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MANUFACTURING CHEMISTS ASSOCIATION 182b CONNECTICUT AVENUE, N. W. WASH INGTON, D. C. 20009 (202)483-6126 May 22, 1976 TO: Vinyl Chloride Technical Panel SUBJECT: Minutes of Last Meeting and Other Items Gentlemen: Enclosed please find the minutes of the last meeting, in addition to the following: 1. Dr. Tamburro's letter of April 20 to Dr. Torkelson describing proposed studies on immunological systems and electron micro scopic evaluation of liver tissue. 2. Summaries of talks presented at the April 28 Panel meeting. 3. References for Dr. Johnston's talk on "Cytogenetic Studies of Bone Marrow Cells Prom Rats Exposed to Vinyl Chloride". 4. "Interim Report on Mortality and Gross Observations on Rats, Mice and Hamsters with Vinyl Chloride", by M. L. Keplinger, et al., (IBT). This is a draft of a paper to be given at the American Association of Industrial Hygienists meeting in Atlanta the week of May 17. INFORMATION At a meeting April 7 called by Mr. R. M. Graziano, Director and Chief Inspector of the Bureau of Explosives, discussion centered on the concern of the Association of American Railroads over the problems of handling VCM in emergencies, particularly as they relate to the recent indication of carcinogenicity. Representatives of VCM producers, the railroads. Bureau of Explosives, trade associations and car leasing companies were present. Methods of establishing a mutual support program similar to that operated by the Chlorine Institute and The National Agricultural chemical Association were reviewed. Representatives of the Society of The Plastics Industry said URL 17081 they would explore the possibility of organizing a response system. Sincerely, MF: ec Enclosures Milton Freifeld Y Project Manager Vinyl Chloride Research UNIVERSITY OF LOUISVILLE Louisville, Kentucky 40202 April 20, 1976 SCHOOL OF MEDICINE DEPARTMENT OF MEDICINE DIGESTIVE DISEASES AND NUTRITION SECTION ITEM 1 HEALTH SCIENCE CENTER WALNUT A PRESTON STREETS Dr. T. R. Torkelson Dow Chemical Company Corporate Medical Department 2030 Dow Center Midland, Michigan 48640 Dear Doctor Torkelson: May I express our delight in having had members of the Manufacturing Chemists Association Research Committee visit us earlier this month; we hope that their visit was both enjoyable and informative. Our post-lunch- : eon meeting helped to clarify a number of points concerning our Manufactur ing Chemists Association Grant proposal and I would like to take this op portunity to submit for the members consideration a modification of our original proposal in light of the expressed interests of the members of the research committee. Since the members expressed interest in supporting proposals A and G, having to do with the study of immunological systems, it seems that - these would require no further elaboration or adjustment. My understand- 32 ing is that the same would be true for proposal G, concerning electron microscopic evaluation of liver tissue. What I would like to do here is (a) amplify on the Immediate clinical applicability of some of the other proposals presented, (b) provide a modified budget for these proposals and (c) suggest that if cuts must be made in the program, the cuts that would be least damaging in terms of ongoing efforts and in terms of a cohesive ness of the overall research program, would be the proposals by Dr. Hoffman (H) and Dr. Slgdestad (1) and parts of Drs. Du (B2 B5 B6 B7) and Wong (EZ) (see revised budget attached). Firstly, proposal C concerning the glyco- saminoglycans in the early detection and etiology of angiosarcoma of the liver presented by Dr. Charles E. Kupchella: this has already produced significant results. Most chemical Injury and cancer appear to be as sociated with "scar" or collagen formation. This collagen formation is as sociated with the increased production of glycosaminoglycans. Dr. Kupchella*s preliminary study indicates that there is a characteristic pattern of glyco- samlnoglycan urinary excretion among long-term vinyl-chloride-exposed in dividuals not seen in individuals with alcoholic liver injury, hepatitis, and cancers not directly involving the liver. These urinary excretion pat terns appear to change as one develops primary liver cancer such as angio sarcoma. At this point, the findings reported by Dr. Kupchella at the Third 801-1 Dr. T.R. Torkelson page -2- t-so i-n u n International Symposium on the Detection and Prevention of Cancer, require additional verification and modifications of this method for use as spot urine testing in large worker population. If future studies continue to verify our present finding, i.e. that this test is indicative of early vinyl chloride injury, it could be applicable throughout the industry where chemically Induced fibrosis may occur. This proposal could yield a sensi tive, simple and inexpensive means of surveillance for early liver injury. A copy of the paper to be presented in New York is enclosed. Concerning the proposal by Dr. Wong, and the related proposal by Dr. Streips; I would like to elucidate on the value of this work in deter mining and directing priorities as to which metabolite should be studied for their potential carcinogenicity. Dr. Wong's ability to synthesize metabolic products of various chemicals allows immediate mutagenic bac terial studies for the identification of those chemicals with greatest car cinogenic capability. In order to control costs and increase benefits for the amount of time and money invested, these mutagenic studies must be per formed in order to realistically develop strategies for blocking the etlologic chemical changes leading to the initiation of angiosarcoma. Considering the overall complexity of studies of this sort, we feel that the combined work of Drs. Wong and Streips provides a reasonably straight for ward approach to unravelling sequences leading to angiosarcoma. : In addition. Dr. Wong's work will develop a method of detecting trace amounts of chemicals and their metabolic products by use of multivarient analysis in biological tissue. Our present system of storing bloods, urines, and tissue on all workers in the medical surveillance program gives us the equivalent of an Immediate clinical trial in a care fully observed worker population with known exposure. This procedure could give us an applicable test system within three years. Finally, Dr. Du's work will make use of animal studies in deter mining the most specific and earliest enzymatic and biochemical alterations produced by chemical exposure. Although some have expressed the view that biochemical alterations have all been identified and worked out, our clini cal experience Indicates the need for studies which will objectively deter mine which screening methods should be applied and to which of the exposed industrial population. The animal studies proposed by Dr. Du will allow us to apply these methods under controlled conditions of exposure, dose and duration, providing useful information in just a few years. This will be far more efficient means of determining the best screening methods to employ based on hard scientific data rather than clinical opinion. Simultaneously, Dr. Wong will utilize these same animal tissues for verifying the sensitivity and specificity of the multivarient analysis system for trace organic element detection in biological tissue. The de tection of a trace product does not by itself prove a causal relationship nor Indicate recent or past exposure. Therefore, it is vital that these studies be done (simultaneously with the studies for sensitivity and specificity) for verification of the relationship of detection to cause of injury. This information will greatly help in the interpretation of our findings from, our stored human blood, urine and tissue samples. Dr. T.R. Torkelson page -3- We would like to again point out the importance of our being able to continue to pursue this multidisciplinary research approach in the study of the vinyl chloride problem. By addressing the vinyl chloride problem as a model and in this systematic manner we feel that this unique combination of investigations will go far toward providing understanding chemical carcinogenesis in general and the etiology of angiosarcoma. Our program can be left essentially intact with the attached budget. The work proposed, we feel, is clearly defined and should produce positive results well in excess of investment. In addition, we would also like to point out that funding of such a program will sharpen our overall skills and further develop this approach applicability to other chemicals and in other situ ations. I believe that the budget proposed is well within the desirability and capability of the Manufacturing Chemists Association's constituency. In essence, we are asking the vinyl chloride Industry to support the program at somewhat less than a level that has been supported by the B.F. Goodrich Company alone for the past two years. Thank you for your serious consideration. Sincerely yours. CHT:noa Enclosures cc: Dr. Zeb G. Bell, Jr. Dr. Walter D. Harris Dr. Maury Johnson Mr. Howard L. Kusnetz Dr. W.E. Rinehart Dr. W. Mayo Smith Mr. R.N. Wheeler Carlo H. Tamburro, M.D. Associate Professor of Medicine Chief, Digestive Diseases & Nutrition Section REVISED BUDGET UNIVERSITY OF LOUISVILLE RESEARCH PROPOSAL RESEARCH TECHNIQUES AND METHODS FOR DETECTION AND PREVENTION OF CARCINOGENESIS IN INDUSTRIAL WORKERS This revised budget is for proposals Al-4, G, D, C, El, B3-4, and F, all of which have the most clinical appli cability. We have excluded proposals Bl, B2, B5, B6, B7, E2, H, and I. INDIVIDUAL BUDGETS Proposal Title Investigator A 1-4 G D C E1 B3&4 F Immunological Systems for the Detection of Vinyl Chloride and Other Chemical Injury P. Fortwengler Tissue Antigenic Systems of Detection E. Espinosa Electron Microscopic Evaluation of Liver Tissue from Chemical Workers R. Schrodt Tissue and Urinary Acid Mucopolysaccharide Changes Related to Vinyl Chloride Injury: Use In Early Detection and Diagnosis C. Kupchella Multivarient Analysis of Biological End Products and Biochemicals to Document Chemical Exposure for Early Diagnosis J. Wong Biochemical Enzymatic Systems for Detection of Vinyl Chloride and Other Chemicals J. Du Assays for Identifi cation of the Carcino genic Potential of Industrial Chemicals U. Strelps i80Ulttf Indirect Costs Subtotals Total Personnel Budget 34,100 9,000 8,000 10,000 27.000 12.000 100,100 65,065 Supplies 16,795 Total 50,895 5.000 7.000 14.000 7,000 7,500 15,500 15.000 25.000 7,332 11.000 34,332 23.000 69,627 169,727 234,792 A. SUMMARY OF BUDGETS Salaries: Supplies: Indirect Cost: 65% 100,100 69,627 65,065 TOTAL: 234,792 B. POSSIBLE ALTERNATE FUNDING SUGGESTION: Salaries: Supplies: Indirect Cost: 30% 100,100 69,627 30,035 TOTAL: 199,757 URL 17089 URINARY AND TISSUE GLYCOSAMINOGLYCAN PATTERNS IN HEPATIC ANGIOSARCOMA Charles E. Kupchella and Carlo H. Tamburro Cancer Center and Department of Medicine University of Louisville School of Medicine Louisville, Kentucky 40201 I. INTRODUCTION The recent discovery of a relationship between vinyl chloride and angiosarcoma of the liver has received much attention (1*3). Although there are now systematic detection programs for vinyl chloride workers (3,4)* there is as yet no specific chemical abnormality that serves as a good indicator of early, vinyl-chloride-induced liver injury and angio sarcoma. Alpha feto-protein has been a relatively valuable serological marker for hepatocellular carcinoma (5), but is has not as yet proven use ful in the detection of angiosarcoma (6). New leads are needed if more specific tests are to be developed for angiosarcoma. The literature suggests that the glycosaminoglycans in the urine and/or blood should be evaluated as a possible aid in early detection. The production of sulfated glycosaminoglycans is characteristic of malig nant vascular tumors of the skin and some pathologists use this feature as a diagnostic aid (7). Barr and Bonin (8) observed a strong positive alcian-blue, glycosaminoglycan staining reaction in human angiosarcoma tissue and suggested than an attempt be made to qualitate and quantitate the production of glycosaminoglycans in the neoplasms, serum, and urine of those at risk. They pointed out that the urinary glycosaminoglycans may have diagnostic significance in angiosarcoma and, if so, a glycosami noglycan spot test might easily be employed as a gross screening test of vinyl chloride production workers. A number of other observations place the glycosaminoglycans in a relevant position with regard to angiosarcoma. Angiosarcoma is accom panied by connective tissue abnormalities (2,9) and changes in tissue, urinary, and blood glycosaminoglycans have been found to occur in many connective-tissue disorders -- including connective tissue disorders of the liver (10-14) -- as well as in hepatic cancer (15-17). Supported in part by grants from the B. F. Goodrich Company and the American Cancer Society (IN-111) and a contract with the National Cancer Institute (NOI-cn-55212). URL 17090 CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO . The purpose of this study was to make a preliminary determination of the glycosaminoglycan patterns in tissue and urine associated with angiosarcoma of the liver and with vinyl-chloride-induced liver injury other than angiosarcoma and to compare these patterns with those in normal controls and those associated with other liver disease. Our goal was to evaluate the use of glycosaminoglycan patterns in the early detec tion of vinyl-chloride-induced liver injury and angiosarcoma an<? to explore the role of the glycosaminoglycans in the etiology of vinyl chloride injury. II. PROCEDURES AND MATERIALS USED Urine specimens were collected as occasional samples from: 9 normal controls; 9 individuals with histories of occupational exposure to vinyl chloride and having abnormal, liver, biochemical studies; 6 with "other" cancers prior to surgery; 3 with angiosarcoma; 8 with active viral hepatitis; 6 with cirrhosis; 2 with lung-liver metastases; and 4 with metabolic disorders of the liver (congenital and indirect hyperbiliru binemia). In one case of angiosarcoma, 24-hr urines were collected on alter nate days beginning 2 weeks prior to death. Urine samples were collected without preservative and frozen at -76 until analysis. Specimens were divided into two 25 ml samples and one 5 ml sample. Urinary creatinine was measured on the 5 ml sample using a Technicon Autoanalyzer. The degree of urinary glycosaminoglycan polymerization was estimated by dialyzing one 25 ml sample for 24 hours in tap water; the sample was then treated identically to an undialyzed sample by the method of DiFerrante (18) using cetylpyridinium chloride as a precipitant. After resolubilization of the glycosaminoglycans In water, duplicate samples were assayed for total uronic acid by the modi fied carbozole reaction of Bitter and Muir (19). The remaining glyco saminoglycans were reprecipitated with cetylpyridinium chloride and separated into the wash, hyaluronic acid, chondroitin sulfate, and heparin fractions as described by Schiller et. al.(20). Each of the fractions was assayed for uronic acid (>ug per mg of creatinine). Autopsy tissue was obtained in 2 cases of hepatic angiosarcoma (tumor tissue and noo-tumor tissue adjacent to tumor), 2 cases of cirrhosis, and in 3 control cases (gun-shot wound victims without liver pathology) and analyzed for glycosaminoglycans by a previously reported modification (21) of the method of Schiller et. al. (20). Uronic acid was determined in each of the hyaluronic acid, chondroitin sulfate, and heparin fractions. Pieces of tissue were subjected to alcian-blue-periodic-acidSchiff staining with and without hyaluronidase and diastase pretreatment. These procedures were carried out according to the methods described by Mowry (22). Ascitic fluid was also obtained at autopsy in one case of angio sarcoma and analyzed for glycosaminoglycans. The fluid was centrifuged GLYCOSAMINOGLYCANS IN ANGIOSARCOMA and the sediment analyzed as tissue above. The supernatant was treated by the method for urine described above. III. RESULTS A summary of the urinary glycosaminoglycan measurement is given in Table I. Normal controls had the least urinary glycosaminoglycans (measured as uronic acid) of all groups. All other groups showed some elevation. The levels in angiosarcoma, hepatitis, cirrhosis, and liver metastases were significantly elevated (P < .05) over normal controls. The cirrhotic group exhibited the greatest variance in urinary glycosami noglycans. No significant differences were found in urinary creatinine levels between groups. There were no significant differences between groups in either the percentage of the total glycosaminoglycans that was dialyzable (Table I) or in the percentage of the unfractionated total that appeared in the hyaluronic acid, chondroitin sulfate, and heparin fractions. Seven of 9 vinyl-chloride-exposed individuals other than those with angiosarcoma had positive chondroitin sulfate fractions with nega tive hyaluronic acid and heparin fractions. This was true in only 3 of 32 other urines evaluated in this same manner. The pattern of daily glycosaminoglycan excretion prior to death due to angiosarcoma in one individual is given in Figure 1. Total tissue glycosaminoglycan levels for angiosarcoma tumors, fibrotic tissue adjacent to tumors, cirrhotic liver tissue and normal liver tissue are shown in Figure 2. Fractional hyaluronic acid, chon droitin sulfate, and heparin levels are given in Figure 3. Histochemically, angiosarcomatous tissue exhibited a strong alcianblue positive staining reaction. Alcian-blue staining was only slightly less in "non-tumor" tissue adjacent to tumor masses. The staining reaction in tissue from normal liver was very weak and only slightly stronger in cirrhotic liver.tissue. The strong alcian-blue reaction in angiosarcomatous tissue did not occur if sections were pretreated with hyaluronidase. Ascitic fluid sediment was uronic-acid-positive in only the hyaluronic acid fraction -- 112 >ug uronic acid per gram of dry, defatted sediment; ascitic fluid supernatant contained 1.7, 1.2, and 0.2 uronic acid per ml in the hyaluronic acid, chondroitin sulfate, and heparin fractions, respectively. IV. DISCUSSION The literature indicates that normal male creatinine excretion is 1.5 g per 24 hoursU3).Thus, our normal mean (Table I) of 3.2 * -4 ^9 cetylpyridinium chloride-precipitable uronic acid per mg creatinine falls in the middle of the normal ranges reported by Varma et. al. (24), URL 11091 f URL 17092 CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO 2.6 - 4.Zug/mg; DiFerrante and Rich (25), 2.9 - 4.Qug/mg; and Kao and Leslie (26), 1.8 - 4.9>ug/mg. Although ou** study was not controlled for age, Goldberg and Cotlier (27) have shown that urinary glycosaminoglycan excretion is constant from ages 20-70. Manley et. al. (28) have shown that the proportion of urinary glycosaminoglycans in the chondroitin sulfate fraction is constant from ages 20-70. Manley et. al. also reported that the chondroitin sulfate fraction is highest at birth and that it gradually drops until age 20, suggesting that urinary chondroitin sulfate reflects tissue growth. The fact that we found no differences between groups in the creatinine concentration is significant in that it indicates that occa sional samples do reflect 24-hour excretion when normalized to creatinine. Precedent for expressing glycosaminoglycan measurements as a function of creatinine content in occasional urine samples has been established by DiFerrante and Rich (25) and Pennock (29). Manley et. al. (28) have shown that the creatinine/uronic acid ratio is steady from ages 20-70. Our results indicate that the liver diseases evaluated are accom panied by elevated urinary glycosaminoglycan excretion. Our tissue data suggests that this reflects liver-tissue glycosaminoglycan elevation and conforms to the reports by others that both hepatic connective tissue disorders (10-14) and hepatic cancer (15) result in increased hepatic glycosaminoglycan levels. It may be significant that the angiosarcoma patients had half the urinary glycosaminoglycan excretion of patients with liver metastases and that our analysis of angiosarcomatous tumor tissue exhibited half the glycosaminoglycan content reported by Kojima et. al. (15) for hepatocellular carcinoma. While our data suggest that liver disease results In a decrease in the proportion of highly polymerized glycosaminoglycans, variance was large within each group and none of the differences between groups were statistically significant. Although we have not completed the characterization of isolated glycosaminoglycan fractions, our data indicate: 1) that the chondroitin sulfates are the primary urinary alycosaminoglycans in both normal controls and in disease states; 2) that the chondroitin sulfates and heparin are the dominant glycosaminoglycans in normal and cirrhotic livers (Figure 3). Chondroitin sulfate is elevated in the fibrotic, nontumor, portions of angiosarcomatous livers while heparin is the predomi nant glycosaminoglycan in tumor tissue. Hyaluronic acid is also apparent ly elevated relative to chondroitin sulfate in angiosarcomatous tumors (Figure 3); and 3) that hyaluronic acid is the sole glycosaminoglycan in ascites fluid sediment. These qualitative data are in general agreement with those reported by others. Goldberg and Cotlier (27), Douglas et. al. (30), and Varma et. al. (24) have reported that the chondroitin sulfates are the predominant urinary glycosaminoglycans. Varma et. al. reported that 2/3 of urinary glycosaminoglycans are chondroitin-4-and chondroitin-6-sulfate and this agrees with our data on normal controls and on those with liver disease. ----- Kojima et. al. (15) reported that in hepatocellular carcinoma 6LYC0SAMIN0GLYCANS IN ANGIOSARCOMA tissue, chondroitin sulfates and hyaluronic acid were increased 33 and 10 times, respectively, over amounts found in healthy livers; the heparin and heparan sulfate proportions dropped. This contrasts with our data on angiosarcoma tissue, i.e. heparin and hyaluronic acid increased 5 and 10 times, respectively, over normal tissue; chondroitin sulfate levels rose but fell in proportion to other glycosaminoglycans. Galambos and Shapira (10) reported that the chondroitin sulfates are dominant in normal livers and in hepatic fibrosis, but Kojima et. al. (15) report that chondroitiinase-resistant and hyaluronidase-resistant glycosaminogly cans are dominant. Kuroda et. al. (31) also reported that heparan sulfate is the dominant glycosaminoglycan in the normal liver. Our histochemical observation that nearly all of the increased alcian-blue positive material in angiosarcomatous livers was susceptible to hyaluronidase digestion suggests that the observed chondroitin sulfate elevation is due to chondroitin-4- and/or chondroitin-6-sulfate. The increases in liver and urinary glycosaminoglycans may well reflect an important role of these substances in the process of fibrogenesis and in tumor growth. Galambos and Shapira (10) reported that hyaluronic acidl was elevated during hepatic fibrogenesis. If a similar fibrotic process is operative in angiosarcoma, it may be that the ob served tumor-tissue heparin increase is reflective of tumor growth. We did observe a four- to six-fold greater heparin level in tumor tissue than in adjacent, non-tumor tissue. The observation that the chondroitin sulfates tend to be the exclusive uronic-acid-positive constituents in the urine of individuals is paradoxical in that those glycosaminoglycan fractions that are most elevated in angiosarcomatous tissue are those that are absent from the urine of individuals who may well have early, vinyl-chloride-induced liver injury. This pattern may be due to the selective action of lyso somal, glycolytic enzymes in the liver and/or may reflect the role of the chondroitin sulfates in early fibrotic changes in the liver. Certainly the potential usefulness of this pattern in early detection warrants the more complete evaluation now ongoing in our laboratory. V. SUMMARY Glycosaminoglycans were measured in urine and tissue of patients with hepatic fibrosis and hepatic cancer including vinyl-chlorideexposure-associated liver injury and angiosarcoma. Angiosarcoma, hepa titis, cirrhosis, and liver metastatic patients exhibited significantly elevated glycosaminoglycan excretion. Angiosarcoma tissue exhibited elevated glycosaminoglycan levels with the greatest increases in the heparin fraction. Histochemically, angiosarcomatous tissue gave a strong alcian-blue staining reaction which could be prevented by pretreatment with hyaluronidase. Although vinyl-chloride-exposure-associated liver injury other than angiosarcoma was not accompanied by a significantly elevated glycosaminoglycan excretion, this condition tended to be associated with a urinary glycosaminoglycan excretion pattern in which the chondroitin sulfate fraction was the only uronic-acid-positive fraction. URL 17093 CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO URL 17094 +i TABLE I Urinary Glycosaminoglycan Levels in aq Uronic Acid Per mg Creatinine by Liver Diseases Category Patient qroup normal control vinyl chloride exposed Cases 9 9 ug uronic acid per mg creatinine (- 1 S.E.) 3.2 * .4 % uronic acid not dialyzable ( 1 S.E.) 65 5 39 6 other cancer other liver disease 6 4 4.5 1.5 5.1 0.8 39-6 37 13 angiosarcoma hepatitis cirrhosis liver metastasis 3 8 6 2 7.6 * 1.6 8.5 1.8 12.7 3 13.8 .9 41 22 52 14 53 9 42 1200 r 3 SJlOOO <ce 2*St gou>iSOO -_r 600 GLYCOSAMINOGLYCAN CONCENTRATION IN ANGIOSARCOMA, CIRRHOSIS, AND NORMAL LIVER TISSUE |g400 mi|S 85 SUhoJ; 200 _J- ?o ------------------- ANGIOSARCOMA-- Turner Non-tumor Turner Adjoeent Cote I Cote I Cate 2 Non-tumor Adjacent Cote 2 CIRRHOSIS Coe* 3 Com 4 Cat* 5 Come 6 Cate 7 CONCENTRATION OF INDIVIDUAL GLYCOSAMINOGLYCAN FRACTIONS ISOLATED FROM HEPATIC ANGIOSARCOMA, CIRRHOSIS, AND NORMAL LIVER TISSUE z z S URL 17095 Cate I Cote l Cate 2 Cote 2 Cote 3 Coee 4 Cota S Ceet 6 DAILY GLYCOSAMINOGLYCAN URINARY OUTPUT MIUQRAMS URONIC AGIO PER 24 HOURS URL 17096 _______I_______I_______I_______ > 16 14 12 10 6 6 4 2 0 PAYS PRIOR TO LIVER DEATH Figure 1 Figure 2 Figure 3 i Urinary glycosaminoglycan output in one angio sarcoma patient during the 16-day period prior to death. Glycosaminoglycan concentration in angiosarcomatous, cirrhotic and normal human liver tissue. Fractional concentrations of glycosaminoglycans in angiosarcomatous, cirrhotic and normal human liver tissue. te o tv ia n CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO VI. REFERENCES 1. Creech, J. L. and Johnson, M. N. Angiosarcoma of the Liver in the Manufacture of Polyvinyl Chloride. J. Occup. Med. 1J5: 150-151, 1974. 2. Falk, H., Creech, J. L., Heath, D. W., Johnson, M. N., and Key, M. M. Hepatic Disease Among Workers at a Vinyl Chloride Polymerization Plant. JAMA 230: 59-63, 1974. 3. Makk, L., Creech, J. L., Whelan, J. G., and Johnson, M. N. Liver Damage and Angiosarcoma in Vinyl Chloride Workers: A Systematic Detection Program. JAMA 230: 64-68, 1974. 4. Creech, J. L., Makk, L., Whelan, J. and Tamburro, C. H. Hepatotoxicity Among Polyvinyl Chloride Production Workers During First Year of Surveillance Program. Gastroenterology 67.: 786, 1974. 5. Kohn, J. and Weaver, P. C. Serum Alpha Fetoprotein in Hepatocellular Carcinoma. Lancet 2: 334-336, 1974. 6. Tamburro, C. H., Makk, L. and Creech, J. L. Unpublished observation. 7. Girard, D., Johnston, W. C., and Grahm, J. H. Cutaneous Angiosar coma. Cancer 25: 868-883, 1970. 8. Barr, R. and Bonin, M. "Letters." JAMA 231(9): 914, 1975. 9. Popper, H., and Thomas, L. B. Alterations of Liver and Spleen Among Workers Exposed to Vinyl Chloride. Ann. NY Acad. Sci. 246: 172-194, 1975. 10. Galambos, J. T., and Shapira, R. Natural History of Hepatitis: IV Glycosaminoglycuronans and Collagen in the Hepatic Connective Tissue. J. Clin. Invest. 52(11): 2952-2962, 1973. 11. Koizumi, T., Nakamura, N., and Abe, H. Changes in Acid Mucopoly saccharide in the Liver In Hepatic Fibrosis. Biochim. Biophys. Acta. 148: 749-756, 1967. 12. Kojima, J. Studies on the Metabolism of Hepatic Connective Tissue in Fibrosis of the Liver. Med. J. Osaka UniY. 16.: 419-429, 1964. 13. Rubin, E. Autoradiographic Characterization of Sulfated Acid Mucopolysaccharides in Experimental Cirrhosis. J. Histochem. Cytochem. 14: 688-689, 1966. 14. Patrick. R. s. and Kennedy, J. S. The Synthesis of Sulfated Mucopolysaccharide at Sites of Hepatic Fibrosis Induced by Carbon Tetrachloride, Amyloidosis, and the Implantation of Catgut. J. Pathol. Bacterio!. 88: 549-555, 1964. 15. Kojima, J.> Nakamura, N., Kanatani, M. and Ohmori, K. The Glycosaminoglycans in Human Hepatic Cancer. Cancer Res. 35(3): 542-547 1975. 86011^ GLYCOSAMINOGLYCANS IN ANGIOSARCOMA 'JRL 17099 16. Anghileri, L. J. Metabolism of Acid Mucopolysaccharides in Hepatoma and in Normal Liver. Oncology 30: 304-317, 1974. 17. Yamamoto, K., and Teryama, H. Comparison of Cell Coat Acid Muco polysaccharides of Normal Liver and Various Ascites Hepatoma Cells. Cancer Res. 33: 2257-2264, 1973. 18. DiFerrante, N. M. The Measurement of Urinary Mucopolysaccharides. Anal. Biochem, 21: 98-106, 1967. 19. Bitter, T., and Muir, H. A Modified Uronic Acid Carbazole Reaction. Anal. Biochem. 330-334, 1962. 20. Schiller, S., Slover, G. A., and Dorfman, A. A Method for the Separation of Acid Mucopolysaccharides: Its Application to the Isolation of Heparin from the Skin of Rats. J. Biol. Chem. 236(4): 983-987, 1961. 21. Kupchella, C., and Steggerda, F. The Distribution of Acid Muco polysaccharides in the Canine Gastrointestinal Mucosa. Trans. NY Acad. Sci. 34: 351-360, 1971. 22. Mowry, R. W. Alcian Blue Techniques for the Histochemical Study of Acidic Carbohydrates. J. Histochem. and Cytochem. 4_: 407, 1956. 23. Sunderman, F. W. and Boerner, F. Normal Values in Clinical Medicine. W. B. Saunders. Philadelphia, p. 353, 1949. 24. Varma, R. S., Varma, R., Allen, W. S., and Wardi, A. H. Urinary Excretion of Acid Mucopolysaccharides In Schizophrenia. Biochem. Med. 11(4): 358-369, 1974. 25. DiFerrante, N. and Rich, C. The Determination of Acid Aminopolysaccharide in Urine. J. Lab. Clin. Med. 48: 491-494, 1956. 26. Kao, K. and Leslie J. -Micro Fractionation and Determination of Urinary Glycosaminoglycans. Biochem. Med. 9(4): 317-326, 1974. 27. Goldberg, 0. and Cotlier, E. Specific Isolation and Analysis of Mucopolysaccharides (Glycosaminoglycans) from Human Urine. Clin. Chim. Acta. 41: 19-27, 1972. 28. Manley, G., Severn, M. and Hawksworth, J. Excretion Patterns of Glycosaminoglycans and Glycoproteins in Normal Human Urine. J. Clin. Pathol. 21: 339-345, 1968. 29. Pennock, C. A. A Modified Screening Test for Glycosaminoglycan Excretion. J. Clin. Path. 22: 310, 1969. CHARLES E. KUPCHELLA AND CARLO H. TAMBURRO 30. Douglas, C., Nowak, J. and Danes, B. Mucopolysaccharides in Urine During Normal Human Development. Pediatr. Res. ]_: 724-727, 1973. 31. Kuroda, J., Saito, S., Seno, N., Nagase, S., and Anno, K. Isolation and Chemical Characterization of Mucopolysaccharides from Rat Tumors. Cancer Res. 34(2): 308-312, 1974. URL 17100 URL 17101 IMMUNOPATHOLOGIC OBSERVATIONS IN LIVER ANGIOSARCOMA Enrique Espinosa, M.D. Department of Pathology University of Louisville School of Medicine Louisville, Kentucky I. INTRODUCTION In hepatic fibrosis and angiosarcoma associated with vinyl chloride exposure of industrial workers, manifestations of the disease could not be detecteu in most cases until the process was far advanced (1). Normal values of liver function tests were reported in a case with significant vinyl chloride hepa tic fibrosis (2) , and only . small percentage of workers of a plant unit where seven cases of liver angiosarcoma were diag nosed had abnormal blood screening tests (3). Thus, conven tional liver function tests do not appear, to be sensitive in dicators of vinyl chloride liver disease. Development of more sensitive mithods for detecting the disease in early stages would be of great importance. An approach to this may be pro vided by immunologic studies. In such a study the question arises whether the fibrotic and angiosarcomatous livers contain antigens that are different from those present in normal tis sue and whether such changes could stimulate an immunologic response. The purpose of this study was to search for anti genic changes in the angiosarcomatous tissue and to test for possible presence of an antibody response in the host. II.. PROCEDURES AND MATERIALS USED A. Patients' Sera and Tissue Specimens Serum samples from B.F. Goodrich Co. workers' with histor ies of vinyl chloride exposure of several years included sam ples from two individuals with liver angiosarcoma, ten with liver dysfunction with fibrosis and ten with normal liver function tests. The patients With angiosarcoma died and the diagnosis was confirmed at autopsy and portions of tumor and neighboring liver tissues were obtained at autopsy. Patients with liver dysfunction with fibrosis included individuals with ENRIQUE ESPINOSA, M.D. abnormalities in liver function tests and fibrosis detected at biopsy. Tissues were also obtained from coroner's autopsies of healthy individuals a few hours after death by gunshot wounds. B. Tissue Extracts and Antisera Liver angiosarcoma and adjacent liver tissue and post-mor tem tissues considered to be normal were frozen and stored at -70C until used. Portions were cut, thawed and homogenized in 2-3 volumes of distilled water in a Potter-Elvehjem grinder in an ice bath until a smooth suspension was obtained. After centrifugation at 20,000 x G for 30 min, the supernatant fluid containing the aqueous extract was lyophilized. Albino rab bits were immunized with the tumor or normal liver extracts in Freund's complete adjuvant and sera collected`and stored fol lowing procedures detailed elsewhere (4). Reaction of these immune sera with human serum or plasma was eliminated by ab sorption with 100 mg of lyophilized, pooled normal human serum /ml antiserum. Antisera were routinely absorbed in this man-: ner prior to use. Additional absorption with tissue extracts was carried out with 100 mg lyophilized extract/ml antiserum. Absorptions followed a procedure described previously (5). C. Treatment of Tissue Extracts Enzymatic treatment of tissue extracts was carried out with Pronase and trypsin as previously described (6). Periodate oxidation was done according to Rajam et al. (7). Ammonium sulfate and cold ethanol fractionations were carried out as detailed (8). D. Immunodiffusion and Immunofluorescence Double .immunodiffusion was carried out in 0.8% agarose in phosphate-buffered saline pH 7.2 (PBS) containing 0.1% sodium azide. Circular wells, 2 mm in diameter, 3 mm apart were used. Immunoelectrophoresis was performed according to Schei- . .degger (9) using 0.8% agarose in 0.025 M Veronal buffer at pH 82 In immunofluorescent studies cryostat sections of rat kid ney, stomach or intestine and liver (4 microns) were used as substrate for antimitochondrial, antismooth-muscle and anti nuclear antibodies. Liver sections from rats exposed 4, 8 and 14 days to 1-2% vinyl chloride for 4 hr/dav were also used. The sections were covered with dilutions of patients' sera for 45 min at room temperature, washed twice in PBS for 10 min and then covered with fluorescein conjugated IgG fraction of rab bit anti-human immunoglobulins'serum (Cappel Laboratories, Inc.) for 45 min and washed as before prior to examination. Cryostat sections .of liver angiosarcoma and liver tissue con sidered to be normal (4 microns) were washed twice in PBS for 10 min to wash off nonfixed- immunoglobulins. After drying. URL 17102 ANGIOSARCOMA sections were stained with fluorescein conjugated IgG frac tions of rabbit anti-human IgG serum and goat anti-human IgM serum (Cappel Laboratories, Inc.). Sections were washed as above and examined under the fluorescent microscope. Represen tative frozen sections were stained with hematoxylin and eosin to allow correlation between immunofluorescence and the histo logical findings. The antigenic preservation of the tissues was indicated by the demonstration of their staining by anti nuclear factor according to the indirect immunofluorescent procedure. E. Elution of Tumor-bound IgG k The tumor and liver tissues were extracted five times with PBS to wash off nonfixed immunoglobulins and then extracted at pH 2.5 to release bound IgG according to a procedure applied in the elution of renal-bound antibody (10). F. Circulating Tissue Antigens Liver-specific antigen LSA (8), tissue antigens of wide organ distribution (4) and bile antigens (11) were tested in the patients* sera by immunodiffusion as described previously. tOVLV III. RESULTS c. 33 A. Angiosarcoma-related Antigen To test for presence of new antigens appearing in liver angiosarcoma, antiangiosarcoma serum was absorbed with human serum and liver extract and tested by immunodiffusion with ex tracts of both normal liver and angiosarcoma tumor at varying concentrations. This absorption eliminated all reactivity with liver extracts prepared from five normal individuals but not with the angiosarcoma extracts where one line of precipi tation remained (Fig. 1) . This line of precipitation could still be seen after additional absorption of the antiserum with kidney extract. In contrast, absorption with the tumor extracts eliminated completely the angiosarcoma-related line of precipitation. Absorption with spleen and lung extracts also eliminated this line of precipitation. Thus, the antigen appeared to be of restricted tissue distribution and not an giosarcoma specific. The'antigen was inactivated by trypsin and Pronase and thus appeared to be a protein or closely asso ciated to protein. Incubation of the tumor extracts for 1 hr at 4C in citrate buffer, pH 2.5, resulted in inactivation of the antigen whereas incubationin phosphate buffer, pH 5.0, neutral or alkaline pH up to pH 10.0, did not affect it. The' antigen was shown to be relatively thermolabile. Incubation of the tumor extracts for 30 min in PBS at 25 and 56C did not affect the antigen whereas incubation at 70C and higher ENRIQUE ESPINOSA, M.D. completely inactivated it. The antigen precipitated mainly at 20-30% saturated ammonium sulfate and at ethanol concentra tions of 30-70% (Table I). B. Absence of a Normal Tissue Antigen in Angiosarcoma Antiliver serum absorbed with human serum gave several arcs of precipitation in immunoelectrophoresis with extracts of normal liver and angiosarcoma tissue (Fig. 2a). These lines could not be seen following additional absorption of the anti serum with normal liver. In contrast, absorption with liver angiosarcoma extract failed to eliminate one of the arcs of precipitation (Fig. 2b). Thus, the tissue antigen related to this arc of precipitation appeared to be absent in the angiosarcomatous tissue whereas the antigens corresponding to the other arcs were present. The absent antigen in angiosarcoma was shown to be present in kidney and lung extracts in addi tion to liver by absorption and direct immunodiffusion tests. Physicochemical characterization studies indicated this anti gen to be unaffected by Pronase and trypsin and inactivated by periodate treatment. The antigen was relatively thermostable withstanding incubation at 70C for 30 min in PBS. The anti-: gen was destroyed following incubation of the liver extract in citrate buffer at pH 2.5 or lower for 1 hr at 4C; incubation at pH 5.0 or higher (up to pH 10.0) did not affect it. This antigen precipitated over a wide range of ammonium sulfate and ethanol concentrations (Table I). C. Angiosarcoma-bound IgG IgG fluorescent staining appeared in a linear pattern in the peripheral portion of the tumor cells suggesting in vivo binding by the tumor of the immunoglobulin. This staining is illustrated in Fig. 3. Fig. 4 shows the angiosarcomatous cells surrounding irregular vascular spaces. Relatively coarse, linear fluorescence was also present along some hepa tic cords and strands of connective tissue. There was no ev idence of IgM. Control post-mortem liver tissue did not show any significant fluorescence of bound IgG. Staining for IgG of the tumor sections *did not change after several washings at pH 7.2 indicating that the IgG was firmly bound to the tumor. In contrast, sections showed marked diminution of staining after washing at pH 2.5. Elution of bound IgG from salineextracted tumor homogenates was thus attempted at acid pH. With the five successive saline extractions .the amount of sa line soluble IgG gradually diminished to nondetectable levels; and at acid pH bound IgG was released from-the Tiomogenate. Similar treatment of liver homogenates did not demonstrate presence of bound IgG (Table II). D. Circulating Autoantibodies and Tissue Antigens Serum autoantibodies to nuclei, mitochondria and smooth URL 17104 ENRIQUE ESPINOSA, M.D. is due to the deposition of a specific antibody, that the elu ted antibody is specific or until the relevant antigen has been identified. Work is in progress to determine the precise significance of the finding of IgG in the tumor. Acknowledgements: The author wishes to thank Drs. W. M. Christopherson, G. R. Schrodt and P. H. Carstens for fruitful discussion and advice about the histologic sections and Drs. C. Tamburro and L. Maak for providing serum samples and au topsy material. I also wish to thank Miss M. VanBraun and Mrs. V, Petrey for skilled technical assistance. This work was supported in part by a grant from B. F. Goodrich, Co. V. SUMMARY Immunodiffusion analyses of human liver angiosarcoma asso ciated with vinyl chloride exposure indicated presence in the tumor of an antigen not detected in normal liver and kidney but found to be present in lung and spleen. This antigen was shown to be a protein, inactivated by Pronase and trypsin, relatively susceptible to heating and to acid pH and precipi tated mainly at 20-30% saturated ammonium sulfate ar.d at 3070% ethanol concentrations. The tumor was shown to contain several antigenic constituents of normal tissue but one normal tissue antigen was not detected. This antigen was character ized as a substance unaffected by Pronase and trypsin and in activated by periodate. It was relatively thermostable, af fected by acid pH and precipitated over a wide range of ammon ium sulfate and ethanol concentrations. Tumor specimens ob tained at autopsy contained bound IgG as shown by immunofluor escence and elution experiments suggesting possible in vivo binding of IgG to the tumor. URL 17106 ANGIOSARCOMA TABLE I Angiosarcoma-related Antigen and Antigen Absent from the Tumor in Ammonium Sulfate and Ethanol Fractions a Presence of Fraction tested Angiosarcoma-related b antigen Antigen absent from c Angiosarcoma Ammonium sulfate: 0-20% saturation + - 20-30% saturation ++ + 30-50% saturation - +++ . 50-70% saturation - ++ Ethanol: 0-20% - ........... ++ . . 20-30% - +++ 30-50% ++ +++ 50-70% d SN ++ ++. ... . + a +++, ++, + indicate strength of doubl-e diffusion reaction in dilution assay. ' ** b^ Detected in angiosarcoma fractions. r* c Detected in liver fractions, d . SN = supqrnate of the 70% ethanol precipitation, dialyzed and lyophilized. _. URL 17107 ENRIQUE ESPINOSA, M.D. URL 17108 TABLE II IgG in Saline and Acid Extracts of Angiosarcoma and Liver Tissues Weight solid extracted from a Preparation tested 1 gm (wet weight) tissue Presence of IgG (mg) Angiosarcoma: Saline extract 1 29.8 +++ Saline extract 2 8.6 ++ Saline extract 3 6.2 + Saline extract 4 5.6 - Saline extract 5 6.1 - Acid extract 6.1 ++ Liver: Saline extract 1 47.4 +++ Saline extract 2 14.5 +++ Saline extract 3 8.6 + Saline extract 4 7.4 - Saline extract 5 Acid extract 6.4 9.0 m -- a Tested by immunodiffusion at concentrations of the eluates ranging up to 2%. Present at concentrations 0.05-0.1% (+++); 0.2-0.5% (+-fc) } l*-2% (+) ; negative at 2%, {-) . ENRIQUE ESPINOSA, M.D. URL 17108 TABLE II IgG in Saline and Acid Extracts of Angiosarcoma and Liver Tissues Weight solid extracted from a Preparation tested 1 gm (wet weight) tissue Presence of IgG (mg) Angiosarcoma: Saline extract 1 29.8 +++ Saline extract 2 8.6 ++ Saline extract 3 Saline extract 4 6.2 5.6 + - Saline extract 5 Acid extract 6.1 6.1 ++ Livers Saline extract 1 47.4 +++ Saline extract 2 14.5 +++ Saline extract 3 Saline extract 4 Saline extract 5 Acid extract 8.6 7.4 9.0 + - a Tested by immunodiffusion at concentrations of the eluates ranging up to 2%. Present at concentrations 0.05-0.1% (+++); 0.2-0.5% (+-fc)? 1-2% (+)? negative at 2%, (-). ANGIOSARCOMA LEGENDS OF FIGURES Fig. 1 Demonstration of angiosarcoma-related antigen. Per ipheral wells have 2-fold serial dilutions of liver angiosarcoma extract (a) and normal liver extract (b). Dilutions are clockwise and start at 100 mg/ml in the upper right well. Central wells in each plate contain rabbit antiangiosarcoma serum absorbed with normal human serum and liver extract. Fig. 2 Demonstration of tissue antigen absent in angiosar coma. (a) Trough contains rabbit antihuman liver serum absorbed with normal human serum. (b) Trough contains the antiliver serum additionally*absorbed with angiosarcoma extract. In both plates top wells have 10% solution of liver extract and lower wells angiosarcoma extract. Anode is to the right. Fig. 3 1 Immunofluorescent staining of liver angiosarcoma by fluorescein conjugated igG fraction of rabbit anti human IgG serum (x 400). Fig. 4 Cryostat section of angiosarcoma tumor stained with hematoxylin-eosin (x 400). URL 17109 REFERENCES HTTKUJU'H "ESPlflOSAV "M1. L). 1.* 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Heath, C.W., Jr., Falk, H., and Creech. J.L. Character istics Of Cases Of Angiosarcoma Of The Liver Among Vinyl Chloride Workers In The United States. Ann. N.Y. Acad. Sci., 246:231-236, 1975. Berk, P.D., Martin, J.F., and Waggoner, J.G. Persistence Of Vinyl Chloride-Induced Liver Injury After Cessation Of Exposure. Ann. N.Y. Acad. Sci., 246:70-77, 1975*. Wyatt, R.H., Kotchen, J.M., Hochstrasser, D.L., Buchanan, J.W., Jr., Campbell, D.R., Slaughter, J.C., and Doll, A.H. An Epidemiologic Study Of Blood Screening Tests And Illness Histories Among Chemical Workers Involved In The Manufacture Of Polyvinyl Chloride. Ann. N.Y. Acad. Sci., 246:80-87, 1975. Espinosa, E. Circulating Tissue Antigens. I. Tissue Antigens In Serum Of Patients With Diseases Involving In jury Of The Liver And Of Other Organs. Clin. Exp. Im munol., 16^153-162, 1974. Espinosa, E., and Kaplan, M.H. Antigenic Analysis Of Human Heart Tissue. Identification Of Antigens With Specificity Restricted To Heart And Skeletal Muscle In Acid Extracts Of Myocardium. J. Immunol., 100:1020-1031, 1968. Espinosa, E., and Kaplan, M.K. Antigenic Analysis Of Human Heart Tissue. Antigens With Restricted Organ Dis tribution In Acid Extracts Of Human Myocardium. J. Im munol., 105:416-425, 1970. Rajam, P.C., Gaudreau, C.J., Grady, A., and Rundlett, S.T. Preparation, Derivation And Partial Characteriza tion Of Organ-Specific Antigens From Human Brain. Im munology , 17:367-385, 1969. Espinosa, E. Circulating Tissue Antigens. II. Studies On An Organ-Specific Antigen Of Human Liver. Lab. Invest., 29:556-561, 1973. Scheidegger, J.J. Une Micro-M^thode De L*Immuno-filectro- phorfese. Internat. Arch. Allergy, Basel, 7:103-110, 1955. Krishnan, C., and Kaplan, M.H. Immunopathologic Studies Of Systemic Lupus Erythematosus. II. Antinuclear Re action of tf-Qobulin Eluted From Homogenates And Isolated Glomeruli Of Kidney From Patients With Lupus Nephritis. J. Clin. Invest., 46:569-579, 1967. Espinosa, E. Circulating Tissue Antigens. III. Identi fication And Characterization Of Antigens Of Limited And Of Wide Body Distribution In Human Gallbladder Bile. Presence In Serum Of Patients With Acute Hepatitis. Submitted for publication. Nairn, R.C., Richmond, H.G., McEntegartY'M.G./ and Fothergill, J.E. Immunological Differences Between Normal And Malignant Cells. Br. Med. J., 2:1335-1340, 1960. Carruthers, C., and Baumler, A. Immunochemical Staining With Fluorescein-Labeled Antibodies As An Aid In The Study Of Skin Cancer Formation. J. Natl. Cancer Inst., 34:191-200, 1965. o vvtt ANGIOSARCOMA 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29 . Fel, VJ. , and Tsikarishvili , t.N. Reduction Of Normal Muscle Antigens In Rat Tumors Of Muscle Origin Induced By Intramuscular Injections Of 20-Methylcholanthrene. Cancer Res., 24:1675-1677, 1964. Baldwin, R.W., and Barker, C.R. Antigenic Deletions In Carcinogen-Induced Rat Hepatoma. Nature, 214:292-293, 1967. ------- Kalnins, V.I., and Stick, H.F. Loss Of Liver Cell Anti gens In Azo-Dye Induced Hepatomas Of Rats. Nature, 200: 189-190, 1963. Hiramoto, R., Bernecky, J., Jurandowski, J., and Press man, D. Immunohistochemical Staining Prooerties Of The N-2-FAA Rat Hepatoma. Cancer Res., 2^:1372-1376, 1961. Abelev, G.I., Khramkova, N.I., and Postnikova, Z.A. The Antigenic Structure Of Mouse Hepatomas. I. Organ-Spe cific Antigens Of The Liver And Immunoelectrophoretic Study Of Their Occurrence In Hepatomas. Neoolasma, 9: 123-130, 1962. `~ Hillemans, H.G. Serological And Immunological Studies On The Pathogenesis Of Cervical Cancer. Z. Naturforsch. , 17B:240-261, 1962. Kay, H.E.M. A And B Antigens, In Normal And Malignant Cells. Br. J. Cancer, 11:409-414, 1957. Davidsohn, I., and Ni, L.Y. Loss Of Isoantigens A, B And H In Carcinoma Of The Lung. Am. J. Pathol., 57:307-314, 1969. Seigler, H.F., Kremer, W.B., Metzgar, R.S., Ward, F.E., Haung, A.T., and Amos, D.B. HL-A Antigenic Loss In Ma lignant Transformation. J. Natl. Cancer Inst., 46:577- 583, 1971. ~ Cryan, W.S., Hide, R.M., and Garb, S. Demonstration By Gel Diffusion Of Antigen In Spontaneous Mouse Tumors. Cancer Res., 26:1458-1465, 1966. Heppner, G.H., and Pierce, G. In Vitro Demonstration Of Tumor-Specific Antigens In Spontaneous Mammary Tumors In Mice. Internat. J. Cancer, 4:212-218, 1969. Isojima, S., Yagi, Y., and Pressman, D. Antigens Common To Rat Hepatoma Induced With 2-Acetylaminofluorene. Cancer Res., 29:140-144, 1969. Kahan, B.D., Holmes, E.C., Reisfeld, R.A., and Morton, D.L. Water Soluble Guinea Pig Transplantation Antigen From Carcinogen-Induced Sarcomas. J. Immunol., 102:28- 36, 1969. Hughes, L.E., and Litton, B. Antigenic Properties of Human Tumors: Delayed Cutaneous Hypersensitivity Reac tions.. Br. Med. J. 1:209-212, 1964. Itakura, K. Studies On Human Cancer Antigens By Gel Dif fusion Methods. Gann, 54:93-104, 1963. McKenna, J.M., Sanderson, R.P., and Blakemore, W.S. Ex traction Of Distinct Antigens From Neoplastic Tissue. Science, 135:370-371, 1962. v tu n a o 7.VU.V TrSt'i ATTENTIONS Mr. Mtlton Fretfeld ITEM 2 4/22/76 A Mortality Study of Workers Exposed to Vinyl Chlorlda April 22. 1976 This Is a report of a study of mortality rates In a population of 10*173 workers*who at soma tlmu In the past ware engaged In the manufac ture of vinyl chloride or its polymers. An earlier report on a portion of tills population showed that these workers experienced an Increased risk of death from cancer of the liver (already found by other Investi gators) and cancers of the lung, brain and lymphatic system. The larger group represented In this study has permitted a more detailed analysis of the relation between the risk of death from these causes and dura tion and level of exposures. The study population was identified by an examination of past and present personnel records In 37 plants which produced either vinyl chloride or polyvinyl chloride. Every man who had been In on exposed job for more than one year* was identified and his work history obtained. Man who had worked In the past and had quit or bean laid off were traced to'determine whether they were still alive as of December 31. 1972* the date chosen as the "dosing date*1 of the study. Of the 10.173 men selected for study. 9b percent were successfully traced, end 707 deaths were found. The study population Included men hired as far back as the late 1930's and Included 2.006 men who had worked 15 years or more. The number of deaths which occurred was compared with the number which would have been expected In a comparable population of United States males for each cause. e iu n O T Tha total number of death* was about 16 percent less than expected, end the total number of cancer deaths was about equal to what would be expected. ^ Some cancers occurred at a lower rate than expected, end these did arot appear to be related to the length or level of exposure. Cancer of the urinary organs and the lymphatic system showed some excess deaths (22 percent end 12 percent) but were not ^related In any systematic way to exposure. There was 44 percent exdess In leukemia deaths* also not / related to the circumstances jf exposure. Lung cancer showed an 8 percent excess in the total study group, but In men who had worked for 16 years or more and had begun work at least 20 years before the closing date of the study, there was a 62 per cent excess. Cancers classed as "Other and Unspecified" showed a 44 pereent ex cess In the study population as a whole, but a 2J0 percent excess In the older group mentioned above. This excess was largely due to an excess of*bra1n cancer* slnca 38 parcant of these cancers wore brain, compared with 20 percent for the general population. URL 17114 There wet a slight excess of digestive cancer in men with high ex posure. and in men with other than low exposure who had worked at least 16 years beginning at least 20 years before the study date. In these older man 50 percent of the digestive cancers were liver cancer com pared with about 9 percent for the general population. ATTENTION: Mr. MU ton Frol Told page 3 * In another analysis, the study population was scored according to the estimated total amount of vinyl chloride to which they were exposed^ man were given a score of 1 for each month of low level exposure, 2 for each month at medium exposure, and 3 for each month at high exposure. There was a somewhat Irregular Increase In mortality from digestive cancer, respiratory cancer and cancer of other and unspecified sites with Increasing scora. For men with scores of 400 or more (equivalent to 11 years of high exposure or 33 years of low exposure, for example) the excess mortality was 88 percent for digestive cancer, 94 percent for respiratory cancer, and 1B3 percent for cancers of other and un specified sites. The study generally confirms the findings of earlier investigators, but provides additional data for men with long service whose employment began many years ago. by Dr. W. R. Gaffey i 1 $ t X \ URL 17115 4 STATEMENT ON VINYL CHLORIDE RESEARCH It is well known that toxic effects are caused by absorption into the body of excessive amounts of any substance. When this occurs the capacity of the body's normal defense mechanisms are exceeded and this may result in more or less serious consequences - as with an excessive intake of alcohol. However, when the amount absorbed does not exceed this capacity the body is capable of dealing with it without toxic effects. Research on vinyl chloride (VC) in rats has demonstrated that high doses of VC are handled in the body differently than low doses. The results indicate that low doses of VC are readily detoxified, but high doses of VC exceed the body's normal defense mechanisms and detoxification is slowed and perhaps changed. Therefore, exposure to high levels of VC in rats results in an increased susceptibility to toxic effects including cancer. From data available, it appears that induction of cancer is correlated with high doses ^jf VC where detoxification is slowed, altered or incomplete, and that cancer is not induced at low doses where the body's normal defense mechanisms remain fully operative. by P. G. Watanabe et al. April 28, 1976 URL 17116 .% Summary of the Studies on the Pharmacokinetics of Vinyl Chloride in Rats and its Possible Relationship to Oncogenicity P. G. Watanabe, G. R. McGowan, J. A. Zempel and P. J. Gehring ABSTRACT Studies on the fate of 14 C-labeled vinyl chloride (VC) following oral administration and inhalation exposure in rats demonstrated that the disposition of VC in the body is a function of the dose. As the dose or exposure level of VC is increased the metabolism or detoxification pathways become saturated. VC is detoxified primarily by conjugation with nonprotein sulfhydryl groups in the liver (primarily glutathione, GSH). Studies to charac terize the depression of liver GSH as a function of magnitude and duration of exposure to VC have shown that a single 7 hour exposure of rats to 100 ppm or greater results in an unequivocal depression of liver GSH. Exposure to 10 ppm VC for 7 hours did not result in any depression = of liver GSH. These results suggest that increasing doses of VC deplete liver GSH and consequently lower the bodys defense mechanism for detoxification of the reactive metabolites of VC. This increased susceptibility may URL 171 f 7 . result in induction of carcinogenesis, and from data available, it appears that a correlation exists between doses of VC which cause tumors and those that saturate metabolic or detoxifying pathways. Conceptually, the results of these investigations indicate that statistical projections utilizing data collected from rats exposed to high doses of VC are invalid for pre dicting the hazard of low level exposure because such projections assume that the dynamics governing the fate of VC in the body are unaltered; and in fact, the data presented demonstrate that the fate of VC in rats is highly dependent on the dose administered. April 28, 1976 VVLV^ The Effects of Maternally Inhaled Vinyl Chloride on Embryonal and Fetal Development in Mice, Rats and Rabbits John, J. A., Smith, F. A., Leong, B.K.J. and Schwetz, B. A. Studies have been conducted to assess the possible deleterious effects of inhaled vinyl chloride on the developing embryo and fetus of mice, rats and rabbits. Groups of pregnant CF-1 mice, Sprague-Dawley rats and New Zealand white rabbits were exposed to 500 ppm vinyl chloride 7 hrs daily during the period of major organogenesis - the days of gestation when the embryo is most sensitive to developmental alterations. Subsequently, other groups of mice were similarly exposed to 50 ppm vinyl chloride and rats and rabbits were exposed to 2500 ppm vinyl chloride. While maternal toxicity was observed, vinyl chloride alone did not cause significant embryonal or fetal toxicity and was not teratogenic in any of the species ;f CO at the concentrations tested. Maternal toxicity was more prominent among mice than among rats and rabbits. Simultaneous exposure of some of the pregnant animals to vinyl chloride by inhalation (mice: 500 and 50 ppm; rats and rabbits; 2500 ppm) plus 15% ethanol in the drinking water resulted in toxic effects greater than those associated with exposure to vinyl chloride alone in the three species. The combination of ethanol and vinyl chloride appeared to enhance the toxicity to the pregnant females more than it enhanced the toxicity to the developing embryo and fetus. CYTOGENETIC STUDIES OF BONE MARROW CELLS FROM RATS EXPOSED TO VINYL CHLORIDE R. V. Johnston, D.V.M., D. J. Mensik, B.S., ' M. N. Pinkerton, B.S., L. B. Whorton, Jr., Ph.D.* Dow Chemical U.S.A, Texas Division - Freeport, Texas 77541 Industrial Health and Medicine Department Biomedical and Comparative Toxicology Research Laboratory The Biomedical and Comparative Toxicology Research Laboratory of the Texas Division of Dow Chemical U.S.A. was asked by the Manufacturing Chemists Association to conduct cytogenetic studies on some of the rats which had been exposed to vinyl chloride at Industrial BIO-TEST Laboratories, Decatur, Illinois. The details of the exposure levels and pathological findings will be reported separately. Preliminary reports have appeared in Chemical Week (115:30, July 17 , 1974) and in the Annals of the New Xork Academy of Sciencest Volume 246 , page 219. The test animals were Charles River CD outbred albino rats which had been exposed to vinyl chloride gas for 7 hours per day, 5 days per week for one year at 0, 50, 200 and 2500 ppm in air. Cytogenetic studies on 5 males and 5 females from each group showed that there was no statistically significant increase in the chromosomal aberration rates in bone marrow cells of the exposed rats. April 28/ 1976 o ? u i- la n ^Associate Professor and Director Division of Biometry Department of Preventive Medicine and Community Health University of Texas Medical Branch Galveston, Texas 77550 jItidMiniai B I O T E S T 2alto*atoMe4., 9*ta. Inhalation Studies in Rats, Mice and Hamsters With Vinyl Chloride At Industrial BIO-TEST Laboratories, Inc. , a study was conducted to determine the effects from inhalation of vinyl chloride monomer to rats, mice and hamsters. The study was supported by the voluntary contributions of thirty-one VCM/PVC producing com panies ancl is administered by the Manufacturing Chemists Association with the advice and guidance of a technical panel of scientists. In this study, 200 rats, hamsters and mice (100 of each sex or 600 animals total per exposure group) were exposed to 50, 200 or 2,500 ppm vinyl chloride vapor 7 hours per day, 5 days per week. Mice were exposed for 9 months while rats and hamsters were exposed for 12 months. All animals were then kept for the remainder of their natural lives (up to 26 months of mice and 30 months for rats and hamsters). There was an increased incidence in neoplasms in the liver of all 3 species at all 3 doses and the incidences are dose-related. In general, the only significant increases in rats or hamsters were in the liver. For mice there appears to be an increased incidence of all neoplasms, but particularly in the lung as well as the liver. URL 17121 !}itdud*ial B I O T E S T Jtabafcit&u&i, Vinyl Chloride Inhalation Study Incidence (percent) of Neoplasms - Females Species and Organ Control Experimental Groups T-I T-H T-ni (50 ppm) (200 ppm) (2500 ppm) T-rv (2500 ppm) RATS Liver 4. 8 39.5 76. 3 78.5 76.7 Kidney 0 1.3 1. 3 2.6 0 Skin 64. 0 56. 0 23. 0 13. 1 25.0 Misc. Pituitary Brain Me s ente ry Adrenals Thymus Lymph Node MICE Liver Lung 17. 9 0 4.3 13.2 36.1 43. 1 9.2 8.9 82.6 60.5 85.9 81.2 6.8 c Xr~> ro ro Skin 2.0 37.0 17.0 20.0 Misc. Kidney Mesentery Lymph Nods Genital 2.1 38.9 5.8 8.2 HAMSTERS Liver Skin Misc. Kidney Mesentery Lymph Node Thymus Genital 11.4 0 11.4 18.2 2.0 9. 1 26.6 15.3 3.9 60, 6 1.0 6.1 * Food Available During Exposure. IJrtdu&t'ual B I O - T E S T XalttnataA+eA, Dnc. < Vinyl Chloride Inhalation Study Incidence (percent) of Neoplasms - Males Species and Organ RATS Liver Kidney Skin Control 6.4 0 19.0 Experimental Groups T-I T-H (50 ppm) (200 ppm) 30.9 7.4 11.2 73. 7 0 5.0 Misc. Pituitary Brain Mesentery Adrenals Thymus Lymph Node Genital 6.4 18.5 10. 5 MICE Liver Lung 1.8 1.8 27.0 30.2 90.2 67. 1 Skin 0 0 1. 0 Misc. Kidney Mesentery Lymph Node Genital 1.8 1. 1 9.3 HAMSTERS Liver Skin 0 23.9 17. 5 0 5.0 4. 0 Misc. Kidney Mese'ntery Lymph Node Thymus Genital 0 15.2 7. 5 -0 0 CD 00 T-m (2500 ppm) 63. 3 0 2. 0 4. 4 tD rsj co 87.7 4. 0 62. 5 1. 1 8.9 ITEM 3 Interim Report on Mortality and Gross Observations in Rats, Mice and Hamsters With Vinyl Chloride M. L. Keplinger, J. W. Goode, D. E. Gordon and J. C. Calandra Industrial BIO-TEST Laboratories, Inc. 1810 Frontage Road Northbrook, Illinois 60062 -it URL H IM 9*ujLtA&ual BIO-TEST Jz&yniAyu&i, Jnc. Interim Report on Mortality and Gross Observations in Rats, Mice and Hamsters With Vinyl Chloride M. L. Keplinger, J. W. Goode, D. E. Gordon and J, C. Calandra Industrial BIO-TEST Laboratories, Inc. 1810 Frontage Road Northbrook, Illinois 60062 At Industrial BIO-TEST Laboratories, Inc. , a study was conducted to determine the effects from inhalation of vinyl chloride monomer to rats, mice and hamsters. The study was supported by the voluntary contributions of thirty VCM/PVC producing companies. It is administered by the Manufacturing Chemists Association (MCA), with the advice and guidance of a technical panel of scientists from the sponsoring firms. The protocol for the study was designed by the technical panel. The study was designed as a life-span study in these species which usually oceans 1-1/2 to 2 years for the "in-life" portion, with tissue examination to follow this period of time. All animals were allowed to live the remainder of their natural lives. Therefore, the total time of the study was longer than 2 years. Vi. I-W O !}nduiiniai BIO-TEST Jtakyia&yuel, 9*tc. 2 In this study, 200 rats, hamsters and mice (100 of each sex or 600 animals total per exposure group) were exposed to 50, 200 or 2,500 ppm vinyl chloride vapor 7 hours per day, 5 days per week. Mice were exposed for 9 months while rats and hamsters were exposed for 12 months. All animals were then kept for the remainder of their natural lives. The rats were COBS Charles River; mice were CDI Swiss Charles River; and the hamsters were Golden Syrian from the same source. Food was removed during exposure, water was present and no bedding was used at any time. The usual procedure in conducting inhalation studies in not to have food present in the chamber during exposure. In at least some of Dr. Maltoni's studies, food was present in the chamber. The question contaminated food was raised as to whether^ would influence the onset or incidence of tumors. Therefore, a separate group of 100 female rats was exposed to 2,500 ppm VCM while food was in the chamber. .All 600 animals per level were exposed in a single chamber. The chambers were operated under dynamic conditions. The chambers were sealed and were operated under slight negative pressure. This was done so that, in case of an accidental leak, room air would flow into the chamber instead of contaminated air flowing out of the chamber. WL 17126 y+idudM&l B I O - T E S T Aa&o*abyueir 9k&. 3 URL 17127 The concentrations in the chamber were analyzed using gasliquid chromatography. An automatic sampling device allowed samples to be drawn from 3 locations in each chamber. The concentrations at all locations in each chamber were monitored every day during the total period of operation of the chamber. Moribund and dead animals received gross autopsies and many gross lesions were photographed. The major organs of all animals were fixed for histological examination. There were no particularly adverse effects on body weights or, elements or enzymes measured in the blood. The data for mortality or survival are presented in Tables I, n and m. It can be seen that the last survivors of mice were sacrificed or died in month 26, while the last survivors of rats or hamsters were sacrificed or died in month 30. While these tables indicate "mortality", it should be noted that the numbers also include those animals sacrificed in extremis. In rats and mice (and somewhat in hamsters) there was a doserelated increase in mortality. This is well illustrated by examining the time to 100% mortality in the tables. For example, in mice the months in which there was 100% mortality were 11 at 2,500 ppm, 14 at 200 ppm, 16 at 50 ppm and 20 (male) or 26 (female) controls. In rats the months were 16 at 2,500 ppm (both fed and fasted), 21 at 200 ppm, 29 (male) or 30 (female) at 50 ppm and 30 for controls. $ttd*t6&ual B I O - T E 5 T JaliMa&yue, 9*tc. 4 URL 17128 Preliminary examination of tissues from mice which had died after about 6 or 7 months of exposure indicated possible tumors in the livers, lungs, mammary glands and/or skin* At that time there was no evidence of tumors in the rats or hamsters. A tabulation of incidences of neoplasms for males and females is presented in Tables IV and V. The males and females were tabulated separately to reveal any differences between the 2 sexes. A comparison of the data indicates that there are no real differences between males and females with regard to type or incidence of neoplasms. The data from these tables indicate an increased incidence in neoplasms in the liver of all 3 species at all 3 doses and the incidences are dose related. In general^the only significant increases in rats or hamsters were in the liver. For mice there appears to be an increased incidence of all neoplasms, but particularly in the lung as well as the liver. In many instances the incidences at the 2 higher levels A (200 and 2500 ppm) are about the same. This might be interpreted as evidence of lack of dose-response. However,these incidences are very high and probably indicate nearing the plateau of the doseresponse curve at 200 ppm. Cttdu&btiaL B I O - T E S T ^ahyiai&uei, Hhc. 5 The incidences (liver) at 50 ppm are well above control values which indicates that this is a definite effect level. A comparison of tumor data of fed and fasted (during exposure) female rats exposed to 2500 ppm VCM leads to the conclusion that there was no significant difference between the 2 groups. For example, the incidences of liver tumors were 77 and 78%. Although the incidence of skin tumors was 25% in the fed animals vs. 13% in the fasted animals, it should be noted that incidence in this group of fasted animals was the lowest of any group (control 64%, 50 ppm 56% and 200 ppm 23%). It was stated previously that there were 100 animals per group (male or female). It will be noted in the tables of incidences of neoplasms that the denominator in most cases is less than 100. There are several reasons for this. As soon as an animal dies, autolysis starts. If an animal is necropsied soon after death the tissues are worth saving for histological examination. If, however, several hours elapse from death to necropsy, the tissues may not be saved if the pathologists feels there is too much postmortem autolysis. The animals were housed in groups. If an animal (particularly mice, hamsters &c rats) dies in a cage with other animals, cannibalism QtuLuUial B I O - T E 5 T lahoMlt&ueA, Hoc. 6 usually occurs unless the dead animal is removed immediately. When the chambers were open (no exposure) and an animal was observed to be dead, it was removed and tissues were saved. If an animal died overnight, it frequently was lost for histology. * There is one other important aspect for inhalation studies compared to other studies such as feeding. The chamber is sealed during exposure. Even if an animal is observed to be dead in the chamber, there is no way to remove it until the end of the exposure. This results in loss of tissues for histology. In most of the chronic inhalation studies now being conducted.1 all animals are housed individually to prevent many of these losses. The denominator for the skin usually is 100 (or slightly less such as 98 or 99). If an animal is cannibalized, the skin usually is left. Even though there is autolysis or degeneration of other organs and tissues, the skin is not changed enough to present meaningful evaluation. Therefore, the denominator for the skin is higher than for other organs or tissues, and does allow accountability of the number of animals in the study. *v It might also be emphasized that in the usual chronic toxicity or carcinogenic study at the present time 50 animals per group are judged adequate to start a study. With life-time studies in rodents such as rats, mice or hamsters, a number of animals B I O - T E $ T JtahyxtbvU&L, y*tc. are expected to be lost for good histological examination. The number of 50 is used to assure that an adequate number, usually 20 to 25, are available for examination at the end of the study. The VCM study was designed with twice as many animals to assure that the usual expected number of 20 to 25 would be available at the end of the study. It will be noted that the denominator in all cases exceeds the 20 to 25, and it far exceeds the 20 to 25 in almost all cases. vevaw !)+ult4it'uai B I O - T 5 S T ^ssIt&iaityuei, 2*tc. 8 TABLE I TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice and Golden Hamsters Summary of Mortality Data - Albino Rats Month Number Cumulative Number of Mortalities at End of Month uc T-I T-II T-III MF MF MF MF T-IV F 1 2 3 4 5 6 7 8 9 10 11 12* 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29 30 0 c 0 0 2 3 4 4 4 5 7 13 16 19 21 23 23 33 38 46 52 57 64 73 7$ 86 92 98 100 0 0 1 2 3 4 4 5 6 7 8 14 . 17 21 23 25 27 28 31 34 42 48 54 65 69 76 84 93 100 0 2 2 4 5 6 8 9 10 11 13 19 23 26 28 31 35 38 43 47 52 63 72 81 85 91 96 100 100 0 0 0 1 1 1 2 3 4 7 9 16 24 30 37 . 45 53 59 66 73 80 89 92 94 97 98 99 99 100 0 1 1 3 6 6 6 10 11 12 14 21 26 33 40 46 55 64 71 77 100(18) 100 100 0 0 0 0 1 1 2 5 5 9 19 37 50 63 75 87 90 94 94 94 100(4) 100 100 0 0 0 1 3 5 6 10 16 24 34 58 73 85 94 100(4) 100 100 100 100 100 100 100 0 0 4 4 4 4 5 8 15 28 52 66 83 86 89 100 100 100 100 100 100 100 100 0 0 0 0 0 0 0 1 4 7 'jj 17 H 40 46 76 86 100(7; 100 100 100 100 100 100 100 ^Included 5 males and 5 females from each group excluding T-IV sacrificed for cytogenic study () Indicates moribund or sick animals sacrificed. JrtJudtuai B I 0 - T E S T JaMAofou&S., 2*tc. 9 Month Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 TABLE IX TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice and Golden Hamsters Summary of Mortality Data - Albino Mice UC M Cumulative Number of Mortalities at End of Month T-I T-II F MF M F T--nr M F 0 0 2 5 8 12 18 18 23 28 30 34 44 54 67 82 82 82 92 100 100 100 100 0 0 0 1 2 2 5 5 8 10 15 16 23 28 39 46 54 58 70 77 81 89 - 92 96 97 100 0 0 6 19 26 30 31 32 38 42 54 57 70 79 88 100 100 100 100 100 100 100 100 0 3 4 5 6 6 n 14 24 31 46 66 80 91 97 100 100 100 100 100 100 100 100 0 0 3 4 4 9 16 37 78 96 98 99 100 100 100 100 100 100 100 100 100 100 0 0 0 1 2 3 12 30 58 82 94 95 97 100 100 100 100 100 100 100 100 100 100 ,0 0 00 10 4 8 :-- 7 7 10 . S 9 13 w 23 30 46 56 75 76 98 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 1Q0 100 100 100 100 100 Undudl'dal 10 URL 19584 TABLE III TEST MATERIAL: Vinyl Chloride (Ethylene Derived) Chronic Vapor Inhalation Toxicity Study - Albino Rats, Albino Mice and Golden Hamsters Summary of Mortality Data - Golden Hamsters Month Number UC M Cumulative Number of Mortalities at End of Month T-I T-H P M F ______ M F T-HI M F 1 5444 7 5 25 2 8 11 10 12 9 16 10 11 3 8 13 12 14 12 18 14 13 4 9 15 16 16 15 19 15 13 5 9 18 16 16 .16 20 15 15 6 13 18 16 16 20 21 16 16 7 13 28 24 19 22 33 18 26 8 14 28 24 19 23 35 19 >L_ 28 9 14 29 24 20 25 36 20 3f--D 28 10 14 30 26 20 26 37 23 --j 29 11 12 15 18 31 36 28 21 34 33 26 37 38 46 28 40 o4_*>. 36 45 13 23 40 43 49 46 49 49 58 14 28 46 49 58 54 53 61 69 15 39 61 53 72 66 55 71 84 16 50 78 58 83 74 59 84 100(3) 17 57 81 61 85 76 66 86 100 18 62 85 62 86 77 75 88 100 19 65 90 68 89 79 84 92 100 20 71 96 73 95 86 89 100(5) 100 21 71 100(2) 80 100(3) 100(13) 100(7) 100 100 22 73 100 82 100 100 100 100 100 23 78 100 85 100 100 100 100 100 24 S3 88 25 88 92 26 90 95 27 .94 98 28 94 99 29 99 100 . 30 100 {) Indicates moribund or sick animals sacrificed. j*tdMfrual B I O - T E 5 T SaJvyia&yu&i, Htv:. TABLE IV Vinyl Chloride Inhalation Study - IBT NO. 663-03222 Projected Incidence of Neoplasms - Males Species Sc Organ Control Experimental Groups T-I T-II (50 ppm) (200 ppm) T-UI (2500 ppm) RATS Liver 5/78 (6.4%) Kidney 0/78 Skin 19/100(19%) Misc. Pituitary Brain Mesentery Adrenals Thymus Lymph Node Genital 5/78 (6.4%) 25/81 (30.9%) 6/81 (7.4%) 11/98(11.2%) 15/81 (18.5%) 56/76 (73. 7%) 57/90 (63. 3%) 0/76 0/90 5/100(5. 0%) 2/100 (2. 0%) 8/76 (10. 5%) 4/90 (4.4%) 33 r~ Cj MICE Liver 1/55 (1.8%) Lung 1/55 (1.8%) Skin 0/100 Misc. Kidney Mesentery Lymph Node Genital 1/55 (1.8%) 17/63 (27%) 19/63 (30.2%) 0/100 7/63 (1. 1%) 74/82 (90.2%) 59/65 (90.8%) 55/82 (67. 1%) 57/65 (87/7%) 1/100 (1.0%) 4/100 (4.0%) 8/86 (9.3%) 7/65 (10. 8%) HAMSTERS Liver 0/53 Skin 0/100 Misc. Kidney Mesentery Lymph Node Thymus Genital 0/53 11/46 (23.9%) 5/100 (5. 0%) 7/46 (15.2%) 7/40 (17.5%) 4/100 (4. 0%) 3/40 (7.5%) 35/56 (62.5%) 1/94 (1. 1%) 5/56 (8.9%) SAdH&ial B I O - T E S T lal&iaitvueA, Hkc. u Species c Organ TABLE V Vinyl Chloride Inhalation Study - IBT NO. 663-03222 Projected Incidence of Neoplasms - Females Control Experimental Groups T-I T-II (50 ppm) (200 ppm) T-IH (2500 ppm) T-IV* (2500 ppn RATS Liver Kidney Skin Misc. Pituitary Brain Mesentery Adrenals Thymus Lymph Node Genital MICE Liver Lung Skin Misc. Kidney Mesentery Lymph Node Genital 4/84 (4.8%) 30/76 (39.5%) 0/84 1/76 (1.3%) 64/100 (64%) 56/100 (56%) 15/84 (17.9%) 10/76 (13. 2%) 0/47 2/47 (4.3%) 2/100 (2.0%) 1/47 (2.1%) 26/72 (36. 1%) 31/72 (43. 1%) 37/100 (37.0%) 28/72 (38. 9%) 58/76 (76. 3%) 62/79(78.5%) 56/73(76.7` 1/76 (1.3%) 2/79(2.6%) 0/73 23/100 (23%) 13/99(13. 1%) 25/100(257 7/76 (9.2%) 7/79(8. 9%) 5/73(6. 8%) 71/86(82. 6%) 73/85 (85. 9%) c~ XI r-- --A ^ 52/86(60.5%) 69/85 (81.2%) 17/100(17.0%) 20/100 (20.0%) 5/86 (5.8%) 7/85 (8.2%) HAMSTERS Liver 4/35 (11.4%) Skin 0/100 Misc. Kidney Mesentery Lymph Node Thymus Genital 4/35 (11.4%) * & -- ;i. 6/33 (18.2%) 2/98 (2.0%) 3/33 (9. 1%) 21/79 (26.6%) 20/33 (60. 6%) 15/98 (15.3%) 1/99 (1.0%) 7/79 (8.9%) 2/33 (6. 1%) URL 17131 ITEM 4 References for "Cytogenetic Studies of Bone Marrow Cells From Rats Exposed to Vinyl Chloride by Dr. R. V. Johnston et al. 1. Kilian, D. J. and Picciano, D* J,, Cytogenetic monitoring and vinyl chloride workmen. Open Meeting on Evaluation of Systems to Detect Mutagenic Activity of Chemicals, July 16 and 17, 1975, NIH, Bethesda, Maryland. 2. Fleig and Thiess, A. M., Chromosome analysis after vinyl chloride exposure, Arbeitsmedizin, Sozialmedizin, Praventivsmedizin 9(12) 280-283 (1974)7 3. Johnston, R. V., Mensik, D. J., Pinkerton, M. N., Whorton, E. B., Cytogenetic studies of bone marrow cells from rats exposed to vinyl chloride, TBM-108-1, B16863, (1975). 4. Ducatman, A., Hirschhorn, Kurt, Selikoff, I. J., Vinyl chloride exposure and human chromosome aberrations, Mutation Research 31:163-168 (1975). 5. Rannug, U. et al. The mutagenicity of vinyl chloride after metabolic activation, Ambio 3:194-197 (1974), 6. Funes-Cravioto, F. et al. Chromosome aberrations in workers exposed to vinyl chlorTSe, The Lancet, February 22, 1975, page 459. 7. Purchase, I.F.H. et al. Chromosomal and dominant lethal effects of vinyl cKloride, The Lancet, (1975), 6. Bartsch, H., Human, rat and mouse liver-mediated muta genicity of vinyl chloride in S. Typhimurium strains, Int. J^ Cancer 15:429-437 (19757. % 9- Malaveille, C., Bartsch, H. et al. Mutagenicity of vinyl chloride, chloroethyleneoxide, cHloroacetaldehyde and chloroethanol. Biochemical and Biophysical Research Communications, Vol. 63:363-370 (1975). UNIROYAL UNIROYAL, Inc. Oxford Management & Research Center Middlebury, Connecticut 06749 8 U U 1 tin June 3, 1976 A. C, Clark Manufacturing Chemists Association 1825 Connecticut Avenue, N.W. Washington, D.C. 20009 Dear Al: Enclosed is a signed copy of the yellow form you sent with your letter of May 20. If Dr. McCleary already sent one in, please disregard this one. Our continued support beyond the presently proposed projects would depend upon the need. I hesitate to commit to an unknown program. Very truly yours, WcdU- W. D. Harris, Ph.D. Corporate Industrial Toxicologist WDH/df Enclosure cc: C. D, McCleary G. A. Anderson