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I /. .. 3E2* #* ***--. - ' irHSfc>-TJ -' f free airway cells to rsive X-ray analysis. alterations in blood/ jes. Environ. Health iterworths. London. 11, 271-291. Jers. Philadelphia, os. Environ. Health ies of fibers: lmpor- mc and Nutritional on of nonheme cel- je stain in electron :opy. J. Exp. Med. identic Press. New jnges in lung tissue roscopy. J. Utrra- spect. 9, 241-252. e study with three A mer. J. Pathol. oy. Soc. Med. 71, environmental research 32, 91-103 (1983) CERTAINTEED CORPORATION CORPORATE LIBRARY INFORMATION CENTER Neoplastic Effect of Vinyl Chloride in Mouse Lung-- Lower Doses and Short-Term Exposure Yasunosuke Suzuki The En\ ironinenlnl Silences Lnhornion. Department of Community Medicine, and Department of Putliolonc. Mount Smut School of Medicine of the City University of Acir York. One Gustave Lew Place. Hen York. Hen )ork 10029 Received September 13. 1982 Neoplastic pulmonary effects of lower doses of vinyl chloride (0 = control. 1. 10. 100. 300. and 600 ppm) and short-term exposure (4 weeks) by inhalation have been studied by light and electron microscopy in 220 mice. Except for dead or seriously sick animals, a large majority of the animals were sacrificed at three different stages: immediately after exposure. 12 weeks later, and 40 or 41 weeks afier exposure. Six mice (4: 600 ppm. 2: 0 ppm) were kept longer than 41 weeks to examine the effects of the chemical after a long-term recovery period. Alveologenic tumors were first observed 10 weeks after exposure to 600 ppm In the subgroups exposed to higher concentrations (600 and 300 ppm) the incidence of tumors was higher and their appearance was earlier than in the subgroups exposed to lower concentra tions (100. 10. and I ppm). These findings indicated a dose-response relationship for inci dence of alveologenic tumors, and the latency period was inversely related to dose. By light and electron microscopy, there was no obvious evidence that tumor cells were derived from Clara cells of the terminal bronchioles. Rather, neoplastic cells in both the tubulopapillarv and adenomatous forms of the pulmonary tumors possessed all or some of the ultrastructural characteristics of type 11 alveolar cells, based on observations of mitochondria, microvilli, osmiophilic lamellar bodies, and other criteria. Type II alveolar cells are therefore consid ered to be the most sensitive in mice to the neoplastic effect of vinyl chloride. I. INTRODUCTION In previous studies, a high incidence of pulmonary tumors as well as non neoplastic alterations of bronchioles and alveoli was observed in mouse lung exposed to vinyl chloride at heavy doses (2500 and 6000 ppm) for a long duration (5 and 6 months) (17. 18). The induced pulmonary tumors corresponded to those reported by Stewart et al. (IS. 16) as alveologenic tumors originating in type II alveolar cells. It has been suggested that the mouse lung is an extremely sensitive indicator for the oncogenicity of vinyl chloride. A dose-response relationship has been demonstrated in the production of alveologenic tumors with vinyl chloride (3,6, 8). In addition, a delayed appearance of the neoplastic effect after periods of recovery, following exposure to vinyl chloride, has been postulated (3. 10. 17). In this study, the neoplastic effect of exposure to lower doses of vinyl chloride (0 = control, 1, 10, 100, 300. and 600 ppm), and short-term exposure (4 weeks), has been studied using light and electron microscopy in 220 mice. Details of the findings are reported here. 91 / NOTICE: This Material may be protected by copyright law. (Title 17 US. Cod ) OOI3-935I/83 $3.00 Cup* right . IV8? h> Academic Prr**. Inc Ml nphls uf reproduction m n> f, - *n reserved CTL030360 92 YASUNOSUKE SUZUKI II. MATERIAL AND METHODS (a) Test material. Vinyl chloride was supplied by the Dovv Chemical Company. Midland. Michigan. The material was greater than 999? pure. Prior to use in this study, the purity was confirmed by gas chromatography. (b) Test animals. Two hundred and twenty CD1 Charles River male mice. 5-6 weeks old at first exposure, were used. Exposure was for 6 hr per day. 5 days per week, for a total of 4 weeks in all animals, but six different doses of vinyl chloride were administered as follows: 600 ppm (40 mice). 300 ppm (30). 100 ppm (30), 10 ppm (30). 1 ppm (30). and 0 ppm (60 = controls). (c) Chambers, vapor generation, and analyses. The chambers used for this study were stainless steel and Rochester-type glass, approximately I m1 in vol ume. The chambers were operated under dynamic airflow conditions with tem perature and humidity controlled to approximately 70F and 509c rh. Vinyl chloride gas was metered at a controlled rate into the airstream where it was further diluted to the desired concentration. The analytical concentration of vinyl chloride in each exposure chamber was continuously monitored by infrared spec troscopy at a wavelength of 10.9 jim. Standards for analysis were made by inject ing a known volume of vinyl chloride gas into a 100-liter Saran gas sampling bag filled with a known volume of air. The nominal concentration of vinyl chloride in the chamber was the ratio of the rate at which the test material was dispensed to the ratio of total airflow through the chamber. These inhalation experiments were performed under the supervision of Dr. M. J. McKenna of the Toxicology Re search Laboratory. Health and Environmental Science. U.S.A. Dow Chemical, Midland. Michigan. (d) Procedure of animal sacrifice. A large majority of the animals were sacri ficed at three different stages: (a) immediately after exposure (a total of 70 mice, 10 each of the 600. 300, 100, 10. and 1 ppm groups, and 20 of the 0 ppm group); (b) 12 weeks after exposure (a total of 61 mice, 9 of the 600, 9 of the 300. 6 of the 100, 9 of the 10. 10 of the 1, and 18 of the 0 ppm groups): and (c) 40 or 41 weeks after exposure (a total of 58 mice, 7 of the 600, 7 of the 300. 9 of the 100. 9 of the 10.9 of the 1. and 17 of the 0 ppm groups). Between the first and second sacrifices. 14 mice (6 of the 600, 1 of the 300, 4 of the 100. 1 of the 10. and 2 of the 0 ppm groups) were found dead or were killed. Eleven mice (4 of the 600, 3 of the 300, I of the 100. 1 of the 10. 1 of the 1. and 1 of the 0 ppm groups) were found dead or were killed between the second and third sacrifices. A small number of animals (4 of the 600 and 2 of the 0 ppm groups) were allowed a long-term postexposure recovery period (42-65 weeks) prior to sacrifice or death. (e) Hisiopathological and electron microscopic procedures. All animals were systematically autopsied. Sacrifice was performed by ether anesthesia. Lungs were inflated with paraformaldahyde (for sacrificed mice) or with 109? neutral buffered Formalin (for mice found dead). Then, to confirm the presence of pulmo nary tumors, and to count the number of the tumors, all lobes of the lungs were thoroughly examined under a dissecting microscope. The lungs, together with a majority of organs, including liver, brain, kidney, adrenal, stomach, intestine, esophagus, testis, and seminal vesicle, were fixed in 109? neutral buffered for- CTL030361 emical Company, rior to use in this r male mice, 5-6 r day. 5 days per of vinyl chloride 100 ppm (30), 10 rs used for this ely 1 m* in vol itions with tem50% rh. Vinyl m where it was itration of vinyl y infrared specmade by injectis sampling bag inyl chloride in as dispensed to oeriments were Toxicology Re)ow Chemical, als were sacrital of 70 mice, 0 ppm group); e 300.6 of the 0 or 41 weeks e 100. 9 of the md sacrifices, of the 0 ppm 3 of the 300, 1 'ound dead or of animals (4 postexposure animals were hesia. Lungs 10% neutral \ce of pulmoe lungs were ether with a h. intestine, 'offered for- NEOPLXST1C EFFECT OF VINSL CHLORIDE EXPOSURE 9? malin and embedded in paraffin after dehydration in alcohol. Five- to six-mierometer sections were made and stained with hematoxylin-eosin. Gomori s silver, periodic acid-SchifFs (PAS). Masson's trichrome. and Van Gieson's picrofuchsin technique. For electron microscopy, small pieces (smaller than 1 mm') were taken from the tissues that were first fixed in paraformaldehyde and then fixed in 1% phosphate-buffered osmic acid. After alcohol dehydration the blocks were embed ded in epoxy resin. Ultrathin sections were obtained with an LKB microtome. The sections were stained with uranyl acetate and lead. Siemens 101 and Hitachi 11DS electron microscope were used for ultrastructural observation. III. RESULTS (A) Induction of Pulmonary Tumors Table 1 summarizes the results of induction of pulmonary tumors in mice ex posed to low doses of vinyl chloride for 4 weeks and sacrificed or found dead at various times after exposure. The first pulmonary tumor was observed in 1 mouse of the 600 ppm group that was found dead 10 weeks after exposure. The tumor was a single alveologenic tumor. At the time of the second sacrifice (12 weeks after exposure), a high incidence of alveologenic tumors was seen in the subgroups exposed to heavier doses [600 and 300 ppm. 88.8% (8 of 9) and 66.6% (6 of 9). respectively] although the tumor was not induced in the subgroups exposed to lower doses (100. 10. and I ppm). Alveologenic tumors induced in the 300 ppm group were all single tumors; however, the tumors of the 600 ppm group consisted of multiple tumors in 6 of 8 (75%) while the remaining 2 of 8 (25%) were single tumors. Between the second and third sacrifices (40 or 41 weeks after exposure), again, animals of the lower doses (100. 10. and 1 ppm) did not develop an> pulmonary tumors, but those of the heavier doses--subgroups (600 and 300 ppm)--showed a high incidence of tumors (4 of 4 [100%] and 2 of 3 [66.6%]. respectively), all of which were multiple neo plasms in the lungs. At the third sacrifice, the pulmonary tumors were induced in all subgroups (600. 300. 100. 10. and 1 ppm) and the incidence of tumor production was 6 of 7 (85.7%) in the 600 ppm. 5 of 7 (71.4%) in the 300 ppm. 6 of 9 (66.7%) in the 100 ppm, 3 of 9 (33.3%) in the 10 ppm, 1 of 9 (11.1%) in the I ppm. and 0 of 17 (0%) in the 0 ppm (control) groups. The percentage of induced tumors which were multiple was 6 of 6(100%) in the 600 ppm. 3 of 5 (60%) in the 300 ppm, 3 of 6(50%) in the 100 ppm, 0 of 3 (0%) in the 10 ppm, and 0 of 1 (0%) in the 1 ppm groups. In the 600 ppm group, sacrificed over 41 weeks after exposure, a multiple al veologenic tumor was found in 4 of 4 (100%). although 2 controls did not develop any pulmonary tumor. These findings demonstrate a dose-response relationship for incidence of al veologenic tumors, and the latency period inversely related to dose. Further, the incidence of multiple alveologenic tumors seems to be related to two factors, dose and the recovery period after exposure. It is not possible to compare accurately the size of the induced tumors. How ever, in general one can say that larger tumors are seen in the heavier-dose group CTL030362 94 YASUNOSUKE SUZUKI TABLE I Induction of Mouse Pulmonary Tumors with Low Dom n of \ isvi Chloride (VC) Exposed for 4 Wlf.k.s Sacrificed or found dead time No. of animals VC-induced pulmonary tumors No. 7, Muliple foci of VC-induced pulmonary tumors No. t-- Spontaneous pulmonary tumors No. 7c Immediately after exposure 600 ppm 10 (9 sac. 1 fid.) 300 ppm 10 (sac) 100 ppm 10 (sac) 10 ppm 10 (sac) 1 ppm 10 (sac) 0 ppm 20 (sac) 0 0 0 0 0 0 00 0 00 0 000 000 00 0 000 00 00 00 00 00 00 Between first and second sacrifices 600 ppm 6 (2 sac. 4 f.d.) 300 ppm 1 (f.d.) 100 ppm 4 (1 sac. 3 f.d.) 10 ppm 1 (sac) 0 ppm 2 (f.d.) 1 <f.d..s) 0 0 0 0 16.6 0 0 0 0 0lof 1) 0 0 0 0 0 0 0 0 0 00 00 00 00 00 12 Weeks after exposure (second sacrifice) 600 ppm 9 (sac) 8 (2 s. 6 ml 300 ppm 9 (sac) 6 (si 100 ppm 6 (sac) 0 10 ppm 9 (sac) 0 1 ppm 10 (sac) 0 0 ppm 18 (sac) 0 88.8 66.6 0 0 0 0 6 (of 8) 0 (of 6) 0 0 0 0 > 0 0 0 0 0 00 00 00 00 00 00 Between the second and third sacrifices 600 ppm 4 (1 sac. 3 f.d.) 4 (m) 300 ppm 3 (1 sac. 2 f.d.) 2 (m) 100 ppm 1 (sac) 0 1 ppm 1 (f.d.) 0 0 ppm 1 (f.d.) 0 100 4 (of 4) ll>0 66 6 2 lof 2) 100 000 000 000 00 00 0 o. 00 00 40 or 41 Weeks after exposure (third sacrifice) 600 ppm 7 isac) 6 (m) 300 ppm 7 (sac) 5 (3 m. 2 s) 100 ppm 9 (sac) 6 (3 m. 3 s) 10 ppm 9 (sac) 3 is) 1 ppm 9 (sac) 1 (S) 0 ppm 17 (sac) 0 83.7 71.4 66.7 33.3 II.1 0 6 (of 6) 3 (of 3) 3 (of 6) 0 (of 3) 0lof 1) 0 100 60 30 0 0 0 1* 14.3 00 1* II.1 00 I* II.1 y 17.6 Over 41 weeks after the exposure 600 ppm 4 (sac) 0 ppm 2 (1 sac. 1 f.d.) 4 (m> 0 100 4 (of 4) too 000 00 00 * The Tirsi mouse hearing a pulmonary tumor, found dead 10 weeks after exposure, sac. * sacri ficed: f.d. found dead, s * a single pulmonary tumor: m = multiple pulmonary tumors. * Spontaneous pulmonary' tumor: a single, small (not identified by either naked eye and a stereo scope. but barely identified by light microscopy of lung section slides) tumor appeared in and after the period of third sacrifice (49 weeks old in age or older). CTL030363 ORIDfc (VC) ontaneous monary tumors No. 9? 00 00 l0 '0 )0 '0 0 0 () 0 0 0 0 0 0 0 0 0 0 0 0 0 14.3 0 II.I 0 III 17.6 0 0 - = sacn- 4 a stereo' *ind after I I NEOPLASTIC EFFECT OF VINYL CHLORIDE EXPOSURE 95 and also when the recovery period was longer. Huge alveologenic tumors were observed in only two mice of the 600 ppm group, found dead 40 weeks after and sacrificed 55 weeks after exposure, and the tumors seen in the groups exposed to smaller doses (100, 10, and 1 ppm) were generally smaller. In addition to the induced pulmonary tumors, spontaneous alveologenic tumors presented in six animals (3 of 0 ppm. 1 of 10 ppm. 1 of 100 ppm. and 1 of 600 ppm) sacrificed 40 and 41 weeks after exposure. These tumors were excluded from the calculations of incidence of vinyl chloride-induced alveologenic tumors. Details of this will be described in the Discussion. (B) Light and Electron Microscopic Observations of the Alveologenic Tumors The histological aspects of the pulmonary tumors were similar to those induced by heavy doses of vinyl chloride with a long exposure. No metastases to other organs were observed. The tumors were usually seen in the peripheral part of the lung; however, occasionally, the tumors were found in more centrally located areas. The tumors were not encapsulated by connective tissue. The neoplastic cells were arranged in the tubular, papillary, and adenomatous fashions. When adenomatous, the cells were small in size, cuboidal in shape, and similar to hyperplastic type II cells which have been postulated as the presursor of the neoplastic cell. When tubular or papillary, the cells were usually large in size and cuboidal or cylindrical in shape, but small, cuboidal cells were also identified. Pleomorphism and anaplastic features were not striking and mitoses were uncom monly observed. In rare cases a huge tumor almost completely occupied a whole lobe of lung. Figure 1 indicates a part of such a huge pulmonary tumor (600 ppm. sacrificed 55 weeks after exposure). Figures 2-4 are light microscopic pictures of higher magnification, taken from either the peripheral (Fig. 2) or the deeper (Figs. 3 and 4) parts of the tumor. In addition to the alveolar (Fig. 2) and tubulo-papillary (Fig. 4) patterns, an intermediate form (Fig. 3) was also observed in the huge tumor. Electron microscopically, the neoplastic cells of these patterns possessed all or some of the ultrastructural characteristics of type II cells, such as microvilli, large round- or rod-shaped mitochondria, osmiophilic lamellar bodies, junctional struc tures, multivesicular bodies, and a basement membrane. Less differentiated cells possessed only a few of those characteristics. Interestingly, the neoplastic cells of the tubulo-papillary pattern, seen in the huge tumor (600 ppm. 55 weeks after exposure) still showed some of the ultrastructural characteristics, such as micro villi. osmophilic lamellar bodies, and multivesicular bodies. Size and shape of the mitochondria of the neoplastic cells varied: however, cristae of mitochondria were usually well developed. Figure 5 is a low-power electron micrograph of the tubulo-papillary pattern seen in the huge tumor for which light microscopy illustrations are shown in Figs. 1-4. A part of Fig. 5 is enlarged in Fig. 6. The electron micrographs clearly indicate that the neoplastic cells forming the tubulopapillary pattern still show some similarities to type II cells: well-developed mi crovilli (Figs. 5 and 6). lamellar bodies (Fig. 5). and cristae-rich mitochondria with a clear matrix (Fig. 6) are characteristic features seen in type II cells. Mitochondria of Clara cells in lerminal bronchioles of the mouse are known to be c o u ritttA tx . l n,; CTL030364 96 YASUNOSUKE SUZUKI U\ Figs. 1-4. Taken from a huge pulmonary tumor seen tn a mouse exposed to 600 ppm vinyl chloride for tTweeks and sacrificed 55 weeks after exposure. Fig. 1. A low-power view, x 78. round in shape, dark in the tone of the mitochondrial matrix, and poor in the development of mitochondrial cristae. Such miiochondria were not present in the neoplastic cells of any patterns seen by us. In addition to the above findings, intracytoplasmic compartments formed by a membrane structure and glycogen granules in a cisterna of the endoplasmic reticulum were frequently observed in the neoplastic cells, as stated in our previous report (17). Hypercellularity of the alveolar lining cells was frequently observed around the induced pulmonary tumors. In the previous study (17). it had been strongly sug gested that such a change was the precondition for the alveologenic tumor produc tion because electron microscopically, the proliferated cells were quite similar to the normal type II cell on the one hand, and also to the well-differentiated cell type CTL030365 *SF5r,-^7 NEOPLASTIC EFFECT OF VINYL CHLORIDE EXPOSURE 97 3 9 4 b 3 4 i <c 'm vinyl chloride ! poor in the iresent in the tve findings, nd glycogen observed in 1 around the trongly sugTior producte similar to led cell type iI of the alveologenic tumor on the other hand. A small proportion of the subgroups exposed to heavier doses (2 of 20 in the 600 ppm and 1 of 10 in the 300 ppm groups) already showed such an alteration immediately after exposure to vinyl chloride. t C) Tumors Other than Pulmonary Tumors Hemangiosarcomas were induced in both the subcutaneous connective tissue of the ear (10 ppm; 29 weeks after exposure) and liver (600 ppm: 65 weeks after). Details of those tumors have been reported elsewhere (19). IV. DISCUSSION Hepatic hemangiosarcoma induced by vinyl chloride. Thorotrast. or arsenics \ 1,2. 7. 9, 11. 12, 21) has been known to be a "signal" |I. J. Selikoff (13)] of human CTL030366 98 YASUNOSUKE SUZUKI cancer. Maltoni and Lefemine (10) have clearly documented that hepatic hemangiosarcomas can be induced in mouse, rat. and hamster exposed to vinyl chloride at various doses and different exposure times. Among vinyl chloride workers, in addition to hepatic hemangiosarcoma. an increased number of deaths due to bronchogenic cancer has been postulated (20, 22). To the present, however, lung tumors have not been induced by vinyl chlo ride in laboratory animals except the mouse. Although alveologenic tumors of the mouse have been frequently produced by vinyl chloride (3. 6. 8. 10. 16, 17). the cell origin and malignancy of the alveologenic tumors are still in controversy (4, 5, 15, 16). Regardless of this controversy, however, the induction of alveologenic tumors by vinyl chloride may be indirectly predictive of risk of human bron chogenic carcinoma from the chemical, since a number of chemical carcinogens, such as polycyclic aromatic hydrocarbons, nitrogen mustard, and chromate com- CTL030367 NEOPLASTIC EFFECT OF VINYL CHLORIDE EXPOSURE 99 I l'(h ! dut hepatic iW'd io vinyl pounds have also been known to induce alveologenic tumors in the mouse, and to be associated with excess bronchogenic carcinoma among workers exposed to the carcinogens. The cell origin of alveologenic neoplasms has been disputed. Type 11 alveolar cells, or terminal bronchiolar cells, or either alveolar and bronchiolar cells have been reported as the progenitor cell of the tumors. Recently Kauffman et al. (4. 5) have divided mouse pulmonary tumors induced by transplacental administration of ethyl nitrosourea into two groups, benign and malignant. The two were histologically characterized by alveolar and tubulopapillary patterns, respectively. According io these investigators, the type II cell is the progenitor of the benign form and the Clara cell of the terminal bronchioles the progenitor of the malignant form. In the present study, however, we have failed to detect Clara cell tumors among the mouse pulmonary tumors induced by CTL030368 100 YASUNOSUKE SUZUKI Fig. 5. A low-power electron micrograph showing neoplastic cells, seen in the tubulo-papillary pattern of the huge tumor shown in Fig. I. x 10,500. Some ultrastractural characteristics of the type II cells, such as microvilli, osmiophilic lamellar bodies, and cristae-rich mitochondria, are shown in the neoplastic cells illustrated in this figure and figure 6. vinyl chloride, although the tumors induced included various patterns; alveolar, tubulo-papillary. and mixed forms. It is known that the Clara cell of the mouse contains unique mitochondria which are round or ovoid in shape, large in size, dense in the mitochondrial matrix, and very poor in mitochondrial cristae. ^030369 NEOPLASTIC EFFECT OF VINYL CHLORIDE EXPOSURE 101 *t * - NL. > 'C u- v. * O -s ubulo-papillary :>cs of the type are shown in ': alveolar, f the mouse ;ryc in siae, I:'l cristae. Fig. 6. A part of Fig. 5 x 21,900. Unlike the type II cell, the Clara cell lacks osmiophilic lamellar bodies and micro villi (14). Such unique mitochondria were not detected in the neoplastic cells of any of the induced tumors, while microvilli, osmiophilic lamellar bodies, cristaerich mitochondria with clear matrix, and multivesicular bodies, which are com monly seen in the type II cell, were often observed in the lumor cells of the tumors we observed. From this, it is suggested that the type 11 cell is the sole progenitor CTL030370 1 102 YASUNOSUKE SUZUKI cell of the tumor. If this suggestion is accepted, the term of "alveologenic tumor" (Stewart) would be accepted as adequate. It is known that alveologenic tumors spontaneously occur in the mouse lung. Stewart et al. reported that the incidence of spontaneous pulmonary tumors was quite high in certain strains, such as A and DD after 10 to 12 months of age. and that these spontaneous tumors could be differentiated from those produced by carcinogens by their small size and their single number, as well as the older age of the individual mouse (15. 16). Taking this into consideration, alveologenic tumors seen in six mice (3 of the 0 ppm. 1 of the 1 ppm, 1 of the 100 ppm. and 1 of the 600 ppm groups) sacrificed 40 and 41 weeks after exposure have been considered spontaneous. They were single in number and were barely identified under a light microscope. In addition, the age of the mice (over 49 weeks old) was old enough to expect production of spontaneous tumors. It has been suggested that the latency of alveologenic tumors after exposure to vinyl chloride is dose related (3. 6. 8. 10). Indeed, in this study, the latency was shorter in the groups exposed to heavier doses. The shortest latent period for tumor induction was 10 weeks after exposure in the 600 ppm group. 12 weeks after exposure in the 300 ppm. and 40 weeks after exposure in the 100. 10. and 1 ppm groups. No multiple tumors were induced in the groups of 10 and 1 ppm. As stated earlier, the size of the induced tumor appears to be related to both the dose and to the recovery time. Previous findings of a number of investigators support the idea that al veologenic tumors induced by vinyl chloride (a) seem to appear earliest among all neoplasms induced by the chemical in all species of laboratory animals (3.6. 8. 10, 17. 18): (b) the incidence of the tumors produced is dose related (3, 6. 8. 10): and (c) the latency of the tumor is also dose-related (3. 6. 8. 10). In this study, evi dence to support all three of these suggestions has been presented. In addition, electron microscopic observations have focused on the type II alveolar cell as the progenitor cell of the tumor. Thus, it is considered to be the cell type most sensitive to the oncogenicity of vinyl chloride. ACKNOWLEDGMENTS The author would like to express his appreciation to Dr. 1. J. Selikoff for his review and Dr. S. Frank for her editorial assistance. The author also thanks Mr. R. Ashley. Mrs. A. Calderaro. Mr. S. Yuen, and Mr. R. Fuller for their technical assistance, and Mrs. J. Roberts for secretarial assistance. This work was supported by Research Grant OH 00681 from the National Institute for Occupational Safety and Health. U.S. Department of Health and Human Services. REFERENCES 1. Baxter. P. J.. Anthony. P. P.. MacSween. R. N.. and Schever. P. J. (1980). Angiosarcoma of the liver: Annual occurrence and aetiology in Great Britain. Bra. J. hid. Med. 37. 213-221. 2. Creech. J. 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D.. SelikofT. 1. J.. and Falk. H. (1975). Vinyl chloride- induced liver disease. N. Engl. J. Med. 292, 17-22. 22. Waxweiler. R. J.. Stringer. W., Jones, J., Wagoner. J. N., Falk. H.. and Carter. C. (1976). Neoplastic risk among workers exposed to vinyl chloride. Ann. N.Y. At ad Set 271. 39--48. CTL030372