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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Volume 534 LIVING IN A CHEMICAL WORLD OCCUPATIONAL AND ENVIRONMENTAL SIGNIFICANCE OF INDUSTRIAL CARCINOGENS Edited by Cesare Maltoni and Irving J. Selikoff The New York Academy of Sciences New York, New York 1988 SL 062845 Long-Term Carcinogenicity Bioassay on Vinylidene Chloride Administered by Inhalation to Sprague-Dawley Rats" New Results GIULIANO COTTI, CESARE MALTONI. AND GIUSEPPE LEFEMINE Institute of Oncology " F. Addarii" Bologna. Italy INTRODUCTION Vinylidene chloride is a monomer mainly used for the production of copoly mers and widely employed for food packaging. The aim of this report is to present recent new results of a long-term carcinoge nicity bioassay of vinylidene chloride on rats. The bioassay was performed at the Bentivoglio (BT) Experimental Unit of the Bologna Institute of Oncology. The experiment was started in 1980 and the biophase ended in 1983. VINYLIDENE CHLORIDE Vinylidene chloride (CH^CCI;) has a molecular weight of 97.0 and its struc tural formula is C=C Cl Cl Data on the production, uses, and occurrence ot the compouno are given in Tables 1, 2, and 3. The findings in the literature on carcinogenicity and other relevant biological effects are shown in Table 4. They conclude that: (1) in rats treated by inhalation, there was limited evidence of an increased incidence of mammary tumors (not dose-related), and possibly of liver and menin geal tumors; (2) in mice vinylidene chloride is carcinogenic, causing kidney adenocarcino mas, and an increased incidence of lung tumors, mammary carcinomas, and ma lignant tumors. In vinylidene-treated mice, limited evidence was found of an increased incidence of lymphomas and leukemias in females that was not dose- This experiment was partly supported by the EEC (Contract 3I3-79-4-ENV-1). 160 ay on i by itsa non of copolyertn carcinogentai Unit of the 1980 and the > and its struc- to are given in city and other hat: f an increased .er and menin- adenocarcinojmas. and mas found of an was not dose- -ENV-l), , i com ft al.: VINYLIDENE CHLORIDE 161 table i. Vinyiidene Chloride: Production Method* of Production Vmylidene chloride may be prepared by several methods. Most of the monomer tUSA. Japanl is produced commercially by the dehydrochlortnation (using sodium and hydroxide or limei of 1. 1,3-trichloroethane 130,000 tons per year World Production Geographical Areas of Production USA (70.000 tons. 1976), Western Europe (30,000 tons), Japan (30.000 tons) table:. Vinyiidene Chloride: Uses Major Uses Production of copolymers with high vinyiidene chloride content (the other major mono mer used is vinyl chlonde). used in the form of film coatings (solvent soluble resins and latexes) and fibers, and for tubes, pipes and pipe liners Other Uses Manufacture of modacnlic fibers (largely based on acrylonitrile); as a chemical intermedi ate in the production of 1.1,1 -tnchloroethane table 3. Vinyiidene Chloride; Occurrence'1 Natural Sources It is not known to occur as a natural product Workplaces Where vinyiidene chlonde or products containing vinyiidene chloride are produced, used, and stocked Atmosphere of submannes Spacecraft Air Water Effluents discharged from chemical manufacturing plants Rivers Wells Other untreated water Finished USA drinking water (tap water) Food Potato chips packed in polypropylene film coated with vinyiidene chlonde copolymers Industrial Materials In trichloroethylene, vinyl chloride and commercial chloroprene Film for packaging From 1ARC1 and Maltoni et al.2 SL 062847 162 ANNALS NEW YORK ACADEMY OF SCIENCES table 4. Vinylidene Chloride; Data on Carcinogenicity and Other Relevant Correlated Biological Effects (1) Carcinogenicity Studies (A) Experimental animals (a) Rat: by inhalation limited evidence of increased incidence of mammary tumors in female SpragueDawley rats1-4 no effects on Sprague-Dawley and Wistar rats' no effects on Sprague-Dawley rats* limited evidence of induction of extrahepatic angiosarcomas in CD rats.'* Con current unwanted exposure to vinyl chloride cannot be ruled out in this study1 no effects on CD rats' Rat: by ingestion (stomach tube), no effects on Sprague-Dawley rats1-* (drinking water): no effects on Sprague-Dawley rats* (stomach tube), limited evidence of increased incidence of liver and meningeal tumors in BD1V rats10 (stomach tube); no effects on F344/N rats11 (b) Mouse: by inhalation in Swiss mice, induction of kidney adenocarcinomas in males, and increased incidence of mammary carcinomas, lung tumors and total malignant tumors m females1-1 induction of liver angiosarcomas and increased incidence of lung adenomas m CD-I mice.11 Concurrent unwanted exposure to vinyl chloride cannot be ruled out in this study* no effects m CD-I mice* Mouse: by ingestion (stomach tube); limited evidence of increased incidence of lymphomas and leu kemias in female B6C3F1 mice" Mouse: by skin painting skin tumor initiator on Ha:lCR Swiss mice" <c) Hamster: by inhalation no effects on Chinese hamsters1-* (B) Humans: occupational exposure no evidence of an increased number of malignant tumors.1114 These studies cannot be considered conclusive (2) Genetic Effects (A) Bacterial systems: mutagen after metabolic activation (B) Other systems: increase in the chromosome aberration rate in Chinese hamsters, following inhalation and ingestion exposure" related. Vinylidene chloride was also shown to be an initiator of skin carcino genesis. (3) in Chinese hamsters no carcinogenic effects of the monomer were found. The reported results in rats do not conclusively prove that vinylidene chloride is carcinogenic in this animal species. Therefore, until the results of the experi ment herewith reported were obtained, the evidence for carcinogenicity of vinyli dene chloride was almost exclusively drawn from the data of the bioassays in mice. SL 062848 MY OF SCIENCES her Relevant n female Sprague- i CD rats.7* Conout in this study1 :r and meningeal and increased ignam tumors in ng adenomas in ; cannot be ruled nphomas and leu- Fhese studies rs. following of skin carcinomer were found, nylidene chloride Its of the experitenicity of vinylithe bioassays in COTT1 et at.: VINYLIDENE CHLORIDE 163 LONG-TERM CARCINOGENICITY BIOASSAY PERFORMED AT THE BENT1VOGLIO (BT) EXPERIMENTAL UNIT OF THE BOLOGNA INSTITUTE OF ONCOLOGY Materials, Methods, Plan, and Conduct of the Experiment Vinylidene chloride was tested in Sprague-Dawley rats by inhalation. Details on the test material and test animals are given in Table 5. The chambers for inhalation exposure were made of statnless steel, with two glass doors, and they measure 135 x 98 x 65 cm. The volume of each chamber is 860 liters. Continuous air flow provided 12-15 ajr changes per hour. Before its introduction the air was filtered, and the chamber arrangement was such that air flowed from one part of the chamber to the other without recirculation. The internal pressure was about 1 mm Hg less than that of the room where the chambers were situated to avoid any possible contamination of the outside envi ronment. The concentrations in air were checked by continuous gas chromato graphic monitoring. The plan of the experiment is shown in Table 6. while details on the conduct of the experiment are presented in Table 7 Systematic and standardized histopathologic examinations were performed in each animal on the subcutaneous lymph nodes, brain and cerebellum, Zymbal glands, interscapular brown fat, salivary glands. Harderian glands, tongue, thy mus and mediastinal lymph nodes, lungs, diaphragm, liver, kidneys, adrenals, spleen, pancreas, esophagus, mesenteric lymph nodes, stomach, various seg ments of the intestine, bladder, uterus, gonads, sternum, and any other organ with pathologic lesions. Results Survival. The treatment with vinylidene chloride did not affect the survival rate of any of the exposed groups. tables. Vinylidene Chloride: Test Compound and Test Animals Test Compound (A) Supplier; Solvay, Belgium (B) Purity: Vinylidene chloride 999.43 g/kg 1,2-dichloroethy lene acetone methylene chloride mono- and dichloroethylene paramethoxyphenol (as stabilizer) Test Animals 0.40 g/kg 0.10 g/kg 0.05 g/kg 0.02 g/kg 200 ppm Male and female Sprague-Dawley rats, breeders 13 weeks old and embryos (12th day of gestauon) at the start of experiment. The animals were of the breed cunenUy used in the BT Experimental Unit for more than 15 years. SL 062849 164 ANNALS NEW YORK ACADEMY OF SCIENCES TABLE t. Vmyltdene Chloride: Plan of the Experiment and Inhalation Schedule in Sprague-Dawley Rats (Exp. BT 4002, 4006) Xmmals Croup No. 1 11 1 11 III Total Concentration (ppmi 100 0 (Control) 100 100 0 (Control) Schedule 4 hd/5 dw/7 wa and then 7 hd/J dw/97 w* -- 4 hd/5 dwj7 w and then 7 hd/5 dw 97 w 4 hd/5 dw/7 w and then 7 hd/5 dw/g w' ___ No. at Age Sex Stan Breeders F 54 Breeders Embryos Embryos Embrvos F M F M*F M F M-F M F M*F M F M-F 60 62 61 123 60 60 120 158 149 307 280 384 664 * Four hours a day for 5 days a week for 7 weeks. * Seven hours a day for 5 days a week for 97 weeks. f Seven hours a day for 5 days a week for 8 weeks. Body weight. In breeders and m male and female offspring treated with vinyli* dene chloride for 104 weeks, a slight decrease in body weight was observed. In offspring treated for 15 weeks, a slight body weight decrease was evident in male but not in female animals. Carcinogenicity. In rats of the strain used, the tumors most frequently ex pected on the basis of the literature and of the historical controls of the BT Experimental Unit, are mammary tumors tbenign and malignant), leukemias, pheochromocytomas and pheochromoblastomas. Moreover, a variety of other miscellaneous tumors are also observed.1617 Table 7. Vinylidene Chloride: Conduct of the Experiment The animals were exposed by: Inhalation, 4-7 hours daily. 5 days weekly, for 104 weeks (breeders) Transplacental route and then inhalation. 4-7 hours daily. 5 days weekly, for 104 weeks or 15 weeks (12-day embryos! All the animals were kept under observation unnl spontaneous death, The status and behavior of the animals were examined Y times daily. The animals were submitted to clinical examination for gross changes every 2 weeks. The animals were weighed every 2 weeks during treaimem and then every 8 weeks. Full necropsy and histopathologic cxaminanon were performed on all the animals. The housing and the diet of the animals were (he same highly standardized ones adopted in the BT Experimental Unit during the last 15 years IENCES rS d * r fi ft S1!! rDt" 551 S fxt 5 3ft <. I-Io- 3< S S 4k U CF U > ^ {Co^<9COOOwii 3* Z) { >0 4a E ^ COTTI i t a/.: VINYLIDENE CHLORIDE TABLE*. Vinylidene Chloride: Tola) Tumors in Rais (Exp. BT 4002, 4006) Group No 1 II 1 II ill Concentrat ion (ppm! 100 0 (Control) 100 100 0 (Coni roll Schedule 4 hd/5 dw/7 w' and then 7 lull 5 dw/97 w4 -- 4 hd/5 dw/7 w and Ihen 7 hd/5 dw/97 w 4 hd/5 dw/7 w and Ihen 7 hd/5 dw/8 w' ___ * Tolal benign and malignant tumors. * Malignant tumors. * Four hours a day for 5 days a week for 7 weeks. 4 Seven hours a day for 3 days a week for 97 weeks. * Seven hours a day for I days a week for 8 weeks. Animals Age Breeders Sen F No at Sian 54 Breeders Embryos Embryos Embryos I- M 1 M+F M F Mr F M F M+E 60 67 61 123 60 60 120 158 149 307 I ^ Percentage of Animals Beanng Tumors TUMI" Ml* 68 5 20 4 No of Malignant Tumors per 100 Animals 22 2 58 3 15 0 62 9 32 2 67 1 295 65.0 30.9 58 3 21 7 78 3 23 3 68 3 22 5 44 9 17.1 78.5 17 4 61 2 17 3 16 7 11 9 14 4 34 1 23 3 23 3 23 3 18 3 17 4 17 9 SL 062851 ANNALS NEW YORK ACADEM Y OF SCIENCES table*. Vinylidene Chloride: Mammary Tumors, Leukemias, Pheochromocytomas and Pheochromoblaslomas in Rats 4006) Percenlage of Animals Bearing Tumors Group No. j II | 11 III Concentration (ppm) 100 0 (Control) 100 100 0 (Control) Schedule 4 hd/5 dw/7 w* and then 7 hd/5 dw/97 w' -- 4 hd/5 dw/ 7w and then 7 hd/5 dw/97 w 4 hd/5 dw/7 w and then 7 hd/5 dw/8 w/ Animals Age Breeders Sex F No. at Start 54 Breeden F Embryos Embryos Embryos M F MF M F M+F M F M+ F 60 62 61 123 60 60 120 138 149 307 Mammary Tumors BMT* MT* 53.7 7.4 400 3 3 11.3 -- 45.9 4.9 28.4 2.4 50 -- 53.3 10.0 29.2 5.0 70 19 56.4 5.4 30.9 3.6 Lcukemias' 18 Phcochromocylomas 7.4 Pheochromoblastemas I8 3 3 18 3 16.1 306 6.5 4.9 11.4 17.9 13 3 28 3 3 3 117 B 3 20.0 7.6 22 B 0.7 188 4 2 20 8 -- -- -- -- 17 -- 08 0.6 0.7 0.6 * Benign and malignant tumors. * Malignant tumors. * The term "leukemia" includes a variety of hemolymphoreticular neoplastic diseases at different sites * Four hours a day for S days a week for 7 weeks. ' Seven hours a day for 3 days a week for 97 weeks. f Seven hours a day for 3 days a week for 8 weeks. SL 062852 C01T1 tt ml.: VINYLIDENE CHLORIDE 167 An increased percentage of rats bearing malignant tumors and an increased number of malignant tumors per 100 animals were found in offspring of both sexes treated with vmylidene chloride for 104 weeks There was only limited evidence of a borderline increase in total malignant tumors in the other exposed groups, i.e., offspring treated for 15 weeks and breeders treated for 104 weeks (Table 8l An increased incidence of '`leukemias" was found in the vinylidene-chlortdeexposed offspring; this increase was related to the length of treatment (Table 9i. No relevant differences were observed in the incidence of other tumors fre quently occurring in untreated rats of the BTcolony (Table 9). No unexpected tumors related to treatment were found. CONCLUSIONS These results demonstrate that under particular experimental conditions ti e., prolonged exposure started during embryonal development, when responsiveness to carcinogenic agents is usually higher), vinylidene chloride has carcinogenic__ ,, effects on rats. too. These effects, in our bioassay, were shown by the increased incidence of malignant tumors and leukemias among the exposed animals. SUMMARY Vinylidene chloride was administered by inhalation. 7 hours daily. 5 days weekly, at the concentration of 100 and 0 ppm, to Sprague-Dawley rats. The treatment was started on 13-week-old breeders, and male and female offspring (12-day embryos). The breeders and some of the offspring were exposed for 104 weeks: the other offspring were exposed for 15 weeks only. An increased incidence was found of malignant tumors and of leukemias, particularly in offspring treated for 104 weeks. REFERENCES 1. International Agency for Research on Cancer (1ARC). 1979, Monographs on the Evaluation of Carcinogenic Risk of Chemicals lo Humans. Some Monomers. Plastics and Synthetic Elastomers, and Acrolein. 19: 439-459. Lyon. France. 2. Maltoni. C.. G. Lefemine, G. Com, P. ChiecoA V. Patella. 1985. Experimental Research on Vinylidene Chloride Carcinogenesis. Archives of Research on Indus trial Carcinogenesis, Vol. 111. Princeton Scientific Publishers. Pnnceton. Nl. 3. Maltoni, C.. G. Corn. L, Morisi A P. Chieco. 1977. Carcinogenicity bioassavs of vinylidene chloride. Research plan and early results. Med. Lavoro 68: 241-262 4. Maltoni, C., G. Com A P. Chieco. 1984. Chronic toxicity and carcinogenicity bioassays of vinylidene chloride. Acta Oncol. 5: 91-146. 5. Viola, P. L. A A. Cafuto. 1977. Carcinogenicity studies on vinylidene chloride. Environ. Health Perspect. 21: 45-47. 6. Ramey, L. W., J. F. Quast, C. G. Humiston. M. F. Balmer A B. A. Schetz. 1977 Interim results of two-year toxicological studies in rais of vinylidene chlonde incor porated in the dnnking water or administered by repeated inhalation. Environ. Health Perspect. 21: 33-43. SL 062853 168 ANNALS NEW YORK ACADEMY OF SCIENCES 7 Lee. C. C . J C Bhandari. J M. Winston, W, B House. P J, Peters. R. L. Dixon A J, S. Woods. 1977. Inhalation toxicity of vinyl chloride and vinylidene chloride. Environ Health Perspect. 21: 25-32. 8. Lee. C C., J. C Bhandari. J. M. Winston, W B House. R. L. Dixon A J. S. Woods. 1978 Carcinogenicity of vinyl chloride and vinylidene chloride. J. Toxicol. Environ. Health 4: 15-30. 9. Hong. C. B., J. M Winston, w. b. House. A. M. El-Hawari, R. D. Short, Jr.. E R. Ellis, E. Castillo. J. H. Hagensen. K. J. Smith A C. C. Lee. 1979. Additional evaluation of the environmental toxicants vinyl chloride (VC) and vinyli dene chloride (VDC): I) Exposure of VC or VDC followed by observation for 12 months in rats and mice. 2) the effect of disulhram (DS) on VC exposure in mice. Contract No. NO1-ES-2-2084 (Continuation of NIH-N1EHS-72-2-2084), Final Re port. Report with limited circulation. 10. Ponomarkov, V. A L. Tomatis. 1980. Long-term testing of vinylidene chloride and chioroprene for carcinogenesis in rats. Oncology 37: 136-141. 11. National Toxicological Program. 1982. NTPTechnical Report on the carcinogen esis bioassay of vinylidene chloride (CAS No. 75-35-4), in F344/N rats and B6C3FI/N mice (gavage studv). NTP-80-82, NIH Publicauon No. 82-1784. 12. Van Duuren. B . B. Goldschmidt. G. Loewengart, A Smith. S. MelChionne, 1. Seidman A D. Roth. 1979. Carcinogenicity of halogenated olehnic and aliphatic hydrocarbons in mice. J. Natl. Cancer Inst. 63: 1433-1438. 13. Orr. M G , W. a. Fishbeck. J. C. Townsend A E. J. Schneider. 1976. A health study of employees exposed to vinylidene chloride. J. Occup. Med. 18: 735-738. 14. Thiess. A. M . R. Frentzel-Beyme A E. Penning. 1979. Mortality study of vinyli dene chloride exposed persons in the BASF. In Proceedings of the Vth Medichem Congress, San Francisco. California. 15. Badische Anilin A Soda Fabrik (BASF). 1976. Unpublished data. 16. Maltoni, C., G. Lefemine. A. Ciliberti, G. Com A D. Carretti. 1984. Experi mental Research on Vinyl Chloride Carcinogenesis. Archives of Research on Indus trial Carcinogenesis. Vol. 11. Princeton Scientific Publishers. Princeton. NJ. 17. Maltoni. C., G. Lefemine. G. Com. D. Carretti, F. Vicini. A. Ciliberti. B. Conti. F. Minardi A G. Perjno. Long-term Carcinogenicity Bioassays. Role in Cancer Control. Biological Basis. Experimental Factors and Interpretation of Results. Archives of Research on Industrial Carcinogenesis, Vol. I. Princeton Scien tific Publishers. Pnnceion. NJ. In press. SL 062854