NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF DIVINYLBENZENE-HP (CAS NO. 1321-74-0) IN F344/N RATS AND B6C3F1 MICE (INHALATION STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 November 2006 NTP TR 534 NIH Publication No. 07-4470 National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES FOREWORD The National Toxicology Program (NTP) is an interagency program within the Public Health Service (PHS) of the Department of Health and Human Services (HHS) and is headquartered at the National Institute of Environmental Health Sciences of the National Institutes of Health (NIEHS/NIH). Three agencies contribute resources to the program: NIEHS/NIH, the National Institute for Occupational Safety and Health of the Centers for Disease Control and Prevention (NIOSH/CDC), and the National Center for Toxicological Research of the Food and Drug Administration (NCTR/FDA). Established in 1978, the NTP is charged with coordinating toxicological testing activities, strengthening the science base in toxicology, developing and validating improved testing methods, and providing information about potentially toxic substances to health regulatory and research agencies, scientific and medical communities, and the public. The Technical Report series began in 1976 with carcinogenesis studies conducted by the National Cancer Institute. In 1981, this bioassay program was transferred to the NTP. The studies described in the Technical Report series are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected substances in laboratory animals (usually two species, rats and mice). Substances selected for NTP toxicity and carcinogenicity studies are chosen primarily on the basis of human exposure, level of production, and chemical structure. The interpretive conclusions presented in NTP Technical Reports are based only on the results of these NTP studies. Extrapolation of these results to other species, including characterization of hazards and risks to humans, requires analyses beyond the intent of these reports. Selection per se is not an indicator of a substance’s carcinogenic potential. The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public review. NTP Technical Reports are indexed in the NIH/NLM PubMed database and are available free of charge electronically on the NTP website (http://ntp.niehs.nih.gov) or in hardcopy upon request from the NTP Central Data Management group at [email protected] or (919) 541-3419. NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF DIVINYLBENZENE-HP (CAS NO. 1321-74-0) IN F344/N RATS AND B6C3F1 MICE (INHALATION STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 November 2006 NTP TR 534 NIH Publication No. 07-4470 National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 2 CONTRIBUTORS National Toxicology Program NTP Pathology Working Group Evaluated and interpreted results and reported findings Evaluated slides and prepared pathology report on rats (July 17, 2003) D.L. Morgan, Ph.D., Study Scientist W.G. Lieuallen, D.V.M., Ph.D., Chairperson G. Pearse, B.V.M.&S., Study Pathologist Pathology Associates, A Charles River Company D.W. Bristol, Ph.D. M.T. Butt, D.V.M. J.R. Bucher, Ph.D. Pathology Associates, A Charles River Company L.T. Burka, Ph.D. J.T. Boyce, D.V.M., Ph.D., Observer R.S. Chhabra, Ph.D. Pathology Associates, A Charles River Company J.R. Hailey, D.V.M. S. Harbo, D.V.M., Observer R.A. Herbert, D.V.M., Ph.D. Battelle Northwest Operations G.E. Kissling, Ph.D. R.A. Herbert, D.V.M., Ph.D. D.E. Malarkey, D.V.M., Ph.D. National Toxicology Program R.R. Maronpot, D.V.M. P.C. Howroyd, M.A., VetMB Experimental Pathology Laboratories, Inc. S.D. Peddada, Ph.D. D.E. Malarkey, D.V.M., Ph.D. J.H. Roycroft, Ph.D. National Toxicology Program C.S. Smith, Ph.D. G. Pearse, B.V.M.&S. G.S. Travlos, D.V.M. National Toxicology Program K.L. Witt, M.S. J.C. Peckham, D.V.M., M.S., Ph.D. Experimental Pathology Laboratories, Inc. Battelle Northwest Operations R.C. Sills, D.V.M., Ph.D. Conducted studies and evaluated pathology findings National Toxicology Program J.A. Dill, Ph.D., Principal Investigator (2-year studies) Evaluated slides and prepared pathology report on mice (March 6, 2003) S.L. Grumbein, D.V.M., Ph.D. R.A. Renne, D.V.M. W.G. Lieuallen, D.V.M., Ph.D., Chairperson B.K. Hayden Pathology Associates, A Charles River Company M.T. Butt, D.V.M. Experimental Pathology Laboratories, Inc. Pathology Associates, A Charles River Company Provided pathology review D. Dixon, D.V.M., Ph.D. National Toxicology Program M.H. Hamlin, II, D.V.M., Principal Investigator R.A. Herbert, D.V.M., Ph.D. P.C. Howroyd, M.A., VetMB National Toxicology Program J.C. Peckham, D.V.M., M.S., Ph.D. G. Pearse, B.V.M.&S. National Toxicology Program Dynamac Corporation J.C. Peckham, D.V.M., M.S., Ph.D. Experimental Pathology Laboratories, Inc. Prepared quality assurance audits Biotechnical Services, Inc. S. Brecher, Ph.D., Principal Investigator Prepared Technical Report Constella Group, Inc. S.R. Gunnels, M.A., Principal Investigator Provided statistical analyses N.N. Buchanan, B.S. L.M. Harper, B.S. P.W. Crockett, Principal Investigator J.I. Powers, M.A.P. L.J. Betz, M.S. D.C. Serbus, Ph.D. M.R. Easterling, Ph.D. K.P. McGowan, M.B.A. J. Matthews, M.S. 3 CONTENTS ABSTRACT . 5 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . 9 TECHNICAL REPORTS REVIEW SUBCOMMITTEE . 10 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . 11 INTRODUCTION . 13 MATERIALS AND METHODS . 19 RESULTS . 31 DISCUSSION AND CONCLUSIONS . 65 REFERENCES . 69 APPENDIX A Summary of Lesions in Male Rats in the 2-Year Inhalation Study of Divinylbenzene-HP . 75 APPENDIX B Summary of Lesions in Female Rats in the 2-Year Inhalation Study of Divinylbenzene-HP . 117 APPENDIX C Summary of Lesions in Male Mice in the 2-Year Inhalation Study of Divinylbenzene-HP . 155 APPENDIX D Summary of Lesions in Female Mice in the 2-Year Inhalation Study of Divinylbenzene-HP . 189 APPENDIX E Genetic Toxicology . 225 APPENDIX F Clinical Pathology Results . 233 APPENDIX G Organ Weights and Organ-Weight-to-Body-Weight Ratios . 241 APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization . 247 APPENDIX I Chemical Characterization and Generation of Chamber Concentrations . 251 APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration . 263 APPENDIX K Sentinel Animal Program . 267 APPENDIX L Physiologically Based Pharmacokinetic Model . 271 4 Divinylbenzene-HP, NTP TR 534 SUMMARY Background Divinylbenzene is used to make vinyl polymers. We studied divinylbenzene to determine if it caused cancer in rats or mice. Methods We exposed groups of 50 male and 50 female rats and mice to air containing divinylbenzene 6 hours per day for 2 years. Rats were exposed to concentrations of 100, 200, or 400 parts per million (ppm) of divinylbenzene in air, and mice were exposed to concentrations of 10, 30, or 100 ppm. Similar groups of 50 animals were exposed to clean air in the same inhalation chambers 6 hours per day as the untreated control groups. Tissues from more than 40 sites were examined for every animal. Results Each of the groups exposed to the highest concentration of divinylbenzene weighed less than their control group. A few rare tumors of the kidney and brain were seen in exposed male rats, and the rate of lung tumors was slightly increased in exposed female mice. Male and female rats exposed to divinylbenzene had degeneration and hyperplasia of the epithelium of the nose. Exposed male and female mice had hyperplasia of the lung and inflammation, degeneration, and hyperplasia of the epithelium of the nose. Conclusions We conclude that the occurrence of carcinomas of the kidney and glial cell tumors of the brain in male rats and adenomas or carcinomas of the lung in female mice may have been related to exposure to divinylbenzene. 5 ABSTRACT H2C HC CH CH2 DIVINYLBENZENE-HP CAS No. 1321-74-0 Chemical Formula: C10H10 Molecular Weight: 130.189 Synonyms: Benzene, diethenyl-(9CI); diethenylbenzene; divinyl benzene; divinylbenzene-HP (high purity); divinylbenzene (m- and p-mixture); divinylbenzene (m-, p-mixture); divinyl benzene, mixed isomers; DVB; DVB-HP; m- (or p-) divinylbenzene; vinylstyrene Divinylbenzene-HP is used for producing vinyl 2-WEEK STUDY IN MICE polymers. Divinylbenzene-HP was nominated for study Groups of five male and five female mice were exposed by the National Cancer Institute because of the potential by whole body inhalation to divinylbenzene-HP at target for worker exposure and the structural similarity of concentrations of 0, 25, 50, 100, 200, or 400 ppm for divinylbenzene to styrene, a potential human carcinogen. 6 hours plus T (12 minutes) per day, 5 days per week Male and female F344/N rats and B6C3F mice were 90 1 for 17 days. All 400 ppm males and females died on or exposed to divinylbenzene-HP (80%) by inhalation for before the second day of the study, and two male and two 2 weeks, 3 months, or 2 years. Genetic toxicology stud­ female 200 ppm mice died early. Mean body weights of ies were conducted in Salmonella typhimurium, Escher­ 100 and 200 ppm males were significantly less than ichia coli, and mouse peripheral blood erythrocytes.