50 ppm of VC became lethargic, last weight quickly, and died. Only a few mice exposed to 50 ppm survived for 12 months. Pulmonary macrophage count was elevated in some mice. There was a high Incidence of bronchiolo-alveolar adenoma, mammary gland tumors including ductular adenocardnoma, squamous and anaplastic cell carcinomas with metastasis to the lung, and hemangioaarcoma in the liver, and, to a lesser extent. In some other organs. The incidence of these tumors quickly increased, and the severity was in direct proportion to the levels of VC and the length of exposure. Malignant lymphoma involving various organs was observed In a few mice. Rats were more resistant to the toxk effects of VC. Exposure to 1000 ppm slighdy depressed the body weight of the females. Exposures of 250 or 1000 ppm caused a number of deaths and hemangiosarcoma in the liver starting the ninth month. Most rats with hepatic hemangiosarcoma also developed hemangiosar coma in the lung. Hemangiosarcoma occasionally occurred in other tissues of one or two rats exposed to 50 ppm or higher level of VC. Exposure of mice to 55 ppm of vinylidene chloride (VDC) also caused a few acute deaths and a few hepatic hemangiosarcomas. Inflammatory, degenerative, and mitotk changes occurred in the liver. No mouse exposed to VDC developed any mammary gland tumors. Several mice had bronchioloalveolar adenoma. Exposure of rats to 55 ppm of VDC slighdy depressed the body weight. Hemangiosarcoma occurred in the mesenteric lymph node or subcutaneow tissue In two rats. Introduction rhythmias (2), and sensitization of the myocardium (3). Relatively high concentrations of VC (10-40% Vinyl chloride (VC) was first prepared more than in air) for 30 min produced narcosis and/or death in a century ago, and its fire and explosion hazards are mice, rats, and guinea pigs (4). The guinea pigs t well known. Acute toxicity of VC was first re were found to be more resistant. The main lesions ported in guinea pigs (/). As a possible anesthetic were congestion of the lung with pulmonary edema * agent in dogs, VC was found to cause incoordinated and hemorrhages in some animals, and congestion muscular activity of the extremities, cardiac ar- of the liver and kidney. Failure of the blood to clot I was also observed. Detectable injury of the liver 'Preliminary results were presented to the 1976 Fall meeting and/or kidney occurred in rats exposed to 100, 250, of the American Society for Pharmacology and Expenmental or 500 ppm but not 50 ppm of VC, 7 hr/day and 5 7 Therapeutics. August 15-19. 1976. Tulane University, New Or days/week, for up to 6 months (5). Changes in liver leans, Louisiana; published in the Pharmacologist, 18: No. 2.245 (Fall 1976) and to the First International Congress on Toxicol and spleen weights, a decrease in leukocytes, and ogy. March 30-April 2, 1977, Toronto, Canada (Abstracts, 1977, an increase in erythrocytes were observed in rats P. 32). exposed to 2% VC, 8 hr/day for 3 months (6). tPharmacology and Toxicology, Midwest Research Institute, Carcinogenic action of VC was first reported in 425 Volker Boulevard, Kansas City, Missouri 64110. 1971. Male rats (Ar/IRE) exposed to 30,000 ppm of * ^Environmental Toxicology Branch. National Institute of En vironmental Health Sciences, P. O. Box 12233, Research VC, 4 hr/day and 5 days/week for 12 months, de ft Triangle Park, North Carolina 27709. veloped tumors of the skin, lungs, and bones (7). V December 1977 25 CMA, 050771 I Furthermore, Zymbal gland carcinomas, nephro outside of the chambers. Mice were housed four to blastoma, hepatic and extrahepatic angiosarcomas six per cage and rats two per cage. Pulverized or were observed in rats and pulmonary tumors, block laboratory chow (Wayne Manufacturing mammary carcinomas, and liver angiosarcomas Company) was provided at all times except during were observed in mice exposed to as low as 50 ppm exposure. Water was available ad libitum. A 12-hr of VC, 4 hr/day, 5 days/week, after 7-12 months light cycle was maintained at all times. The temper (8). Angiosarcoma of the liver and other hepatic ature in the chamber and in the room averaged 24 diseases, notably portal fibrosis, were identified 1.3C. The relative humidity ranged 25-60% at the among VC polymerization workers (9-12). An stan of the experiment and was later regulated at 50 epidemiological study (13) indicated that cancers at 10%. Four animals of each species, sex, and ex multiple sites might be developed in VC and posure level were terminated for various laboratory poly(vinyl chloride) workers. tests, gross and histopathologic examinations at the The present study was undertaken to determine end of 1, 2, 3, 6, and 9 months; the surviving ani the toxic and carcinogenic effects of 50,250, or 1000 mals were terminated at the end of 12 months. ppm of VC in rats and mice and to define any possi ble biochemical changes relating to any histological and neoplastic lesions. In addition, one extra Laboratory Evaluations chamber was available and used to compare the ef fects of 55 ppm of vinylidene chloride (VDC). All animals were observed throughout the study for adverse signs. Feed consumption was recorded Material and Methods weekly and body weight biweekly at a uniform time of day. Hean (mouse) or aortic (rat) blood from four males and four females of each group of Inhalation Chambers and Monitoring each species was collected under anesthesia (ether for mice and sodium pentobarbital for rats) at in The inhalation chambers are cubical type and terim and final terminations. Hematology (RBC, re made of stainless steel, with a volume of 3.5 m3. ticulocyte, platelet, WBC and differential counts, Three chambers were used for 50,250, or 1000 ppm nucleated RBC, hematocrit, hemoglobin, of VC; one chamber was used for 55 ppm of VDC, methemoglobin, and Heinz bodies) and clinical and one chamber was used for uncontaminated blood chemistry (SGPT and BUN) were performed air as control. VC gas (99.8% pure, Matheson on all samples. For rats, prothrombin time, SGOT, Products) was metered with rotameters into the alkaline phosphatase, bilirubin, creatinine, LDH, chamber air supply. VDC (99% pure, Aldrich o-HBDH, immunoglobin IgA, IgB-A, IgB-B, and Company) was heated to 37'C to generate the IgM (14), total protein (15), albumin (16), globulin vapor. The VDC lines and rotameter were heated (by difference), and collagen contents in liver and to 40C to prevent condensation. Chamber air was lung (17, 18) were also measured. Macrophage initially sampled with a syringe and monitored using counts of pulmonary washings and cytogenic a gas chromatograph (Varian-2700) with a flame analysis of bone marrow cultures (19) were per ionization detector. An automatic sampling system formed on the control, and animals receiving 1000 was later used. Periodically, a sample was directed ppm VC and 55 ppm VDC animals. Limbs from the to the gas chromatograph and the readout was pro longest exposed animals were examined for os cessed by a Varian CDS 111 electronic integrator. teoporosis or malacia using a senograph x-ray The integrator was programmed to measure peak machine. They were examined for the presence of area and to calculate chamber concentrations by any bone tumors, any changes in bone density, cor external standards. tical thickness or striations within the bone cortex, any loss of bone cortex, or any unusually trabecular Experimental Design pattern of the bone. Other specific studies including l4C-thymidine incorporation into DNA (20), he Albino CD-I mice and CD rats (Charles River patic aminolevulinic acid (ALA) synthetase (21), Breeding Lab) about 2 months old were used at the urinary ALA assays (22) and a-fetoprotein (23) I start in these studies. For each species. 360 animals were performed in mice and /or rats. were divided into five groups, each consisting of 36 When moribund or at termination, all animals males and 36 females. Each group of both species were euthanized for necropsy after the collection of was exposed to 50, 250, or 1000 ppm of VC, 55 ppm blood. Gross examination, especially for any ap of VDC, or uncontaminated air for 6 hr/day and 5 pearance of abnormal growth or other lesions, was days/week. All animals lived in the same stainless carefully performed on all tissues including the steel cages with wire bottoms during exposure and brain, pituitary, thyroids, respiratory tract, alimen- 26 Environmental Health Perspectives ary n. oancrea -iody ~a' ;on; s adja nt '.he van ;ion'"C All1 te amiiiwd Re u Mic- C ic deaths.. corr'-oi.: the igh the ..iii exposec rep' ?e* for e i miu ar Durir die<' jt \ Th cl lethargy occurre to D< me hs VC det domina na> ur tur rs were n Tl 1. * Treatmei Co -ol VC, 50 VC 25< VC IOi Ve. 55 D tm CMA 050772 ^ ^ used founS ilvcrized orl lufacturingi cept during] <rn. A 12-hrl '"he temper? eraged 24 1 -60% at thel ulated at 50* ix, and exJ laboratory tions at the. viving aniy ! months, j the study s recorded a uniform rat) blood i group of :sia (ether ats) at in- >:RBC, reil counts, loglobin, 1 clinical erformed . SGOT, c. LDH, B-B, and globulin liver and rophage 'togenic ere pering 1000 from the for osh x-ray ience of ity, corcortex, tbecular icluding 10), he>e (21), in (23) tnimals :tion of my ap is, was ng the ilimen- ectives lary c .nal, urogenital organs, thymus, heart, liver, pane:-ms, spleen, mesenteric lymph nodes, and nod> cavities. The brain, liver, kidneys, spleen and con.i. were removed and weighed. Tumors with jdja< .t normal tissues and the whole or portions of the ious tissues were fixed, processed, sec tion and stained for microscopic examination. All rnal and internal tumors were carefully ex am and identified histologically. Its M ral Observations. There were five early di apparently due to the acute effect of the test Ci unds. Two males and one female exposed to tl hest level of VC were found dead between tl rd and ninth days of exposure; two males e .d to VDC died on the 13th day. They were n id with healthy mice from the same shipment f- remainder of the experiment. Thereafter, all r. appeared in good health. ring the sixth month, a few mice exposed to VC d ;r were terminated before their imminent death. "1 clinical signs included rough hair coat, I rgy, anorexia and rapid weight loss. No death i i-red in the control group or the group exposed l DC. During the seventh through the ninth i ths, the general health of the mice exposed to deteriorated. Additional clinical signs were ab( anal distention and/or the appearance of exterr tumor masses, especially mammary gland i ors in the females. As shown in Table I, there v e numerous deaths or unscheduled termina- tions, proportional to exposure levels of VC. By the end of the ninth month, all males and females in the group exposed to 1000 ppm and all females exposed to 250 ppm died or were terminated. Additional mice exposed to 250 or 50 ppm died or were termi nated during the 10th through 12th months. In the control group, two males died during the eighth and ninth months. One death was due to injury from fighting, the other mouse was found dead with au tolysis and the cause of death was unknown. Of the mice exposed to 55 ppm of VDC, two males were terminated during the ninth month and one female during the 10th month. They all had tumors in the liver. Body Weight. The weight gains of the male and female mice exposed to 1000 ppm of VC or 55 ppm of VDC were comparable to those of the respective controls during the first 8 months (Fig. 1). Although not shown, the body weights of the mice exposed to 50 or 250 ppm of VC were also not significantly different from the controls. During the ninth month, the body weights of both the males and the females exposed to 1000 ppm began to decline, followed by sudden death. T- SIt l. Number or deaths and unscheduled terminations of mice exposed to VC or VDC. Treatment Control VC, 50 ppm VC, 250 ppm VC. 1000 ppm VC. 55 ppm Sex M F M F M F M F M F December 1977 Deaths or terminations during exposure month 6 7 8 9 10 II 12 --, 1 1 - _ _ 11 12 _1 34 23 29 _ I1 1 4 4 --2 1 1 42 1 1 10 -- -- -- -- 53 _ 6 4 ----" _2 1 Figure I. Body weights of male and female mice exposed to VC or VDC: () control; (A) 1000 ppm VC; (o) 55 ppm VDC. Laboratory Tests. No persistent change was found in the following laboratory results of the male and female mice exposed to VC or VDC as com pared with those of the respective controls: hematology, clinical blood chemistry, cytogenic 27 CMA. 050773 analysis of bone marrow cultures, x-ray examina tions of extremities, and serum a-fetoprotein. However, the pulmonary macrophage count in the male mice exposed to 1000 ppm of VC, but not at lower levels, was greater than that of the control mice at the end of the ninth month. The only female mouse in the 250 or 1000 ppm of VC group that was examined at the end of the ninth month also had an elevation of pulmonary macrophage count. At the 12th month, one of two males exposed to 50 ppm also had an elevated macrophage count. There was no survival in the group exposed to 250 or 1000 ppm. The mice with elevated macrophage count also had bronchiolo-alveolar adenoma. A study of DNA synthesis was undertaken to determine if the observation of an increased number of mitotic figures in the liver of a few mice exposed to VC or VDC could be confirmed at the biochemi cal level. DNA synthesis, as measured by 14C-thymidine incorporation into DNA, was sig nificantly increased in the livers of male mice ex posed to 50 ppm of VC for 11 months (Table 2). However, the livers of these mice did not show any increase in the number of mitotic figures or any evidence of neo- or preneoplastic lesions. mitotic figures or polyploidy; microfoci of mononuclear cells; focal degeneration and necrosis. The incidence and severity of these lesions pro gressed with the lengths of exposure. Some of these mice also had hemangiosarcoma. Tumors. Nearly all the mice that died or were terminated ahead of schedule and many mice that were terminated on schedule at various times de veloped one or more types of tumors. Bronchiolo alveolar adenoma first occurred in mice exposed to 1000 or 250 ppm of VC during the second month. The adenoma was characterized by papillary prolif eration of alveolar epithelium forming small and well-demarcated, but not capsulated, round nodules. The incidence (Fig. 2) and severity of this tumor increased in direct proportion to the level of VC and to the length of exposure. Only one control male mouse had a bronchiolo-alveolar adenoma during the ninth month. A few small nodules of bronchiolo-alveolar adenoma occurred in six mice exposed to 55 ppm of VDC. Table 2. uC-Thymldine Incorporation into Hepatic DNA of male mice exposed to VC for 11 months. VC level, ppm "C activity, dpm mg DNA* 0 2886 = 240(8) 50 4232 * 463(7)* `Mean SE (number of observations). `Significantly different from the control [two-sample rank test (24)]. Lesions in Early Deaths. Microscopic exami nation of the five unscheduled deaths (two males and one female exposed to 1000 ppm of VC for 3 to 9 days, and two males exposed to 55 ppm of VDC for 13 days) revealed a number of lesions. These included an acute toxic hepatitis, characterized by focal to marked congestion, and marked diffused coagulation type necrosis of hepatocytes beginning in the centrilobular area. Marked tubular necrosis cha:acterized by pyknosis and eosinophilic granula tion of the cytoplasma in the renal cortex was also observed. Lesions in Late Death. During the eighth to ninth months of exposure, several mitotic figures were observed in the liver of a number of mice ex posed to 50 or 1000 ppm of VC. This observation was not apparent in mice terminated at other times. Mice exposed to VDC for 6 to 12 months had sev eral changes in the liver. There were enlarged and basophilic hepatocytes with enlarged nuclei, many of which had large round eosinophilic inclusions; Figure 2. Incidence of bronchiolo-alveolar adenoma in male and female mice exposed to VC or VDC: () 1000 ppm VC: (A) 2JO ppm VC; (O) 50 ppm VC; (V) 55 ppm VDC; (o) controls. Hemangiosarcoma occurred in the livers of mice exposed to 1000 or 250 ppm of VC starting the sixth month. The hemangiosarcoma was characterized by moderate to severe proliferation of endothelial cells lining the sinusoids, dilation of the sinusoids, focal hemorrhage forming small to large cavernous blood spaces, invasion of the hepatic parenchyma with neoplastic cells, and mild to severe necrosis. In addition, hemangiosarcoma was occasionally found in other tissues including mammary gland, i 28 Environmental Health Perspectives '.eart, -pi d _ke u en;an ence Cr er n y 55 r ny co re e f' ` ilivar FlGURk. mice V ( at The mar m tun rs mo...n. VC lex tun rs cim ia tic cell creasei in , ;i adc o cinomn mice. 1 troi iii } iii, posed i dup-'c 250 i'p and .hi ninth t the ?1 tiss :. Decern CMA 050774 and female (------ ) liver; to markilization, livers of v controls i is change me early sex, two im of VC and two 1 the 13th hepatitis il cortex. 1th. Duro various , became / died or ith. After the mice jmber of ml tumor rs in the nale and rspectives exposed to 250 ppm died or were termil' Only a few mice exposed to 50 ppm survived . months. Two male control mice and two L md one female exposed to 55 ppm of VDC uring the experiment. ! onary macrophage count was elevated in lice exposed to VC. The macrophage count vated earlier in mice exposed to 1000 or 250 m in mice exposed to 50 ppm. The mice with J macrophage count also had bronchiolor adenoma. However, not all mice with olo-alveolar adenoma had the elevation of hage count. The relationship of macrophage nd this pulmonary tumor needs further intion. oderate number of mitotic figures were obin the liver of mice exposed to VC during the month. DNA synthesis, as measured by midine incorporation into DNA, was sign: tly increased in the livers of male mice exp- to 50 ppm of VC for 11 months. However, ti totic figures were not observed in the livers of ll mice, nor was there any evidence of neoplasti .d preneoplastic lesions. Further studies are u way to determine if the histopathological obs- tion could be related to changes at the b cmical level. -lumber of lesions occurred in the liver of mice e- ed to 55 ppm of VDC. The lesions included .e. zed and basophilic hepatocytes, enlarged nucl wth eosinophilic inclusions, mitotic figures or P- iloidy, microfoci of mononuclear cells, focal ds iteration, and necrosis. The significance of th. e lesions as related to the hemangiosarcoma or o; r liver tumors may be of a "preneoplastic" naUir.. Since only a few mice, as compared to the nu, ,ner of those with these preneoplastic lesions. de\doped the definite neoplasms, further study tsith mice exposed to high levels of VDC is needed to establish any definite conclusions. Exposure to 50, 250 or 1000 ppm of VC. 6 hr/day and 5 days/week, caused bronchiolo-alveolar adenomas, mammary gland tumors, and heman- gioxarcomas. The squamous and anaplastic cell carcinomas of the mammary gland metastasized to the lung. Hemangiosarcomas first occurred in the hver and then in other organs. The incidence of these tumors quickly increased, especially in mice exposed to 1000 or 250 ppm. The severity was in direct proportion to the levels of VC and the length of exposure. In addition, a few mice exposed to VC also developed malignant lymphomas. Bron chioloalveolar adenomas and hepatic hemangiosar comas also occurred in some mice exposed to 55 Ppm of VDC. The pathogenesis of these tumors was described elsewhere (25). December 1977 Rats were more resistant to the toxic and car cinogenic effects of VC or VDC. Exposure to 50. 250. or 1000 ppm of VC or 55 ppm of VDC did not cause any acute deaths, or any persistant changes in hematology or clinical laboratory results. However, exposure to 1000 ppm of VC or 55 ppm of VDC slightly depressed the body weight of the females or both sexes. In addition, a number of deaths oc curred in the rats exposed to 250 or 1000 ppm of VC or 55 ppm of VDC starting the 9th month. Exposure to 250 or 1000 ppm of VC caused hemangiosarcoma in the liver starting' the ninth month. In contrast to the mice, many of the rats with hepatic hemangiosarcomas also developed hemangiosarcomas in the lung. Two rats exposed to 55 ppm of VDC developed hemangiosarcomas in the mesenteric lymph node or subcutaneous tissue. The livers of most rats exposed to VDC had a mild or markedly severe focal, disseminated vacuoliza tion, probably fatty change. The significance of this change as related to the exposure of VDC is not understood. This research was supported by the National Institute of En vironmental Health Sciences under Contract No. N0I-ES-2-2084. We are indebted to Dr. Robert D, Short. Jr.. Senior Toxicologist. Midwest Research Institute, for his super vision of studies on ,JC*thymidine uptake, hepatic ALA synthetti'e and urinary ALA assays; to Dr. John R. Hodgson, Head. Biochemical and Developmental Pharmacology, Midwest Re search Institute, for his supervision of studies on collagen con tents and cytogenic analysis: io Dr Norman L. Martin, As sociate Professor of Diagnostic Radiology. The University of Kansas Medical Center. Kansas City, Kansas, for his assistance and interpretation on x-ray examination of extremities, io Dr, Stewart Sell. Professor. School of Medicine. University of California. San Diego. California, for his assistance on a-fetoprotein assay: to Miss Judith D. Girvin. M. T. (ASCP certified), for her supervision on the hematology and clinical laboratory tests, and to Mrs. Ellen R. Ellis, hisiologist. for her supervision on the histology preparation. REFERENCES 1. Patty, F. A., ct al. Acute response of guinea pigs to vapors of some new commercial organic compounds. V: Vinyl chloride. Pub. Health Rept. 4J: 1963 11930). 2. Osier. R. H-, et al. Anesthesia. XXVII. Narcosis with vinyl chloride. Anesthesiology 8: 339 (1947). 3. Carr, J., et al. Anesthesia. XXIV. Chemical constitution of hydrocarbons and cardiac automaticity. J. Pharmacol. 97: 1 (1949). 4. Mastromatteo, E., et al. Acute inhalation toxicity of vinyl chlonde io laboratory animals. Amer. Ind, Hyg. Assoc J 21: 391 (I960). 3. Torkerson, T. R., et al. The toxicity of vinyl chloride as determined by repeated exposure of laboratory animals. Amer. Ind. Hyg. Assoc. J. 22: 334 (1961). 6. Lester. D.. et al. Effects of single and repeated exposures of humans and rats to vinyl chloride. Amer. Ind. Hyg, Assoc. J 24: 263 (1963). 7. Viola, P. L-, et al. Oncogenic response of rat skin, lungs. 31 CMA. 050777 and bones to vinyl chloride. Cancer Res. 31: 516 (1971). 8. Maltoni. C.. and Lefemine, G. Carcinogenicity bioassays or vinyl chloride. Current results. Ann. N. Y. Acad. Sci. 246: 195 (1975). 9. Creech, J. L., Jr., and Johnson. M. N. Angiosarcoma of liver in the manufacture of polyvinyl chloride. J. Occup. Med. 16: 150 (1974). 10. Falk, H., et a). Hepatic disease among workers at a vinyl chloride polymerization plant. J. Amer. Med. Assoc. 230:59 (1974). 11. Makk. L., et at. Liver damage and liver angiosarcoma in vinyl chloride workers. J. Amer. Med. Assoc. 230: 64 (1974). 12. Lee, F. 1., and Harry, D. S. Angiosarcoma of the liver in a vinyl chloride worker. Lancet 1: 1316 (1974). 13. Tabershaw, 1. R., and Gaffey, W. R. Mortality study of workers in the manufacture of vinyl chloride and its poly mers. J. Occup. Med. 16: 509 (1974). 14. Mancini,G.,etal. Immunochemical quantitation ofantigens by single radial immunodiffusion. Immunochemistry 2: 235 (1964). 15. Kingsley, G. R. The direct biurer method for the determina tion of serum protein as applied to photoelectric and visual colorimetry. J. Lab. Clin Med. 27 : 840 (1942). 16. Doumas, B. T., et al. Albumin standards and the measure ment of serum albumin with Bromcresol Green. Clin. Chem. Acta 31: 87 (1971). 17. Houck, J. C.. and Jacob, R. A. Chemical dissection of rat skin. Proc. Soc. Exptl. Biol. Med. 105: 324 (I960). 18. Martin, C. J., and Axelrod. A. E. A modified method for determination of hydroxyproline. Proc. Soc. Exptl. Biol. Med. 83 : 461 (1953). 19. Tjio, J. H., and Wang. J. Direct chromosome preparations of bone marrow cells. In: Human Chromosome Methodol ogy. J. J, Yunis, Ed., Academic Press. New York. 1965. 20. Burton, K. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 315 (1956). 21. Woods. J. S. Studies on the role of heme in the regulation of 6-aminolevulinic acid synthetase during fetal hepatic de velopment. Mol. Pharmacol. 10: 389 (1974), 22. Davis, J. R., and Andelman. S. L. Urinary delta-aminolevulinic acid (ALA) levels in lead poisoning II: a modified method for the rapid determination of urinary delta-aminolevulinic acid using disposable ion-exchange chromatography columns. Arch. Environ. Health 15: 53 (1967). 23. Sell, S.. and Gord, D. Rat a-fetoprmein--111 refinement of radioimmunoassay for detection of I ng rat a,F. Im munochemistry 10' 439 (1973). 24. Mann. H. B.. and Whitney. J. H. On a test of whether one or two random variables is statistically larger than the other. Ann. Math. Stat. 18: 50 (1947). 25. Lee, C. C., et al. Carcinogenicity of vinyl chloride and vinylidene chloride. J. Toxicol. Environ. Health, in press. irue T( x Vin D.ir R^f by L. M F, ! a *if * i t Ini oc ) Vinyli HSC =( oth' mi are use*, where c hun n e bee ise VDC in of VDC C rre on ' tyli Toxico Rest ch. Decern tx 32 Environmental Health Perspectives CMA. 050778