Document 3Qge5rzywMj2VkLLzmoDxwxbn

NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF CUMENE (CAS NO. 98-82-8) IN F344/N RATS AND B6C3F1 MICE (INHALATION STUDIES) Scheduled Peer Review Date: May 16-17, 2007 NOTICE This DRAFT Technical Report is distributed solely for the purpose of predissemination peer review under the applicable information quality guidelines. It has not been formally disseminated by the NTP. It does not represent and should not be construed to represent NTP determination or policy. NTP TR 542 NIH Publication No. 07-5885 National Toxicology Program 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 cdm@niehs.nih.gov or (919) 541-3419. NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF CUMENE (CAS NO. 98-82-8) IN F344/N RATS AND B6C3F1 MICE (INHALATION STUDIES) Scheduled Peer Review Date: May 16-17, 2007 NOTICE This DRAFT Technical Report is distributed solely for the purpose of predissemination peer review under the applicable information quality guidelines. It has not been formally disseminated by the NTP. It does not represent and should not be construed to represent NTP determination or policy. NTP TR 542 NIH Publication No. 07-5885 National Toxicology Program National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 2 CONTRIBUTORS National Toxicology Program Evaluated and interpreted results and reported findings P.C. Chan, Ph.D., Study Scientist J.C. Peckham, D.V.M., M.S., Ph.D., Study Pathologist D.W. Bristol, Ph.D. J.R. Bucher, Ph.D. L.T. Burka, Ph.D. R.S. Chhabra, Ph.D. R.A. Herbert, D.V.M., Ph.D. A.P. King-Herbert, D.V.M. G.E. Kissling, Ph.D. D.E. Malarkey, D.V.M., Ph.D. S.D. Peddada, Ph.D. J.H. Roycroft, Ph.D. C.S. Smith, Ph.D. G.S. Travlos, D.V.M. K.L. Witt, M.S. Battelle Toxicology Northwest Conducted studies and evaluated pathology findings J.A. Dill, Ph.D., Principal Investigator S.L. Grumbein, D.V.M., Ph.D. B.K. Hayden R.A. Renne, D.V.M. Experimental Pathology Laboratories, Inc. Provided pathology review M.H. Hamlin, II, D.V.M., Principal Investigator K.J. Cimon, D.V.M., M.S. J.C. Peckham, D.V.M., M.S., Ph.D. Dynamac Corporation Prepared quality assurance audits S. Brecher, Ph.D., Principal Investigator NTP Pathology Working Group Evaluated slides and prepared pathology report on rats (March 3, 2005) A.W. Suttie, B.V.Sc., Ph.D., Chairperson ILS, Inc. M.F. Cesta, D.V.M. National Toxicology Program P.C. Chan, Ph.D. National Toxicology Program K.J. Cimon, D.V.M., M.S. Experimental Pathology Laboratories, Inc. E. Dick, D.V.M., Observer U.S. Army Institute of Surgical Research S.A. Elmore, D.V.M. ILS, Inc. G.C. Hard, D.V.M., Ph.D. National Toxicology Program R.A. Herbert, D.V.M., Ph.D. National Toxicology Program P.B. Little, D.V.M. Pathology Associates, A Charles River Company D.E. Malarkey, D.V.M., Ph.D. National Toxicology Program G. Pearse, B.V.M. & S. National Toxicology Program J.C. Peckham, D.V.M., M.S., Ph.D. Experimental Pathology Laboratories, Inc. R.C. Sills, D.V.M., Ph.D. National Toxicology Program NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 NTP Pathology Working Group Evaluated slides and prepared pathology report on mice (January 27, 2005) M.A. Hanes, D.V.M., Chairperson ILS, Inc. M.F. Cesta, D.V.M. National Toxicology Program K.J. Cimon, D.V.M., M.S. Experimental Pathology Laboratories, Inc. S.A. Elmore, D.V.M., M.S., Ph.D. ILS, Inc. G.P. Flake, M.D. National Toxicology Program R.A. Herbert, D.V.M., Ph.D. National Toxicology Program P.B. Little, D.V.M. Pathology Associates, A Charles River Company D.E. Malarkey, D.V.M., Ph.D. National Toxicology Program G. Pearse, B.V.M. & S. National Toxicology Program J.C. Peckham, D.V.M., M.S., Ph.D. Experimental Pathology Laboratories, Inc. R.C. Sills, D.V.M., Ph.D. National Toxicology Program K. Yoshizawa, D.V.M., Ph.D. National Toxicology Program Constella Group, Inc. Provided statistical analyses P.W. Crockett, Ph.D., Principal Investigator L.J. Betz, M.S. K.P. McGowan, M.B.A. Biotechnical Services, Inc. Prepared Technical Report S.R. Gunnels, M.A., Principal Investigator L.M. Harper, B.S. D.C. Serbus, Ph.D. G.E. Simmons, M.A. 3 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 4 CONTENTS ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . . . . . . . . . . . . . . . . 15 TECHNICAL REPORTS REVIEW SUBCOMMITTEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . . . . . . . . . . . . 17 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 APPENDIX A Summary of Lesions in Male Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1 APPENDIX B Summary of Lesions in Female Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1 APPENDIX C Summary of Lesions in Male Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1 APPENDIX D Summary of Lesions in Female Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1 APPENDIX E Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1 APPENDIX F Clinical Pathology Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1 APPENDIX G Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . . . . . . . . . . . G-1 APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization . . . . . . . . . . . . . . H-1 APPENDIX I Chemical Characterization and Generation of Chamber Concentrations . . . . . . . . . . . . I-1 APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-1 APPENDIX K Sentinel Animal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-1 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 5 APPENDIX L Characterization of K-ras and p53 Mutations in Lung Neoplasms of Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L-1 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 6 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft ABSTRACT H H3C C CH3 7 CUMENE CAS No. 98-82-8 Chemical Formula: C9H12 Molecular Weight: 120.19 Synonyms: Cumol; isopropylbenzene; isopropylbenzol; (1-methyl/ethyl)benzene; 2-phenylpropane Cumene is produced in a modified Friedel-Crafts reaction process that uses acidic catalysts to alkylate benzene with propylene. Cumene is the principal chemical used in the production of phenol and acetone. Cumene is used to produce styrene, acetophenone, "-methylstyrene, diisopropylbenzene, and dicumylperoxide; as a thinner; as a constituent of some petroleum-based solvents; in gasoline blending, diesel fuel, and high-octane aviation fuel; and as a raw material for peroxides and oxidation catalysts. Because cumene is a good solvent for fats and resins, it has been suggested as a replacement for benzene in many industrial applications. Cumene occurs naturally in petroleum and in a variety of foodstuffs. Cumene was nominated for study by the NIEHS because of its high production volume, presence in gasoline and other fuels, potential for human exposure, and lack of existing carcinogenicity test data. Male and female F344/N rats and B6C3F1 mice were exposed to cumene (greater than 99.9% pure) by inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat bone marrow, and mouse peripheral blood. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 8 Cumene, NTP TR 542 2-WEEK STUDY IN RATS Groups of five male and five female rats were exposed to cumene vapor at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 days. All rats exposed to 4,000 ppm died on day 1, and two male and three female rats exposed to 2,000 ppm died by day 4. Mean body weights of 2,000 ppm rats were significantly less than those of the chamber controls. Rats exposed to 2,000 ppm that died early were severely lethargic following daily exposure. Liver and kidney weights of all exposed groups were increased. Accumulation of minimal to mild hyaline droplets was observed in the renal tubular cortex of males exposed to concentrations of 250 to 2,000 ppm. 2-WEEK STUDY IN MICE Groups of five male and five female mice were exposed to cumene vapor at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 17 days. All mice exposed to 4,000 ppm died on day 1; all mice exposed to 2,000 ppm died on day 2, and four female mice exposed to 1,000 ppm died by day 4. Mean body weights of all exposed groups were similar to those of the chamber controls. Mice exposed to 2,000 ppm were severely lethargic after the first exposure. The four female mice exposed to 1,000 ppm that died early exhibited signs of lethargy and ataxia. Liver weights, both relative and absolute, were increased in all groups of surviving males and in 250 and 500 ppm female groups. 3-MONTH STUDY IN RATS Groups of 10 male and 10 female rats were exposed to cumene vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Kidney and liver weights of 250 ppm or greater males and liver weights of 1,000 ppm females were significantly greater than those of the chamber controls. There were significant differences between exposed and chamber NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 9 control females in the relative length of time spent in the estrous stages. The amount of "2u-globulin in the right kidneys was significantly increased in male rats exposed to 125 ppm or greater. The incidences of medullary granular casts in males exposed to 250 ppm or greater were significantly increased. The severities of renal tubule cortex hyaline droplet accumulation and regeneration increased with increasing exposure concentration in male rats. 3-MONTH STUDY IN MICE Groups of 10 male and 10 female mice were exposed to cumene vapor at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. Eight 1,000 ppm females died during week 1 of the study. Mean body weights of males exposed to 500 or 1,000 ppm were significantly less than those of the chamber controls. The eight 1,000 ppm female mice that died during the first week of the study exhibited clinical signs of acute toxicity, including lethargy or ataxia. Liver weights of mice exposed to 500 or 1,000 ppm were significantly increased. The weight of the cauda epididymis and the spermatid count were significantly decreased in 1,000 ppm males. 2-YEAR STUDY IN RATS Groups of 50 male and 50 female rats were exposed to cumene vapor at concentrations of 0, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Survival of all exposed groups of rats was similar to that of the chamber controls. Mean body weights of 1,000 ppm females were slightly less than those of the chamber controls during the second year of the study but were similar to the chamber controls at the end of the study. Incidences of adenoma of the respiratory epithelium in the nose occurred with a positive trend in males and were significantly increased in all exposed groups of males and in 250 ppm females. Incidences of hyperplasia of basal Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 10 Cumene, NTP TR 542 cells in the olfactory epithelium in the nose of all exposed groups and hyperplasia of the respiratory epithelium in the nose of all exposed groups of males and 1,000 ppm females were significantly increased. The incidences of renal tubule adenoma in all exposed groups of males, renal tubule carcinoma in 500 and 1,000 ppm males, and renal tubule adenoma or carcinoma (combined) in all exposed groups of males were increased; the difference from chamber controls for the combined incidence was significant at 500 ppm. The incidences of hyperplasia of the renal tubule and transitional epithelium of the renal pelvis in 500 and 1,000 ppm males and mineralization of the renal papilla in all exposed groups of males were significantly greater than those of the chamber controls. 2-YEAR STUDY IN MICE Groups of 50 male and 50 female mice were exposed to cumene vapor at concentrations of 0, 125 (female mice only), 250, 500, or 1,000 (male mice only) ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. An exposure concentration-related decrease in survival occurred in male mice, and the survival of 1,000 ppm males was significantly less than that of the chamber controls. Mean body weights of 1,000 ppm males were generally less than those of the chamber controls after week 8 of the study, and those of 500 ppm females were less from week 28 until week 76 of the study. The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) in all exposed groups of mice occurred with positive trends and were significantly greater than those in the chamber controls. The incidences of alveolar epithelial bronchiole metaplasia and bronchiole hyperplasia were significantly increased in all exposed groups of mice. p53 and K-ras mutations were found in 52% and 87% of lung neoplasms in exposed mice compared to 0% and 14% in the chamber controls, respectively. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 11 In female mice, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) occurred with positive trends and were significantly increased in the 500 ppm group. In male mice, there were significant increases in the incidences of eosinophilic foci of the liver. In the nose, the incidences of olfactory epithelium atrophy, basal cell hyperplasia of the olfactory epithelium, atypical hyperplasia of the olfactory epithelium, hyperplasia of olfactory epithelium glands, and suppurative inflammation were generally significantly increased in 500 and 1,000 ppm males and 500 ppm females. The incidences of squamous metaplasia of the respiratory epithelium were significantly increased in 500 ppm females. The incidence of basal cell hyperplasia was also significantly increased in 250 ppm females. The incidences of epithelial hyperplasia of the forestomach in the 500 and 1,000 ppm groups of males and the incidences of ulceration and inflammation of the forestomach in 1,000 ppm males were significantly increased. GENETIC TOXICOLOGY Cumene was not mutagenic in S. typhimurium strain TA97, TA98, TA100, or TA1535, when tested with and without liver S9 activation enzymes. Cumene induced small but significant increases in micronucleated polychromatic erythrocytes in bone marrow of male rats treated by intraperitoneal injection. In contrast, no increase in micronucleated erythrocytes was observed in peripheral blood of male or female mice exposed to cumene by inhalation for 3 months. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 12 Cumene, NTP TR 542 CONCLUSIONS Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity* of cumene in male F344/N rats based on increased incidences of respiratory epithelial adenoma in the nose and renal tubule adenoma or carcinoma (combined). There was some evidence of carcinogenic activity of cumene in female F344/N rats based on the incidences of respiratory epithelium adenoma in the nose. There was clear evidence of carcinogenic activity of cumene in male B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. There was clear evidence of carcinogenic activity of cumene in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Increased incidences of hepatocullar adenoma or carcinoma (combined) in female mice were also considered to be related to exposure to cumene. Exposure to cumene resulted in nonneoplastic lesions in the nose and kidney of male rats; the nose of female rats; the lung, nose, liver, and forestomach of male mice; and the lung and nose of female mice. __________ * Explanation of Levels of Evidence of Carcinogenic Activity is on page 15. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 13 Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Cumene Male F344/N Rats Female F344/N Rats Male B6C3F1 Mice Female B6C3F1 Mice Concentrations in air Body weights Survival rates Nonneoplastic effects 0, 250, 500, or 1,000 ppm 0, 250, 500, or 1,000 ppm 0, 250, 500, or 1,000 ppm 0, 125, 250, or 500 ppm Exposed groups similar to Exposed groups similar to 1,000 ppm males less than Exposed groups similar to the chamber control group the chamber control group the chamber control group the chamber control group 26/50, 23/50, 27/50, 24/50 21/50, 27/50, 31/50, 32/50 38/50, 34/50, 30/50, 23/50 37/50, 36/50, 39/50, 35/50 Nose: olfactory epithelium, hyperplasia, basal cell (0/50, 19/50, 27/49, 26/50); respiratory epithelium, hyperplasia (0/50, 15/50, 16/49, 23/50) Kidney: renal tubule, hyperplasia (0/50, 3/50, 8/50, 6/50); papilla, mineralization (5/50, 35/50, 44/50, 41/50); pelvis, transitional epithelium, hyperplasia (3/50, 5/50, 14/50, 15/50) Nose: olfactory epithelium, hyperplasia, basal cell (0/50, 14/48, 25/50, 31/50); respiratory epithelium, hyperplasia (0/50, 0/48, 4/50, 6/50) Lung: alveolar epithelium, bronchiole, metaplasia (5/50, 43/50, 42/50, 39/50); bronchiole, hyperplasia (0/50, 11/50, 17/50, 18/50) Lung: alveolar epithelium, bronchiole, metaplasia (0/50, 42/50, 49/50, 47/50); bronchiole, hyperplasia (0/50, 17/50, 10/50, 14/50) Nose: olfactory epithelium, atrophy (4/50, 13/50, 11/49, 38/48); olfactory epithelium, hyperplasia, basal cell (0/50, 0/50, 15/49, 33/48); olfactory epithelium, hyperplasia, atypical (0/50, 0/50, 5/49, 11/48); olfactory epithelium, glands, hyperplasia (3/50, 11/50, 9/49, 23/48); inflammation, suppurative (2/50, 2/50, 9/49, 6/48) Liver: eosinophilic focus (6/50, 5/50, 16/50, 14/50) Nose: olfactory epithelium, atrophy (4/50, 11/50, 9/50, 18/50); olfactory epithelium, hyperplasia, basal cell (0/50, 1/50, 11/50, 25/50); olfactory epithelium, hyperplasia, atypical (0/50, 0/50, 2/50, 10/50); olfactory epithelium, glands, hyperplasia (1/50, 4/50, 4/50, 11/50); respiratory epithelium, metaplasia, squamous (0/50, 0/50, 1/50, 6/50); inflammation, suppurative (0/50, 1/50, 3/50, 7/50) Forestomach: epithelium, hyperplasia (2/50, 7/50, 8/50, 13/49); ulcer (1/50, 4/50, 6/50, 6/49); inflammation (0/50, 2/50, 1/50, 5/49) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 14 Cumene, NTP TR 542 Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Cumene Male F344/N Rats Female F344/N Rats Male B6C3F1 Mice Female B6C3F1 Mice Neoplastic effects Nose: respiratory epithelium, adenoma (0/50, 7/50, 18/49, 10/50) Nose: respiratory epithelium, adenoma (0/50, 5/48, 4/50, 3/50) Kidney: renal tubule, adenoma or carcinoma (2/50, 5/50, 8/50, 7/50) Lung: alveolar/bronchiolar adenoma (13/50, 31/50, 31/50, 29/50); alveolar/bronchiolar carcinoma (9/50, 19/50, 32/50, 33/50); alveolar/bronchiolar adenoma or carcinoma (19/50, 38/50, 42/50, 43/50) Lung: alveolar/bronchiolar adenoma (1/50, 26/50, 36/50, 38/50); alveolar/bronchiolar carcinoma (3/50, 16/50, 20/50, 34/50); alveolar/bronchiolar adenoma or carcinoma (4/50, 31/50, 42/50, 46/50) Level of evidence of carcinogenic activity Clear evidence Genetic toxicology Salmonella typhimurium gene mutations: Micronucleated erythrocytes Rat bone marrow in vivo: Mouse peripheral blood in vivo: Some evidence Clear evidence Liver: hepatocellular adenoma (18/50, 23/50, 27/50, 29/50); hepatocellular adenoma or carcinoma (25/50, 26/50, 29/50, 36/50) Clear evidence Negative in strains TA97, TA98, TA100, and TA1535, with and without S9 Positive Negative NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 15 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies. Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic activity. For each separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings. These categories refer to the strength of the experimental evidence and not to potency or mechanism. Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related (i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy. Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear evidence. Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of neoplasms that may be chemical related. No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in malignant or benign neoplasms. Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations, cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity. For studies showing multiple chemical-related neoplastic effects that if considered individually would be assigned to different levels of evidence categories, the following convention has been adopted to convey completely the study results. In a study with clear evidence of carcinogenic activity at some tissue sites, other responses that alone might be deemed some evidence are indicated as "were also related" to chemical exposure. In studies with clear or some evidence of carcinogenic activity, other responses that alone might be termed equivocal evidence are indicated as "may have been" related to chemical exposure. When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline between two adjacent levels. These considerations should include: adequacy of the experimental design and conduct; occurrence of common versus uncommon neoplasia; progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to assume that benign neoplasms of those types have the potential to become malignant; combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue; latency in tumor induction; multiplicity in site-specific neoplasia; metastases; supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same lesion in another sex or species); presence or absence of dose relationships; statistical significance of the observed tumor increase; concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm; survival-adjusted analyses and false positive or false negative concerns; structure-activity correlations; and in some cases, genetic toxicology. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 16 Cumene, NTP TR 542 NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS TECHNICAL REPORTS REVIEW SUBCOMMITTEE The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on cumene on May 16-17, 2007, are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP studies: to ascertain that all relevant literature data have been adequately cited and interpreted, to determine if the design and conditions of the NTP studies were appropriate, to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, to judge the significance of the experimental results by scientific criteria, and to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. Nancy Kerkvliet, Ph.D., Chairperson Department of Environmental and Molecular Toxicology Oregon State University Corvallis, OR Christopher Bradfield, Ph.D. McArdle Laboratory for Cancer Research University of Wisconsin Madison, WI Kenny Crump, Ph.D. Environ International Ruston, LA Prescott Deininger, Ph.D. Tulane University Medical Center New Orleans, LA Jon Mirsalis, Ph.D. SRI International Menlo Park, CA Harish Sikka, Ph.D. Environmental Toxicology and Chemistry Laboratory State University of New York College at Buffalo Buffalo, NY Keith Soper, Ph.D. Merck Research Laboratories West Point, PA Vernon Walker, D.V.M., Ph.D. Lovelace Respiratory Institute Albuquerque, NM Special Ad Hoc Reviewers Russell C. Cattley, V.M.D., Ph.D. Amgen Thousand Oaks, CA Raymond F. Novak, Ph.D. Institute of Environmental Health Sciences Wayne State University Detroit, MI Michael V. Pino, D.V.M., Ph.D. Drug Safety Evaluation Sanofi-aventis Bridgewater, NJ NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 17 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS NOTE: A summary of the Technical Reports Review Subcommittee's remarks will appear in a future draft of this report. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 18 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft INTRODUCTION H H3C C CH3 19 CUMENE CAS No. 98-82-8 Chemical Formula: C9H12 Molecular Weight: 120.19 Synonyms: Cumol; isopropylbenzene; isopropylbenzol; (1-methyl/ethyl)benzene; 2-phenylpropane CHEMICAL AND PHYSICAL PROPERTIES Cumene is a volatile, colorless liquid with a sharp, penetrating aromatic or gas-like odor (Merck Index, 1989; Cavender, 1994; NIOSH, 2005). Cumene has a boiling point of 152.4 C (Lide, 1995), a melting point of 96.0 C (Lide, 1995), and a vapor pressure of 3.2 mm Hg at 20 C (Verschueren, 1983). Cumene is insoluble in water but is miscible with acetone, benzene, and ethanol (Lide, 1995). Cumene forms cumene hydroperoxide upon prolonged exposure to the air (NIOSH, 2005). PRODUCTION, USE, AND HUMAN EXPOSURE Cumene as a pure chemical intermediate is produced in a modified Friedel-Crafts reaction process that uses acidic catalysts to alkylate benzene with propylene. The majority of cumene is manufactured with a solid phosphoric acid catalyst; the remainder is made with an aluminum chloride catalyst (Schulz et al., 1993). The annual production of cumene in the United States was 4.49 billion pounds in 1993 and 5.63 billion pounds in 1995 (Anonymous, 1995; Kirschner, 1996; HSDB, 2003). Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 20 Cumene, NTP TR 542 Cumene is the principal chemical used in the production of phenol and acetone via the chemical intermediate cumene hydroperoxide (hydroperoxide is cleaved to phenol and acetone in an acidic environment). Cumene is used to produce styrene, acetophenone, "-methylstyrene, diisopropylbenzene, and dicumylperoxide. Cumene can also be used as a thinner for paints, enamels, and lacquers; as a constituent of some petroleum-based solvents such as naphtha; in gasoline blending, diesel fuel, and high-octane aviation fuel; and as a raw material for peroxides and oxidation catalysts such as polymerization catalysts for acrylic and polyester-type resins. It is also a good solvent for fats and resins and, as such, has been suggested as a replacement for benzene in many industrial applications (Parmeggiani, 1983; Verschueren, 1983; Mannsville, 1985; Merck Index, 1989; ACGIH, 1993; Anonymous, 1993; Hawley's, 1993; Schulz et al., 1993; HSDB, 2003). Cumene occurs naturally in petroleum crudes and coal tar (Verschueren, 1983). It also occurs in a variety of natural substances, including essential oils from plants, marsh grasses, and a variety of foodstuffs. Trace quantities have been detected in papaya, sapodilla fruit, and Australian honey. Cumene has been detected but not quantified in fried chicken, tomatoes, Concord grapes, cooked rice, oat groats, baked potatoes, Beaufort cheese, fried bacon, dried legumes (beans, split peas, lentils), southern pea seeds, and Zinfandel wine (HSDB, 2003). The potential for worker exposure exists during production and processing from petroleum refining. General population exposure to cumene results from inhalation of air contaminated with cumene from evaporation of petroleum products and from cigarette smoke; additional exposure may result from ingestion of food (HSDB, 2003). Cumene is released into the environment as a result of cumene production, processing, and transport, petroleum refining and the evaporation and combustion of petroleum products, the transportation and distribution of motor fuel, and the use of a variety of products containing cumene. Cumene has been detected in air samples from Los Angeles, CA, at concentrations as high as 144 g/m3 and in groundwater, surface water, and drinking water (Jackson, 1985; USEPA, 1997; HSDB, 2003). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 21 The threshold-limit value-time-weighted average recommended by the American Conference of Governmental Industrial Hygienists (2005) for cumene is 50 ppm (246 mg/m3); a short-term exposure limit (STEL) has not been determined. The Occupational Safety and Health Administration permissible exposure limit is 50 ppm (245 mg/m3), with a skin designation, averaged over an 8-hour work shift; a STEL has not been determined (29 CFR, Part 1910.1000). The exposure limit recommended by the National Institute for Occupational Safety and Health for cumene is 50 ppm (245 mg/m3), with a skin notation, averaged over a 10-hour work shift (NIOSH, 2005). The United States Environmental Protection Agency (1997) has assigned cumene to carcinogen category D (not classifiable or inadequate human or animal data) and determined an inhalation reference concentration of 0.4 mg/m3 using uncertainty factors to reflect a daily exposure concentration without appreciable risk of deleterious effects during a lifetime. ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION Metabolism, disposition, and pharmacokinetic studies of cumene in rats following oral, intravenous injection, or nose-only inhalation administration have demonstrated that the chemical was well absorbed. Following absorption, a small quantity (approximately 5%) of the cumene was exhaled unchanged, but the major portion was oxidized at the benzylic carbon to dimethylphenylcarbinol (Figure 1), with subsequent oxidation to 2-phenyl-2propanol, 2-phenylpropanoic acid, 2-hydroxy-2-phenylpropanoic acid, 2-phenyl-1,2-propanediol, and phenyllactic acid. A minor pathway is oxidation of a methyl group to 2-phenyl-1,2-propanol. A minor metabolite may be phenylmalonic acid (Senczuk and Litewka, 1976; NRC, 1981; Parmeggiani, 1983; Gosselin et al., 1984; Lee, 1987; Sato and Nakajima, 1987; Slauter and Jeffcoat, 1989, 1990, 1992; Ishida and Matsumoto, 1992; Cavender, 1994; USEPA, 1997). These metabolites were excreted mainly in the urine in conjugated form. Oxidation occurs in both hepatic and extrahepatic tissues, including the lung (Sato and Nakajima, 1987). Cumene does not bioaccumulate in tissues (ACGIH, 1993). There is no known biotransformation product that would suggest toxicity. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 22 Cumene, NTP TR 542 OH OH OH Cumene Dimethylphenylcarbinol ? OO OH HO ? O HO OH 2-Phenyl-1,2-propanediol O OH O OH OH 2-Phenylpropanoic acid Phenylmalonic Acid 2-Phenyllactic acid 2-Hydroxy-2- phenylpropanoic acid FIGURE 1 Known Metabolites of Cumene NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 23 TOXICITY Experimental Animals Table 1 lists LC50 and LD50 values for cumene in rats, mice, and rabbits (Cavender, 1994; Cushman et al., 1995). TABLE 1 Acute Toxicity Values for Cumene Species Rats Rats Mice Mice Rabbits Route of Administration Inhalation (4-hour) Oral Inhalation (7-hour) Oral Dermal LC50/LD50 8,000 ppm 1.4 g/kg (male) 2,000 ppm 12.75 g/kg 12.3 mL/kg Mice exposed to cumene vapor exhibited dilation of cutaneous blood vessels, grades of central nervous system depression, narcosis, depression of respiration, and death, depending on the concentration and duration of exposure. The narcosis was characterized by slow induction and long duration relative to benzene and toluene. Cumene is an eye and skin irritant (ACGIH, 1993; HSDB, 2003). When rats were dosed by repeated gastric intubation with 154, 462, or 769 mg cumene/kg body weight for 194 days, no changes in body weights, hematology, or histopathology of the liver or kidney were found, but at 462 and 769 mg/kg, increases in kidney weights were observed (ACGIH, 1993). Subcutaneous administration of 1 mL/kg daily for 2 weeks did not lower the femoral bone marrow cell population of rats (ACGIH, 1993). In an inhalation study, exposure of rabbits to 1,323 ppm (6,496 mg/m3) cumene for up to 180 days resulted in no changes in behavior or body weight gain (Fabre et al., 1955). In the same study, inhalation exposure of rats to 509 ppm (2,499 mg/m3) for 180 days produced a decrease in body weight gain limited to the initial part of the study and congestion of the lung, liver, spleen, kidney, and adrenal gland; higher exposure concentrations [814 ppm (3,997 mg/m3) and 1,323 ppm (6,496 mg/m3)] killed the rats within 16 hours of exposure. Daily Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 24 Cumene, NTP TR 542 inhalation exposure of rats to 500 ppm for 5 months resulted in no significant changes in the peripheral blood; however, hyperemia and congestion were noted in the lung, liver, and kidney of exposed animals (ACGIH, 1993). Jenkins et al. (1970) used inhalation techniques to expose rats, guinea pigs, dogs, and monkeys to 244 ppm (1,195 mg/m3) for 8 hours per day, 5 days per week for 30 exposures or to 3.7 ppm (18 mg/m3) or 30 ppm (146 mg/m3) continuously for 90 to 130 days and reported essentially negative findings. Cushman et al. (1995) exposed groups of 21 male and 21 female Fischer 344 rats to cumene vapor at concentrations of 0, 100, 500, or 1,200 ppm for 6 hours per day, 5 days per week for 13 weeks. A subsequent 13-week study with a 4-week recovery period was conducted in groups of 15 male and 15 female rats at exposure concentrations of 0, 50, 100, 500, or 1,200 ppm. No exposure-related changes in body weights, mortality, a functional observation battery, auditory brain stem responses, brain measurements, or nervous system histopathology were observed. Motor activity decreases seen only in 500 and 1,200 ppm males in the first study were not replicated in the second study. The 500 and/or 1,200 ppm groups showed transient decreases in body weight gain and feed consumption, increases in water consumption, and changes in several hematologic and clinical chemistry parameters. No exposure-related ophthalmologic findings or effects on spermatogenesis occurred. Liver, kidney, and adrenal gland weights were increased in the 500 and 1,200 ppm groups. Renal proximal tubule cell hypertrophy, hyperplasia, and hyaline droplet formation were observed in 500 and 1,200 ppm males. Daily inhalation exposure to 500 ppm cumene for 150 days was reported to induce hyperemia of the lung, liver, and kidney (species and sex not specified), but no changes occurred in the peripheral blood or bone marrow of animals exposed to 1,300 to 1,400 ppm for 180 days (Parmeggiani, 1983). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 25 Humans Cumene is an eye, skin, and mucous membrane irritant (NIOSH, 2005). Short-term exposure to cumene may cause dizziness, headache, drowsiness, slight incoordination, and unconsciousness (HSDB, 2003). Prolonged contact with liquid cumene may cause erythema or blisters (NIOSH/OSHA, 1981; Gosselin et al., 1984). REPRODUCTIVE AND DEVELOPMENTAL TOXICITY Experimental Animals Timed-pregnant Sprague-Dawley rats (25 per group) were exposed to cumene vapor for 6 hours per day on gestational days 6 through 15 at target concentrations up to 1,200 ppm (Neeper-Bradley, 1989a). In this study, maternal toxicity was evidenced at 1,200 ppm by significant reductions in body weight gain and treatment-related clinical signs of toxicity (perioral wetness and encrustation), decreased feed consumption, and increased relative liver weight; gestational parameters and fetal body weights per litter were unaffected by the exposure. In a subsequent study, this investigator found that pregnant rabbits exposed to 2,300 ppm cumene vapor during organogenesis evidenced consistent maternal toxicity (reductions in weight gain and feed consumption and increased relative liver weight) (Neeper-Bradley, 1989b). Gestational parameters such as the number of corpora lutea; the total, nonviable, or viable implantations per litter; sex ratio; pre- or postimplantation loss; fetal body weights per litter; and malformations exhibited no significant changes. Humans No studies of reproductive or developmental effects of cumene in humans were found in a review of the literature. CARCINOGENICITY No reports of carcinogenic effects of cumene in experimental animals or evidence of carcinogenic effects in humans in epidemiology studies or case reports were found in the literature. However, the NTP has conducted carcinogenicity studies of ethylbenzene (a structurally related chemical) administered by inhalation and reported Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 26 Cumene, NTP TR 542 induction of renal tubule neoplasms in male and female F344/N rats, testicular adenoma in male rats, alveolar/bronchiolar neoplasms in male B6C3F1 mice, and hepatocellular neoplasms in B6C3F1 female mice (NTP, 1999). In contrast, ethylbenzene was not carcinogenic in male or female CD (Sprague-Dawley) rats gavaged daily with 500 mg/kg, 4 to 5 days per week for 104 weeks (Maltoni et al., 1985). GENETIC TOXICITY There are few published reports on the mutagenicity of cumene, and the limited information available suggests that cumene is not mutagenic in standard assays. Cumene was reported to be nonmutagenic in Salmonella typhimurium tester strains with and without metabolic activation (Simmon et al., 1977; Florin et al., 1980), and results obtained with cumene in several industry-sponsored in vitro and in vivo genetic toxicity studies in mammalian cell test systems also indicated no potential for mutagenic or clastogenic activity (GLSC, 1985a,b; Curren, 1987; Putman, 1987; Yang, 1987). As noted earlier, cumene is structurally related to ethylbenzene. NTP (1999) studies have demonstrated that ethylbenzene is not mutagenic in S. typhimurium tester strains with or without S9 activation enzymes (Zeiger, et al., 1992), but treatment of cultured mouse lymphoma L5178Y cells with ethylbenzene in the absence of S9 resulted in significantly increased mutation frequencies at the tk+/- locus (McGregor et al., 1988). No induction of sister chromatid exchanges or chromosomal aberrations was observed in cultured Chinese hamster ovary cells treated with ethylbenzene with or without S9 enzymes (NTP, 1999), and no increases in the frequencies of micronucleated erythrocytes were seen in peripheral blood of male or female B6C3F1 mice exposed to ethylbenzene by inhalation for 3 months (Witt et al., 2000). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 27 STUDY RATIONALE Cumene was nominated for study by the NIEHS because of its high production volume, presence as a component in gasoline and other fuels, potential for human exposure, and lack of existing carcinogenicity test data. Inhalation was chosen for these studies because this is the primary route of human exposure. Cumene was studied for toxic and carcinogenic effects in rats and mice exposed by whole body inhalation for 2 weeks, 3 months, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat bone marrow, and mouse peripheral blood. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 28 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 29 MATERIALS AND METHODS PROCUREMENT AND CHARACTERIZATION OF CUMENE Cumene was obtained from Sunoco, Inc. (Philadelphia, PA), in one lot (200556852) that was used in the 2-week, 3-month, and 2-year studies. Identity and purity analyses were conducted by the study laboratory at Battelle Toxicology Northwest (Richland, WA), by the analytical chemistry laboratory at Midwest Research Institute (Kansas City, MO), and by Chemir/Polytech Laboratories, Inc. (Maryland Heights, MO), (Appendix I). Reports on analyses performed in support of the cumene studies are on file at the National Institute of Environmental Health Sciences. Lot 200556852 of the chemical, a colorless liquid with a sharp, penetrating, aromatic odor, was identified as cumene by the analytical chemistry laboratory and Chemir/Polytech Laboratories, Inc., using 1H-nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and/or infrared spectroscopy. For lot 200556852, Karl Fischer titration indicated a water content ranging from approximately 50 to 220 ppm; elemental analyses for carbon and hydrogen were in agreement with the theoretical values for cumene. Gas chromatography by one system detected no impurities greater than 0.05%, and the purity was determined to be approximately 100%. Using gas chromatography by another system, the area percent purity for the major cumene peak was 99.9%, and no peaks were detected with an area percent greater than 0.1%. The overall purity of lot 200556852 was determined to be greater than 99.9%. To ensure stability, the bulk chemical was stored at controlled room temperature in the original shipping containers (55-gallon metal drums). Stability was monitored by the study laboratory during the 2-week, 3-month, and 2-year studies with gas chromatography. No degradation of the bulk chemical was detected. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 30 Cumene, NTP TR 542 VAPOR GENERATION AND EXPOSURE SYSTEM The design of the vapor generation and exposure system was influenced by the relatively high boiling point of cumene (approximately 152 C) and the need to reach relatively high concentrations. Therefore, with the exception of individual chamber inlets, all vapor transport lines and dilution air were heated to the minimum temperature needed to move vapor to the chambers without condensation. A bulk supply of cumene was held in an 8-gallon stainless steel chemical reservoir and pumped through a preheater into the top of a heated glass column filled with glass beads to increase the surface area for evaporation. Heated nitrogen entering the column from below vaporized the chemical as it conveyed it out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Cumene vapor was transported to the exposure room through transport lines at an elevated temperature to prevent condensation. In the distribution manifold cabinet, the vapor was mixed with additional heated air before it entered a short vapor distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate, all of which were monitored by the exposure operator. The pressure in the distribution manifold was fixed to ensure constant flow through the manifold and into the chambers as the flow of vapor to each chamber was adjusted. Electronically actuated metering valves controlled the flow to each chamber. In addition, an exposure-shutoff valve, mounted in series with each chamber-metering valve, controlled vapor delivery to each chamber. Vapor was diverted to the exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To start the exposure, the valves were opened to allow the flow of vapor to reach the chamber-metering valves and move into individual temperature-controlled delivery lines to each chamber. The vapor was then injected into the chamber inlet duct where it was diluted with conditioned chamber air to achieve the desired exposure concentration. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 31 The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3. A condensation particle counter (Model 3022A, TSI, Inc., St. Paul, MN) was used to count the particles in all chambers before and during generation to ensure that cumene vapor, and not aerosol, was produced. No particle counts greater than 200 particles/cm3 were detected. VAPOR CONCENTRATION MONITORING Concentrations of cumene in the exposure chambers were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 (2-week and 3-month studies) or 26 (2-year studies) minutes during each 6-hour exposure period using Hasteloy-C stream-select and gas-sampling valves (Valco Instruments Co., Houston, TX) in a separate, heated valve oven. The sample lines composing each sample loop were made from Teflon tubing and were connected to the exposure chamber relative humidity sampling lines at a location close to the gas chromatograph. A vacuum regulator maintained a constant vacuum in the sample loop to compensate for variations in sample line pressure. An in-line flow meter between the vacuum regulator and the gas chromatograph allowed digital measurement of sample flow. Summaries of the chamber vapor concentrations are given in Tables I2 through I4. The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of cumene in nitrogen supplied by a permeation tube standard generator (Kin-Tek Model 491, Kin-Tek Laboratories, Inc., La Marque, TX). The on-line gas chromatograph was calibrated prior to the start of each study, three times during the 2-week studies, and monthly during the 3-month and 2-year studies by a comparison of chamber concentration data to data from grab samples that were collected with charcoal sampling tubes (ORBOTM-101, Supelco, Bellefonte, PA), extracted with toluene containing 1,2,4-trimethylbenzene as an internal standard, and analyzed by an off-line gas chromatograph. The volumes of gas were sampled from each chamber at a constant Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 32 Cumene, NTP TR 542 flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standard solutions of cumene and the internal standard (1,2,4-trimethylbenzene) in toluene. CHAMBER ATMOSPHERE CHARACTERIZATION Buildup and decay rates for chamber vapor concentrations were determined with (all studies) and without (3-month and 2-year studies) animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. The uniformity of cumene vapor concentration in the inhalation exposure chambers without animals present was evaluated before the 3-month and 2-year studies began; concentration uniformity with animals present in the chambers was measured once during the 2-week studies, once during the 3-month studies, and approximately quarterly in the 2-year studies. The vapor concentration was measured using an on-line gas chromatograph. Chamber concentration uniformity was maintained throughout the studies. The persistence of cumene in the chambers after vapor delivery ended was determined by monitoring the postexposure vapor concentration in the 4,000 ppm chambers in the 2-week studies and the 1,000 ppm chambers in the 3-month and 2-year studies, with (all studies) and without (3-month and 2-year studies) animals present in the chambers. In the 2-week studies, the concentration decreased to 1% of the target concentration within 77 minutes. In the 3-month studies, the concentration decreased to 1% of the target concentration within 25 minutes without animals present and within 28 minutes with animals present. In the 2-year studies, the NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 33 concentration decreased to 1% of the target concentration within 25 (rats) and 24 (mice) minutes without animals present and within 35 (rats) and 27 (mice) minutes with animals present. No evidence of degradation of cumene was noted in any part of the exposure system. With the exception of one peak noted in the distribution line samples taken during the 3-month studies, no impurity peaks were resolved with an area greater than 0.1% of the total peak area, and no additional impurities were detected with the polar gas chromatography analyses. The relative purity of all generator reservoir and vapor trap samples exceeded 99% compared to the bulk chemical, and these samples were 99.97% pure by area percent. 2-WEEK STUDIES Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY). On receipt, the rats and mice were approximately 4 weeks old. Animals were quarantined for 11 days and were approximately 6 weeks old on the first day of the studies. Before the studies began, four male and six female rats and five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. At terminal sacrifice, serum was collected from five male and five female chamber control rats and mice, and serologic analyses were performed using the protocols of the NTP Sentinel Animal Program (Appendix K). Groups of five male and five female rats and mice were exposed to cumene at concentrations of 0, 250, 500, 1,000, 2,000, or 4,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 16 (rats) or 17 (mice) days. Feed was available ad libitum except during exposure periods; water was available ad libitum. Rats and mice were housed individually. Clinical findings were recorded twice daily on exposure days for rats and mice. The animals were weighed initially, on days 6 and 13, and at the end of the studies. Details of the study design and animal maintenance are summarized in Table 2. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 34 Cumene, NTP TR 542 Necropsies were performed on all animals. The heart, right kidney, liver, lung, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 m, and stained with hematoxylin and eosin. Complete histopathologic examinations were performed on all chamber control and 1,000 (mice only), 2,000, and 4,000 ppm animals, and tissues were examined to a no-effect level in the remaining exposure groups. Table 2 lists the tissues and organs routinely examined. 3-MONTH STUDIES The 3-month studies were conducted to evaluate the cumulative toxic effects of repeated exposure to cumene and to determine the appropriate exposure concentrations to be used in the 2-year studies. Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY). On receipt, the rats and mice were approximately 4 weeks old. Animals were quarantined for 11 (male rats and male and female mice) or 12 (female rats) days and were approximately 6 weeks old on the first day of the studies. Before the studies began, five male and five female rats and mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Serologic analyses were performed by the study laboratory on five male and five female sentinel rats and mice 3 weeks after arrival at the study laboratory and five male and five female chamber control rats and mice at the end of the studies using the protocols of the NTP Sentinel Animal Program (Appendix K). Groups of 10 male and 10 female rats and mice were exposed to cumene at concentrations of 0, 62.5, 125, 250, 500, or 1,000 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week (except holidays) for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. One additional exposure day was scheduled during the last exposure week to give the rats at least 2 consecutive days of exposure before terminal sacrifice. Feed was available ad libitum except during exposure NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 35 periods; water was available ad libitum. Rats and mice were housed individually. Clinical findings were recorded weekly. The animals were weighed initially, weekly, and at the end of the studies. Details of the study design and animal maintenance are summarized in Table 2. Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry analyses; blood was collected from the retroorbital sinus of mice at the end of the study for hematology analyses. Blood samples for hematology analyses were placed in tubes containing potassium EDTA. Erythrocyte, leukocyte, and platelet counts; hemoglobin; packed red cell volume; mean cell volume; mean cell hemoglobin; and mean cell hemoglobin concentration were determined using an ABX Cobas Helios hematology analyzer (ABX Co., Irvine, CA). Manual hematocrit values were determined using a microcentrifuge (Heraeus Haemofuge, Germany) and a Damon/IEC capillary reader (International Equipment Company, Needham Heights, MA) for comparison to Helios values for packed cell volume. Blood smears for mice and rats were stained with Romanowsky-type aqueous stain in a Wescor 7100 Aerospray Slide Stainer (Wescor, Inc., Logan, UT). Leukocyte differential counts for rats and mice were based on classifying a minimum of 100 white cells. Reticulocytes were stained with new methylene blue and enumerated as a reticulocyte:erythrocyte ratio using the Miller disc method (Brecher and Schneiderman, 1950). Blood samples for clinical chemistry analyses were placed in tubes containing separator gel and allowed to clot. After clot retraction occurred, the samples were centrifuged, and the serum was aliquoted for assay of serum chemistry analytes using a Roche Cobas Fara (Roche Diagnostics, Branchburg, NJ). Table 2 lists the parameters measured. At the end of the 3-month studies, samples were collected for sperm count and motility and vaginal cytology evaluations on rats and mice exposed to 0, 125 (female mice only), 250, 500, or 1,000 (rats and male mice) ppm. The parameters evaluated are listed in Table 2. Male animals were evaluated for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk (rats) or modified Tyrode's buffer Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 36 Cumene, NTP TR 542 (mice) was applied to slides, and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, the left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65 C. Sperm density was then determined microscopically with the aid of a hemacytometer. Four sperm morphology slides were prepared for each animal evaluated. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemacytometer. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females were moistened with saline if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). The left kidney was removed from all male and female core study rats at terminal necropsy, sectioned in half longitudinally, placed in a cassette, and fixed with 10% neutral buffered formalin for approximately 24 hours. After fixation, one half of the left kidney was processed and embedded in paraffin. A cross-section of small intestine was included in the embedding paraffin as a positive control for the cell proliferation study. After embedding, the left kidney was cut into three 5-m thick sections. The first section was stained with hematoxylin and eosin for histopathology (males and females). The second section was stained with Mallory-Heidenhain for evaluation for hyaline droplets (males and females). The third section was stained with proliferating cell nuclear antigen (PCNA) complexed with avidin and biotin for determination of cell proliferation indices (males). The right kidneys of all core study male rats were frozen and stored at approximately 70 C. For male rats, the slides stained with PCNA were evaluated to determine the number of proximal tubule cells in the S-phase and the labeling index. Evaluation was done using a 20 objective and an ocular grid. Counting NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 37 started at the second grid in from the outer edge of the cortex of the kidney. After one grid was counted, the slide was moved toward the medulla, and every other field encountered by the grid was counted. This procedure was repeated until at least 2,000 proximal tubule nuclei (labeled and unlabeled) were counted. If 2,000 proximal tubule nuclei were counted but the entire grid had not been counted, the remainder of the grid was counted. If 2,000 proximal tubule nuclei had not been counted by the time the outer medulla was reached, the slide was moved two grids laterally, and the counting process was resumed at the second grid in from the edge of the cortex. The frozen kidneys from core study male rats were evaluated for "2u-globulin and soluble protein. Each right kidney was thawed; a volume of sodium/potassium phosphate buffer (pH ~7.2) equivalent to twice the recorded fresh weight of the sample was added, and the sample was homogenized for 30 to 60 seconds using an UltraTurrax tissue homogenizer (Tekmar Co., Cincinnati, OH). The homogenate was centrifuged at approximately 3,000 g for 15 minutes at 4 C. The protein content of each supernatant was measured in a 1:50 dilution (in phosphate-buffered saline-Tween) using the Bicinchoninic Acid Protein Assay Reagent kit (Pierce Chemical Co., Rockford, IL). Analysis of "2u-globulin in supernatants prepared from kidney homogenates was conducted using a competitive indirect enzyme-linked immunosorbent assay (ELISA). Ascites fluid containing anti-"2u-globulin monoclonal antibodies was developed by Chemical Industry Institute of Toxicology (Research Triangle Park, NC). The amount of "2u-globulin was measured by comparing the relative fluorescent signal intensity in the study samples to that observed with known amounts of "2u-globulin present in calibration standards. Calibration standards and ELISA control standards (negative and positive) were plated in predetermined wells on 96-well microtiter plates. Calibration standards and study samples were assayed in triplicate. Necropsies were performed on all core study animals. The heart, right kidney, liver, lung, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin (eyes were fixed in Davidson's solution for up to 10 days and then transferred to 10% neutral buffered formalin), Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 38 Cumene, NTP TR 542 processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 m, and stained with hematoxylin and eosin. Complete histopathologic examinations were performed on all chamber control, 500 (female mice only), and 1,000 ppm animals, and tissues were examined to a no-effect level in the remaining exposure groups. Table 2 lists the tissues and organs routinely examined. 2-YEAR STUDIES Study Design Groups of 50 male and 50 female rats and mice were exposed to cumene at concentrations of 0, 125 (female mice only), 250, 500, or 1,000 (rats and male mice) ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week (except holidays) for 105 weeks. Source and Specification of Animals Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Laboratory Animals and Services (Germantown, NY) for use in the 2-year studies. Rats were quarantined for 11 days and mice for 10 days before the beginning of the studies. Five male and five female rats and mice were randomly selected for parasite evaluation and gross observation of disease. Rats and mice were approximately 6 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program (Appendix K). Animal Maintenance Rats and mice were housed individually. Feed was available ad libitum except during exposure periods; water was available ad libitum. Cages, racks, and chambers were changed weekly. Cages were rotated weekly in chambers. Refer to Table 2 for more information about animal maintenance. Refer to Appendix J for information about feed composition and contaminants. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 39 Clinical Examinations and Pathology All animals were observed twice daily. For rats, clinical findings were recorded every 4 weeks through week 93, every 2 weeks thereafter, and at the end of the study. For mice, clinical findings were recorded weekly through week 13, then every 4 weeks through week 93, every 2 weeks thereafter, and at the end of the study. Rats and mice were weighed initially, weekly for the first 13 weeks, then every 4 weeks through week 93, every 2 weeks thereafter, and at the end of the studies. Complete necropsies and microscopic examinations were performed on all rats and mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin (eyes were fixed in Davidson's solution for up to 72 hours and then transferred to 10% neutral buffered formalin), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 m, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. Tissues examined microscopically are listed in Table 2. Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual animal records and tables were compared for accuracy; the slide and tissue counts were verified, and the histotechnique was evaluated. For the 2-year studies, a quality assessment pathologist evaluated slides from all tumors and all potential target organs, which included the eye, forestomach, lung, and nose of rats and mice, the kidney of male and female rats and male mice, the urinary bladder of male rats, and the liver of mice. The quality assessment report and the reviewed slides were submitted to the NTP Pathology Working Group (PWG) chairperson, who reviewed the selected tissues and addressed any inconsistencies in the diagnoses made by Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 40 Cumene, NTP TR 542 the laboratory and quality assessment pathologists. Representative histopathology slides containing examples of lesions related to chemical administration, examples of disagreements in diagnoses between the laboratory and quality assessment pathologists, or lesions of general interest were presented by the chairperson to the PWG for review. The PWG consisted of the quality assessment pathologist and other pathologists experienced in rodent toxicologic pathology. This group examined the tissues without any knowledge of dose groups or previously rendered diagnoses. When the PWG consensus differed from the opinion of the laboratory pathologist, the diagnosis was changed. Final diagnoses for reviewed lesions represent a consensus between the laboratory pathologist, reviewing pathologist(s), and the PWG. Details of these review procedures have been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses of the pathology data, the decision of whether to evaluate the diagnosed lesions for each tissue type separately or combined was generally based on the guidelines of McConnell et al. (1986). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 41 TABLE 2 Experimental Design and Materials and Methods in the Inhalation Studies of Cumene 2-Week Studies 3-Month Studies 2-Year Studies Study Laboratory Battelle Toxicology Northwest (Richland, WA) Battelle Toxicology Northwest (Richland, WA) Battelle Toxicology Northwest (Richland, WA) Strain and Species F344/N rats B6C3F1 mice F344/N rats B6C3F1 mice F344/N rats B6C3F1 mice Animal Source Taconic Laboratory Animals and Services (Germantown, NY) Taconic Laboratory Animals and Services (Germantown, NY) Taconic Laboratory Animals and Services (Germantown, NY) Time Held Before Studies 11 days Rats: 11 days (males) or 12 days (females) Mice: 11 days Rats: 11 days Mice: 10 days Average Age When Studies Began 6 weeks 6 weeks 6 weeks Date of First Exposure April 24, 2000 Rats: July 24 (males) or 25 (females), 2000 Mice: July 24, 2000 Rats: June 4, 2001 Mice: June 11, 2001 Duration of Exposure 6 hours plus T90 (12 minutes) per day, 5 days per week, for 16 (rats) or 17 (mice) days 6 hours plus T90 (12 minutes) per day, 5 days per week, excluding holidays, for 14 weeks 6 hours plus T90 (12 minutes) per day, 5 days per week, excluding holidays, for 105 weeks Date of Last Exposure Rats: May 9, 2000 Mice: May 10, 2000 Necropsy Dates Rats: May 10, 2000 Mice: May 11, 2000 Rats: October 23 (males) or 24 (females), 2000 Mice: October 25 (males) or 26 (females), 2000 Rats: October 24 (males) or 25 (females), 2000 Mice: October 26 (males) or 27 (females), 2000 Rats: June 1-4, 2003 Mice: June 8-12, 2003 Rats: June 2-5, 2003 Mice: June 9-13, 2003 Average Age at Necropsy 8 weeks Size of Study Groups 5 males and 5 females Method of Distribution Animals were distributed randomly into groups of approximately equal initial mean body weights. Animals per Cage 1 19 weeks 110 weeks Rats: 10 males and 10 females (core study), 10 males and 10 females (clinical pathology study) Mice: 10 males and 10 females 50 males and 50 females Same as 2-week studies Same as 2-week studies 11 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 42 Cumene, NTP TR 542 TABLE 2 Experimental Design and Materials and Methods in the Inhalation Studies of Cumene 2-Week Studies 3-Month Studies 2-Year Studies Method of Animal Identification Tail tattoo Diet NTP-2000 irradiated pelleted diet (Zeigler Brothers, Inc., Gardners, PA); available ad libitum (except during animal exposure periods); changed weekly Tail tattoo Same as 2-week studies Water Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum Same as 2-week studies Cages Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); changed weekly Same as 2-week studies Cageboard Untreated paper cage pan liner; changed daily Same as 2-week studies Chamber Air Supply Filters Single HEPA (Environmental Filter, Santa Rosa, CA), changed annually; charcoal (RSE, Inc., New Baltimore, MI), new at study start; Purafil (Environmental Systems, Lynwood, WA), new at study start Same as 2-week studies Chambers Stainless steel, excreta pan at each of six levels (Lab Products, Inc., Seaford, DE); chambers changed weekly; excreta pans changed daily Same as 2-week studies Chamber Environment Temperature: 75 3 F Relative humidity: 55% 15% Room fluorescent light: 12 hours/day Chamber air changes: 15 2/hour Exposure Concentrations 0, 250, 500, 1,000, 2,000, and 4,000 ppm Temperature: 75 3 F Relative humidity: 55% 15% Room fluorescent light: 12 hours/day Chamber air changes: 15 2/hour 0, 62.5, 125, 250, 500, and 1,000 ppm Tail tattoo Same as 2-week studies, except wafer form Same as 2-week studies Same as 2-week studies Untreated paper cage pan liner (Techboard, Sheperd Specialty Papers, Kalamazoo, MI); changed daily Same as 2-week studies, except single HEPA was open stock Same as 2-week studies Temperature: 75 3 F Relative humidity: 55% 15% Room fluorescent light: 12 hours/day Chamber air changes: 15 2/hour Rats: 0, 250, 500, and 1,000 ppm Mice: 0, 125 (females only), 250, 500, and 1,000 (males only) ppm NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 43 TABLE 2 Experimental Design and Materials and Methods in the Inhalation Studies of Cumene 2-Week Studies 3-Month Studies 2-Year Studies Type and Frequency of Observation Observed twice daily; animals were weighed initially, on days 6 and 13, and at the end of the studies; clinical findings were recorded twice daily on exposure days. Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies; clinical findings were recorded weekly. Observed twice daily; for rats, clinical findings were recorded every 4 weeks through week 93, every 2 weeks thereafter, and at the end of the study; for mice, clinical findings were recorded weekly through week 13, then every 4 weeks through week 93, every 2 weeks thereafter, and at the end of the study; animals were weighed initially, weekly for the first 13 weeks, then every 4 weeks through week 93, every 2 weeks thereafter, and at the end of the studies. Method of Sacrifice Carbon dioxide asphyxiation Same as 2-week studies Same as 2-week studies Necropsy Necropsies were performed on all animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus. Necropsies were performed on core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus. Necropsies were performed on all animals. Clinical Pathology None Blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of mice at the end of the study for hematology. Hematology: hematocrit; packed red cell volume; hemoglobin; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials. Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids None Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 44 Cumene, NTP TR 542 TABLE 2 Experimental Design and Materials and Methods in the Inhalation Studies of Cumene 2-Week Studies 3-Month Studies 2-Year Studies Histopathology Histopathology was performed on 0, 2,000, and 4,000 ppm rats and 0, 1,000, 2,000, and 4,000 ppm mice. In addition to gross lesions and tissue masses, the following tissues were examined: kidney, liver, lung, and nose. These tissues were examined to a no-effect level in the remaining exposure groups. Complete histopathology was performed on 0 and 1,000 ppm core study rats and 0, 500 (females only), and 1,000 ppm mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, gallbladder (mice), harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung (with mainstem bronchus), lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the kidney of male rats in the remaining groups, the liver of 500 ppm male mice, and the forestomach of 250 ppm female mice were examined. Complete histopathology was performed on all rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, gallbladder (mice), harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung (with bronchus), lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. Sperm Motility and Vaginal Cytology None At the end of the studies, sperm samples were collected from male animals in the 0, 250, 500, and 1,000 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females exposed to 0, 125 (mice), 250, 500, or 1,000 (rats) ppm for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated. None Renal Toxicity Study None At the end of the study, concentrations of "2u-globulin and soluble protein were measured in the right kidney of core study rats; the left kidneys were used for evaluation of hyaline droplets, assessment of cell proliferation indices, and histopathology. None NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 45 STATISTICAL METHODS Survival Analyses The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals found dead of other than natural causes or missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox's (1972) method for testing two groups for equality and Tarone's (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Calculation of Incidence The incidences of neoplasms or nonneoplastic lesions are presented in Tables A1, A4, B1, B4, C1, C4, D1, and D4 as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site examined microscopically. For calculation of statistical significance, the incidences of most neoplasms (Tables A2, B2, C2, and D2) and all nonneoplastic lesions are given as the numbers of animals affected at each site examined microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues (e.g., harderian gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms had multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators consist of the number of animals on which a necropsy was performed. Tables A2, B2, C2, and D2 also give the survivaladjusted neoplasm rate for each group and each site-specific neoplasm. This survival-adjusted rate (based on the Poly-3 method described below) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, only to site-specific, lesion-free animals that do not reach terminal sacrifice. Analysis of Neoplasm and Nonneoplastic Lesion Incidences The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure that modifies the Cochran-Armitage linear trend test to take survival differences into account. More specifically, this Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 46 Cumene, NTP TR 542 method modifies the denominator in the quantal estimate of lesion incidence to approximate more closely the total number of animal years at risk. For analysis of a given site, each animal is assigned a risk weight. This value is one if the animal had a lesion at that site or if it survived until terminal sacrifice; if the animal died prior to terminal sacrifice and did not have a lesion at that site, its risk weight is the fraction of the entire study time that it survived, raised to the kth power. This method yields a lesion prevalence rate that depends only upon the choice of a shape parameter for a Weibull hazard function describing cumulative lesion incidence over time (Bailer and Portier, 1988). Unless otherwise specified, a value of k=3 was used in the analysis of site-specific lesions. This value was recommended by Bailer and Portier (1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific neoplasms in control F344 rats and B6C3F1 mice (Portier et al., 1986). Bailer and Portier (1988) showed that the Poly-3 test gave valid results if the true value of k was anywhere in the range from 1 to 5. A further advantage of the Poly-3 method is that it does not require lesion lethality assumptions. Variation introduced by the use of risk weights, which reflect differential mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended by Bieler and Williams (1993). Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1P with the letter N added (e.g., P=0.99 is presented as P=0.01N). Analysis of Continuous Variables Two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, renal toxicity, spermatid, and epididymal NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 47 spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trendsensitive test (Williams' or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett's or Dunn's test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis. Average severity values were analyzed for significance with the MannWhitney U test (Hollander and Wolfe, 1973). Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations. Historical Control Data The concurrent control group represents the most valid comparison to the treated groups and is the only control group analyzed statistically in NTP bioassays. However, historical control data are often helpful in interpreting potential treatment-related effects, particularly for uncommon or rare neoplasm types. For meaningful comparisons, the conditions for studies in the historical database must be generally similar. One significant factor affecting the background incidence of neoplasms at a variety of sites is diet. In 1995, the NTP incorporated a new diet (NTP-2000) that contains less protein and more fiber and fat than the NIH-07 diet previously used in toxicity and carcinogenicity studies (Rao, 1996, 1997). The current NTP historical database contains all studies that use the NTP-2000 diet with histopathology findings completed within the most recent 5-year period. A second potential source of variability is route of administration. In general, the historical database for a given study will include studies using the same route of administration, and the overall incidences of neoplasms for all routes of administration are included for comparison, including the present study. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 48 Cumene, NTP TR 542 QUALITY ASSURANCE METHODS The 3-month and 2-year studies were conducted in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 2-year studies were submitted to the NTP Archives, these studies were audited retrospectively by an independent quality assurance contractor. Separate audits covered completeness and accuracy of the pathology data, pathology specimens, final pathology tables, and a draft of this NTP Technical Report. Audit procedures and findings are presented in the reports and are on file at NIEHS. The audit findings were reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the preparation of this Technical Report. GENETIC TOXICOLOGY The genetic toxicity of cumene was assessed by testing the ability of the chemical to induce mutations in various strains of Salmonella typhimurium and increases in the frequency of micronucleated erythrocytes in rat bone marrow and mouse peripheral blood. Micronuclei (literally "small nuclei" or Howell-Jolly bodies) are biomarkers of induced structural or numerical chromosomal alterations and are formed when acentric fragments or whole chromosomes fail to incorporate into either of two daughter nuclei during cell division (Schmid, 1975; Heddle et al., 1983). The protocols for these studies and the results are given in Appendix E. The genetic toxicity studies have evolved from an earlier effort by the NTP to develop a comprehensive database permitting a critical anticipation of a chemical's carcinogenicity in experimental animals based on numerous considerations, including the molecular structure of the chemical and its observed effects in short-term in vitro and in vivo genetic toxicity tests (structure-activity relationships). The short-term tests were originally developed to clarify proposed mechanisms of chemical-induced DNA damage based on the relationship between electrophilicity and mutagenicity (Miller and Miller, 1977) and the somatic mutation theory of cancer (Straus, 1981; Crawford, 1985). However, it should be noted that not all cancers arise through genotoxic mechanisms. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 49 DNA reactivity combined with Salmonella mutagenicity is highly correlated with induction of carcinogenicity in multiple species/sexes of rodents and at multiple tissue sites (Ashby and Tennant, 1991). A positive response in the Salmonella test was shown to be the most predictive in vitro indicator for rodent carcinogenicity (89% of the Salmonella mutagens are rodent carcinogens) (Tennant et al., 1987; Zeiger et al., 1990). Additionally, no battery of tests that included the Salmonella test improved the predictivity of the Salmonella test alone. However, these other tests can provide useful information on the types of DNA and chromosomal damage induced by the chemical under investigation. The predictivity for carcinogenicity of a positive response in acute in vivo bone marrow chromosome aberration or micronucleus tests appears to be less than that in the Salmonella test (Shelby et al., 1993; Shelby and Witt, 1995). However, clearly positive results in long-term peripheral blood micronucleus tests have high predictivity for rodent carcinogenicity (Witt et al., 2000); negative results in this assay do not correlate well with either negative or positive results in rodent carcinogenicity studies. Because of the theoretical and observed associations between induced genetic damage and adverse effects in somatic and germ cells, the determination of in vivo genetic effects is important to the overall understanding of the risks associated with exposure to a particular chemical. Most organic chemicals that are identified by the International Agency for Research on Cancer as human carcinogens, other than hormones, are genotoxic. The vast majority of these are detected by both the Salmonella assay and rodent bone marrow cytogenetics tests (Shelby, 1988; Shelby and Zeiger, 1990). Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 50 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 51 RESULTS RATS 2-WEEK STUDY Rats exposed to 4,000 ppm cumene died during or after the first exposure on day 1, and half (2/5 males and 3/5 females) of the rats exposed to 2,000 ppm died by day 4 (Table 3). Final mean body weights and body weight gains of 2,000 ppm rats were significantly less than those of the chamber controls. Rats exposed to 2,000 ppm that died early were severely ataxic and lethargic on the day of exposure and into the following morning. Rats exposed TABLE 3 Survival and Body Weights of Rats in the 2-Week Inhalation Study of Cumene Concentration (ppm) Survivala Mean Body Weightb (g) Initial Final Change Final Weight Relative to Controls (%) Male 0 250 500 1,000 2,000 4,000 5/5 82 2 126 3 45 3 5/5 84 2 130 3 46 2 5/5 83 3 128 6 46 4 053///555cd 84 2 85 3 86 2 127 4 92 3** -- 43 3 5 4** -- 103 102 100 73 -- Female 0 250 500 1,000 2,000 4,000 5/5 79 2 108 2 29 0 5/5 78 2 110 2 32 1 5/5 77 2 111 2 33 1 52//55e 80 2 79 3 110 2 86 4** 30 2 3 1** 0/5d 79 3 -- -- 102 103 102 80 -- *a * b Significantly different (P#0.01) from the chamber control group by Dunnett's test Number of animals surviving at 2 weeks/number initially in group Weights and weight changes are given as mean standard error. Subsequent calculations are based on animals surviving to the end of the c d e study. Day of death: 2, 4 Day of deaths: 1 Day of death: 2, 3, 4 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 52 Cumene, NTP TR 542 to 1,000 ppm and those that survived exposure to 2,000 ppm exhibited varying degrees of lethargy or ataxia, although the rats appeared to develop a tolerance, and the severity of clinical effects lessened by the end of week 1. Rats exposed to 500 ppm exhibited mild ataxia only after the initial exposure on day 1. Significant increases in absolute and relative weights of the liver and relative weights of the kidney occurred in all exposed groups (Table G1). The absolute kidney weights of 250 and 1,000 ppm males and 250, 500, and 1,000 ppm females were significantly increased. Absolute and relative thymus weights were significantly decreased in the 2,000 ppm groups. No exposure-related gross lesions were observed. Accumulation of minimal to mild hyaline droplets was observed in the renal tubular cortex of males exposed to concentrations of 250 to 2,000 ppm, and the incidences were greatest in the 250 and 1,000 ppm groups [chamber controls, 0/5; 250 ppm, 3/5 (1.3); 500 ppm, 2/5 (1.5); 1,000 ppm, 3/5 (1.0); 2,000 ppm, 1/5 (1.0); 4,000 ppm, 0/5]. The lack of hyaline droplets in 2,000 ppm males that died on days 2 and 4 and 4,000 ppm males that died on day 1 was considered the result of the short period of exposure. Exposure Concentration Selection Rationale: In male rats, increased incidences of hyaline droplet accumulation in the renal cortex were caused by cumene exposure. There was no evidence of other renal tubule epithelium damage. Based on the mortalities at 2,000 and 4,000 ppm, organ weight changes, and the lack of lesions in tissues examined, cumene exposure concentrations selected for the 3-month inhalation study in rats were 62.5, 125, 250, 500, and 1,000 ppm. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 53 3-MONTH STUDY All rats survived to the end of the study (Table 4). Final mean body weights and body weight gains of all exposed groups were similar to those of the chamber controls. Male rats exposed to 1,000 ppm exhibited ataxia for several days but recovered and appeared to develop a tolerance by day 3. TABLE 4 Survival and Body Weights of Rats in the 3-Month Inhalation Study of Cumene Concentration (ppm) Survivala Mean Body Weightb (g) Initial Final Change Final Weight Relative to Controls (%) Male 0 62.5 125 250 500 1,000 10/10 10/10 10/10 10/10 10/10 10/10 89 3 91 3 90 3 91 3 87 3 89 2 302 8 303 6 313 5 319 4 303 5 312 5 213 9 212 6 223 6 228 5 216 6 223 5 100 104 105 100 103 Female 0 62.5 125 250 500 1,000 10/10 10/10 10/10 10/10 10/10 10/10 84 2 84 2 81 1 83 2 85 2 83 2 190 2 184 3 190 4 187 3 182 3 184 3 105 3 100 3 109 4 104 3 97 3 101 3 97 100 99 96 97 a Number of animals surviving at 3 months/number initially in group b Weights and weight changes are given as mean standard error. Differences from the chamber control group are not significant by Dunnett's test. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 54 Cumene, NTP TR 542 The hematology and clinical chemistry data for rats in the 3-month inhalation study of cumene are listed in Tables 5 and F1. The most consistent changes involved markers of hepatic injury/function. Serum total bile acid concentrations were significantly increased in essentially all exposed groups on days 3 and 23 of the study. The bile acid increases occurred in an exposure concentration-related manner (ranging from approximately 20% in the 62.5 ppm groups to approximately 65% in the 1,000 ppm groups). This effect was, however, transient and by study termination affected only the 500 and 1,000 ppm males. Markers of hepatocyte injury, alanine aminotransferase and sorbitol dehydrogenase activities, demonstrated an exposure concentration-related decrease in serum activity. Alanine aminotransferase appeared to be most affected, demonstrating some effect at all time points; the sorbitol dehydrogenase effect was limited to week 14. The magnitude of the effect intensified with time, and by study termination, the 250, 500, and 1,000 ppm male and female groups demonstrated decreases in serum activity of these two enzymes (up to an approximate 58% decrease in alanine aminotransferase activity in the 1,000 ppm males and females). Serum alkaline phosphatase activity (a marker of hepatobiliary function) also demonstrated minimal exposure concentration-related decreases; the decreases were significant in male rats exposed to 250 ppm or greater at week 14 and in 500 and 1,000 ppm females at all time points. No exposurerelated lesions were observed in the livers of exposed rats. However, the relative liver weights increased with increasing exposure concentrations in essentially all male and female groups (Tables 6 and G2). The liver weight effects, coupled with the effects detected in the clinical chemistry, could suggest hepatic effects that altered liver function, resulting in some perturbation in bile acid uptake or excretion and liver enzyme production/turnover or, possibly, inhibition. Interestingly, serum albumin concentration (a nonspecific marker of hepatic function) was either unaffected or slightly (approximately 10%) increased in exposed males and females (Tables 5 and F1). The liver synthesizes albumin, and these results suggest that albumin synthesis was unaffected by exposure to cumene. No exposure-related gross lesions were observed. Relative kidney and liver weights of all exposed groups of males and relative liver weights of 125 ppm or greater females and relative kidney weights of 250 ppm or greater females were greater than those of the chamber controls (Tables 6 and G2). In addition, the absolute weights of the kidney were significantly increased in 125 ppm or greater males, and the absolute weights of the liver were NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 55 TABLE 5 Selected Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm n 10 Male Albumin (g/dL) Day 3 4.0 0.1 Day 23 3.8 0.1 Week 14 3.8 0.1 Alanine aminotransferase (IU/L) Day 3 64 2 Day 23 44 1 Week 14 113 6 Alkaline phosphatase (IU/L) Day 3 733 14 Day 23 496 15 Week 14 309 7 Sorbitol dehydrogenase (IU/L) Day 3 13 1 Day 23 13 0 Week 14 26 1 Bile acids (mol/L) Day 3 33.8 1.1 Day 23 32.1 2.9 Week 14 28.6 1.7 10 10 3.8 0.1 3.7 0.1 3.9 0.0 61 1 39 1* 113 11 759 16 490 11 294 4 12 1 13 0 24 2 39.9 0.9** 33.2 1.4 31.2 1.3 3.6 0.1* 3.8 0.1 3.8 0.0 61 1 39 1** 110 12 779 14 506 15 293 10 12 1 13 0 22 1 47.1 0.7** 37.7 1.4* 32.7 1.5 10 3.6 0.1** 3.8 0.1 3.9 0.0 64 2 38 1** 70 4** 794 24 485 15 283 9* 11 1 12 1 20 1** 50.9 1.7** 41.1 1.0** 30.9 0.9 10 3.8 0.1 3.9 0.1 4.1 0.1** 60 2 35 1** 61 3** 771 24 456 27 275 7** 12 0 12 0 17 1** 50.4 2.0** 43.4 1.7** 32.4 0.9* 10 3.8 0.1 3.9 0.0 4.2 0.0** 56 1** 35 0** 50 2** 674 15 470 12 250 6** 11 1 12 0 17 1** 55.6 4.1** 44.2 1.4** 35.9 2.2** Female Albumin (g/dL) Day 3 3.6 0.1 Day 23 3.8 0.0 Week 14 4.2 0.1 Alanine aminotransferase (IU/L) Day 3 50 1 Day 23 37 1 Week 14 80 8 Alkaline phosphatase (IU/L) Day 3 642 18 Day 23 372 6 Week 14 272 9 Sorbitol dehydrogenase (IU/L) Day 3 13 1 Day 23 12 1 Week 14 22 1 Bile acids (mol/L) Day 3 27.2 1.3 Day 23 23.0 0.7 Week 14 29.7 2.6 3.6 0.1 3.8 0.0 4.0 0.1 51 2 37 1 64 6 636 23 388 11 275 8 13 0 11 0 21 1 32.6 1.9* 27.6 1.5** 24.3 1.3 3.6 0.1 3.7 0.0 4.3 0.1 48 2 34 1 63 4 618 19 370 8 262 12 13 0 11 0 19 0 34.4 1.8** 29.1 0.9** 26.2 1.6 3.5 0.1 3.9 0.1 4.2 0.1 48 2 35 1 53 4** 608 12 362 9 267 7 12 0 12 0 19 1 38.5 1.8** 31.5 1.1** 32.1 4.3 3.6 0.0 3.8 0.1 4.3 0.1 47 2 34 1 53 3** 585 15* 348 7* 243 7* 12 0 11 0 18 1** 42.5 0.9** 38.5 2.9** 25.7 2.3 3.7 0.1 3.8 0.1 4.6 0.1** 42 2** 33 1 33 1** 474 10** 319 7** 207 6** 12 0 12 0 15 1** 38.0 1.7** 38.4 3.6** 23.8 0.9 * Significantly different (P#0.05) from the chamber control group by Dunn's or Shirley's test *a * P#0.01 Data are given as mean standard error. Ratios were calculated and statistical tests were performed on unrounded data. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 56 Cumene, NTP TR 542 TABLE 6 Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm n Male Necropsy body wt R. Kidney Absolute Relative Liver Absolute Relative 10 312 8 0.923 0.024 2.962 0.032 9.518 0.327 30.481 0.348 10 10 10 313 6 322 5 331 4 0.980 0.025 1.010 0.031*b 3.128 0.051** 3.131 0.056**b 1.059 0.021** 3.194 0.036** 10.123 0.267 10.260 0.264 32.279 0.278* 31.792 0.430* 11.170 0.302** 33.660 0.558** 10 314 5 1.070 0.017** 3.411 0.045** 11.589 0.282** 36.895 0.563** 10 323 5 1.152 0.023** 3.561 0.029** 12.637 0.288** 39.068 0.549** Female Necropsy body wt 195 2 190 3 194 4 R. Kidney Absolute Relative Liver Absolute Relative 0.637 0.016 3.263 0.061 5.553 0.130 28.442 0.389 0.636 0.010 3.355 0.049 5.669 0.148 29.858 0.458 0.649 0.018 3.322 0.057 5.885 0.204 30.094 0.634* 190 3 185 3 187 4 0.655 0.017 3.439 0.057* 5.959 0.137 31.289 0.412** 0.645 0.011 3.486 0.044** 5.979 0.133 32.286 0.386** 0.675 0.011 3.612 0.040** 6.923 0.227** 36.958 0.724** * Significantly different (P#0.05) from the chamber control group by Williams' test a** P#0.01 Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean standard error). b n=9 increased in 250 ppm or greater males and 1,000 ppm females. There were no significant differences between exposed and chamber control males in reproductive tissue evaluations (Table H1). Exposed females differed significantly from the chamber control females in the relative length of time spent in estrus and proestrus (Table H2). In cell proliferation analyses of the left kidney, the mean numbers of proximal tubule cells in the S-phase were significantly increased in the 500 and 1,000 ppm groups; however, the number of cells labeled and the labeling index were not significantly different from the chamber control group (Table 7). Concentrations of soluble protein NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 57 TABLE 7 Renal Toxicity Data for Male Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm n 10 Cells labeled Cells counted Labeling index (%)b Soluble protein (mg/mL) "2u-Globulin (nmol/g kidney) "2u-Globulin (ng/g soluble protein) 60.60 4.25 2,095 25 2.894 0.204 21.22 1.27 172.2 22.3 76.46 9.24 10 78.90 5.60 2,190 28 3.607 0.266 23.43 0.51 328.1 69.6 130.98 27.27 10 10 10 10 63.80 6.03 2,100 29 3.051 23.93 00..93305c 383.4 46.3**c 150.90 19.23*c 54.90 4.50 2,171 31 2.536 0.219 25.36 0.69* 420.7 50.1** 154.35 16.98** 43.70 6.21 2,268 42 1.944 0.306 25.51 0.73** 79.40 6.65 2,223 45 3.614 0.346 26.16 1.09** 363.2 41.4** 575.2 74.8** 133.01 14.25** 209.79 31.34** * Significantly different (P#0.05) from the chamber control group by Shirley's test *a * b P#0.01 Data are presented as mean standard error. Labeling index was calculated as the number of labeled cells divided by the total number of cells counted times 100. A minimum of 2,000 cells c were counted. n=9 and "2u-globulin in the right kidney from male rats demonstrated increases in all exposed groups. Soluble protein levels were significantly increased in male rats exposed to 250 ppm or greater. Amounts and concentrations of "2u-globulin were significantly increased in groups exposed to 125 ppm or greater. These findings are consistent with the hyaline droplet accumulations observed microscopically (Table 8). TABLE 8 Incidences of Nonneoplastic Lesions in the Kidney of Male Rats in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Number Examined Microscopically 10 Cortex Renal Tubule, Accumulation, Hyaline Dropleta 10 Cortex Renal Tubule, Regeneration 8 Medulla, Casts Granular 0 (1.1)b (1.0) 10 10 (1.4) 6 (1.2) 0 10 10 (1.9) 8 (1.5) 2 (1.0) 10 10 (2.4) 10 (1.8) 8** (1.5) *a * b Significantly different (P#0.01) from the chamber control group by the Fisher exact test Number of animals with lesion Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked 10 10 (3.0) 10 (2.1) 10** (2.5) 10 10 (2.9) 10 (2.1) 9** (2.2) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 58 Cumene, NTP TR 542 The severities of hyaline droplet accumulation in the renal cortical tubules of exposed male rats were increased, and the incidences and severities of renal cortical tubule regeneration were generally slightly increased (Table 8). These changes were accompanied by significantly increased incidences of medullary granular casts in groups exposed to 250 ppm or greater. Hyaline droplet accumulation was observed in all male rats and was minimal to mild in the chamber control and 62.5 ppm groups, minimal to moderate at 125 ppm, and mild to moderate at 250 ppm or greater. In males, minimal tubular regeneration occurred in the chamber controls; minimal to mild lesions were observed in groups exposed to 62.5 or 125 ppm, and moderate tubular regeneration occurred in groups exposed to 250 ppm or greater. In males, minimal casts were observed in the 125 ppm group; minimal to moderate casts occurred in the 250 ppm group, and minimal to marked casts occurred in groups exposed to 500 or 1,000 ppm. In hematoxylin and eosin stained sections, hyaline droplets appeared as brightly eosinophilic globules of varying size in the renal cortical tubular epithelium. In Mallory-Heidenhain stained sections, hyaline droplets were magenta and were more easily visualized for severity evaluations. Tubular regeneration was characterized by tubules lined by more basophilic epithelium and having larger nuclei than the surrounding tubules. Affected tubules typically occurred in clusters. The granular casts were generally present at the corticomedullary junction and caused dilation of the tubular lumen with lightly eosinophilic granular acellular material. Exposure Concentration Selection Rationale: In male rats, granular casts in the renal tubules of the medulla and increased severities of renal tubule regeneration and hyaline droplet accumulation in the cortex were caused by cumene exposure. The granular casts and renal tubule regeneration are indicative of some renal tubule epithelium damage. Based on the lack of mortalities and body weight effects, minimal organ weight changes, and the lack of lesions in other tissues, cumene exposure concentrations selected for the 2-year inhalation study in rats were 250, 500, and 1,000 ppm. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 59 2-YEAR STUDY Survival Estimates of 2-year survival probabilities for male and female rats are shown in Table 9 and in the Kaplan-Meier survival curves (Figure 2). Survival of all exposed groups of rats was similar to that of the chamber controls. TABLE 9 Survival of Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Male Animals initially in study Moribund Natural deaths Animals surviving to study termination PMeercaennstuprrvoivbaalb(ildiatyyso)fbsurvival at end of studya Survival analysisc 50 20 4 26 52 676 P=0.994N 50 24 3 23 46 665 P=0.511 50 21 2 27 54 671 P=1.000N 50 24 2 24 48 684 P=0.946 Female Animals initially in study Moribund Natural deaths Animals surviving to study termination Percent probability of survival at end of study Mean survival (days) Survival analysis 50 23 6 21 42 673 P=0.061N 50 18 5 27 54 677 P=0.357N 50 17 2 31 62 685 P=0.109N 50 15 3 32 64 684 P=0.071N a b c Kaplan-Meier determinations Mean of all deaths (uncensored, censored, and terminal sacrifice) The result of the life table trend test (Tarone, 1975) is in the chamber control column, and the results of the life table pairwise comparisons (Cox, 1972) with the chamber controls are in the exposed group columns. A negative trend or lower mortality in an exposed group is indicated by N. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 60 Cumene, NTP TR 542 FIGURE 2 Kaplan-Meier Survival Curves for Male and Female Rats Exposed to Cumene by Inhalation for 2 Years NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 61 Body Weights and Clinical Findings Mean body weights of exposed groups of males were similar to those of the chamber controls throughout the study; mean body weights of 1,000 ppm females were slightly less than those of the chamber controls during the second year of the study but were similar to the chamber controls at the end of the study (Tables 10 and 11; Figure 3). There were no clinical findings related to exposure to cumene. Seizures occurred in a few exposed males (chamber control, 0/50; 250 ppm, 1/50; 500 ppm, 4/50; 1,000 ppm, 1/50) and in a few chamber control and exposed females (6/50, 8/50, 5/50, 5/50). The seizures were clonic and of short duration. They were most frequently observed and recorded during daily animal care activities. The first seizure episode was observed in a female rat at 32 weeks of exposure. No evidence of brain lesions was found to account for the cause or effect of the clonic seizures noted clinically in the animals. Similar, sporadic seizures have been observed in F344/N rats in six other NTP inhalation or dermal studies at three different laboratories. In all of these studies, the single common factor was that the animals were housed individually. No such episodes have been observed in concurrent dosed feed, gavage, or drinking water studies in which animals are group housed. In the individually housed animals, most seizures were observed early in the day when technical and maintenance activities were commencing following the animals' dark cycle period. No deaths were associated with the seizures, and there were no correlations with body weight, feed consumption or composition, or histopathologic lesions in this or the other studies. Thus, these transient events were not considered to have affected the toxicologic or carcinogenic evaluations of this study. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 62 Cumene, NTP TR 542 TABLE 10 Mean Body Weights and Survival of Male Rats in the 2-Year Inhalation Study of Cumene Days on Study Chamber Control Av. Wt. No. of (g) Survivors 250 ppm Av. Wt. Wt. (% of No. of (g) controls) Survivors 500 ppm 1,000 ppm Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of (g) controls) Survivors (g) controls) Survivors 1 107 9 145 16 176 23 202 30 224 37 245 44 260 51 275 58 288 65 300 72 311 79 321 86 328 114 363 142 391 170 415 198 432 226 446 254 461 282 473 310 480 338 490 366 496 394 507 422 513 450 519 478 523 506 531 534 534 562 536 591 540 618 540 646 539 660 541 674 532 688 529 702 527 716 531 50 106 99 50 50 144 99 50 50 175 99 50 50 204 101 50 50 226 101 50 50 247 101 50 50 261 100 50 50 275 100 50 50 289 100 50 50 302 100 50 50 311 100 50 50 321 100 50 50 328 100 50 50 360 99 50 50 385 99 50 50 411 99 50 50 427 99 50 50 442 99 50 50 455 99 50 50 467 99 50 49 474 99 50 49 482 98 50 49 490 99 50 4499a 500 509 99 50 99 50 49 514 99 49 48 510 98 49 47 523 99 48 47 521 98 46 46 528 99 44 44 534 99 40 40 529 98 38 36 534 99 32 35 524 97 32 35 525 99 29 34 541 102 25 32 533 101 25 27 536 101 23 106 99 50 142 98 50 174 99 50 203 101 50 227 101 50 248 101 50 264 101 50 278 101 50 292 101 50 305 101 50 316 102 50 326 102 50 336 103 50 368 101 50 394 101 50 419 101 50 434 101 50 449 101 49 464 101 49 476 101 49 485 101 49 490 100 49 499 100 49 508 100 49 517 101 49 518 100 49 520 100 48 527 99 47 529 99 46 534 100 44 532 99 43 537 99 42 534 99 36 535 99 34 534 100 32 529 100 30 527 100 28 527 99 27 105 97 137 94 169 96 198 98 221 98 243 99 258 99 272 99 287 100 301 100 310 100 321 100 330 101 362 100 388 99 408 98 423 98 441 99 456 99 465 98 473 99 484 99 492 99 500 99 504 98 504 97 511 98 517 97 515 97 523 98 524 97 517 96 524 97 517 96 514 96 515 97 509 97 500 94 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 49 49 48 46 45 44 38 37 35 31 30 28 Mean for weeks 1-13 245 14-52 439 53-103 527 245 100 434 99 522 99 247 442 525 a Number of animals weighed was less than number of animals surviving. 101 101 100 242 99 433 99 512 97 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 63 TABLE 11 Mean Body Weights and Survival of Female Rats in the 2-Year Inhalation Study of Cumene Days on Study Chamber Control Av. Wt. No. of (g) Survivors 250 ppm Av. Wt. Wt. (% of No. of (g) controls) Survivors 500 ppm 1,000 ppm Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of (g) controls) Survivors (g) controls) Survivors 1 89 50 89 100 50 88 99 50 88 99 50 9 110 50 110 100 50 110 100 50 107 98 50 16 124 50 124 100 49 125 101 50 123 99 50 23 135 50 138 102 49 138 102 50 137 101 50 30 147 50 150 102 49 149 101 50 149 101 50 37 157 50 160 102 49 159 101 50 158 101 50 44 164 50 167 102 49 166 101 50 166 101 50 51 171 50 172 101 49 171 100 50 171 100 50 58 174 50 176 101 49 175 100 50 175 100 50 65 180 50 182 101 49 180 100 50 180 100 50 72 183 50 186 102 49 185 101 50 183 100 50 79 188 50 191 102 49 189 101 50 188 100 50 86 191 50 193 101 49 193 101 50 190 100 50 114 204 50 208 102 49 205 100 50 202 99 50 142 215 50 217 101 49 216 100 50 211 98 50 170 227 50 231 102 49 227 100 50 222 98 50 198 234 50 236 101 49 233 100 50 227 97 50 226 241 50 243 101 49 240 100 50 233 97 50 254 251 282 259 50 50 253 101 260 100 4499a 248 255 99 50 98 50 239 95 50 246 95 49 310 269 50 270 100 49 262 98 50 251 94 49 338 281 50 282 100 49 269 96 50 259 92 49 366 294 50 293 100 49 282 96 49 269 91 49 394 302 49 304 101 49 292 97 49 279 92 49 422 312 49 316 101 49 306 98 49 291 93 47 450 318 49 320 101 49 312 98 49 298 94 47 478 323 49 326 101 49 317 98 49 303 94 47 506 336 46 338 101 49 326 97 49 314 94 47 534 341 45 344 101 48 330 97 45 320 94 47 562 346 43 345 100 46 336 97 45 327 95 46 591 352 43 354 100 44 341 97 44 334 95 46 618 355 41 361 102 42 343 97 42 333 94 45 646 354 38 362 102 40 346 98 39 339 96 40 660 349 38 364 104 39 348 100 39 341 98 39 674 350 33 366 104 35 350 100 36 342 98 39 688 348 30 363 104 35 349 100 35 344 99 37 702 343 30 370 108 32 351 102 34 348 101 33 716 344 26 373 108 28 356 104 32 351 102 32 Mean for weeks 1-13 155 14-52 242 53-103 335 157 101 244 101 344 103 156 239 330 a Number of animals weighed was less than number of animals surviving. 101 99 99 155 100 232 96 321 96 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 64 Cumene, NTP TR 542 FIGURE 3 Growth Curves for Male and Female Rats Exposed to Cumene by Inhalation for 2 Years NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 65 Pathology and Statistical Analyses This section describes the statistically significant or biologically noteworthy changes in the incidences of neoplasms and nonneoplastic lesions of the nose and kidney. Summaries of the incidences of neoplasms and nonneoplastic lesions, statistical analyses of primary neoplasms that occurred with an incidence of at least 5% in at least one animal group, and historical incidences for the neoplasms mentioned in this section are presented in Appendix A for male rats and Appendix B for female rats. Nose: Incidences of adenoma of the respiratory epithelium occurred with a positive trend in males and were significantly increased in all exposed groups of males and in 250 ppm females; the incidences in all exposed groups of rats exceeded the ranges for historical chamber controls in inhalation studies and historical controls (all routes) (Tables 12, A2, A3a, B2, and B3). The incidences of multiple adenomas in the respiratory epithelium of exposed groups of males increased with increasing exposure concentration, and the incidence in the 1,000 ppm group was significantly increased. Microscopically, the adenomas were rounded masses or papillary projections arising from the turbinates or the lateral wall and protruding into the nasal cavity at section Levels I and II. The adenomas consisted of cords or clusters of basophilic cuboidal epithelial cells that often formed acinar patterns. Incidences of hyperplasia of basal cells in the olfactory epithelium were significantly increased in all exposed groups (Tables 12, A4, and B4). Basal cell hyperplasia was characterized by increased numbers of basal cells crowded along the basement membrane of the olfactory epithelium, most often affecting cells lining the nasal septum. In more severe lesions, affected basal cells infiltrated the overlying epithelium and occasionally formed small rosettes within the olfactory epithelium. The incidences of hyperplasia of the respiratory epithelium were significantly increased in all exposed groups of males, and the incidence of this lesion was significantly increased in 1,000 ppm females (Tables 12, A4, and B4). Hyperplasia of the respiratory epithelium and adenoma form a morphologic continuum. Hyperplastic lesions Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 66 Cumene, NTP TR 542 TABLE 12 Incidences of Neoplasms and Nonneoplastic Lesions of the Nose in Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Male Number Examined Microscopically OlfBaacstoalryCEelplaithelium, Hyperplasia, Respiratory Epithelium, Hyperplasia Goblet Cell, Hyperplasia 50 0 0 3 (1.7) Glands, Respiratory Epithelium, Adenoma Respiratory Epithelium, Adenoma, Multiple 0 0 Respiratory Epithelium, Adenoma (includes multiple and all sites)c ATOevdrejmurasintleladrlarrtaaettdeefe First incidence (days) 0/50 (0%) 0.0% 0--/2g6 (0%) Poly-3 testh P=0.004 50 19** (1.1)b 15** (2.0) 11* (2.3) 0 1 7/50 (14%) 17.6% 5/23 (22%) 639 P=0.006 49 27** (1.0) 16** (2.9) 7 (2.3) 1 2 18/49 (37%) 43.2% 13/27 (48%) 638 P<0.001 50 26** (1.0) 23** (2.7) 5 (2.0) 0 6* 10/50 (20%) 23.3% 7/24 (29%) 674 P<0.001 Female Number Examined Microscopically Olfactory Epithelium, Hyperplasia, Basal Cell Respiratory Epithelium, Hyperplasia Respiratory Epithelium, Adenomai Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 50 0 0 0/50 (0%) 0.0% 0/21 (0%) -- P=0.320 48 14** (1.0) 0 5/48 (10%) 12.2% 5/27 (19%) 730 (T) P=0.030 50 25** (1.0) 4 (3.0) 4/50 (8%) 9.3% 4/31 (13%) 730 (T) P=0.066 50 31** (1.1) 6* (2.3) 3/50 (6%) 6.9% 2/32 (6%) 638 P=0.130 (T)Terminal sacrifice * Significantly different (P#0.05) from the chamber control group by the Poly-3 test a** b c P#0.01 Number of animals with lesion Average severity grade of lesions in affected animals: Historical incidence for 2-year inhalation studies with 1=minimal, 2=mild, 3=moderate, 4=marked chamber control groups (mean standard deviation): 1/447 (0.2% 0.7%), d e f g h range 0%-2%; all routes: 2/1,439 (0.1% 0.5%), range 0%-2% Number of animals with neoplasm per number of animals with nose examined microscopically Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Not applicable; no neoplasms in animal group Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence is the P value corresponding to pairwise comparison between the chamber controls and that exposed group. The Poly-3 test accounts for differential i mortality in animals that do not reach terminal sacrifice. Historical incidence for inhalation studies: 0/496; all routes: 0/1,343 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 67 consisted of minimal to marked increases in populations of homogeneous respiratory epithelial cells with less distinct acinar formation than adenomas. The incidence of goblet cell hyperplasia was significantly increased in 250 ppm males (Tables 12 and A4). Goblet cell hyperplasia was characterized by minimal to moderate increases in the number of mucous cells in respiratory epithelium. Hyperplasia was often accompanied by enlargement or hypertrophy of the mucous cells. The hyperplasia resulted in thickening of the respiratory epithelium and an undulating surface. The affected cells were taller, with many large cells containing abundant amounts of mucin. The nuclei appeared more numerous. Kidney: The incidences of renal tubule adenoma were increased in all exposed groups of males compared to the chamber control group and exceeded the historical range for chamber controls in inhalation studies (Tables 13, A2, and A3b). The incidences of renal tubule carcinoma were increased in 500 and 1,000 ppm males and exceeded the historical chamber control range; one 500 ppm male rat had a bilateral renal tubule carcinoma. The incidence of renal tubule adenoma or carcinoma (combined) was significantly increased in 500 ppm males, and the incidences of these combined lesions in all exposed groups of males exceeded the range for historical chamber controls. Six of the renal tubule adenomas were large enough to be observed grossly at necropsy and were described as nodules or lesions varying in size from 1 to 3 mm in diameter. Microscopically, renal tubule adenomas were typically well circumscribed, discrete, rounded or oval masses greater than five times the diameter of a normal renal tubule, composed of solid aggregates, small nests or tubule-like structures of neoplastic epithelial cells without obvious lumens. The neoplastic cells were round to polygonal in shape with pale-staining, foamy cytoplasm. Larger adenomas had areas of cells with central vacuolation. Seven of the renal tubule cell carcinomas were observed at necropsy and were described as masses or nodules varying in size from 2 mm to 1.0 to 1.5 cm in diameter or as a dilated, thickened pelvis. Microscopically, renal tubule carcinomas were large, less discrete masses of neoplastic cells that were locally invasive and composed of a mixture of round cells with large vesicular nuclei and abundant, pale eosinophilic cytoplasm forming sheets and large nests resembling tubules. Some Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 68 Cumene, NTP TR 542 TABLE 13 Incidences of Neoplasms and Nonneoplastic Lesions of the Kidney in Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Male Number Renal ETxuabmulien,eHd yMpiecrrpolsacsoiapaically Papilla, Mineralization Pelvis, Transitional Epithelium, Hyperplasia Nephropathy RenATOaevdlrejmTurasuintlbelaudrllaerrtaa,ettdAeefedenomac FPoirlsyt-i3ncteidstegnce (days) 50 0 5 (1.0) 3 (1.7) 47 (2.3) 1/50 (2%) 2.4% 1/26 (4%) 729 (T) P=0.219 Renal Tubule, Carcinoma, Bilateral Renal Tubule, Carcinoma (includes bilateral)h Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Renal Tubule, Adenoma or Carcinomai Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Renal Tubule, Lipomaj 0 1/50 (2%) 2.4% 1/26 (4%) 729 (T) P=0.180 2/50 (4%) 4.8% 2/26 (8%) 729 (T) P=0.087 1 50 3 (3.3)b 35** (1.7) 5 (1.8) 47 (2.6) 4/50 (8%) 10.0% 1/23 (4%) 665 P=0.165 0 1/50 (2%) 2.5% 1/23 (4%) 729 (T) P=0.749 5/50 (10%) 12.5% 2/23 (9%) 665 P=0.198 0 50 8** (2.6) 44** (2.1) 14** (2.4) 47 (2.9) 5/50 (10%) 12.1% 2/27 (7%) 679 P=0.099 1 3/50 (6%) 7.3% 2/27 (7%) 639 P=0.302 8/50 (16%) 19.2% 4/27 (15%) 639 P=0.044 0 50 6* (2.2) 41** (2.1) 15** (2.0) 50 (2.7) 4/50 (8%) 9.3% 2/24 (8%) 635 P=0.187 0 3/50 (6%) 7.0% 2/24 (8%) 618 P=0.314 7/50 (14%) 16.2% 4/24 (17%) 618 P=0.087 1 Female Number Examined Microscopically Nephropathy 50 38 (1.4) 50 37 (1.5) 50 41 (1.9) 50 44 (1.9) (T)Terminal sacrifice * Significantly different (P#0.05) from the chamber control group by the Poly-3 test a** b c P#0.01 Number of animals with lesion Average severity grade of lesions in affected animals: Historical incidence for 2-year inhalation studies with 1=minimal, 2=mild, 3=moderate, 4=marked chamber control groups (mean standard deviation): 4/449 (0.9% 1.0%), d e f g range 0%-2%; all routes: 8/1,436 (0.6% 0.8%), range 0%-2% Number of animals with neoplasm per number of animals with kidney examined microscopically Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence is the P value corresponding to pairwise comparison between the chamber controls and that exposed group. The Poly-3 test accounts for differential h i j mortality in animals that do not reach terminal sacrifice. Historical incidence for inhalation studies: 2/449 (0.4% 0.9%), range 0%-2%; all routes: 2/1,436 (0.1% 0.5%), range 0%-2% Historical incidence for inhalation studies: 6/449 (1.3% 1.4%), range 0%-4%; all routes: 10/1,436 (0.7% 1.0%), range 0%-4% Historical incidence for inhalation studies: 1/449 (0.2% 0.7%), range 0%-2%; all routes: 2/1,436 (0.1% 0.5%), range 0%-2% NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 69 carcinomas had small numbers of vacuolated, signet ring, neoplastic cells. A few carcinomas had areas of necrosis or tubular structures with necrotic centers surrounded by a thin fibrous stroma. Cellular atypia and pleomorphism were usually present. One chamber control and one 1,000 ppm male each had a lipoma, a benign tumor (Tables 13 and A1). The lipoma in the chamber control male occupied the majority of the renal medulla and was composed of sheets of vacuolated cells with a few residual renal tubules and cystic spaces within the tumor. The lipoma in the 1,000 ppm male also was primarily composed of vacuolated cells but located in the cortex. These lipomas are infrequent tumors in F344/N rats and unrelated to exposure. The tumor in the chamber control male is the only lipoma in the current historical control data for inhalation studies, and only one other lipoma is included for all routes of exposure combined (Tables 13 and A3b). Incidences of renal tubule hyperplasia were increased in all exposed groups of male rats and were significantly increased in the 500 and 1,000 ppm groups (Tables 13 and A4). Microscopically, renal tubular cell hyperplasia consisted of mild to marked foci having single to multiple cortical tubules composed of multiple layers of epithelial cells with pale-staining, foamy cytoplasm that partially filled the lumen and enlarged the tubules. The epithelial cells were variably enlarged with distinct cell borders, expanded eosinophilic cytoplasm, variable nuclear size, and multiple, enlarged nucleoli. Affected tubules were generally, but not always, larger than normal but less than five times the diameter of a normal renal tubule. Renal tubule hyperplasia, as defined in the current study, was distinguished from regenerative epithelial changes commonly seen as a part of nephropathy and was considered a preneoplastic lesion. Renal tubule hyperplasia, adenoma, and carcinoma are part of a morphologic continuum. The incidences of mineralization of the renal papilla were significantly increased in all exposed groups of males (Tables 13 and A4). Microscopically, the mineralization consisted of elongated profiles of dark basophilic or black Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 70 Cumene, NTP TR 542 granular material within the lumens of the tubules. This linear pattern is consistent with mineralization associated with "2u-globulin nephropathy. Significantly increased incidences of minimal to marked hyperplasia of the transitional epithelium of the renal pelvis occurred in 500 and 1,000 ppm males (Tables 13 and A4). Hyperplasia of the transitional epithelium was characterized by increased numbers of cells in the epithelium lining the pelvis, often with formation of papillary projections. These are common findings in rats and often increase with increased severities of nephropathy. Although the incidences of minimal to marked nephropathy in exposed groups of males and females were not significantly different from those in the chamber controls, the severity of nephropathy generally increased with increasing exposure concentration (Tables 13, A4, and B4). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 71 MICE 2-WEEK STUDY All mice exposed to 4,000 ppm died on day 1; all mice exposed to 2,000 ppm died by the morning of day 2, and four female mice exposed to 1,000 ppm died by day 4 (Table 14). There were no exposure-related changes in mean body weights among survivors in either sex. Mice exposed to 2,000 ppm were severely lethargic after the first exposure. The four female mice exposed to 1,000 ppm that died early exhibited signs of lethargy and ataxia. Ataxia was most apparent during the first few days of exposure in week 1, and the severity of lethargy and ataxia was greater in females. TABLE 14 Survival and Body Weights of Mice in the 2-Week Inhalation Study of Cumene Concentration (ppm) Survivala Mean Body Weightb (g) Initial Final Change Final Weight Relative to Controls (%) Male 0 250 500 1,000 2,000 4,000 5/5 21.9 0.3 25.3 0.5 3.4 0.4 5/5 22.6 0.8 26.1 0.5 3.4 0.6 5/5 23.1 0.8 25.1 0.5 2.0 0.6 005///555cd 23.1 0.5 23.0 0.5 22.7 0.6 25.9 0.8 -- -- 2.7 0.3 -- -- 103 99 102 -- -- Female 0 250 500 1,000 2,000 4,000 5/5 18.6 0.4 21.3 0.3 2.7 0.3 5/5 19.2 0.6 21.5 0.6 2.4 0.6 0015////5555ecd 19.3 0.3 18.9 0.4 18.9 0.4 18.7 0.7 21.6 0.6 22.6 0.6 -- -- 2.4 0.5 2.4 0.5 -- -- 101 101 106 -- -- a Number of animals surviving at 2 weeks/number initially in group b Weights and weight changes are given as mean standard error. Subsequent calculations are based on animals surviving to the end of the c d e study. Differences from the chamber control group are not significant by Dunnett's test. Day of deaths: 2 Day of deaths: 1 Day of deaths: 3, 3, 3, 4 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 72 Cumene, NTP TR 542 Absolute and relative liver weights were increased in all groups of surviving male mice and the 250 and 500 ppm females (Table G3). Absolute and relative kidney weights were generally increased in both sexes of surviving mice compared to the chamber control groups; the increases were significant in 250 ppm males. No microscopic changes were present in either sex that would account for the increased liver or kidney weights or for the early deaths. Thymus weights were decreased in 1,000 ppm males. Exposure Concentration Selection Rationale: Based on the mortalities at 2,000 and 4,000 ppm and signs of central nervous system effects, cumene exposure concentrations selected for the 3-month inhalation study in mice were 62.5, 125, 250, 500, and 1,000 ppm. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 73 3-MONTH STUDY With the exception of eight 1,000 ppm females, all mice survived to the end of the study (Table 15). Final mean body weights and body weight gains of males exposed to 250 ppm or greater were generally less than those of the chamber controls. Ataxia was observed for several days in 1,000 ppm males, but the animals recovered and appeared to develop a tolerance by day 3. The eight 1,000 ppm female mice that died during the first week of the study exhibited clinical signs of acute toxicity, including lethargy or ataxia. TABLE 15 Survival and Body Weights of Mice in the 3-Month Inhalation Study of Cumene Concentration (ppm) Survivala Mean Body Weightb (g) Initial Final Change Male 0 62.5 125 250 500 1,000 10/10 10/10 10/10 10/10 10/10 10/10 23.4 0.3 23.1 0.3 23.2 0.5 22.9 0.2 22.9 0.3 23.2 0.3 37.5 0.6 36.1 0.8 35.8 0.8 35.3 0.7* 34.5 0.8** 34.1 0.6** 14.1 0.7 13.0 0.7 12.6 0.8 12.4 0.6 11.6 0.8* 10.9 0.7** Final Weight Relative to Controls (%) 96 96 94 92 91 Female 0 62.5 125 250 500 1,000 10/10 10/10 10/10 10/10 120//1100c 19.3 0.3 19.0 0.2 19.5 0.2 19.3 0.3 19.3 0.3 19.1 0.3 30.6 1.0 29.7 1.0 29.6 0.8 29.6 0.8 28.3 0.5 29.2 0.4 11.3 0.8 10.7 0.9 10.1 0.8 10.3 0.9 9.0 0.3 8.9 1.1 97 97 97 92 95 * Significantly different (P#0.05) from the chamber control group by Williams' test a** b P#0.01 Number Weights of animals surviving at 3 months/number initially and weight changes are given as mean standard in group error. Subsequent calculations are based on animals surviving to the end c of the study. Week of deaths: 1 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 74 Cumene, NTP TR 542 There were no toxicologically relevant changes in the hematology variables for mice (Table F2). Significant increases in absolute liver weights occurred in mice exposed to 500 or 1,000 ppm, and significant increases in relative liver weights occurred in groups exposed to 125 ppm or greater (Tables 16 and G4). Although the weight of the cauda epididymis and the spermatid count were significantly decreased in 1,000 ppm males, there were no other significant differences between exposed and chamber control groups in reproductive tissue evaluations in males or vaginal cytology parameters in females (Tables H3 and H4). No exposure-related gross lesions were observed. TABLE 16 Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Male n 10 10 10 Necropsy body wt 38.3 0.7 37.7 0.9 37.0 0.8 Liver Absolute Relative 1.531 0.027 1.601 0.049 1.607 0.041 40.048 0.559 42.490 0.894 43.485 0.551* 10 36.1 0.8 10 35.8 0.9* 10 34.7 0.6** 1.591 0.048 1.705 0.048* 1.913 0.070** 44.052 0.746** 47.668 0.795** 55.103 1.501** Female n 10 10 10 Necropsy body wt 32.4 1.1 31.0 1.2 31.4 1.1 Liver Absolute Relative 1.453 0.037 1.430 0.047 1.495 0.053 45.016 1.043 46.200 0.778 47.622 0.576* 10 31.5 1.1 10 29.8 0.7 2 30.8 1.3 1.552 0.045 1.593 0.042* 1.910 0.110** 49.380 0.521** 53.510 0.663** 62.071 1.054** * Significantly different (P#0.05) from the chamber control group by Williams' test a** P#0.01 Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean standard error). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 75 Incidences of minimal to mild liver necrosis were significantly increased in male mice exposed to 1,000 ppm (Table 17). Incidences of focal chronic inflammation were significantly increased in females exposed to 62.5, 125, 250, or 500 ppm. Necrosis varied from tiny foci of lymphocytes with a few deeply eosinophilic individual necrotic hepatocytes to foci having coagulative or liquefactive necrosis with scattered neutrophils and lymphocytes involving hepatocytes and adjacent liver tissues. Chronic inflammation consisted of lesions where the predominant features were clusters of lymphocytes and a few neutrophils. Liver necrosis and focal chronic inflammation in male and female mice were considered to be parts of a pathologic continuum. Sporadic incidences of minimal to mild squamous epithelial hyperplasia and mild inflammation of the mucosa occurred in the forestomach of exposed groups of female mice (Table 17). Squamous epithelial hyperplasia consisted of minimal focal to diffuse thickening of the squamous epithelium. The affected epithelium was five to six cell layers thick as compared to the three to four cell layers for normal squamous epithelium. Acute and chronic inflammation of the forestomach was associated with hyperplasia in 500 and 1,000 ppm female mice. This inflammation was minimal, confined to the lamina propria, and consisted of a mixture of inflammatory cells, primarily neutrophils and macrophages. The forestomach lesions had low incidences and were of uncertain relationship to cumene exposure. Marked necrosis of the thymus was observed in the eight female mice exposed to 1,000 ppm cumene that died during the first week of the study (Table 17). The changes in the thymus were characterized by thymic lymphocytes with small, shrunken, dense nuclei. In multifocal areas, nuclei of the lymphocytes were fragmented or disintegrated. The thymic necrosis was considered to be a nonspecific terminal event associated with glucocorticoid release and not the cause of death; the cause of death was not explained. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 76 Cumene, NTP TR 542 TABLE 17 Incidences of Selected Nonneoplastic Lesions in Mice in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Male Livera Inflammation, Chronic Focalb Necrosis 10 4 0 (1.0)c 10 3 (1.0) 1 (1.0) 10 2 (1.0) 1 (2.0) 10 3 (1.0) 1 (1.0) 10 5 (1.0) 1 (1.0) 10 4 (1.0) 5* (1.2) Female Liver Inflammation, Chronic Focal Necrosis 10 1 (1.0) 4 (1.3) Forestomach Hyperplasia, Squamous Inflammation, Acute Inflammation, Chronic Active 10 0 0 0 Thymus Necrosis 10 0 10 10** (1.0) 0 10 1 (1.0) 0 0 10 0 10 10** (1.0) 0 10 0 0 0 10 0 10 9** (1.0) 0 10 0 0 2 (1.0) 10 0 10 7** (1.0) 2 (1.5) 10 2 (2.0) 0 2 (1.0) 10 0 10 2 (1.0) 0 10 1 (1.0) 1 (1.0) 0 10 8** (4.0) * Significantly different (P#0.05) from the chamber control group by the Fisher exact test *a * b c P#0.01 Number Number Average of animals with tissue examined microscopically of animals with lesion severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked Exposure Concentration Selection Rationale: Based on slight decreases in body weights in males and minimal effects on organ weights and incidences of lesions, cumene exposure concentrations selected for the 2-year inhalation study in male mice were 250, 500, and 1,000 ppm. Because of a lower survival rate for 1,000 ppm females, incidences of thymic necrosis at 1,000 ppm, and incidences of liver and forestomach lesions, cumene exposure concentrations selected for the 2-year inhalation study in female mice were 125, 250, and 500 ppm. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 77 2-YEAR STUDY Survival Estimates of 2-year survival probabilities for male and female mice are shown in Table 18 and in the Kaplan-Meier survival curves (Figure 4). An exposure concentration-related decrease in survival occurred in male mice, and the survival of 1,000 ppm males was significantly less than that of the chamber controls. Survival of exposed groups of female mice was similar to that of the chamber controls. TABLE 18 Survival of Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Male Animals initially in study Moribund Natural deaths Animals surviving to study termination PMeercaennstuprrvoivbaalb(ildiatyyso)fbsurvival at end of studya Survival analysisc 50 7 5 38 76 709 P=0.001 50 9 7 34 68 696 P=0.475 50 13 7 30 60 693 P=0.150 50 20 7 23 46 665 P=0.004 Chamber Control Female Animals initially in study 50 Moribund Natural deaths Animals surviving to study termination Percent probability of survival at end of study Mean survival (days) 8 5 37 74 685 Survival analysis P=0.996 125 ppm 50 10 4 36 72 698 P=1.000 250 ppm 50 8 3 39 78 712 P=0.728N 500 ppm 50 12 3 35d 70 714 P=0.982 a b c Kaplan-Meier determinations Mean of all deaths (uncensored, censored, and terminal sacrifice) The result of the life table trend test (Tarone, 1975) is in the chamber control column, and the results of the life table pairwise comparisons d (Cox, 1972) with the chamber controls are in the exposed group columns. A lower mortality in an exposed group is indicated by N. Includes one animal that died during the last week of the study Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 78 Cumene, NTP TR 542 FIGURE 4 Kaplan-Meier Survival Curves for Male and Female Mice Exposed to Cumene by Inhalation for 2 Years NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 79 Body Weights and Clinical Findings Mean body weights of 1,000 ppm males were generally less than those of the chamber controls after week 8 of the study, and those of 500 ppm females were less from week 28 until week 76 of the study (Tables 19 and 20; Figure 5). There were no clinical findings related to exposure to cumene; however, thinness and abnormal breathing were observed somewhat more frequently in 1,000 ppm males and 500 ppm females late in the study. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 80 Cumene, NTP TR 542 TABLE 19 Mean Body Weights and Survival of Male Mice in the 2-Year Inhalation Study of Cumene Days on Study Chamber Control Av. Wt. No. of (g) Survivors 250 ppm Av. Wt. Wt. (% of No. of (g) controls) Survivors 500 ppm 1,000 ppm Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of (g) controls) Survivors (g) controls) Survivors 1 23.7 8 25.6 15 26.9 22 27.8 29 28.7 36 30.1 43 31.1 50 31.8 57 32.8 64 33.8 71 34.4 78 35.0 85 35.6 113 39.2 141 42.4 169 45.3 197 46.0 225 48.5 253 49.7 281 50.8 309 51.4 337 52.2 365 52.7 393 53.2 421 53.5 449 53.6 477 53.8 505 53.9 533 53.5 561 52.8 589 52.8 617 52.5 645 52.7 659 52.4 673 53.4 687 52.8 701 52.2 715 52.1 50 23.8 100 50 23.7 100 50 23.6 99 50 50 25.1 98 50 25.5 100 50 25.3 99 50 50 26.2 97 50 26.4 98 50 26.6 99 50 50 27.2 98 50 27.5 99 50 27.3 98 50 50 28.1 98 50 28.4 99 50 28.3 98 50 50 29.0 97 50 29.2 97 50 28.9 96 50 50 29.9 96 50 30.0 96 50 29.8 96 50 50 30.5 96 50 30.8 97 50 30.3 96 50 50 31.3 95 50 31.6 96 50 31.1 95 50 50 32.3 95 50 32.2 95 50 31.4 93 50 50 32.7 95 50 32.7 95 50 32.0 93 50 50 33.4 96 50 33.4 95 50 32.6 93 50 50 34.3 96 50 34.1 96 50 33.2 93 50 50 37.5 96 50 37.5 96 50 35.5 91 50 50 40.3 95 50 40.3 95 50 38.3 90 50 50 43.1 95 50 43.2 96 50 41.1 91 50 50 44.6 97 50 45.1 98 50 42.8 93 50 50 45.4 94 50 47.0 97 50 44.6 92 50 50 47.8 96 50 48.3 97 50 46.2 93 50 50 48.7 96 50 49.4 97 50 47.5 94 50 50 50.1 98 50 50.5 98 50 48.6 95 50 50 51.0 98 50 51.2 98 50 49.8 95 50 50 51.5 98 50 51.7 98 50 50.5 96 50 50 51.8 97 50 52.4 98 49 51.0 96 49 50 52.2 98 50 52.5 98 49 51.5 96 47 50 52.3 98 50 53.0 99 49 51.6 96 47 50 52.5 98 49 53.0 98 49 51.5 96 46 50 52.7 98 48 53.1 99 48 52.0 96 45 50 51.6 96 48 52.6 98 47 51.8 97 44 49 52.4 99 46 52.1 99 47 50.7 96 43 48 52.1 99 46 52.7 100 45 50.6 96 42 48 51.8 99 44 52.5 100 44 49.6 95 39 45 50.9 97 43 52.4 100 43 50.1 95 36 45 50.2 96 41 53.2 102 41 50.4 96 35 40 50.3 94 40 52.6 99 39 49.7 93 34 40 50.3 95 38 52.7 100 37 48.7 92 32 39 49.4 95 36 51.1 98 36 48.3 93 27 39 49.2 95 35 50.3 97 34 49.3 95 24 Mean for weeks 1-13 30.6 14-52 47.3 53-103 53.0 29.5 96 45.4 96 51.3 97 29.7 97 45.8 97 52.4 99 29.3 96 43.8 93 50.5 95 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 81 TABLE 20 Mean Body Weights and Survival of Female Mice in the 2-Year Inhalation Study of Cumene Days on Study Chamber Control Av. Wt. No. of (g) Survivors 125 ppm Av. Wt. Wt. (% of No. of (g) controls) Survivors 250 ppm 500 ppm Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of (g) controls) Survivors (g) controls) Survivors 1 19.6 8 20.7 15 21.2 22 22.2 29 23.3 36 24.4 43 25.2 50 25.7 57 26.3 64 27.3 71 26.9 78 27.6 85 28.9 113 32.7 141 36.0 169 38.3 197 41.0 225 44.4 253 46.4 281 48.3 309 51.3 337 53.7 365 55.6 393 57.3 421 58.6 449 59.9 477 60.2 505 60.7 533 59.0 561 59.6 589 59.6 617 58.7 645 57.5 659 58.1 673 58.0 687 57.8 701 56.3 715 56.0 50 19.7 100 50 19.8 101 50 19.8 101 50 50 20.6 99 50 20.6 99 50 20.7 100 50 50 21.5 101 50 21.9 103 50 21.7 103 50 50 22.3 101 50 22.5 102 50 22.6 102 50 50 23.4 101 50 23.6 101 50 23.7 102 50 50 24.5 100 50 24.6 101 50 24.8 102 50 50 25.5 101 50 25.6 101 50 25.7 102 50 50 26.2 102 50 26.3 102 50 26.5 103 50 50 26.6 101 50 27.0 103 50 26.8 102 50 50 27.1 99 50 27.5 101 50 26.9 99 50 50 27.9 104 50 27.7 103 50 27.6 103 50 50 27.2 99 50 28.3 103 50 28.1 102 50 50 29.6 102 50 29.4 102 50 28.6 99 50 50 32.9 101 50 32.7 100 50 31.6 97 50 50 37.0 103 50 36.2 101 50 34.4 96 50 50 41.2 108 50 39.6 103 50 36.7 96 50 49 43.2 105 50 41.7 102 50 38.7 94 50 49 46.0 104 50 44.5 100 50 40.8 92 50 49 48.7 105 50 46.2 100 50 42.4 92 50 49 51.3 106 50 48.7 101 50 43.9 91 50 47 53.9 105 50 51.0 100 50 47.0 92 50 47 56.5 105 50 52.9 98 50 49.0 91 50 47 58.2 105 50 55.0 99 50 51.4 92 50 47 60.0 105 49 56.2 98 50 52.7 92 50 47 61.2 104 49 57.1 97 50 54.3 93 50 47 62.5 104 49 58.2 97 50 55.5 93 50 47 63.5 105 49 59.1 98 50 56.8 94 50 47 64.0 106 47 59.7 98 49 57.2 94 50 47 63.4 107 47 58.7 100 48 58.0 98 49 45 62.5 105 46 58.5 98 48 57.5 97 49 45 62.7 105 46 58.9 99 48 59.3 100 48 44 60.7 103 45 56.7 97 48 58.7 100 48 44 59.6 104 43 57.3 100 46 58.4 102 48 41 60.1 104 41 57.4 99 45 58.2 100 48 41 60.3 104 40 57.2 99 45 56.9 98 46 40 59.7 103 40 56.4 98 43 56.4 98 44 38 58.3 104 39 55.1 98 41 54.7 97 42 38 57.8 103 37 54.6 98 39 55.1 98 38 Mean for weeks 1-13 24.6 14-52 43.6 53-103 58.3 24.8 101 45.6 105 60.9 104 25.0 102 43.7 100 57.3 98 24.9 101 40.5 93 56.3 97 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 82 Cumene, NTP TR 542 FIGURE 5 Growth Curves for Male and Female Mice Exposed to Cumene by Inhalation for 2 Years NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 83 Pathology and Statistical Analyses This section describes the statistically significant or biologically noteworthy changes in the incidences of neoplasms and/or nonneoplastic lesions of the lung, nose, liver, and forestomach. Summaries of the incidences of neoplasms and nonneoplastic lesions, statistical analyses of primary neoplasms that occurred with an incidence of at least 5% in at least one animal group, and historical incidences for the neoplasms mentioned in this section are presented in Appendix C for male mice and Appendix D for female mice. Lung: The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) in all exposed groups of mice occurred with positive trends, were significantly greater than those in the chamber controls, and exceeded the ranges for historical chamber controls in inhalation studies and historical controls (all routes) (Tables 21, C2, C3a, D2, and D3a). Significantly increased incidences of multiple alveolar/bronchiolar adenoma and multiple alveolar/bronchiolar carcinoma occurred in all exposed groups. Microscopically, most of the alveolar/bronchiolar adenomas had papillary patterns. Tumor margins were usually well-demarcated with compression of the surrounding parenchyma. Alveolar/bronchiolar carcinomas varied from well-differentiated neoplasms with papillary patterns to poorly circumscribed, infiltrative tumors consisting of densely packed pleomorphic cells having multiple layers of nuclei. Often, prominent alveolar infiltrates of macrophages and occasional multinucleate giant cells were associated with these carcinomas. The incidences of alveolar epithelial bronchiole metaplasia and bronchiole hyperplasia were significantly increased in all exposed groups of mice, and the severity increased in all exposed groups (Tables 21, C4, and D4). Microscopically, alveolar epithelial bronchiole metaplasia occurs when the flat epithelium of the alveolar ducts and adjacent alveolar septa is replaced by cells similar in appearance to those normally lining terminal bronchioles. Minimal to marked alveolar epithelial bronchiole metaplasia in the exposed mice was characterized by increases in the numbers of plump cuboidal epithelial cells having large hyperchromatic nuclei that lined the alveolar walls adjacent to and extending from the terminal bronchioles. Minimal to marked bronchiole hyperplasia resulted in Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 84 Cumene, NTP TR 542 TABLE 21 Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Male Number Examined Microscopically Alveolar Epithelium, Bronchiole, Metaplasiaa Bronchiole, Hyperplasia 50 5 (1.4)b 0 Alveolar/bronchiolar Adenoma, Multiple 1 Alveolar/bronchiolar Adenoma (includes multiple)c Overall rated 13/50 (26%) Adjusted ratee 27.5% Terminal ratef 10/38 (26%) First incidence (days) Poly-3 testg 628 P<0.001 Alveolar/bronchiolar Carcinoma, Multiple 0 Alveolar/bronchiolar Carcinoma (includes multiple)h Overall rate 9/50 (18%) Adjusted rate 19.1% Terminal rate 6/38 (16%) First incidence (days) 631 Poly-3 test P<0.001 Alveolar/bronchiolar Adenoma or Carcinomai Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 19/50 (38%) 39.8% 14/38 (37%) 628 P<0.001 50 43** (2.9) 11** (2.1) 12** 31/50 (62%) 66.7% 25/34 (74%) 551 P<0.001 8** 19/50 (38%) 41.5% 15/34 (44%) 551 P=0.014 38/50 (76%) 81.4% 31/34 (91%) 551 P<0.001 Chamber Control Female Number Examined Microscopically Alveolar Epithelium, Bronchiole, Metaplasia Bronchiole, Hyperplasia 50 0 0 Alveolar/bronchiolar Adenoma, Multiple 0 Alveolar/bronchiolar Adenoma (includes multiple)j Overall rate 1/50 (2%) Adjusted rate 2.3% Terminal rate 1/37 (3%) First incidence (days) 731 (T) Poly-3 test P<0.001 125 ppm 50 42** (2.6) 17** (2.7) 13** 26/50 (52%) 56.3% 21/36 (58%) 555 P<0.001 50 42** (3.1) 17** (3.2) 15** 31/50 (62%) 66.9% 23/30 (77%) 512 P<0.001 20** 32/50 (64%) 70.5% 25/30 (83%) 565 P<0.001 42/50 (84%) 89.5% 30/30 (100%) 512 P<0.001 250 ppm 50 39** (3.0) 18** (2.8) 20** 29/50 (58%) 67.9% 20/23 (87%) 480 P<0.001 17** 33/50 (66%) 71.3% 12/23 (52%) 420 P<0.001 43/50 (86%) 92.1% 21/23 (91%) 420 P<0.001 500 ppm 50 49** (2.9) 10** (2.8) 20** 36/50 (72%) 74.5% 31/39 (80%) 495 P<0.001 50 47** (3.3) 14** (2.8) 30** 38/50 (76%) 77.9% 29/35 (83%) 565 P<0.001 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 85 TABLE 21 Incidences of Neoplasms and Nonneoplastic Lesions of the Lung in Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm 500 ppm Female (continued) Alveolar/bronchiolar Carcinoma, Multiple 0 Alveolar/bronchiolar Carcinoma (includes multiple)k Overall rate 3/50 (6%) Adjusted rate 6.7% Terminal rate 2/37 (5%) First incidence (days) 533 Poly-3 test P<0.001 Alveolar/bronchiolar Adenoma or Carcinomal Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 4/50 (8%) 8.9% 3/37 (8%) 533 P<0.001 6* 16/50 (32%) 35.3% 13/36 (36%) 646 P<0.001 31/50 (62%) 66.8% 25/36 (69%) 555 P<0.001 7** 20/50 (40%) 41.9% 15/39 (39%) 618 P<0.001 42/50 (84%) 86.0% 34/39 (87%) 495 P<0.001 19** 34/50 (68%) 69.5% 24/35 (69%) 513 P<0.001 46/50 (92%) 92.4% 33/35 (94%) 513 P<0.001 (T)Terminal sacrifice * Significantly different (P#0.05) from the chamber control group by the Poly-3 test a** b c P#0.01 Number of animals with lesion Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked Historical incidence for 2-year inhalation studies with chamber control groups (mean standard deviation): 92/449 (20.5% 5.3%), d e f g range 12%-26%; all routes: 263/1,498 (17.9% 6.1%), range 6%-28% Number of animals with neoplasm per number of animals with lung examined microscopically Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence is the P value corresponding to pairwise comparison between the chamber controls and that exposed group. The Poly-3 test accounts for differential h i mortality in animals that do not reach terminal sacrifice. Historical incidence for inhalation studies: 64/449 (14.2% 4.6%), range 10%-24%; all routes: 161/1,498 (10.9% 5.6%), range 2%-24% Historical incidence for inhalation studies: 146/449 (32.5% 5.9%), range 26%-44%; all routes: 401/1,498 (27.2% 7.8%), j k l range 12%-44% Historical incidence for inhalation studies: 19/449 (4.2% 2.5%), range 2%-8%; all routes: 77/1,596 (4.9% 2.7%), range 0%-12% Historical incidence for inhalation studies: 15/449 (3.4% 3.9%), range 0%-12%; all routes: 57/1,596 (3.6% 3.1%), range 0%-12% Historical incidence for inhalation studies: 34/449 (7.6% 4.0%), range 2%-14%; all routes: 129/1,596 (8.2% 3.9%), range 2%-18% papillary projections into the bronchiolar lumens by cuboidal epithelial cells lining the bronchioles. When these lesions had marked hyperplasia, the epithelium filled the bronchi and resembled early alveolar/bronchiolar adenomas. Nose: The incidences of olfactory epithelium atrophy in all exposed groups of males and 125 and 500 ppm females were significantly greater than those in the chamber controls (Tables 22, C4, and D4). Microscopically, Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 86 Cumene, NTP TR 542 TABLE 22 Incidences of Selected Nonneoplastic Lesions of the Nose in Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Male NuOmlbfaecrtEorxyamEpinitehdelMiuimcr,oAsctoroppichaylaly Olfactory Epithelium, Hyperplasia, Basal Cell Olfactory Epithelium, Hyperplasia, Atypical Olfactory Epithelium, Glands, Hyperplasia Inflammation, Suppurative 50 4 (1.3)b 0 0 3 (1.0) 2 (2.0) 50 13* (1.1) 0 0 11* (1.0) 2 (1.5) 49 11* (1.2) 15** (1.0) 5* (1.6) 9* (1.1) 9* (1.1) Chamber Control Female Number Examined Microscopically 50 Olfactory Epithelium, Atrophy 4 (1.0) Olfactory Epithelium, Hyperplasia, Basal Cell 0 Olfactory Epithelium, Hyperplasia, Atypical 0 Olfactory Epithelium, Glands, Hyperplasia 1 (1.0) Respiratory Epithelium, Metaplasia, Squamous 0 Inflammation, Suppurative 0 125 ppm 50 11* (1.1) 1 (1.0) 0 4 (1.0) 0 1 (1.0) 250 ppm 50 9 (1.1) 11** (1.0) 2 (1.0) 4 (1.0) 1 (2.0) 3 (1.3) * Significantly different (P#0.05) from the chamber control group by the Poly-3 test *a * b P#0.01 Number of animals with lesion Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked 1,000 ppm 48 38** (1.4) 33** (1.1) 11** (1.7) 23** (1.0) 6 (1.5) 500 ppm 50 18** (1.2) 25** (1.1) 10** (1.2) 11** (1.0) 6* (1.2) 7* (1.3) minimal to mild olfactory epithelium atrophy consisted of small focal lesions involving primarily the epithelium lining the dorsal meatus in Level III nasal sections and occasionally the ethmoid turbinates. The epithelium was thin, with decreased numbers of olfactory neurons; often, metaplasia of the olfactory epithelium to ciliated columnar epithelium and diffuse loss of Bowman's glands in the adjacent lamina propria occurred. The incidences of basal cell hyperplasia of the olfactory epithelium were significantly increased in 500 and 1,000 ppm males and 250 and 500 ppm females (Tables 22, C4, and D4). Minimal to moderate basal cell hyperplasia of the olfactory epithelium occurred in Level III nasal sections. Minimal hyperplasia was characterized by increased numbers of basal cells crowded along the basement membrane of the olfactory epithelium. In addition to increased basal cells along the basement membrane, mild lesions had infiltration of the NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 87 adjacent lamina propria by basal cells, with occasional formation of small rosettes within the epithelium. In moderate lesions, the increase in basal cells was accompanied by disruption of the olfactory epithelium. The incidences of atypical hyperplasia of the olfactory epithelium were significantly increased in groups of mice exposed to 500 ppm or greater (Tables 22, C4, and D4). Minimal to moderate atypical hyperplasia of the olfactory epithelium consisted of proliferation of atypical basal cells with disruption and infiltration of the adjacent lamina propria of ethmoid turbinates by irregular polyhedral cells. These cells had hyperchromatic, round to oval nuclei and scant basophilic cytoplasm. Frequently, rosettes of columnar cells with basally located nuclei around a central lumen were formed. Moderate atypical basal cell hyperplasia in four 1,000 ppm male mice had features of preneoplastic lesions. The incidences of hyperplasia of olfactory epithelium glands were significantly increased in all exposed groups of males and in 500 ppm females (Tables 22, C4, and D4). Minimal focal hyperplasia of olfactory epithelium glands consisted of a few dilated Bowman's glands lined by multiple layers of epithelial cells. The incidence of squamous metaplasia of the respiratory epithelium was significantly increased in 500 ppm females (Tables 22 and D4). Minimal to mild squamous metaplasia of the respiratory epithelium was characterized by replacement of the normal cuboidal to columnar epithelium along the tips of the turbinates in nasal section Level I by squamous epithelial cells. The incidences of suppurative inflammation were significantly increased in 500 ppm males and females (Tables 22, C4, and D4). Minimal to mild suppurative inflammation resulted from infiltration of the lamina propria by various inflammatory cells, predominantly neutrophils, and was associated with necrosis or squamous metaplasia of the overlying epithelium. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 88 Cumene, NTP TR 542 Liver: In females, the incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) occurred with positive trends and were significantly increased in the 500 ppm group; incidences of these lesions in all exposed groups of females and the incidence of hepatocellular carcinoma in 500 ppm females exceeded the historical ranges for chamber controls but were generally within the historical ranges for controls (all routes) (Tables 23, D2, and D3b). Microscopically, the hepatocellular adenomas and hepatocellular carcinomas had the typical appearance of these tumors as reported in B6C3F1 mice. Hepatocellular adenomas were usually discrete masses having solid growth patterns that caused compression of the surrounding normal hepatic parenchyma. They consisted of hepatocytes having clear, eosinophilic, or basophilic cytoplasm, and were sometimes difficult to distinguish from hepatocellular foci. However, the lack of normal lobular architecture and presence of plates of neoplastic hepatocytes that intersected the surrounding normal liver plates at sharp angles, rather than merging with them as seen in foci, were characteristics used to differentiate adenomas from foci. Hepatocellular carcinomas were large, poorly demarcated masses that generally had irregular borders due to growth into the surrounding normal parenchyma. The neoplastic hepatocytes often were somewhat atypical in appearance, but the major distinguishing feature of carcinomas was the presence of abnormal patterns of growth. The most common abnormal growth pattern was formation of trabeculae of neoplastic hepatocytes that were three or more cell layers thick, while less commonly, the neoplastic cells formed glandular structures or solid masses. Several growth patterns were often seen within a single neoplasm. Areas of hemorrhage or necrosis were sometimes present. Metastases of carcinomas to the lungs occurred in all groups of mice of both sexes and were often multiple. The incidences of eosinophilic foci were significantly increased in male mice exposed to 500 or 1,000 ppm (Tables 23 and C4). Eosinophilic foci consisted of well-demarcated collections of enlarged hepatocytes with abundant, dark, homogeneous, eosinophilic cytoplasm. These hepatocytes were arranged in normal hepatic cords that merged with the surrounding normal hepatocytes. Usually, little or no compression of the surrounding normal hepatocytes occurred, although some degree of compression was occasionally evident in larger foci. Eosinophilic foci, hepatocellular adenomas, and hepatocellular carcinomas are thought to represent a morphologic continuum. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 89 TABLE 23 Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Male Number Examined Microscopically Eosinophilic Focusa 50 6 Hepatocellular Hepatocellular Adenoma, Multiple Adenoma (includes multiple)b 17 34 Hepatocellular Hepatocellular Carcinoma, Multiple Carcinoma (includes multiple)c HeATOpaevdtrejomurcasientllelaldurlalrrataaertteeeAgfdenoma or Carcinomad FPoirlsyt-i3ncteidstehnce (days) 3 13 40/50 (80%) 81.0% 30/38 (79%) 551 P=0.250 50 5 20 33 1 18 42/50 (84%) 85.8% 28/34 (82%) 453 P=0.355 Chamber Control Female Number Examined Microscopically Eosinophilic focus 50 8 Hepatocellular Adenoma, Multiple Hepatocellular Adenoma (includes multiple)i Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 9 18/50 (36%) 40.5% 17/37 (46%) 654 P=0.040 Hepatocellular Hepatocellular Carcinoma, Carcinoma Multiple (includes multiple)k 2 10 125 ppm 50 11 13 23/50 (46%) 50.0% 17/36 (47%) 609 P=0.243 1 7 50 16** 22 37 4 21 43/50 (86%) 87.2% 26/30 (87%) 381 P=0.284 250ppm 50 7 9 27/50 (54%)j 56.4% 22/39 (56%) 618 P=0.091 2 6 1,000 ppm 50 14* 26 35 7 17 41/50 (82%) 87.1% 20/23 (87%) 391 P=0.286 500 ppm 50 14 10 29/50 (58%) 59.8% 19/35 (54%) 662 P=0.046 0 12 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 90 Cumene, NTP TR 542 TABLE 23 Incidences of Neoplasms and Nonneoplastic Lesions of the Liver in Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Female (continued) Hepatocellular Adenoma or Carcinomal Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 25/50 (50%) 55.6% 22/37 (60%) 607 P=0.024 26/50 (52%) 56.5% 20/36 (56%) 609 P=0.549 29/50 (58%)j 60.4% 23/39 (59%) 618 P=0.395 36/50 (72%) 74.1% 25/35 (71%) 662 P=0.043 * Significantly different (P#0.05) from the chamber control group by the Poly-3 test a** b P#0.01 Number of animals with lesion Historical incidence for 2-year inhalation studies with chamber control groups (mean standard deviation): 196/449 (43.7% 10.9%), c range 30%-68%; all routes: 633/1,496 (43.3% 14.2%), range 14%-70% Historical incidence for inhalation studies: 107/449 (23.8% 4.6%), range 18%-32%; all routes: 382/1,496 (26.0% 9.1%), d range 8%-48% Historical incidence for inhalation studies: 264/449 (58.8% 9.6%), range 50%-80%; all routes: 874/1,496 (59.6% 15.4%), e f g h range 20%-85% Number of animals with neoplasm per number of animals with liver examined microscopically Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence is the P value corresponding to pairwise comparison between the chamber controls and that exposed group. The Poly-3 test accounts for differential i mortality in animals that do not reach terminal sacrifice. Historical incidence for inhalation studies: 109/447 (24.4% 8.7%), range 12%-36%; all routes: 402/1,593 (25.8% 15.8%), j k l range 2%-62% One animal with adenoma also had hepatoblastoma. Historical incidence for inhalation studies: 48/447 (10.7% 4.1%), range 6%-20%; all routes: 159/1,593 (10.2% 6.6%), range 0%-28% Historical incidence for inhalation studies: 145/447 (32.4% 8.8%), range 22%-50%; all routes: 505/1,593 (32.4% 17.5%), range 8%-64% NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 91 Forestomach: The incidences of epithelial hyperplasia were significantly increased in groups of males exposed to 500 or 1,000 ppm, and the incidences of ulceration and inflammation were significantly increased in the 1,000 ppm group (Tables 24 and C4). These three lesions were often present in the forestomach of the same mouse. Epithelial hyperplasia was characterized by a diffuse thickening of the squamous epithelium due to increased numbers of cell layers, primarily of prickle cells. Ulcers of the forestomach resulted after damage to the mucosal surface, with loss of squamous epithelium extending through the basement membrane. The ulcers were usually accompanied by inflammation. The margins of the ulcers often had epithelial hyperplasia. Inflammation of the forestomach had variable numbers of mixed inflammatory cells, with congestion and various degrees of increased fibrous connective tissue. TABLE 24 Incidences of Nonneoplastic Lesions of the Forestomach in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm NuEmpbiethreElixuamm,iHneydpeMrpiclarsoisacaopically Ulcer Inflammation 50 2 (2.0)b 1 (3.0) 0 50 7 (2.1) 4 (2.8) 2 (2.0) 50 8* (2.3) 6 (2.8) 1 (2.0) * Significantly different (P#0.05) from the chamber control group by the Poly-3 test a** b P#0.01 Number of animals with lesion Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked 1,000 ppm 49 13** (2.1) 6* (2.8) 5* (2.0) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 92 Cumene, NTP TR 542 GENETIC TOXICOLOGY Cumene (1 to 333 g/plate) was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535 when tested with and without induced rat or hamster liver S9 activation enzymes (Table E1). In vivo, cumene induced small but significant increases in micronucleated polychromatic erythrocytes in bone marrow of male rats treated by intraperitoneal injection (Table E2). Two trials were performed in rats. In the first trial, doses ranging from 78.13 to 2,500 mg/kg were administered three times at 24-hour intervals, and results were positive, based both on the trend (P=0.011) and the response at the 1,250 mg/kg dose. The data from the 2,500 mg/kg dose were excluded from analysis because only two animals survived and a minimum of three animals is required for a valid dose point. The second confirmatory trial also produced a positive response, although the trend test was not significant (P=0.085). Micronucleated erythrocytes were elevated at all four doses in trial 2; the responses at the 312 and 1,250 mg/kg levels were statistically significant (P<0.006). The percentage of polychromatic erythrocytes in the bone marrow fluctuated unrelated to dose and likely represented variation within the normal range of 40% to 60% polychromatic erythrocytes among the total erythrocyte population in the bone marrow. In contrast to the results in male rats, no increase in micronucleated erythrocytes was observed in peripheral blood of male or female mice exposed to cumene by inhalation (62.5 to 1,000 ppm) for 3 months (Table E3). For both male and female mice, no significant changes in the percentage of polychromatic erythrocytes were observed over the exposure range tested, indicating an absence of treatment-related toxicity to the bone marrow. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 93 DISCUSSION AND CONCLUSIONS Cumene was nominated for study by the National Institute of Environmental Health Sciences because of its high production volume, presence in gasoline and other fuels, potential for human exposure, and lack of existing carcinogenicity test data. Cumene is a central nervous system depressant (Andrews and Snyder, 1993). At high exposure concentrations in the 2-week, 3-month, and 2-year rat and mouse studies, cumene induced ataxia, but surviving animals recovered and developed tolerance after a few days. Microscopic alterations in the peripheral and central nervous systems were not observed. Cushman et al. (1995) reported changes in the functional observational battery test in male and female rats 1 hour after a single 6-hour exposure to 1,200 ppm cumene; the changes included gait abnormalities, increased horizontal activity, decreased mean rectal temperature, and decreased toe-pinch withdrawal reflexes. The alterations in behavioral or motor activity disappeared within 24 hours. The findings were typical of those commonly observed following exposure to alkylbenzenes, such as toluene and ethylbenzene (Andrews and Snyder, 1993; Tegeris and Balster, 1994). The highest exposure concentration selected for male and female rats and male mice in the 2-year studies was 1,000 ppm. Deaths, presumably from central nervous system depression, occurred at higher exposure concentrations in the 2-week studies, and deaths occurred at 1,000 ppm in the 3-month female mouse study, resulting in the selection of 500 ppm as the highest exposure concentration for the 2-year female mouse study. In the 3-month rat study, the severities of renal cortical (proximal) tubular cell hyaline droplet accumulation and regeneration (hypertrophy, hyperplasia) in males increased with increasing exposure concentration. The incidences of medullary granular casts in the 250, 500, and 1,000 ppm male rats were significantly increased, and the severity of this lesion was generally exposure-concentration-related. Levels of "2u-globulin in the kidney also were significantly greater in male rats exposed to 125 ppm or greater compared to the chamber controls. The absolute Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 94 Cumene, NTP TR 542 and relative kidney weights of exposed groups of male rats were increased. Relative kidney weights of female rats exposed to 250 ppm or greater and the absolute kidney weight of 1,000 ppm females were significantly increased, but no accompanying histopathology changes were observed. These changes were consistent with "2u-globulin nephropathy. In the 2-year rat study, incidences of renal tubule adenoma or carcinoma (combined) were increased in exposed males, and the increase was significant at 500 ppm. The renal tubular neoplasms were considered related to cumene exposure because spontaneous incidences of renal neoplasms in untreated male F344/N rats in inhalation studies are very low. Along with renal neoplasms, the incidences of renal papilla mineralization and renal pelvis transitional epithelium hyperplasia were also increased in exposed male rats. Renal tubule hyperplasia was considered a preneoplastic lesion. The pathogenesis underlying the renal lesions in male rats in the 2-year study is likely similar to that observed with many structurally related chemicals, such as ethylbenzene. Cumene or a metabolite is thought to bind to "2u-globulin causing the protein to resist lysosomal degradation. The pathogenesis process probably involves accumulation of protein droplets in the S2 segment of the proximal tubules, necrosis secondary to lysosomal overload, and increased cell proliferation. The continuing necrosis eventually overwhelms the capacity of the kidney to remove necrotic debris in tubular lumens, resulting in granular casts and mineralization of the papillary tubules. The histopathologic changes ultimately lead to the development of renal neoplasm. The pathogenesis of this syndrome has been widely discussed in the literature (Swenberg et al., 1989; Montgomery and Seely, 1990; Melnick, 1992; Hard et al., 1993; Goldstein and Schnellmann, 1996; Lehman-Mckeeman, 1997; Kohn and Melnick, 1999; Schnellmann, 2001). These changes were not seen in female rats or male or female mice. In the nose of 2-year rats, cumene induced significantly increased incidences of olfactory epithelium basal cell hyperplasia in all exposed groups and hyperplasia of the respiratory epithelium in all exposed groups of male rats and in 1,000 ppm females. Respiratory epithelium adenomas were observed in all exposed groups. A positive trend in the incidences of respiratory epithelium adenoma occurred in male rats but not in female rats. The NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 95 incidences of adenoma in the 1,000 ppm males and females were lower than those seen at 500 ppm. However, if the incidences of respiratory epithelium hyperplasia and adenoma are considered together, exposure concentrationrelated increase trends are apparent. Hyperplasia of the respiratory epithelium and adenoma form a morphologic continuum. It is notable that the areas of respiratory epithelial hyperplasia and adenomas were discrete foci with few or no abnormalities in the adjacent respiratory epithelium. Male rats appeared to be more sensitive than females to the progression from hyperplasia to adenoma caused by cumene. Interestingly, hyperplasia of the respiratory epithelium in the nose was not observed in rats in the 3-month study nor in the 3-month or 2-year mouse studies. In the 2-year mouse study, cumene exposure induced olfactory epithelium lesions in the nose, including atrophy, basal cell hyperplasia, atypical hyperplasia, and gland hyperplasia, in males and females. Olfactory atypical basal cell hyperplasia may have represented a preneoplastic change. However, no nasal olfactory epithelium neoplasms were found in this study. A significant increase in the incidence of squamous metaplasia of the respiratory epithelium occurred in 1,000 ppm females. Chemical-related neoplasms and nonneoplastic lesions in the lung included increased incidences in all exposed groups of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, alveolar/bronchiolar epithelium metaplasia, and bronchiole hyperplasia. The incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) in male and female mice exceeded the historical control range at all exposure concentrations, indicating an unequivocal carcinogenic effect. Alveolar/bronchiolar epithelial hyperplasia is considered a preneoplastic change and may progress to adenoma and then carcinoma (Foley et al., 1991; Dixon et al., 1999). In the 2-year mouse study, cumene exposure affected the livers of females to a greater extent than males. Exposure concentration-related increases in the incidences of hepatocellular adenoma or carcinoma (combined) were observed in female mice, and the increase in the 500 ppm group was significant. In males exposed to 500 or 1,000 ppm, the incidences of eosinophilic focus were increased. Interestingly, ethylbenzene, which is structurally similar to cumene, also caused an increased incidence of hepatocellular neoplasms only in female mice (Chan, 1998; NTP, 1999). Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 96 Cumene, NTP TR 542 In the 2-year mouse study, increased incidences of forestomach epithelial hyperplasia and inflammation were observed in exposed males. The increases were probably related to irritating effects of cumene, and similar lesions are commonly seen in NTP inhalation studies, presumably resulting from ingestion of the chemical from fur or clearance from the lung through microciliary action. Cumene is an alkylbenzene structurally similar to benzene, toluene, styrene, xylene, and ethylbenzene (Andrews and Snyder, 1993). Like other alkylbenzenes, cumene may be metabolized by oxidation of the side chain or by ring oxidation. Both biotransformations are catalyzed by cytochromes P450. However, a pathway for metabolic activation of cumene to an intermediate capable of reacting with protein or DNA is not obvious. There are no structural alerts. Cumene is a relatively small aromatic molecule, probably a substrate for CYP2E1 and CYP2F2. CYP2F2 has been located in Clara cells in mouse lung using immunohistochemical techniques. There was little immunological reaction for CYP2F in Clara cells from rat or hamster lungs. This observation correlates with rates of naphthalene biotransformation in the three species (Buckpitt et al., 1995). The biotransformations of styrene have been extensively studied. While epoxidation of the vinyl group is the major biotransformation pathway, a ring-hydroxylated product, 4-vinylphenol, is also formed. Vinylphenol is more toxic than styrene oxide but requires further biotransformation to be toxic (Cruzan et al., 2002). The phenol is more toxic to mouse lung than rat lung. The toxicity can be prevented by treatment with a CYP2F inhibitor, 5-phenyl-1-pentyne (Cruzan et al., 2002). The observation that mice exhale more 14CO2 than rats when dosed with ring-labeled [14C]-styrene implies that the aromatic ring is broken and is consistent with more extensive metabolism in mice (Boogaard et al., 2000). With this as a background, a pathway for activation of cumene can be proposed (Figure 6). Ring-hydroxylation to isopropylphenol is the proposed first step. There are at least three possible activated intermediates from further biotransformation of the phenol: oxidation to a quinone methide, ring-opening to a muconaldehyde, or hydroxylation to a catechol and oxidation to a quinone. Biotransformation of cumene by CYP2F2 in mouse lung and nasal tissue and CYP2F4 in rat nasal tissue could lead to the observed species difference. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 97 ?? Cumene OH OH Isopropylphenol ? OH OH Catechol ?? ? Dimethylcarbonyl O Quinone methide O Quinone O O O Muconaldehyde FIGURE 6 Possible Reactive Metabolites of Cumene Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 98 Cumene, NTP TR 542 Studies of the absorption, distribution, metabolism, and excretion of cumene are ongoing and may provide more insight on the biotransformation and bioactivation. Preliminary results have confirmed the major urinary metabolites as described in Figure 1. Exhaled organic volatiles are composed of an approximately 1:1 mixture of cumene and "-methylstyrene. "-Methylstyrene has not previously been reported as a metabolite of cumene; however, the urinary metabolites 2-phenyl-1,2-propanediol and 2-hydroxyphenylpropanoic acid are likely to be derived from the epoxide of "-methylstyrene. Whether "-methylstyrene requires ring-hydroxylation as an activating step in producing reactive intermediates as described for styrene is not known; however, certain aspects of the toxicity/carcinogenicity of inhaled "-methylstyrene (NTP, 2007) are similar to those of cumene. For example, in rats, cumene at 1,000 ppm induced olfactory epithelial hyperplasia and respiratory epithelial neoplasms in males and females and "2u-globulin nephropathy and renal tubular neoplasms in males. "-Methylstyrene at 1,000 ppm also induced olfactory epithelial hyperplasia in males and females, although it did not appear to affect the respiratory epithelium, as did cumene, and caused "2u-globulin nephropathy and renal tubular neoplasms in males. In mice, cumene at 500 and 1,000 ppm induced olfactory epithelial hyperplasia and other nasal lesions in males and females, hepatocellular neoplasms in females, and hepatic eosinophilic foci in males. "-Methylstyrene at 600 ppm also induced olfactory epithelial hyperplasia in males and females and hepatocellular neoplasms in females. In contrast, the marked carcinogenic effects in the lung of male and female mice caused by cumene did not occur with "-methylstyrene. Thus, even though cumene can be metabolized to "-methylstyrene the toxic and carcinogenic effects of cumene are more widespread than those of "-methylstyrene. Therefore, "-methylstyrene can not be the sole active contributor to the carcinogenicity of cumene. It is not known how "-methylstyrene interacts with cellular macromolecules in its carcinogenesis process, although a metabolic activation via styrene and styrene oxide has been suggested. There is no evidence that "-methylstyrene binds to DNA or protein. "-Methylstyrene is not mutagenic in Salmonella tests with or without S9. "-Methylstyrene did not induce micronuclei in erythrocytes in male and female mice (NTP, 2007). The present studies demonstrated that cumene is a multisite carcinogen both in mice, inducing neoplasms in the lung and liver, and in rats, inducing neoplasms in the nose and kidney. Multisite, multispecies carcinogens are NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 99 frequently genotoxic carcinogens, but standard assays for genotoxicity did not detect cumene as a genotoxicant. Cumene was not mutagenic in the Ames test, and it did not induce micronuclei, indicative of chromosomal damage, in erythrocytes of mice exposed to cumene for 3 months by inhalation (Appendix E). Although positive results in the Ames assay and the 3-month mouse erythrocyte micronucleus assay are highly predictive of carcinogenicity, negative results are not predictive of noncarcinogenicity (Zeiger et al., 1990; Witt et al., 2000). Because of the extensive metabolism that cumene undergoes in vivo, it is possible that the proximate carcinogen is not generated in the in vitro Ames test, even with the addition of exogenous metabolic activation enzymes. The significantly increased frequencies of K-ras and p53 mutations seen in lung neoplasms of mice exposed to cumene in this study (Appendix L) strongly suggest that genetic changes related to cumene exposure are implicated as early events in the neoplastic response. CONCLUSIONS Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity* of cumene in male F344/N rats based on increased incidences of respiratory epithelial adenoma in the nose and renal tubule adenoma or carcinoma (combined). There was some evidence of carcinogenic activity of cumene in female F344/N rats based on the incidences of respiratory epithelium adenoma in the nose. There was clear evidence of carcinogenic activity of cumene in male B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. There was clear evidence of carcinogenic activity of cumene in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Increased incidences of hepatocellular adenoma or carcinoma (combined) in female mice were also considered to be related to exposure to cumene. Exposure to cumene resulted in nonneoplastic lesions in the nose and kidney of male rats; the nose of female rats; the lung, nose, liver, and forestomach of male mice; and the lung and nose of female mice. __________ * Explanation of Levels of Evidence of Carcinogenic Activity is on page 15. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 100 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 101 REFERENCES The Aldrich Library of 13C and 1H FT-NMR Spectra (1993). 1st ed. (C.J. Pouchert and J. Behnke, Eds.), Vol. 2, p. 2B. 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Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. (1992). Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ. Mol. Mutagen. 19 (Suppl. 21), 2-141. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 108 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft A-1 APPENDIX A SUMMARY OF LESIONS IN MALE RATS IN THE 2-YEAR INHALATION STUDY OF CUMENE TABLE A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2 TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-6 TABLE A3a Historical Incidence of Kidney Neoplasms in Control Male F344/N Rats . . . . . . . . . . . . . . . A-11 TABLE A3b Historical Incidence of Adenoma of the Nose in Control Male F344/N Rats . . . . . . . . . . . . A-12 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-13 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-2 Cumene, NTP TR 542 TABLE A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Animals examined microscopically 50 50 20 24 43 26 23 50 50 50 21 2 27 50 Alimentary System Esophagus Intestine large, cecum Intestine large, colon Intestine large, rectum Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Liver Carcinoma, metastatic, prostate Hepatocellular adenoma Hepatocellular carcinoma Mesentery Carcinoma, metastatic, prostate Pancreas Carcinoma, metastatic, prostate Stomach, forestomach Stomach, glandular Tongue Papilloma Tooth (50) (50) (50) (49) (49) (49) (49) (50) (50) (49) (50) (50) (49) (49) (49) (49) (47) (48) (48) (47) (48) (50) (50) (50) 1 (2%) (7) (13) (10) (50) (50) (50) (50) (50) (50) (50) (50) (50) (1) (3) (1) 1 (33%) (1) Cardiovascular System Blood vessel Heart Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, prostate Pericardium, carcinoma, metastatic, lung (1) (50) 1 (2%) (1) (50) (1) (50) 1 (2%) Endocrine System Adrenal cortex Adenoma Adrenal medulla Pheochromocytoma benign Pheochromocytoma malignant Bilateral, pheochromocytoma benign Islets, pancreatic Adenoma Carcinoma Parathyroid gland Pituitary gland Adenoma (50) (50) 7 (14%) 1 (2%) (50) 1 (2%) 2 (4%) (45) (50) 36 (72%) (50) 2 (4%) (50) 6 (12%) 1 (2%) 1 (2%) (50) 2 (4%) 1 (2%) (49) (50) 38 (76%) (50) 3 (6%) (50) 9 (18%) 1 (2%) (50) 2 (4%) 2 (4%) (49) (49) 25 (51%) 1,000 ppm 50 24 2 24 50 (50) (49) (50) (50) (49) (49) (49) (50) 1 (2%) 2 (4%) (6) 1 (17%) (50) 1 (2%) (50) (50) (3) (50) 1 (2%) 1 (2%) (50) (50) 8 (16%) 2 (4%) 3 (6%) (50) 2 (4%) 1 (2%) (48) (50) 30 (60%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Endocrine System (continued) Thyroid gland C-cell, adenoma C-cell, carcinoma Follicular cell, adenoma Follicular cell, carcinoma General Body System Peritoneum Carcinoma, metastatic, prostate Carcinoma, metastatic, urinary bladder Genital System Epididymis Carcinoma, metastatic, prostate Preputial gland Carcinoma Prostate Carcinoma Carcinoma, metastatic, urinary bladder Seminal vesicle Carcinoma, metastatic, prostate Testes Bilateral, interstitial cell, adenoma Interstitial cell, adenoma Hematopoietic System Bone marrow Lymph node Deep cervical, carcinoma, metastatic, skin Lymph node, bronchial Lymph node, mandibular Lymph node, mediastinal Carcinoma, metastatic, thyroid gland Lymph node, mesenteric Spleen Carcinoma, metastatic, prostate Thymus Integumentary System Mammary gland Carcinoma Carcinoma, multiple Fibroadenoma Skin Basal cell adenoma Basal cell carcinoma Keratoacanthoma Keratoacanthoma, multiple Squamous cell papilloma Trichoepithelioma Sebaceous gland, adenoma (50) 3 (6%) 1 (2%) 2 (4%) (2) (50) (50) (50) (50) (50) 18 (36%) 18 (36%) (50) (7) (7) (1) (34) 1 (3%) (50) (50) (49) (50) 1 (2%) 1 (2%) (50) 1 (2%) 4 (8%) 1 (2%) (50) 7 (14%) 2 (4%) 1 (2%) (2) (50) (50) 3 (6%) (50) (50) (50) 24 (48%) 14 (28%) (50) (10) 1 (10%) (12) (3) (32) 1 (3%) (50) (50) (49) (50) 1 (2%) (50) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) (4) 1 (25%) (50) (50) 1 (2%) (50) 1 (2%) (50) (50) 27 (54%) 13 (26%) (50) (8) (11) (34) (50) (50) (50) (50) 2 (4%) 3 (6%) (50) 1 (2%) 2 (4%) 2 (4%) 1 (2%) A-3 1,000 ppm (50) 4 (8%) 2 (4%) 1 (2%) 2 (4%) (2) 1 (50%) (50) 1 (2%) (50) (50) 1 (2%) (50) 1 (2%) (50) 37 (74%) 9 (18%) (50) (14) (8) (1) (34) (50) (50) 1 (2%) (50) (50) 2 (4%) (50) 1 (2%) 2 (4%) 1 (2%) 1 (2%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-4 Cumene, NTP TR 542 TABLE A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Integumentary System (continued) Skin (continued) Sebaceous gland, carcinoma Subcutaneous tissue, carcinoma, metastatic, harderian gland Subcutaneous tissue, fibroma Subcutaneous tissue, fibrosarcoma Subcutaneous tissue, lipoma Subcutaneous tissue, schwannoma, benign (50) 3 (6%) (50) 1 (2%) 1 (2%) 3 (6%) 1 (2%) (50) 3 (6%) 1 (2%) Musculoskeletal System Bone Cranium, carcinoma, metastatic, Zymbal's gland Skeletal muscle Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, lung Carcinoma, metastatic, prostate Carcinoma, metastatic, urinary bladder Rhabdomyosarcoma (50) 1 (2%) (1) 1 (100%) (50) (2) 1 (50%) (50) (2) 1 (50%) 1 (50%) Nervous System Brain Granular cell tumor malignant (50) (50) (50) 1 (2%) Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Carcinoma, metastatic, prostate Carcinoma, metastatic, skin Carcinoma, metastatic, thyroid gland Carcinoma, metastatic, urinary bladder Pheochromocytoma malignant, metastatic, adrenal medulla Nose Glands, respiratory epithelium, adenoma Respiratory epithelium, adenoma Respiratory epithelium, adenoma, multiple Pleura Alveolar/bronchiolar carcinoma, metastatic, lung Trachea (50) (50) 1 (2%) 1 (2%) 1 (2%) (50) (5) (50) (50) (50) 2 (4%) 1 (2%) (50) 6 (12%) 1 (2%) (3) (50) (50) (50) 2 (4%) 1 (2%) 1 (2%) 1 (2%) (49) 1 (2%) 15 (31%) 2 (4%) (5) 1 (20%) (50) Special Senses System Eye Carcinoma, metastatic, harderian gland Harderian gland Carcinoma Zymbal's gland Carcinoma (50) (50) (1) 1 (100%) (50) 1 (2%) (50) 1 (2%) (49) (50) 1,000 ppm (50) 4 (8%) 1 (2%) 1 (2%) (50) (1) 1 (100%) (50) (50) (50) 2 (4%) 2 (4%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (50) 4 (8%) 6 (12%) (6) (50) (50) (50) (2) 2 (100%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Urinary System Kidney Carcinoma, metastatic, prostate Lipoma Bilateral, renal tubule, carcinoma Renal tubule, adenoma Renal tubule, carcinoma Ureter Carcinoma, metastatic, prostate Urethra Carcinoma, metastatic, prostate Urinary bladder Transitional epithelium, carcinoma Transitional epithelium, papilloma (50) 1 (2%) 1 (2%) 1 (2%) (50) (50) 4 (8%) 1 (2%) (1) (50) (50) 1 (2%) 5 (10%) 2 (4%) (1) (49) 2 (4%) Systemic Lesions Multiple organsb Histiocytic sarcoma Leukemia mononuclear Lymphoma malignant Mesothelioma malignant (50) 26 (52%) 3 (6%) (50) 32 (64%) 2 (4%) (50) 31 (62%) 3 (6%) Neoplasm Summary Total animals with primary neoplasmsc Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms 50 50 135 163 48 48 95 117 36 37 40 46 44 56 a b c Number of animals examined microscopically at the site and the number of animals with neoplasm Number of animals with any tissue examined microscopically Primary neoplasms: all neoplasms except metastatic neoplasms 50 166 47 117 38 49 3 8 A-5 1,000 ppm (50) 1 (2%) 1 (2%) 4 (8%) 3 (6%) (1) 1 (100%) (1) 1 (100%) (50) 1 (2%) 2 (4%) (50) 1 (2%) 28 (56%) 1 (2%) 1 (2%) 50 176 50 125 39 51 4 16 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-6 Cumene, NTP TR 542 TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Adrenal Cortex: Adenoma OATevdrejmurasintleladrlarrtaaettaeecb First incidence (days) Poly-3 testd 0/50 (0%) 0.0% 0--/2e6 (0%) P=0.519N Adrenal Medulla: Benign Pheochromocytoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 7/50 (14%) 16.7% 5/26 (19%) 647 P=0.149 Adrenal Medulla: Benign or Malignant Pheochromocytoma Overall rate 7/50 (14%) Adjusted rate 16.7% Terminal rate 5/26 (19%) First incidence (days) 647 Poly-3 test P=0.054 Kidney (Renal Tubule): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 1/50 (2%) 2.4% 1/26 (4%) 729 (T) P=0.219 Kidney (Renal Tubule): Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 1/50 (2%) 2.4% 1/26 (4%) 729 (T) P=0.180 Kidney (Renal Tubule): Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 2/50 (4%) 4.8% 2/26 (8%) 729 (T) P=0.087 Lung: Alveolar/bronchiolar Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 1/50 (2%) 2.4% 1/26 (4%) 729 (T) P=0.104 Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 2/50 (4%) 4.8% 2/26 (8%) 729 (T) P=0.125 2/50 (4%) 5.1% 1/23 (4%) 665 P=0.226 7/50 (14%) 17.4% 4/23 (17%) 624 P=0.583 7/50 (14%) 17.4% 4/23 (17%) 624 P=0.583 4/50 (8%) 10.0% 1/23 (4%) 665 P=0.165 1/50 (2%) 2.5% 1/23 (4%) 729 (T) P=0.749 5/50 (10%) 12.5% 2/23 (9%) 665 P=0.198 0/50 (0%) 0.0% 0/23 (0%) -- P=0.511N 2/50 (4%) 5.1% 2/23 (9%) 729 (T) P=0.675 3/50 (6%) 7.3% 2/27 (7%) 626 P=0.117 9/50 (18%) 21.5% 6/27 (22%) 600 P=0.387 10/50 (20%) 23.8% 6/27 (22%) 600 P=0.293 5/50 (10%) 12.1% 2/27 (7%) 679 P=0.099 3/50 (6%) 7.3% 2/27 (7%) 639 P=0.302 8/50 (16%) 19.2% 4/27 (15%) 639 P=0.044 1/50 (2%) 2.4% 1/27 (4%) 729 (T) P=0.758 3/50 (6%) 7.3% 2/27 (7%) 670 P=0.496 1,000 ppm 0/50 (0%) 0.0% 0/24 (0%) ----f 11/50 (22%) 25.9% 8/24 (33%) 688 P=0.221 13/50 (26%) 30.5% 9/24 (38%) 688 P=0.103 4/50 (8%) 9.3% 2/24 (8%) 635 P=0.187 3/50 (6%) 7.0% 2/24 (8%) 618 P=0.314 7/50 (14%) 16.2% 4/24 (17%) 618 P=0.087 3/50 (6%) 7.0% 1/24 (4%) 674 P=0.314 5/50 (10%) 11.7% 3/24 (13%) 674 P=0.226 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Mammary Gland: Fibroadenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Mammary Gland: Fibroadenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Nose: Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Pancreatic Islets: Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Pituitary Gland (Pars Distalis): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Preputial Gland: Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Preputial Gland: Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Skin: Keratoacanthoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/50 (0%) 0.0% 0/26 (0%) -- P=0.174 2/50 (4%) 4.8% 1/26 (4%) 628 P=0.479 0/50 (0%) 0.0% 0/26 (0%) -- P=0.004 3/50 (6%) 7.2% 2/26 (8%) 689 P=0.578 36/50 (72%) 76.2% 20/26 (77%) 296 P=0.030N 0/50 (0%) 0.0% 0/26 (0%) -- P=0.363N 0/50 (0%) 0.0% 0/26 (0%) -- P=0.363N 4/50 (8%) 9.5% 1/26 (4%) 613 P=0.464N 1/50 (2%) 2.5% 1/23 (4%) 729 (T) P=0.489 1/50 (2%) 2.5% 1/23 (4%) 729 (T) P=0.522N 7/50 (14%) 17.6% 5/23 (22%) 639 P=0.006 3/50 (6%) 7.6% 3/23 (13%) 729 (T) P=0.636 38/50 (76%) 80.4% 18/23 (78%) 480 P=0.396 3/50 (6%) 7.5% 1/23 (4%) 639 P=0.111 3/50 (6%) 7.5% 1/23 (4%) 639 P=0.111 2/50 (4%) 5.1% 2/23 (9%) 729 (T) P=0.369N 3/50 (6%) 7.3% 3/27 (11%) 729 (T) P=0.115 5/50 (10%) 12.2% 5/27 (19%) 729 (T) P=0.205 18/49 (37%) 43.2% 13/27 (48%) 638 P<0.001 4/50 (8%) 9.7% 3/27 (11%) 679 P=0.492 25/49 (51%) 58.4% 17/27 (63%) 625 P=0.048N 1/50 (2%) 2.4% 1/27 (4%) 729 (T) P=0.497 1/50 (2%) 2.4% 1/27 (4%) 729 (T) P=0.497 2/50 (4%) 4.9% 2/27 (7%) 729 (T) P=0.350N A-7 1,000 ppm 2/50 (4%) 4.7% 2/24 (8%) 729 (T) P=0.241 2/50 (4%) 4.7% 2/24 (8%) 729 (T) P=0.691N 10/50 (20%) 23.3% 7/24 (29%) 674 P<0.001 3/50 (6%) 7.1% 3/24 (13%) 729 (T) P=0.657N 30/50 (60%) 62.9% 13/24 (54%) 453 P=0.110N 0/50 (0%) 0.0% 0/24 (0%) -- -- 0/50 (0%) 0.0% 0/24 (0%) -- -- 3/50 (6%) 7.1% 1/24 (4%) 677 P=0.496N Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-8 Cumene, NTP TR 542 TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Skin: Squamous Cell Papilloma or Keratoacanthoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 4/50 (8%) 9.5% 1/26 (4%) 613 P=0.482N 2/50 (4%) 5.1% 2/23 (9%) 729 (T) P=0.369N 3/50 (6%) 7.3% 3/27 (11%) 729 (T) P=0.516N 3/50 (6%) 7.1% 1/24 (4%) 677 P=0.496N Skin: Trichoepithelioma, Basal Cell Adenoma, or Basal Cell Carcinoma Overall rate 1/50 (2%) Adjusted rate 2.4% Terminal rate 1/26 (4%) First incidence (days) 729 (T) Poly-3 test P=0.342 1/50 (2%) 2.5% 1/23 (4%) 729 (T) P=0.749 3/50 (6%) 7.2% 1/27 (4%) 554 P=0.305 2/50 (4%) 4.7% 2/24 (8%) 729 (T) P=0.506 Skin: Squamous Cell Papilloma, Keratoacanthoma, Trichoepithelioma, Basal Cell Adenoma, or Basal Cell Carcinoma Overall rate 5/50 (10%) 3/50 (6%) 6/50 (12%) Adjusted rate 11.8% 7.6% 14.4% Terminal rate 2/26 (8%) 3/23 (13%) 4/27 (15%) First incidence (days) 613 729 (T) 554 Poly-3 test P=0.478 P=0.394N P=0.491 5/50 (10%) 11.8% 3/24 (13%) 677 P=0.626N Skin: Fibroma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.2% 2/26 (8%) 632 P=0.423 3/50 (6%) 7.6% 2/23 (9%) 711 P=0.635 3/50 (6%) 7.3% 2/27 (7%) 688 P=0.654 4/50 (8%) 9.4% 2/24 (8%) 677 P=0.508 Skin: Fibroma or Fibrosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.2% 2/26 (8%) 632 P=0.271 3/50 (6%) 7.6% 2/23 (9%) 711 P=0.635 3/50 (6%) 7.3% 2/27 (7%) 688 P=0.654 5/50 (10%) 11.7% 3/24 (13%) 677 P=0.364 Testes: Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 36/50 (72%) 80.0% 24/26 (92%) 558 P=0.006 38/50 (76%) 84.6% 22/23 (96%) 536 P=0.370 40/50 (80%) 85.7% 25/27 (93%) 460 P=0.311 46/50 (92%) 96.1% 24/24 (100%) 541 P=0.007 Thyroid Gland (C-Cell): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.1% 1/26 (4%) 571 P=0.485N 7/50 (14%) 17.4% 3/23 (13%) 589 P=0.133 1/50 (2%) 2.4% 0/27 (0%) 702 P=0.318N 4/50 (8%) 9.4% 3/24 (13%) 677 P=0.500 Thyroid Gland (C-Cell): Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.1% 1/26 (4%) 571 P=0.432 9/50 (18%) 22.0% 4/23 (17%) 541 P=0.048 2/50 (4%) 4.9% 1/27 (4%) 702 P=0.516N 6/50 (12%) 14.1% 5/24 (21%) 677 P=0.240 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Thyroid Gland (Follicular Cell): Adenoma or Carcinoma Overall rate 2/50 (4%) Adjusted rate 4.8% Terminal rate 2/26 (8%) First incidence (days) 729 (T) Poly-3 test P=0.348 Urinary Bladder: Papilloma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/50 (0%) 0.0% 0/26 (0%) -- P=0.030 All Organs: Mononuclear Leukemia Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 26/50 (52%) 57.8% 12/26 (46%) 600 P=0.490N All Organs: Malignant Mesothelioma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.1% 2/26 (8%) 599 P=0.253N All Organs: Benign Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 48/50 (96%) 97.5% 26/26 (100%) 296 P=0.261 All Organs: Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 36/50 (72%) 77.5% 19/26 (73%) 495 P=0.410 1/50 (2%) 2.5% 0/23 (0%) 686 P=0.518N 0/50 (0%) 0.0% 0/23 (0%) -- -- 32/50 (64%) 69.7% 14/23 (61%) 519 P=0.160 2/50 (4%) 5.0% 0/23 (0%) 575 P=0.522N 48/50 (96%) 98.8% 23/23 (100%) 480 P=0.633 37/50 (74%) 78.1% 16/23 (70%) 445 P=0.576 0/50 (0%) 0.0% 0/27 (0%) -- P=0.240N 2/49 (4%) 4.8% 0/27 (0%) 213 P=0.238 31/50 (62%) 65.5% 13/27 (48%) 460 P=0.289 3/50 (6%) 7.2% 1/27 (4%) 634 P=0.659 47/50 (94%) 97.5% 27/27 (100%) 460 P=0.770 38/50 (76%) 77.3% 17/27 (63%) 213 P=0.588N A-9 1,000 ppm 3/50 (6%) 7.0% 1/24 (4%) 677 P=0.511 3/50 (6%) 7.1% 2/24 (8%) 677 P=0.122 28/50 (56%) 60.0% 11/24 (46%) 515 P=0.501 1/50 (2%) 2.4% 1/24 (4%) 729 (T) P=0.302N 50/50 (100%) 100% 24/24 (100%) 453 P=0.400 39/50 (78%) 80.4% 17/24 (71%) 515 P=0.462 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-10 Cumene, NTP TR 542 TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm All Organs: Benign or Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 50/50 (100%) 100% 26/26 (100%) 296 -- 50/50 (100%) 100% 23/23 (100%) 445 -- 50/50 (100%) 100% 27/27 (100%) 213 -- 50/50 (100%) 100% 24/24 (100%) 453 -- (aT)NTeurmmbinearlosfancreiofipcleasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, kidney, lung, nose, pancreatic islets, pituitary gland, preputial gland, testes, thyroid gland, and urinary bladder; for other tissues, denominator is number of animals b c d necropsied. Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not e f reach terminal sacrifice. A negative trend or a lower incidence in an exposed group is indicated by N. Not applicable; no neoplasms in animal group Value of statistic cannot be computed. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE A3a Historical Incidence of Adenoma of the Nose in Control Male F344/N Ratsa Study Incidence in Controls Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide Overall Historical Incidence: Inhalation Studies Total (%) Mean standard deviation Range Overall Historical Incidence: All Routes Total (%) Mean standard deviation Range a Data as of March 2, 2007 0/50 0/49 0/50 0/50 0/50 0/49 0/50 1/50 0/49 1/447 (0.2%) 0.2% 0.7% 0%-2% 2/1,439 (0.1%) 0.1% 0.5% 0%-2% A-11 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-12 Cumene, NTP TR 542 TABLE A3b Historical Incidence of Kidney Neoplasms in Control Male F344/N Ratsa Study Lipoma Incidence in Controls Renal Tubule Renal Tubule Adenoma Carcinoma Renal Tubule Adenoma or Carcinoma Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide 1/50 0/50 0/50 0/50 0/50 0/49 0/50 0/50 0/50 Overall Historical Incidence: Inhalation Studies Total (%) Mean standard deviation Range 1/449 (0.2%) 0.2% 0.7% 0%-2% Overall Historical Incidence: All Routes Total (%) Mean standard deviation Range 2/1,436 (0.1%) 0.1% 0.5% 0%-2% a Data as of March 2, 2007 1/50 1/50 0/50 0/50 0/50 0/49 1/50 0/50 1/50 4/449 (0.9%) 0.9% 1.0% 0%-2% 8/1,436 (0.6%) 0.6% 0.8% 0%-2% 1/50 0/50 0/50 0/50 0/50 0/49 0/50 1/50 0/50 2/449 (0.5%) 0.4% 0.9% 0%-2% 2/1,436 (0.1%) 0.1% 0.5% 0%-2% 2/50 1/50 0/50 0/50 0/50 0/49 1/50 1/50 1/50 6/449 (1.3%) 1.3% 1.4% 0%-4% 10/1,436 (0.7%) 0.7% 1.0% 0%-4% NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 A-13 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Animals examined microscopically 50 50 20 24 43 26 23 50 50 50 50 21 24 22 27 24 50 50 Alimentary System Esophagus Foreign body Intestine large, cecum Intestine large, colon Diverticulum Intestine large, rectum Intestine small, duodenum Intestine small, ileum Intestine small, jejunum Inflammation, chronic active Thrombosis Ulcer Liver Angiectasis Basophilic focus Clear cell focus Degeneration, cystic Eosinophilic focus Eosinophilic focus, multiple Fatty change, diffuse Hepatodiaphragmatic nodule Inflammation, suppurative Mixed cell focus Necrosis Vacuolization cytoplasmic Bile duct, hyperplasia Hepatocyte, regeneration Oval cell, hyperplasia Periportal, inflammation, chronic Mesentery Hemorrhage Necrosis Fat, hemorrhage Fat, necrosis Pancreas Hyperplasia Vacuolization cytoplasmic Acinus, atrophy Acinus, hyperplasia (50) (49) (49) 1 (2%) (49) (49) (49) (48) 1 (2%) 1 (2%) 1 (2%) (50) 7 (14%) 12 (24%) 3 (6%) 3 (6%) 1 (2%) 3 (6%) 6 (12%) 7 (14%) 1 (2%) (7) 6 (86%) 1 (14%) (50) 2 (4%) (50) 1 (2%) (49) (50) (50) (49) (47) (47) (50) 1 (2%) 4 (8%) 6 (12%) 1 (2%) 1 (2%) 1 (2%) 8 (16%) 1 (2%) 3 (6%) 2 (4%) 4 (8%) (13) 1 (8%) 10 (77%) 1 (8%) (50) 1 (2%) 2 (4%) a Number of animals examined microscopically at the site and the number of animals with lesion (50) (49) (50) (50) (49) (48) (48) (50) 8 (16%) 11 (22%) 4 (8%) 3 (6%) 1 (2%) 2 (4%) 3 (6%) 3 (6%) 3 (6%) 12 (24%) 2 (4%) (10) 10 (100%) (50) 2 (4%) 1 (2%) (50) (49) (50) (50) (49) (49) (49) (50) 6 (12%) 11 (22%) 6 (12%) 3 (6%) 1 (2%) 3 (6%) 2 (4%) 7 (14%) 1 (2%) (6) 1 (17%) 3 (50%) 1 (17%) (50) 1 (2%) 2 (4%) 3 (6%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-14 Cumene, NTP TR 542 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Alimentary System (continued) Stomach, forestomach Hemorrhage Inflammation, suppurative Necrosis Ulcer Epithelium, hyperplasia Stomach, glandular Erosion Hyperplasia, lymphoid Mineralization Ulcer Tongue Epithelium, hyperplasia Tooth Inflammation (50) 1 (2%) 1 (2%) 3 (6%) 6 (12%) (50) 2 (4%) 1 (2%) 3 (6%) (1) 1 (100%) (50) 7 (14%) 11 (22%) (50) 1 (2%) (3) 2 (67%) (1) 1 (100%) (50) 2 (4%) 1 (2%) 7 (14%) (50) 1 (2%) 1 (2%) 1 (2%) (1) 1 (100%) (50) 8 (16%) 10 (20%) (50) 2 (4%) 1 (2%) (3) 3 (100%) Cardiovascular System Blood vessel Mineralization Heart Cardiomyopathy Atrium, thrombosis Valve, cardiomyopathy Valve, hemorrhage Valve, thrombosis Ventricle, hypertrophy (1) 1 (100%) (50) 9 (18%) 3 (6%) 1 (2%) 1 (2%) (1) (50) 7 (14%) 6 (12%) (1) (50) 12 (24%) 4 (8%) 1 (2%) (50) 11 (22%) 4 (8%) 1 (2%) Endocrine System Adrenal cortex Hyperplasia Hypertrophy Vacuolization cytoplasmic Adrenal medulla Hyperplasia Bilateral, hyperplasia Islets, pancreatic Hyperplasia Parathyroid gland Hyperplasia Pituitary gland Cyst Hemorrhage Hyperplasia Pars intermedia, hyperplasia Thyroid gland C-cell, hyperplasia Follicular cell, cyst Follicular cell, hyperplasia (50) 17 (34%) 8 (16%) (50) 11 (22%) (50) 1 (2%) (45) 1 (2%) (50) 1 (2%) 6 (12%) 1 (2%) (50) 7 (14%) (50) 16 (32%) 5 (10%) (50) 13 (26%) (50) 1 (2%) (49) 1 (2%) (50) 1 (2%) 1 (2%) 7 (14%) (50) 11 (22%) 1 (2%) (50) 10 (20%) 1 (2%) 7 (14%) (50) 18 (36%) (50) (49) (49) 2 (4%) 1 (2%) 13 (27%) (50) 6 (12%) 1 (2%) (50) 10 (20%) 4 (8%) (50) 12 (24%) 1 (2%) (50) (48) (50) 3 (6%) 6 (12%) (50) 8 (16%) General Body System Peritoneum (2) (2) (4) (2) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 A-15 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Genital System Epididymis Preputial gland Cyst Hyperplasia Inflammation, suppurative Prostate Hyperplasia Inflammation, suppurative Seminal vesicle Dilatation Hyperplasia Testes Necrosis Artery, inflammation, chronic active Bilateral, interstitial cell, hyperplasia Germinal epithelium, atrophy Interstitial cell, hyperplasia (50) (50) 1 (2%) (50) 42 (84%) (50) (50) 6 (12%) 12 (24%) (50) (50) (50) 1 (2%) 32 (64%) (50) 1 (2%) 1 (2%) (50) 1 (2%) 5 (10%) 18 (36%) (50) (50) 2 (4%) 1 (2%) (50) 1 (2%) 27 (54%) (50) (50) 1 (2%) 2 (4%) 3 (6%) 19 (38%) (50) (50) 1 (2%) (50) 2 (4%) 28 (56%) (50) (50) 1 (2%) 6 (12%) 9 (18%) Hematopoietic System Bone marrow Lymph node Deep cervical, ectasia Deep cervical, hemorrhage Deep cervical, hyperplasia, lymphoid Pancreatic, ectasia Pancreatic, hyperplasia, lymphoid Pancreatic, inflammation, granulomatous Lymph node, bronchial Ectasia Hyperplasia, lymphoid Lymph node, mandibular Ectasia Metaplasia, osseous Lymph node, mediastinal Angiectasis Hemorrhage Hyperplasia, lymphoid Inflammation, suppurative Lymph node, mesenteric Hemorrhage Spleen Accessory spleen Fibrosis Hematopoietic cell proliferation Hemorrhage Necrosis Thymus (50) (50) (7) (10) 1 (10%) (7) 1 (14%) 1 (14%) (1) 1 (100%) 1 (100%) (34) 1 (3%) 1 (3%) (50) (50) 2 (4%) 4 (8%) 1 (2%) (49) (12) 1 (8%) 1 (8%) (3) 2 (67%) (32) 1 (3%) (50) 2 (4%) (50) 2 (4%) 4 (8%) 3 (6%) (49) (50) (8) 1 (13%) 1 (13%) 1 (13%) 1 (13%) (11) 1 (9%) (34) 2 (6%) (50) (50) 7 (14%) 2 (4%) 1 (2%) (50) (50) (14) 1 (7%) 1 (7%) 2 (14%) 1 (7%) (8) (1) (34) 1 (3%) (50) 1 (2%) (50) 1 (2%) 2 (4%) 1 (2%) 4 (8%) (50) Integumentary System Mammary gland Galactocele Epithelium, hyperplasia (50) 2 (4%) 1 (2%) (50) (50) 2 (4%) 1 (2%) (50) 1 (2%) 1 (2%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-16 Cumene, NTP TR 542 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Integumentary System (continued) Skin Cyst epithelial inclusion Hyperkeratosis Inflammation, chronic Ulcer (50) 1 (2%) 1 (2%) (50) 1 (2%) (50) 1 (2%) (50) 2 (4%) 1 (2%) 1 (2%) Musculoskeletal System Bone Hyperostosis Skeletal muscle (50) (50) (50) (50) 1 (2%) (1) (2) (2) (1) Nervous System Brain Compression Hemorrhage Necrosis Thrombosis (50) 7 (14%) 5 (10%) (50) 16 (32%) 7 (14%) 1 (2%) 1 (2%) (50) 6 (12%) 3 (6%) (50) 6 (12%) 1 (2%) Respiratory System Larynx Foreign body Inflammation, suppurative Inflammation, chronic Epiglottis, hyperplasia Epiglottis, metaplasia, squamous Respiratory epithelium, hyperplasia Lung Congestion Hemorrhage Inflammation Inflammation, suppurative Inflammation, granulomatous Inflammation, chronic Thrombosis Alveolar epithelium, hyperplasia Alveolar epithelium, metaplasia, squamous Alveolar epithelium, metaplasia, mucous Alveolus, emphysema Alveolus, foreign body Alveolus, infiltration cellular, histiocyte Alveolus, proteinosis Artery, mineralization Artery, thrombosis Bronchiole, hyperplasia Bronchiole, inflammation, chronic Interstitium, fibrosis Nose Foreign body Hyperplasia, basal cell Inflammation, suppurative Inflammation, chronic (50) 6 (12%) 4 (8%) 1 (2%) 1 (2%) 1 (2%) 3 (6%) (50) 1 (2%) 3 (6%) 1 (2%) 3 (6%) 13 (26%) 9 (18%) 2 (4%) 1 (2%) (50) 5 (10%) 7 (14%) 5 (10%) (50) 3 (6%) 7 (14%) 1 (2%) 2 (4%) (50) 7 (14%) 1 (2%) 1 (2%) 3 (6%) 1 (2%) 11 (22%) 1 (2%) 7 (14%) 1 (2%) 1 (2%) 2 (4%) (50) 5 (10%) 8 (16%) 1 (2%) (50) 1 (2%) 1 (2%) 1 (2%) (50) 3 (6%) 1 (2%) 6 (12%) 10 (20%) 4 (8%) 1 (2%) 1 (2%) (49) 3 (6%) 8 (16%) (50) 3 (6%) 3 (6%) 2 (4%) 1 (2%) (50) 5 (10%) 10 (20%) 1 (2%) 1 (2%) 1 (2%) 14 (28%) 1 (2%) 1 (2%) (50) 4 (8%) 1 (2%) 6 (12%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 A-17 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Respiratory System (continued) Nose (continued) Epithelium, nasolacrimal duct, metaplasia, squamous Glands, olfactory epithelium, hyperplasia Glands, respiratory epithelium, dilatation Glands, respiratory epithelium, hyperplasia Goblet cell, olfactory epithelium, hyperplasia Goblet cell, hyperplasia Nasolacrimal duct, inflammation, suppurative Olfactory epithelium, degeneration Olfactory epithelium, degeneration, hyaline Olfactory epithelium, hyperplasia, basal cell Olfactory epithelium, inflammation, chronic Olfactory epithelium, metaplasia Olfactory epithelium, necrosis Olfactory epithelium, ulcer Respiratory epithelium, degeneration, hyaline Respiratory epithelium, hyperplasia Respiratory epithelium, inflammation, chronic Respiratory epithelium, necrosis Squamous epithelium, hyperplasia Squamous epithelium, inflammation Vomeronasal organ, inflammation, suppurative Pleura Inflammation, chronic Mesothelium, hyperplasia Trachea Epithelium, hyperplasia Glands, cyst (50) 1 (2%) 1 (2%) 3 (6%) 2 (4%) 3 (6%) 7 (14%) 1 (2%) 1 (2%) (5) 4 (80%) 1 (20%) (50) (50) 2 (4%) 3 (6%) 11 (22%) 2 (4%) 2 (4%) 19 (38%) 4 (8%) 1 (2%) 1 (2%) 15 (30%) (3) 3 (100%) (50) 1 (2%) (49) 1 (2%) 1 (2%) 2 (4%) 2 (4%) 1 (2%) 7 (14%) 3 (6%) 1 (2%) 1 (2%) 27 (55%) 5 (10%) 2 (4%) 16 (33%) 1 (2%) 1 (2%) 1 (2%) (5) 4 (80%) (50) (50) 1 (2%) 2 (4%) 4 (8%) 5 (10%) 26 (52%) 1 (2%) 5 (10%) 1 (2%) 23 (46%) 2 (4%) 1 (2%) (6) 6 (100%) (50) 1 (2%) Special Senses System Eye Degeneration Inflammation, chronic active Bilateral, lens, cataract Bilateral, retina, atrophy Ciliary body, iris, inflammation, suppurative Cornea, inflammation, suppurative Cornea, inflammation, chronic Lens, cataract Retina, atrophy Harderian gland Inflammation, suppurative Inflammation, chronic Zymbal's gland (50) 1 (2%) 2 (4%) 5 (10%) 4 (8%) (50) 1 (2%) (1) (50) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) (49) 1 (2%) 1 (2%) 2 (4%) (50) (50) 1 (2%) 1 (2%) 4 (8%) 2 (4%) (50) (2) Urinary System Kidney Atrophy Infarct Infarct, multiple Nephropathy Bilateral, renal tubule, cyst (50) 47 (94%) (50) 1 (2%) 1 (2%) 47 (94%) (50) 2 (4%) 47 (94%) 1 (2%) (50) 1 (2%) 1 (2%) 50 (100%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION A-18 Cumene, NTP TR 542 TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Urinary System (continued) Kidney (continued) Bilateral, infarct Capsule, dilatation Glomerulus, inflammation, suppurative Papilla, mineralization Pelvis, transitional epithelium, hyperplasia Pelvis, dilatation Renal tubule, accumulation, hyaline droplet Renal tubule, cyst Renal tubule, hyperplasia Renal tubule, hypertrophy Renal tubule, mineralization Ureter Urethra Transitional epithelium, hyperplasia Urinary bladder Calculus gross observation Hemorrhage Inflammation, chronic Transitional epithelium, hyperplasia (50) 5 (10%) 3 (6%) 1 (2%) (50) 1 (2%) (50) 1 (2%) 1 (2%) 35 (70%) 5 (10%) 1 (2%) 3 (6%) (1) (50) 1 (2%) (50) 1 (2%) 44 (88%) 14 (28%) 1 (2%) 1 (2%) 3 (6%) 8 (16%) (1) 1 (100%) (49) 1 (2%) 1 (2%) 4 (8%) (50) 41 (82%) 15 (30%) 1 (2%) 1 (2%) 2 (4%) 6 (12%) 1 (2%) (1) (1) (50) 2 (4%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft B-1 APPENDIX B SUMMARY OF LESIONS IN FEMALE RATS IN THE 2-YEAR INHALATION STUDY OF CUMENE TABLE B1 TABLE B2 TABLE B3 TABLE B4 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-6 Historical Incidence of Adenoma of the Nose in Control Female F344/N Rats . . . . . . . . . . B-9 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-10 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-2 Cumene, NTP TR 542 TABLE B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Animals examined microscopically 50 50 23 18 65 21 27 50 50 50 17 2 31 50 Alimentary System Intestine large, cecum Intestine large, colon Leiomyosarcoma Intestine large, rectum Adenoma Sarcoma stromal, metastatic, uterus Liver Cholangiocarcinoma Fibrous histiocytoma, metastatic, skin Mesentery Rhabdomyosarcoma Oral mucosa Squamous cell carcinoma Pharyngeal, squamous cell carcinoma Pancreas Rhabdomyosarcoma Stomach, forestomach Squamous cell papilloma Stomach, glandular Tongue Tooth (46) (48) (48) 1 (2%) (50) 1 (2%) (18) (50) (50) (50) (1) (45) (46) (46) (50) (18) (49) (50) (49) (2) (2) (50) (50) 1 (2%) (50) (50) (17) (1) 1 (100%) (50) (50) 1 (2%) (50) (2) Cardiovascular System Heart (50) (50) (50) Osteosarcoma, metastatic, bone 1 (2%) Pericardium, alveolar/bronchiolar, carcinoma, metastatic, lung 1 (2%) Endocrine System Adrenal cortex Adenoma Osteosarcoma, metastatic, bone Adrenal medulla Pheochromocytoma benign Pheochromocytoma complex Pheochromocytoma malignant Islets, pancreatic Adenoma Carcinoma Parathyroid gland Pituitary gland Adenoma Carcinoma (50) (50) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) (49) (50) 35 (70%) 1 (2%) (49) (49) 1 (2%) (49) (45) (50) 33 (66%) 1 (2%) (50) 1 (2%) (50) 1 (2%) 1 (2%) (50) 1 (2%) (48) (50) 28 (56%) 1,000 ppm 50 15 3 32 50 (49) (50) (50) 1 (2%) (50) 1 (2%) (9) 1 (11%) (1) 1 (100%) (50) 1 (2%) (50) 1 (2%) (50) (3) (50) (50) 1 (2%) (50) (50) 1 (2%) (47) (49) 25 (51%) 1 (2%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Endocrine System (continued) Thyroid gland Fibrous histiocytoma, metastatic, skin C-cell, adenoma C-cell, carcinoma Follicular cell, adenoma Follicular cell, carcinoma (50) 1 (2%) 3 (6%) (49) 4 (8%) 2 (4%) (50) 2 (4%) 1 (2%) 1 (2%) General Body System Peritoneum Carcinoma, metastatic, ovary (1) 1 (100%) Genital System Clitoral gland Adenoma Carcinoma Carcinoma, multiple Ovary Cystadenocarcinoma Granulosa cell tumor benign Leiomyosarcoma, metastatic, uterus Osteosarcoma, metastatic, bone Uterus Carcinoma Leiomyosarcoma Polyp stromal Polyp stromal, multiple Sarcoma stromal Cervix, polyp stromal Vagina Polyp Epithelium, polyp (50) 1 (2%) 1 (2%) 1 (2%) (50) (50) 13 (26%) 1 (2%) (2) 1 (50%) 1 (50%) (49) 2 (4%) (49) 1 (2%) (49) 1 (2%) 7 (14%) 1 (2%) 1 (2%) (50) (50) 1 (2%) 1 (2%) (50) 8 (16%) 1 (2%) Hematopoietic System Lymph node Deep cervical, squamous cell carcinoma, metastatic, skin Lymph node, bronchial Lymph node, mandibular Carcinoma, metastatic, Zymbal's gland Lymph node, mediastinal Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, thyroid gland Squamous cell carcinoma, metastatic, skin Lymph node, mesenteric Carcinoma, metastatic, ovary Spleen Fibrous histiocytoma, metastatic, skin Rhabdomyosarcoma Thymus Thymoma benign (3) (7) (4) (30) (48) (50) 1 (2%) (48) (3) (9) (1) (28) (49) (50) (46) (5) 1 (20%) (8) (1) (26) 1 (4%) 1 (4%) 1 (4%) (49) 1 (2%) (50) (50) B-3 1,000 ppm (50) 2 (4%) 2 (4%) 2 (4%) (50) 1 (2%) (50) 1 (2%) (50) 1 (2%) 7 (14%) 1 (2%) (2) (5) (4) 1 (25%) (31) 2 (6%) (50) (50) 1 (2%) (50) 1 (2%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-4 Cumene, NTP TR 542 TABLE B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Integumentary System Mammary gland Adenoma Carcinoma Carcinoma, multiple Fibroadenoma Fibroadenoma, multiple Skin Basal cell carcinoma Fibrous histiocytoma Keratoacanthoma Squamous cell carcinoma Squamous cell papilloma Subcutaneous tissue, fibroma Subcutaneous tissue, hemangiopericytoma Subcutaneous tissue, schwannoma benign (50) 8 (16%) 2 (4%) 17 (34%) 7 (14%) (50) 1 (2%) (50) 1 (2%) 8 (16%) 1 (2%) 11 (22%) 11 (22%) (50) 2 (4%) 1 (2%) (50) 1 (2%) 1 (2%) 24 (48%) 8 (16%) (50) 1 (2%) 2 (4%) 1 (2%) 1 (2%) Musculoskeletal System Bone Cranium, osteosarcoma Vertebra, osteosarcoma Skeletal muscle Alveolar/bronchiolar carcinoma, metastatic, lung Fibrous histiocytoma, metastatic, skin Rhabdomyosarcoma (50) 1 (2%) (1) 1 (100%) (50) 1 (2%) (50) 1 (2%) (1) 1 (100%) Nervous System Brain Astrocytoma malignant Carcinoma, metastatic, pituitary gland Glioma malignant Granular cell tumor benign Meningioma benign Oligodendroglioma malignant Spinal cord (50) 1 (2%) 1 (2%) 1 (2%) (49) 2 (4%) 1 (2%) (1) (50) 1 (2%) (1) Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Carcinoma, metastatic, ovary Carcinoma, metastatic, uterus Fibrous histiocytoma, metastatic, skin Osteosarcoma, metastatic, bone Squamous cell carcinoma, metastatic, skin Mediastinum, carcinoma, metastatic, uterus Nose Nasopharyngeal duct, carcinoma, metastatic, oral mucosa Respiratory epithelium, adenoma Turbinate, chondroma (50) (50) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) (49) (50) 1 (2%) 1 (2%) (48) 5 (10%) (50) (50) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (50) 4 (8%) 1,000 ppm (50) 1 (2%) 5 (10%) 21 (42%) 8 (16%) (50) 1 (2%) 1 (2%) 1 (2%) (50) (1) 1 (100%) (50) 1 (2%) 1 (2%) (1) (50) (50) 1 (2%) (50) 1 (2%) 3 (6%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Respiratory System (continued) Pleura (15) (16) (16) Alveolar/bronchiolar carcinoma, metastatic, lung 1 (6%) Carcinoma, metastatic, ovary 1 (6%) Trachea (50) (49) (50) Special Senses System Eye Harderian gland Zymbal's gland Carcinoma (50) (50) (1) 1 (100%) (49) (49) (50) (50) (1) 1 (100%) Urinary System Kidney Urinary bladder Leiomyosarcoma (50) (50) 1 (2%) (50) (49) (50) (50) Systemic Lesions Multiple organsb Histiocytic sarcoma Leukemia mononuclear Lymphoma malignant (50) 12 (24%) (50) 1 (2%) 25 (50%) (50) 23 (46%) Neoplasm Summary Total animals with primary neoplasmsc Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms 50 48 117 122 44 42 81 81 24 32 36 41 43 84 a b c Number of animals examined microscopically at the site and the number of animals with neoplasm Number of animals with any tissue examined microscopically Primary neoplasms: all neoplasms except metastatic neoplasms 48 118 43 80 30 38 5 16 B-5 1,000 ppm (20) (50) (50) (50) (1) 1 (100%) (50) (50) (50) 13 (26%) 1 (2%) 47 111 43 78 25 33 6 6 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-6 Cumene, NTP TR 542 TABLE B2 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Adrenal Medulla: Benign, Complex, or Malignant Pheochromocytoma OATevdrejmurasintleladrlarrtaaettaeecb 3/50 (6%) 7.3% 1/21 (5%) First incidence (days) Poly-3 testd 716 P=0.096N Clitoral Gland: Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.2% 1/21 (5%) 501 P=0.158N Mammary Gland: Fibroadenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 24/50 (48%) 54.9% 11/21 (52%) 501 P=0.122 Mammary Gland: Fibroadenoma or Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 24/50 (48%) 54.9% 11/21 (52%) 501 P=0.138 Mammary Gland: Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 10/50 (20%) 23.9% 5/21 (24%) 558 P=0.038N Mammary Gland: Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 10/50 (20%) 23.9% 5/21 (24%) 558 P=0.063N Mammary Gland: Fibroadenoma, Adenoma, or Carcinoma Overall rate 31/50 (62%) Adjusted rate 69.9% Terminal rate 16/21 (76%) First incidence (days) 501 Poly-3 test P=0.444 Nose: Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/50 (0%) 0.0% 0/21 (0%) -- P=0.320 1/49 (2%) 2.4% 1/27 (4%) 730 (T) P=0.300N 2/49 (4%) 4.8% 1/27 (4%) 709 P=0.502N 22/50 (44%) 49.9% 15/27 (56%) 571 P=0.396N 23/50 (46%) 51.9% 15/27 (56%) 571 P=0.472N 9/50 (18%) 21.2% 8/27 (30%) 704 P=0.484N 10/50 (20%) 23.4% 8/27 (30%) 673 P=0.580N 29/50 (58%) 65.3% 20/27 (74%) 571 P=0.400N 5/48 (10%) 12.2% 5/27 (19%) 730 (T) P=0.030 2/50 (4%) 4.7% 2/31 (7%) 730 (T) P=0.479N 0/50 (0%) 0.0% 0/31 (0%) -- P=0.113N 32/50 (64%) 70.4% 22/31 (71%) 530 P=0.087 32/50 (64%) 70.4% 22/31 (71%) 530 P=0.087 2/50 (4%) 4.7% 1/31 (3%) 715 P=0.011N 2/50 (4%) 4.7% 1/31 (3%) 715 P=0.011N 32/50 (64%) 70.4% 22/31 (71%) 530 P=0.576 4/50 (8%) 9.3% 4/31 (13%) 730 (T) P=0.066 1,000 ppm 0/50 (0%) 0.0% --0/3e2 (0%) P=0.109N 1/50 (2%) 2.3% 1/32 (3%) 730 (T) P=0.292N 29/50 (58%) 63.4% 19/32 (59%) 619 P=0.267 29/50 (58%) 63.4% 19/32 (59%) 619 P=0.267 5/50 (10%) 11.2% 1/32 (3%) 542 P=0.101N 6/50 (12%) 13.4% 1/32 (3%) 542 P=0.162N 33/50 (66%) 70.5% 20/32 (63%) 542 P=0.570 3/50 (6%) 6.9% 2/32 (6%) 638 P=0.130 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE B2 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Pituitary Gland (Pars Distalis): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 35/50 (70%) 77.1% 17/21 (81%) 501 P=0.013N 33/50 (66%) 71.4% 17/27 (63%) 536 P=0.342N 28/50 (56%) 61.4% 19/31 (61%) 528 P=0.070N Pituitary Gland (Pars Distalis): Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 36/50 (72%) 78.9% 17/21 (81%) 501 P=0.012N 34/50 (68%) 72.7% 17/27 (63%) 536 P=0.319N 28/50 (56%) 61.4% 19/31 (61%) 528 P=0.045N Skin: Squamous Cell Papilloma, Keratoacanthoma, or Squamous Cell Carcinoma Overall rate 0/50 (0%) 0/50 (0%) Adjusted rate 0.0% 0.0% Terminal rate 0/21 (0%) 0/27 (0%) First incidence (days) ---- Poly-3 test P=0.106 -- 3/50 (6%) 6.8% 1/31 (3%) 516 P=0.132 Skin: Squamous Cell Papilloma, Keratoacanthoma, Basal Cell Carcinoma, or Squamous Cell Carcinoma Overall rate 0/50 (0%) 0/50 (0%) 4/50 (8%) Adjusted rate 0.0% 0.0% 9.1% Terminal rate 0/21 (0%) 0/27 (0%) 2/31 (7%) First incidence (days) Poly-3 test -- P=0.114 ----f 516 P=0.069 Thyroid Gland (C-Cell): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.3% 2/21 (10%) 716 P=0.295N 4/49 (8%) 9.5% 2/27 (7%) 655 P=0.513 2/50 (4%) 4.7% 2/31 (7%) 730 (T) P=0.479N Thyroid Gland (C-Cell): Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 7.3% 2/21 (10%) 716 P=0.495 4/49 (8%) 9.5% 2/27 (7%) 655 P=0.513 3/50 (6%) 7.0% 3/31 (10%) 730 (T) P=0.641N Uterus: Stromal Polyp Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 13/50 (26%) 31.1% 10/21 (48%) 547 P=0.070N 9/50 (18%) 21.1% 7/27 (26%) 696 P=0.210N 8/50 (16%) 18.2% 6/31 (19%) 528 P=0.125N Uterus: Stromal Polyp or Stromal Sarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 14/50 (28%) 33.5% 10/21 (48%) 547 P=0.081N 9/50 (18%) 21.1% 7/27 (26%) 696 P=0.148N 9/50 (18%) 20.3% 6/31 (19%) 528 P=0.124N B-7 1,000 ppm 25/49 (51%) 56.2% 17/32 (53%) 542 P=0.023N 26/49 (53%) 58.1% 17/32 (53%) 542 P=0.022N 2/50 (4%) 4.6% 1/32 (3%) 683 P=0.250 2/50 (4%) 4.6% 1/32 (3%) 683 P=0.250 2/50 (4%) 4.6% 2/32 (6%) 730 (T) P=0.476N 4/50 (8%) 9.3% 4/32 (13%) 730 (T) P=0.529 7/50 (14%) 15.9% 4/32 (13%) 628 P=0.076N 8/50 (16%) 17.9% 4/32 (13%) 411 P=0.075N Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-8 Cumene, NTP TR 542 TABLE B2 Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm All Organs: Mononuclear Leukemia Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Benign Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Benign or Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 12/50 (24%) 27.9% 3/21 (14%) 492 P=0.277N 44/50 (88%) 93.2% 21/21 (100%) 374 P=0.519N 24/50 (48%) 53.0% 7/21 (33%) 485 P=0.304N 50/50 (100%) 100% 21/21 (100%) 374 P=0.132N 25/50 (50%) 55.5% 14/27 (52%) 547 P=0.006 42/50 (84%) 89.8% 24/27 (89%) 536 P=0.399N 33/50 (66%) 70.8% 19/27 (70%) 514 P=0.053 48/50 (96%) 98.0% 26/27 (96%) 514 P=0.496N 23/50 (46%) 50.2% 12/31 (39%) 530 P=0.023 43/50 (86%) 89.7% 28/31 (90%) 516 P=0.390N 30/50 (60%) 62.1% 15/31 (48%) 339 P=0.242 48/50 (96%) 96.0% 29/31 (94%) 339 P=0.237N 13/50 (26%) 29.0% 7/32 (22%) 542 P=0.548 43/50 (86%) 91.8% 30/32 (94%) 542 P=0.559N 25/50 (50%) 52.7% 13/32 (41%) 267 P=0.573N 47/50 (94%) 95.6% 30/32 (94%) 267 P=0.200N (aT) Terminal sacrifice Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal gland, b c d clitoral gland, nose, pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not e f reach terminal sacrifice. A negative trend or a lower incidence in an exposed group is indicated by N. Not applicable; no neoplasms in animal group Value of statistic cannot be computed. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE B3 Historical Incidence of Adenoma of the Nose in Control Female F344/N Ratsa Study Incidence in Controls Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide Overall Historical Incidence: Inhalation Studies Total Overall Historical Incidence: All Routes Total (%) a Data as of March 2, 2007 0/50 0/50 0/50 0/50 0/49 0/49 0/49 0/49 0/50 0/496 0/1,343 B-9 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-10 Cumene, NTP TR 542 TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Animals examined microscopically 50 50 23 18 65 21 27 50 50 50 50 17 15 23 31 32 50 50 Alimentary System Intestine large, cecum Necrosis Intestine large, colon Artery, inflammation, chronic Intestine large, rectum Liver Angiectasis Basophilic focus Clear cell focus Eosinophilic focus Hepatodiaphragmatic nodule Inflammation, granulomatous Mixed cell focus Necrosis Vacuolization cytoplasmic Bile duct, hyperplasia Centrilobular, congestion Periportal, vacuolization cytoplasmic Mesentery Necrosis Oral mucosa Pancreas Fibrosis Hemorrhage Thrombosis Artery, inflammation, chronic Stomach, forestomach Erosion Inflammation, suppurative Inflammation, chronic Ulcer Epithelium, cyst Epithelium, erosion Epithelium, hyperplasia Submucosa, fibrosis Stomach, glandular Erosion Ulcer Epithelium, hyperplasia Tongue Epithelium, hyperplasia Tooth Peridontal tissue, inflammation (46) (48) (48) (50) 1 (2%) 27 (54%) 15 (30%) 6 (12%) 1 (2%) 11 (22%) 1 (2%) (18) 18 (100%) (50) (50) 5 (10%) 11 (22%) (50) 1 (2%) (1) 1 (100%) (45) (46) (46) (50) 31 (62%) 9 (18%) 6 (12%) 1 (2%) 4 (8%) 1 (2%) (18) 16 (89%) (49) 1 (2%) 1 (2%) (50) 1 (2%) 4 (8%) 1 (2%) 13 (26%) (49) (2) 2 (100%) (2) 2 (100%) a Number of animals examined microscopically at the site and the number of animals with lesion (50) (50) (50) (50) 2 (4%) 36 (72%) 6 (12%) 4 (8%) 2 (4%) 1 (2%) 4 (8%) 1 (2%) (17) 17 (100%) (1) (50) (50) 1 (2%) 1 (2%) 3 (6%) 1 (2%) 12 (24%) 1 (2%) (50) 1 (2%) (2) 2 (100%) (49) 1 (2%) (50) 1 (2%) (50) (50) 32 (64%) 5 (10%) 1 (2%) 6 (12%) 1 (2%) 3 (6%) 2 (4%) (9) 8 (89%) (1) (50) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) 8 (16%) (50) 1 (2%) (3) 3 (100%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 B-11 TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Cardiovascular System Heart Cardiomyopathy Inflammation, chronic Atrium, thrombosis (50) 1 (2%) (50) 2 (4%) 2 (4%) (50) 2 (4%) 2 (4%) (50) 1 (2%) 1 (2%) Endocrine System Adrenal cortex Hemorrhage Hyperplasia Necrosis Vacuolization cytoplasmic Adrenal medulla Islets, pancreatic Parathyroid gland Hyperplasia Pituitary gland Cyst Hemorrhage Hyperplasia Thyroid gland Ultimobranchial cyst C-cell, hyperplasia Follicular cell, hyperplasia (50) 1 (2%) 9 (18%) 2 (4%) 11 (22%) (50) (50) (49) (50) 7 (14%) 2 (4%) 10 (20%) (50) 1 (2%) 13 (26%) (49) 9 (18%) 18 (37%) (49) (49) (45) 1 (2%) (50) 2 (4%) 2 (4%) 13 (26%) (49) 13 (27%) (50) 13 (26%) 16 (32%) (50) (50) (48) (50) 2 (4%) 2 (4%) 6 (12%) (50) 15 (30%) 1 (2%) (50) 12 (24%) 1 (2%) 7 (14%) (50) (50) (47) (49) 2 (4%) 16 (33%) (50) 15 (30%) General Body System Peritoneum (1) Genital System Clitoral gland Cyst Hyperplasia Inflammation, chronic Ovary Cyst Cyst, multiple Uterus Hemorrhage Pigmentation Endometrium, hyperplasia Vagina Epithelium, hyperplasia, adenomatous (50) 2 (4%) (50) 3 (6%) (50) 1 (2%) 4 (8%) (2) 1 (50%) (49) 2 (4%) 5 (10%) 1 (2%) (49) 7 (14%) (49) 1 (2%) 7 (14%) (50) 2 (4%) 7 (14%) 1 (2%) (50) 7 (14%) 1 (2%) (50) 4 (8%) 1 (2%) 2 (4%) (50) 1 (2%) 5 (10%) 3 (6%) (50) 6 (12%) (50) 1 (2%) 5 (10%) Hematopoietic System Lymph node Pancreatic, infiltration cellular, histiocyte Pancreatic, pigmentation Lymph node, bronchial Ectasia Hemorrhage Hyperplasia, lymphoid Inflammation (3) (7) 2 (29%) 1 (14%) (3) (9) 1 (11%) (5) 1 (20%) 1 (20%) (8) (2) (5) 1 (20%) 1 (20%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-12 Cumene, NTP TR 542 TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Hematopoietic System (continued) Lymph node, mandibular Ectasia Hyperplasia, lymphoid Lymph node, mediastinal Angiectasis Hemorrhage Hyperplasia, histiocytic Hyperplasia, lymphoid Inflammation, suppurative Lymph node, mesenteric Ectasia Hemorrhage Hyperplasia, lymphoid Infiltration cellular, histiocyte Necrosis Spleen Atrophy Fibrosis Hematopoietic cell proliferation Necrosis Capsule, fibrosis Thymus Hyperplasia, tubular (4) (30) (48) 2 (4%) (50) 1 (2%) (48) (1) (28) (49) 1 (2%) 1 (2%) (50) 1 (2%) (46) (1) (26) 1 (4%) 1 (4%) 1 (4%) 1 (4%) (49) 1 (2%) (50) 3 (6%) 2 (4%) (50) 1 (2%) (4) 1 (25%) 1 (25%) (31) 1 (3%) 2 (6%) 1 (3%) (50) 1 (2%) (50) 1 (2%) 2 (4%) 2 (4%) (50) Integumentary System Mammary gland Galactocele Hyperplasia Inflammation, suppurative Epithelium, hyperplasia Skin Cyst epithelial inclusion Hyperkeratosis Inflammation, chronic Ulcer Sebaceous gland, hemorrhage Subcutaneous tissue, fibrosis Subcutaneous tissue, inflammation, suppurative (50) 3 (6%) 1 (2%) (50) 1 (2%) (50) 1 (2%) (50) 1 (2%) 1 (2%) 3 (6%) 1 (2%) 1 (2%) (50) 1 (2%) (50) (50) 2 (4%) 1 (2%) (50) 1 (2%) 1 (2%) 2 (4%) 1 (2%) Musculoskeletal System Bone (50) (50) (50) (50) Hyperostosis 1 (2%) Maxilla, fracture 1 (2%) Maxilla, inflammation, chronic active 1 (2%) Skeletal muscle (1) (1) (1) Nervous System Brain Compression Demyelination Gliosis Hemorrhage (50) 12 (24%) 1 (2%) (49) 9 (18%) 3 (6%) (50) 4 (8%) 2 (4%) (50) 5 (10%) 1 (2%) 1 (2%) 3 (6%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 B-13 TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Nervous System (continued) Brain (continued) Inflammation, chronic active Necrosis Thrombosis Medulla, meninges, inflammation, suppurative Medulla, neuron, necrosis Neuron, degeneration Spinal cord Gliosis Inflammation, chronic active (50) 1 (2%) 1 (2%) (49) 1 (2%) (1) (50) 1 (2%) (1) (50) 1 (2%) 1 (2%) 1 (2%) (1) 1 (100%) 1 (100%) Respiratory System Larynx Foreign body Inflammation, suppurative Inflammation, chronic Respiratory epithelium, hyperplasia Lung Cyst, squamous Hemorrhage Inflammation, suppurative Inflammation, chronic Thrombosis Alveolar epithelium, hyperplasia Alveolar epithelium, metaplasia, squamous Alveolus, infiltration cellular, histiocyte Alveolus, proteinosis Bronchiole, hyperplasia Bronchiole, inflammation, chronic Interstitium, fibrosis Nose Foreign body Hyperplasia, basal cell Inflammation, suppurative Inflammation, chronic Inflammation, chronic active Glands, respiratory epithelium, dilatation Goblet cell, hyperplasia Nasolacrimal duct, inflammation, suppurative Nasolacrimal duct, inflammation, chronic Olfactory epithelium, degeneration, hyaline Olfactory epithelium, hyperplasia Olfactory epithelium, hyperplasia, basal cell Olfactory epithelium, inflammation, granulomatous Olfactory epithelium, metaplasia Olfactory epithelium, necrosis Respiratory epithelium, degeneration, hyaline Respiratory epithelium, hyperplasia Respiratory epithelium, metaplasia, squamous Turbinate, necrosis Pleura Inflammation, chronic Mesothelium, hyperplasia Trachea Glands, cyst (50) 2 (4%) 3 (6%) 2 (4%) (50) 2 (4%) 1 (2%) 11 (22%) 3 (6%) 16 (32%) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) 1 (2%) 4 (8%) 4 (8%) 1 (2%) 2 (4%) 1 (2%) (15) 15 (100%) (50) (49) 1 (2%) 3 (6%) 1 (2%) (50) 1 (2%) 10 (20%) 1 (2%) 6 (12%) 3 (6%) 1 (2%) (48) 4 (8%) 3 (6%) 3 (6%) 6 (13%) 3 (6%) 1 (2%) 14 (29%) 1 (2%) 1 (2%) 1 (2%) (16) 15 (94%) 1 (6%) (49) (50) 2 (4%) 4 (8%) 2 (4%) (50) 2 (4%) 8 (16%) 1 (2%) 5 (10%) 11 (22%) (50) 2 (4%) 1 (2%) 4 (8%) 1 (2%) 1 (2%) 2 (4%) 1 (2%) 25 (50%) 1 (2%) 3 (6%) 4 (8%) 1 (2%) (16) 14 (88%) (50) 1 (2%) (50) 1 (2%) 2 (4%) 1 (2%) (50) 1 (2%) 16 (32%) 7 (14%) 1 (2%) 14 (28%) 2 (4%) 1 (2%) 3 (6%) (50) 3 (6%) 1 (2%) 2 (4%) 1 (2%) 5 (10%) 1 (2%) 31 (62%) 2 (4%) 6 (12%) (20) 20 (100%) (50) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION B-14 Cumene, NTP TR 542 TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Special Senses System Eye Bilateral, lens, cataract Bilateral, retina, atrophy Cornea, epithelium, hyperplasia Cornea, inflammation Cornea, inflammation, suppurative Lens, cataract Retina, atrophy Harderian gland Inflammation, chronic Zymbal's gland (50) 1 (2%) 1 (2%) 1 (2%) 5 (10%) 3 (6%) (50) 1 (2%) (1) (49) 1 (2%) 2 (4%) 5 (10%) (49) (50) 1 (2%) 5 (10%) 8 (16%) (50) 1 (2%) (1) (50) 1 (2%) 1 (2%) 3 (6%) 2 (4%) (50) 1 (2%) (1) Urinary System Kidney Infarct, multiple Nephropathy Artery, inflammation, chronic active Papilla, mineralization Pelvis, transitional epithelium, hyperplasia Pelvis, transitional epithelium, mineralization Pelvis, dilatation Renal tubule, accumulation, hyaline droplet Renal tubule, cyst Renal tubule, pigmentation Urinary bladder Transitional epithelium, hyperplasia (50) 38 (76%) 6 (12%) 1 (2%) 23 (46%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) (50) 37 (74%) 3 (6%) 1 (2%) 27 (54%) (49) 2 (4%) (50) 1 (2%) 41 (82%) 4 (8%) 6 (12%) 27 (54%) 1 (2%) 1 (2%) (50) 2 (4%) (50) 44 (88%) 1 (2%) 6 (12%) 1 (2%) 22 (44%) 1 (2%) (50) 1 (2%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft C-1 APPENDIX C SUMMARY OF LESIONS IN MALE MICE IN THE 2-YEAR INHALATION STUDY OF CUMENE TABLE C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2 TABLE C2 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6 TABLE C3a Historical Incidence of Alveolar/bronchiolar Neoplasms in Control Male B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-9 TABLE C3b Historical Incidence of Hepatocellular Neoplasms in Control Male B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-10 TABLE C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-11 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-2 Cumene, NTP TR 542 TABLE C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Animals examined microscopically 50 50 79 57 38 34 50 50 50 13 7 30 50 Alimentary System Gallbladder Intestine large, cecum Mast cell tumor malignant, metastatic, uncertain primary site Intestine large, colon Leiomyoma Intestine small, duodenum Carcinoma Intestine small, ileum Intestine small, jejunum Carcinoma Liver Hemangioma Hemangiosarcoma Hepatoblastoma Hepatocellular adenoma Hepatocellular adenoma, multiple Hepatocellular carcinoma Hepatocellular carcinoma, multiple Hepatocholangiocarcinoma Sarcoma, metastatic, uncertain primary site Mesentery Mast cell tumor malignant, metastatic, uncertain primary site Pancreas Sarcoma, metastatic, uncertain primary site Stomach, forestomach Mast cell tumor malignant, metastatic, uncertain primary site Sarcoma, metastatic, uncertain primary site Squamous cell carcinoma Squamous cell papilloma Stomach, glandular Sarcoma, metastatic, uncertain primary site Tongue Squamous cell carcinoma Tooth Mast cell tumor malignant, metastatic, uncertain primary site (43) (49) (50) (49) (49) (48) 1 (2%) (50) 1 (2%) 1 (2%) 17 (34%) 17 (34%) 10 (20%) 3 (6%) (3) (50) (50) 1 (2%) 1 (2%) (50) (1) 1 (100%) (15) 1 (7%) (32) (47) 1 (2%) (46) 1 (2%) (44) 1 (2%) (46) (46) 1 (2%) (50) 1 (2%) 1 (2%) 13 (26%) 20 (40%) 17 (34%) 1 (2%) (7) 1 (14%) (50) (50) 1 (2%) (48) (8) 1 (13%) (37) (44) (45) (43) (43) (43) (50) 1 (2%) 4 (8%) 15 (30%) 22 (44%) 17 (34%) 4 (8%) (6) (49) (50) 1 (2%) (48) (8) 1,000 ppm 50 20 7 23 50 (38) (44) (48) (44) (44) (44) 2 (5%) (50) 1 (2%) 3 (6%) 9 (18%) 26 (52%) 10 (20%) 7 (14%) 1 (2%) 1 (2%) (3) (49) 1 (2%) (49) 1 (2%) (48) 1 (2%) (6) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hemangiosarcoma Hepatocellular carcinoma, metastatic, liver Endocrine System Adrenal cortex Alveolar/bronchiolar carcinoma, metastatic, lung Sarcoma, metastatic, uncertain primary site Subcapsular, adenoma Adrenal medulla Pheochromocytoma benign Pheochromocytoma malignant Islets, pancreatic Pituitary gland Pars distalis, adenoma Thyroid gland Follicular cell, adenoma General Body System None Genital System Epididymis Preputial gland Prostate Testes Interstitial cell, adenoma Hematopoietic System Bone marrow Lymph node Pancreatic, sarcoma, metastatic, uncertain primary site Renal, carcinoma, metastatic, uncertain primary site Lymph node, bronchial Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Sarcoma, metastatic, uncertain primary site Lymph node, mandibular Carcinoma, metastatic, uncertain primary site Mast cell tumor metastatic, uncertain primary site Lymph node, mediastinal Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocellular carcinoma, metastatic, liver Sarcoma, metastatic, uncertain primary site Lymph node, mesenteric Sarcoma, metastatic, uncertain primary site (50) (50) 1 (2%) (50) (50) (48) 1 (2%) (50) (50) (49) (50) (50) 1 (2%) (50) (45) (34) 1 (3%) (37) (48) (50) (50) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) (50) (49) (50) (50) (50) (49) (50) (50) (41) (35) (42) (47) (50) 1 (2%) 1 (2%) 1 (2%) (49) 2 (4%) (48) 1 (2%) (49) (49) (49) (49) (50) (48) (49) (49) (1) (45) (22) (40) 1 (3%) (46) C-3 1,000 ppm (50) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) (50) (49) (49) 1 (2%) (50) 3 (6%) (50) (50) (49) (50) (49) (2) 1 (50%) 1 (50%) (35) 1 (3%) 1 (3%) 1 (3%) (25) 1 (4%) (39) 2 (5%) 1 (3%) 1 (3%) 1 (3%) (44) 1 (2%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-4 Cumene, NTP TR 542 TABLE C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Hematopoietic System (continued) Spleen Hemangiosarcoma Sarcoma, metastatic, uncertain primary site Thymus Alveolar/bronchiolar carcinoma, metastatic, lung Mast cell tumor malignant, metastatic, uncertain primary site Sarcoma, metastatic, uncertain primary site Integumentary System Skin Hepatocellular carcinoma, metastatic, liver Subcutaneous tissue, fibrosarcoma Subcutaneous tissue, fibrous histiocytoma Subcutaneous tissue, sarcoma Musculoskeletal System Bone Carcinoma, metastatic, uncertain primary site Skeletal muscle Alveolar/bronchiolar carcinoma, metastatic, lung Nervous System Brain Carcinoma, metastatic, uncertain primary site Respiratory System Larynx Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Carcinoma, metastatic, harderian gland Carcinoma, metastatic, uncertain primary site Hepatocellular carcinoma, metastatic, liver Sarcoma, metastatic, uncertain primary site Nose Pleura Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Trachea Alveolar/bronchiolar carcinoma, metastatic, lung Special Senses System Eye Harderian gland Adenoma Carcinoma Bilateral, adenoma (50) (45) 1 (2%) (50) 1 (2%) 1 (2%) (50) (50) (50) (50) 12 (24%) 1 (2%) 9 (18%) 1 (2%) 7 (14%) (50) (50) (49) (48) 8 (17%) 4 (8%) (50) (44) (50) 1 (2%) 1 (2%) 2 (4%) (50) (50) (49) (50) 19 (38%) 12 (24%) 11 (22%) 8 (16%) 8 (16%) (50) (50) (50) (50) 2 (4%) 3 (6%) (49) (38) (49) 1 (2%) (50) (1) 1 (100%) (50) (50) (50) 16 (32%) 15 (30%) 12 (24%) 20 (40%) 2 (4%) 11 (22%) 1 (2%) (49) (49) (49) (49) 3 (6%) 2 (4%) 1 (2%) 1,000 ppm (50) 4 (8%) 1 (2%) (44) 1 (2%) 1 (2%) (50) 3 (6%) (50) 1 (2%) (50) 1 (2%) (50) (50) 9 (18%) 20 (40%) 16 (32%) 17 (34%) 1 (2%) 1 (2%) 7 (14%) 1 (2%) (48) (2) 1 (50%) 1 (50%) (50) 1 (2%) (48) (50) 4 (8%) 2 (4%) 1 (2%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Urinary System Kidney Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocellular carcinoma, metastatic, liver Mast cell tumor malignant, metastatic, uncertain primary site Capsule, sarcoma, metastatic, uncertain primary site Urinary bladder Hemangiosarcoma (50) 1 (2%) 1 (2%) (50) (50) 1 (2%) 1 (2%) (48) (50) 1 (2%) (49) 1 (2%) Systemic Lesions Multiple organsb Histiocytic sarcoma Lymphoma malignant (50) 2 (4%) (50) 1 (2%) 1 (2%) (50) 1 (2%) Neoplasm Summary Total animals with primary neoplasmsc Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms Total animals with malignant neoplasms- uncertain primary site 48 50 93 119 43 45 60 69 26 34 33 50 9 10 14 16 11 a b c Number of animals examined microscopically at the site and the number of animals with neoplasm Number of animals with any tissue examined microscopically Primary neoplasms: all neoplasms except metastatic neoplasms 50 140 46 75 47 65 15 19 1 C-5 1,000 ppm (50) 1 (2%) 1 (2%) 1 (2%) (48) (50) 50 140 44 74 45 66 12 40 2 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-6 Cumene, NTP TR 542 TABLE C2 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Harderian Gland: Adenoma OATevdrejmurasintleladrlarrtaaettaeecb FPoirlsyt-i3ncteidstednce (days) Harderian Gland: Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Harderian Gland: Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatoblastoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Carcinoma or Hepatoblastoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 8/50 (16%) 17.2% 8/38 (21%) 729 (T) P=0.400N 4/50 (8%) 8.5% 3/38 (8%) 551 P=0.297N 11/50 (22%) 23.4% 10/38 (26%) 551 P=0.322N 34/50 (68%) 70.6% 28/38 (74%) 551 P=0.135 13/50 (26%) 27.1% 7/38 (18%) 583 P=0.184 40/50 (80%) 81.0% 30/38 (79%) 551 P=0.250 1/50 (2%) 2.1% 0/38 (0%) 659 P=0.118 14/50 (28%) 29.1% 7/38 (18%) 583 P=0.071 2/50 (4%) 4.5% 2/34 (6%) 729 (T) P=0.052N 3/50 (6%) 6.7% 3/34 (9%) 729 (T) P=0.529N 5/50 (10%) 11.2% 5/34 (15%) 729 (T) P=0.102N 33/50 (66%) 69.9% 23/34 (68%) 533 P=0.560N 18/50 (36%) 38.1% 9/34 (27%) 453 P=0.177 42/50 (84%) 85.8% 28/34 (82%) 453 P=0.355 1/50 (2%) 2.2% 1/34 (3%) 729 (T) P=0.750 18/50 (36%) 38.1% 9/34 (27%) 453 P=0.236 4/50 (8%) 9.0% 3/30 (10%) 565 P=0.195N 2/50 (4%) 4.5% 2/30 (7%) 729 (T) P=0.367N 6/50 (12%) 13.4% 5/30 (17%) 565 P=0.168N 37/50 (74%) 77.9% 25/30 (83%) 381 P=0.276 21/50 (42%) 43.3% 7/30 (23%) 381 P=0.071 43/50 (86%) 87.2% 26/30 (87%) 381 P=0.284 4/50 (8%) 9.0% 3/30 (10%) 702 P=0.163 24/50 (48%) 49.4% 9/30 (30%) 381 P=0.030 1,000 ppm 5/50 (10%) 12.2% 2/23 (9%) 556 P=0.358N 2/50 (4%) 5.0% 1/23 (4%) 705 P=0.412N 7/50 (14%) 17.0% 3/23 (13%) 556 P=0.315N 35/50 (70%) 79.5% 20/23 (87%) 526 P=0.218 17/50 (34%) 37.8% 4/23 (17%) 391 P=0.190 41/50 (82%) 87.1% 20/23 (87%) 391 P=0.286 3/50 (6%) 7.4% 2/23 (9%) 610 P=0.256 20/50 (40%) 44.0% 6/23 (26%) 391 P=0.096 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE C2 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm Liver: Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma Overall rate 41/50 (82%) 42/50 (84%) Adjusted rate 82.6% 85.8% Terminal rate 30/38 (79%) 28/34 (82%) First incidence (days) 551 453 Poly-3 test P=0.227 P=0.437 Lung: Alveolar/bronchiolar Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 13/50 (26%) 27.5% 10/38 (26%) 628 P<0.001 31/50 (62%) 66.7% 25/34 (74%) 551 P<0.001 Lung: Alveolar/bronchiolar Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 9/50 (18%) 19.1% 6/38 (16%) 631 P<0.001 19/50 (38%) 41.5% 15/34 (44%) 551 P=0.014 Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 19/50 (38%) 39.8% 14/38 (37%) 628 P<0.001 38/50 (76%) 81.4% 31/34 (91%) 551 P<0.001 Skin: Fibrous Histiocytoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 1/50 (2%) 2.2% 1/38 (3%) 729 (T) P=0.199 2/50 (4%) 4.5% 2/34 (6%) 729 (T) P=0.486 Skin: Fibrous Histiocytoma, Fibrosarcoma, or Sarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 2/50 (4%) 4.3% 1/38 (3%) 583 P=0.416 3/50 (6%) 6.7% 2/34 (6%) 652 P=0.479 Spleen: Hemangiosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/50 (0%) 0.0% --0/3e8 (0%) P=0.002 0/50 (0%) 0.0% 0/34 (0%) ----f Thyroid Gland (Follicular Cell): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/50 (0%) 0.0% 0/38 (0%) -- P=0.010 0/50 (0%) 0.0% 0/34 (0%) -- -- 44/50 (88%) 89.0% 26/30 (87%) 381 P=0.264 31/50 (62%) 66.9% 23/30 (77%) 512 P<0.001 32/50 (64%) 70.5% 25/30 (83%) 565 P<0.001 42/50 (84%) 89.5% 30/30 (100%) 512 P<0.001 1/50 (2%) 2.3% 1/30 (3%) 729 (T) P=0.749 1/50 (2%) 2.3% 1/30 (3%) 729 (T) P=0.521N 0/49 (0%) 0.0% 0/30 (0%) -- -- 0/49 (0%) 0.0% 0/30 (0%) -- -- C-7 1,000 ppm 42/50 (84%) 88.5% 20/23 (87%) 391 P=0.291 29/50 (58%) 67.9% 20/23 (87%) 480 P<0.001 33/50 (66%) 71.3% 12/23 (52%) 420 P<0.001 43/50 (86%) 92.1% 21/23 (91%) 420 P<0.001 3/50 (6%) 7.4% 2/23 (9%) 609 P=0.258 3/50 (6%) 7.4% 2/23 (9%) 609 P=0.433 4/50 (8%) 9.9% 3/23 (13%) 556 P=0.045 3/50 (6%) 7.5% 2/23 (9%) 680 P=0.095 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-8 Cumene, NTP TR 542 TABLE C2 Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm All Organs: Hemangiosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Hemangioma or Hemangiosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Benign Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Benign or Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/50 (0%) 0.0% 0/38 (0%) -- P=0.015 1/50 (2%) 2.2% 1/38 (3%) 729 (T) P=0.054 43/50 (86%) 87.8% 34/38 (90%) 551 P=0.091 27/50 (54%) 55.0% 18/38 (47%) 551 P<0.001 48/50 (96%) 96.0% 36/38 (95%) 551 P=0.114 1/50 (2%) 2.2% 0/34 (0%) 654 P=0.493 1/50 (2%) 2.2% 0/34 (0%) 654 P=0.753 45/50 (90%) 93.2% 32/34 (94%) 533 P=0.275 34/50 (68%) 69.8% 21/34 (62%) 453 P=0.094 50/50 (100%) 100% 34/34 (100%) 453 P=0.237 2/50 (4%) 4.5% 2/30 (7%) 729 (T) P=0.226 2/50 (4%) 4.5% 2/30 (7%) 729 (T) P=0.482 46/50 (92%) 94.5% 30/30 (100%) 381 P=0.192 47/50 (94%) 95.2% 28/30 (93%) 381 P<0.001 50/50 (100%) 100% 30/30 (100%) 381 P=0.237 4/50 (8%) 9.9% 3/23 (13%) 556 P=0.045 4/50 (8%) 9.9% 3/23 (13%) 556 P=0.141 44/50 (88%) 95.6% 23/23 (100%) 480 P=0.132 45/50 (90%) 90.8% 19/23 (83%) 391 P<0.001 50/50 (100%) 100% 23/23 (100%) 391 P=0.237 (aT)NTeurmmbinearlosfancreiofipcleasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, spleen, and b c d thyroid gland; for other tissues, denominator is number of animals necropsied. Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not e f reach terminal sacrifice. A negative trend or a lower incidence in an exposed group is indicated by N. Not applicable; no neoplasms in animal group Value of statistic cannot be computed. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 C-9 TABLE C3a Historical Incidence of Alveolar/bronchiolar Neoplasms in Control Male B6C3F1 Micea Study Adenoma Incidence in Controls Carcinoma Adenoma or Carcinoma Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide 13/50 8/50 12/49 9/50 8/50 10/50 13/50 6/50 13/50 Overall Historical Incidence: Inhalation Studies Total (%) Mean standard deviation Range 92/449 (20.5%) 20.5% 5.3% 12%-26% Overall Historical Incidence: All Routes Total (%) Mean standard deviation Range 263/1,498 (17.6%) 17.9% 6.1% 6%-28% a Data as of March 2, 2007 9/50 8/50 5/49 5/50 5/50 7/50 6/50 7/50 12/50 64/449 (14.3%) 14.2% 4.6% 10%-24% 161/1,498 (10.8%) 10.9% 5.6% 2%-24% 19/50 15/50 16/49 14/50 13/50 17/50 17/50 13/50 22/50 146/449 (32.5%) 32.5% 5.9% 26%-44% 401/1,498 (26.8%) 27.2% 7.8% 12%-44% Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-10 Cumene, NTP TR 542 TABLE C3b Historical Incidence of Hepatocellular Neoplasms in Control Male B6C3F1 Micea Study Adenoma Incidence in Controls Carcinoma Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide Overall Historical Incidence: Inhalation Studies Total (%) Mean standard deviation Range Overall Historical Incidence: All Routes Total (%) Mean standard deviation Range a Data as of March 2, 2007 34/50 22/50 22/50 17/50 24/50 21/49 18/50 23/50 15/50 196/449 (43.7%) 43.7% 10.9% 30%-68% 633/1,496 (42.3%) 43.3% 14.2% 14%-70% 13/50 10/50 13/50 12/50 10/50 10/49 9/50 16/50 14/50 107/449 (23.8%) 23.8% 4.6% 18%-32% 382/1,496 (25.5%) 26.0% 9.1% 8%-48% Adenoma or Carcinoma 40/50 28/50 30/50 27/50 28/50 26/49 25/50 34/50 26/50 264/449 (58.8%) 58.8% 9.6% 50%-80% 874/1,496 (58.4%) 59.6% 15.4% 20%-85% NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 C-11 TABLE C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Terminal sacrifice Animals examined microscopically 50 50 79 57 38 34 50 50 Alimentary System Gallbladder Degeneration, hyaline Hyperplasia Intestine large, cecum Infiltration cellular, histiocyte Inflammation, acute Intestine large, colon Intestine small, duodenum Intestine small, ileum Necrosis Peyer's patch, inflammation, acute Intestine small, jejunum Peyer's patch, hyperplasia Liver Angiectasis Basophilic focus Clear cell focus Eosinophilic focus Hepatodiaphragmatic nodule Inflammation, granulomatous Mixed cell focus Necrosis Tension lipidosis Centrilobular, necrosis Mesentery Thrombosis Fat, necrosis Pancreas Atrophy Duct, cyst Stomach, forestomach Inflammation Ulcer Epithelium, erosion Epithelium, hyperplasia Stomach, glandular Mineralization Necrosis Tongue Tooth Malformation (43) 1 (2%) 1 (2%) (49) (50) (49) (49) 1 (2%) (48) (50) 1 (2%) 7 (14%) 24 (48%) 6 (12%) 1 (2%) 2 (4%) 2 (4%) 1 (2%) 1 (2%) (3) 1 (33%) 3 (100%) (50) 1 (2%) (50) 1 (2%) 2 (4%) (50) (1) (15) 14 (93%) (32) (47) (46) (44) (46) (46) 1 (2%) (50) 3 (6%) 5 (10%) 11 (22%) 5 (10%) 3 (6%) 4 (8%) 1 (2%) 4 (8%) (7) 6 (86%) (50) 1 (2%) (50) 2 (4%) 4 (8%) 1 (2%) 7 (14%) (48) 1 (2%) 2 (4%) (8) 7 (88%) a Number of animals examined microscopically at the site and the number of animals with lesion 50 13 7 30 50 (37) (44) (45) (43) (43) 1 (2%) (43) (50) 1 (2%) 4 (8%) 20 (40%) 16 (32%) 1 (2%) 1 (2%) 4 (8%) 2 (4%) (6) 6 (100%) (49) (50) 1 (2%) 6 (12%) 8 (16%) (48) (8) 8 (100%) 50 20 7 23 50 (38) (44) 1 (2%) 1 (2%) (48) (44) (44) (44) (50) 5 (10%) 11 (22%) 14 (28%) 1 (2%) 1 (2%) 1 (2%) (3) 3 (100%) (49) (49) 5 (10%) 6 (12%) 1 (2%) 13 (27%) (48) 1 (2%) (6) 6 (100%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-12 Cumene, NTP TR 542 TABLE C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Cardiovascular System Heart Cardiomyopathy Inflammation, acute Mineralization Necrosis Thrombosis Artery, inflammation, chronic active Endocrine System Adrenal cortex Atrophy Degeneration Hyperplasia Hypertrophy Adrenal medulla Hyperplasia Islets, pancreatic Hyperplasia Hypertrophy Pituitary gland Pars distalis, hyperplasia Thyroid gland Follicular cell, hyperplasia General Body System None Genital System Epididymis Angiectasis Granuloma sperm Preputial gland Ectasia Inflammation, chronic active Prostate Angiectasis Hyperplasia Infiltration cellular, polymorphonuclear Artery, inflammation, chronic active Testes Atrophy Hematopoietic System Bone marrow Thrombosis Lymph node Iliac, infiltration cellular, mixed cell Lymph node, bronchial Lymph node, mandibular Infiltration cellular, plasma cell Lymph node, mediastinal (50) 10 (20%) 1 (2%) 1 (2%) (50) 14 (28%) 29 (58%) (50) 2 (4%) (50) 1 (2%) (48) 1 (2%) (50) 7 (14%) (50) 18 (36%) 1 (2%) (50) 1 (2%) 15 (30%) 16 (32%) (50) 1 (2%) (50) 3 (6%) (49) 1 (2%) (50) 7 (14%) (50) 13 (26%) 1 (2%) 1 (2%) (49) 12 (24%) 14 (29%) (48) (49) 1 (2%) 1 (2%) (49) 3 (6%) (49) 7 (14%) (50) 17 (34%) 2 (4%) 1 (2%) 1 (2%) 2 (4%) 1 (2%) (50) 1 (2%) 8 (16%) 9 (18%) (50) (49) (49) 1 (2%) (50) 11 (22%) (50) (49) 1 (2%) (50) 1 (2%) 1 (2%) (50) 5 (10%) (50) (45) (34) 1 (3%) (37) (50) 1 (2%) 2 (4%) (50) 1 (2%) (49) 1 (2%) (50) 3 (6%) (50) 1 (2%) (41) (35) (42) (49) (50) 1 (2%) (48) (49) 1 (2%) (49) (1) 1 (100%) (45) (22) (40) (50) 1 (2%) (50) (49) 1 (2%) 1 (2%) (50) 1 (2%) (49) (2) (35) (25) (39) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 C-13 TABLE C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Hematopoietic System (continued) Lymph node, mesenteric Infiltration cellular, mixed cell Infiltration cellular, plasma cell Spleen Hematopoietic cell proliferation Infiltration cellular, plasma cell Thymus Artery, inflammation (48) 1 (2%) (50) 3 (6%) 1 (2%) (45) Integumentary System Skin Cyst epithelial inclusion Inflammation, acute Inflammation, chronic active Ulcer Epidermis, abscess (50) 1 (2%) Musculoskeletal System Bone Hyperostosis Skeletal muscle (50) 1 (2%) Nervous System Brain Degeneration Gliosis Necrosis Ventricle, infiltration cellular, polymorphonuclear (50) Respiratory System Larynx Metaplasia, squamous Lung Thrombosis Alveolar epithelium, bronchiole, metaplasia Alveolar epithelium, hyperplasia Alveolus, infiltration cellular, histiocyte Bronchiole, hyperplasia Bronchus, inflammation, acute Bronchus, necrosis Nose Inflammation, suppurative Polyp, inflammatory Glands, olfactory epithelium, hyperplasia Olfactory epithelium, accumulation, hyaline droplet Olfactory epithelium, atrophy Olfactory epithelium, hyperplasia, atypical Olfactory epithelium, hyperplasia, basal cell Respiratory epithelium, accumulation, hyaline droplet Respiratory epithelium, metaplasia, squamous Vomeronasal organ, inflammation, suppurative (50) (50) 5 (10%) 3 (6%) 2 (4%) (50) 2 (4%) 1 (2%) 3 (6%) 1 (2%) 4 (8%) 1 (2%) 1 (2%) (47) (50) 1 (2%) (44) (50) 1 (2%) 2 (4%) (50) (50) 1 (2%) (49) (50) 1 (2%) 43 (86%) 3 (6%) 3 (6%) 11 (22%) (50) 2 (4%) 11 (22%) 13 (26%) 1 (2%) (46) 1 (2%) 1 (2%) (49) 2 (4%) (38) 1 (3%) (49) 6 (12%) (50) (1) (50) 1 (2%) (50) (50) 1 (2%) 42 (84%) 4 (8%) 17 (34%) 1 (2%) (49) 9 (18%) 9 (18%) 11 (22%) 5 (10%) 15 (31%) 1 (2%) 1 (2%) (44) (50) (44) (50) 1 (2%) 3 (6%) 1 (2%) 1 (2%) (50) (50) 1 (2%) 1 (2%) (50) 1 (2%) (50) 1 (2%) 39 (78%) 1 (2%) 18 (36%) 1 (2%) (48) 6 (13%) 23 (48%) 38 (79%) 11 (23%) 33 (69%) 5 (10%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION C-14 Cumene, NTP TR 542 TABLE C4 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Inhalation Study of Cumene Chamber Control 250 ppm 500 ppm 1,000 ppm Respiratory System (continued) Pleura Trachea Necrosis Special Senses System Eye Cataract Cornea, inflammation, chronic active Cornea, mineralization Harderian gland Hyperplasia Hypertrophy Inflammation, chronic Necrosis Urinary System Kidney Infarct Inflammation, suppurative Metaplasia, osseous Mineralization Nephropathy Artery, inflammation, chronic active Capsule, fibrosis Perirenal tissue, thrombosis Renal tubule, cyst Renal tubule, hyperplasia Renal tubule, necrosis Renal tubule, pigmentation Urinary bladder (50) (49) 1 (2%) (48) 2 (4%) 1 (2%) (50) 1 (2%) 3 (6%) 1 (2%) 47 (94%) 1 (2%) 1 (2%) (50) (50) (50) 1 (2%) 2 (4%) (50) 2 (4%) (50) 4 (8%) 1 (2%) 1 (2%) 44 (88%) 1 (2%) 2 (4%) (48) (49) (49) 1 (2%) (49) 1 (2%) (50) 3 (6%) 45 (90%) 1 (2%) (49) (2) (50) 1 (2%) (48) 1 (2%) 4 (8%) 1 (2%) (50) 1 (2%) 1 (2%) 1 (2%) (50) 7 (14%) 1 (2%) 1 (2%) 2 (4%) 41 (82%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (48) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft D-1 APPENDIX D SUMMARY OF LESIONS IN FEMALE MICE IN THE 2-YEAR INHALATION STUDY OF CUMENE TABLE D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2 TABLE D2 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-6 TABLE D3a Historical Incidence of Alveolar/bronchiolar Neoplasms in Control Female B6C3F1 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-10 TABLE D3b Historical Incidence of Liver Neoplasms in Control Female B6C3F1 Mice . . . . . . . . . . . . . D-11 TABLE D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-12 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-2 Cumene, NTP TR 542 TABLE D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumenea Chamber Control 125 ppm 250 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Died last week of study Terminal sacrifice Animals examined microscopically 50 50 8 10 54 37 36 50 50 50 8 3 39 50 Alimentary System Esophagus Squamous cell carcinoma Gallbladder Intestine large, cecum Intestine large, colon Intestine small, duodenum Carcinoma Intestine small, ileum Intestine small, jejunum Liver Hemangiosarcoma Hepatoblastoma Hepatocellular adenoma Hepatocellular adenoma, multiple Hepatocellular carcinoma Hepatocellular carcinoma, multiple Hepatocholangiocarcinoma Ito cell tumor benign Mesentery Hemangiosarcoma Sarcoma Pancreas Carcinoma, metastatic, uncertain primary site Salivary glands Stomach, forestomach Squamous cell papilloma Epithelium, squamous cell carcinoma Stomach, glandular (50) (38) (46) (47) (46) (46) (46) (50) 9 (18%) 9 (18%) 8 (16%) 2 (4%) (11) (49) 1 (2%) (50) (49) 1 (2%) (49) (50) (41) (47) (50) (47) 1 (2%) (47) (47) (50) 1 (2%) 10 (20%) 13 (26%) 6 (12%) 1 (2%) 1 (2%) (12) 1 (8%) (50) (50) (50) 1 (2%) (50) (50) (44) (49) (50) (49) (49) (49) (50) 1 (2%) 18 (36%) 9 (18%) 4 (8%) 2 (4%) (11) 1 (9%) (50) (50) (50) 2 (4%) (50) Cardiovascular System Heart Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocholangiocarcinoma, metastatic, liver (50) 1 (2%) (50) 1 (2%) (50) Endocrine System Adrenal cortex Hepatocholangiocarcinoma, metastatic, liver Adrenal medulla Pheochromocytoma malignant (50) (50) (50) 1 (2%) (49) (50) (50) 2 (4%) 500 ppm 50 12 3 1 34 50 (50) 1 (2%) (37) (47) (48) (48) (47) (47) (50) 19 (38%) 10 (20%) 12 (24%) 1 (2%) (9) (50) (50) (50) 2 (4%) (49) (50) 1 (2%) 1 (2%) (49) (49) 1 (2%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm Endocrine System (continued) Islets, pancreatic Adenoma Carcinoma Pituitary gland Pars distalis, adenoma Pars intermedia, adenoma Thyroid gland Carcinoma, metastatic, uncertain primary site Bilateral, follicular cell, carcinoma C-cell, adenoma Follicular cell, adenoma Follicular cell, carcinoma General Body System None (49) 1 (2%) (50) 8 (16%) 2 (4%) (50) 1 (2%) 1 (2%) (50) 1 (2%) (49) 6 (12%) (50) 1 (2%) 1 (2%) 4 (8%) (50) 1 (2%) (50) 6 (12%) 1 (2%) (50) Genital System Ovary Carcinoma, metastatic, uncertain primary site Cystadenocarcinoma Cystadenoma Luteoma Teratoma benign Yolk sac carcinoma Uterus Leiomyoma Polyp stromal Bilateral, polyp stromal Endometrium, carcinoma (48) 5 (10%) 1 (2%) (50) 3 (6%) 3 (6%) (50) 1 (2%) 2 (4%) 1 (2%) (50) (50) 1 (2%) 1 (2%) (50) 1 (2%) 1 (2%) 1 (2%) 1 (2%) Hematopoietic System Bone marrow Hemangiosarcoma Lymph node Axillary, hemangiosarcoma, metastatic, spleen Renal, alveolar/bronchiolar carcinoma, metastatic, lung Renal, hepatocholangiocarcinoma, metastatic, liver Lymph node, bronchial Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Lymph node, mandibular Lymph node, mediastinal Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocholangiocarcinoma, metastatic, liver Lymph node, mesenteric Carcinoma, metastatic, uncertain primary site Spleen Hemangiosarcoma (49) (4) (39) 1 (3%) (35) (44) 1 (2%) (46) (49) (50) (9) 1 (11%) (36) (42) (40) 1 (3%) (46) (50) (50) 2 (4%) (5) 1 (20%) 1 (20%) (40) (38) (42) 1 (2%) (48) (50) 3 (6%) D-3 500 ppm (50) 1 (2%) (48) 10 (21%) (50) 1 (2%) 3 (6%) (49) 1 (2%) 3 (6%) (50) 2 (4%) (50) (5) (46) 1 (2%) (42) (40) 2 (5%) (48) 1 (2%) (50) 1 (2%) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-4 Cumene, NTP TR 542 TABLE D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm Hematopoietic System (continued) Thymus Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocholangiocarcinoma, metastatic, liver Integumentary System Mammary gland Carcinoma Fibroadenoma Skin Basal cell carcinoma Squamous cell carcinoma Subcutaneous tissue, fibrous histiocytoma Subcutaneous tissue, hemangioma Subcutaneous tissue, hemangiosarcoma Subcutaneous tissue, sarcoma Musculoskeletal System Bone Hemangiosarcoma Skeletal muscle Carcinoma, metastatic, uncertain primary site Hemangiosarcoma Hepatocholangiocarcinoma, metastatic, liver (49) 1 (2%) (50) 1 (2%) (50) 1 (2%) 1 (2%) (50) (1) 1 (100%) (50) 1 (2%) (50) 1 (2%) (50) 2 (4%) 1 (2%) (50) (1) 1 (100%) (48) (50) (50) 1 (2%) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) (1) 1 (100%) Nervous System Brain Alveolar/bronchiolar carcinoma, metastatic, lung Respiratory System Larynx Squamous cell papilloma Lung Alveolar/bronchiolar adenoma Alveolar/bronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma Alveolar/bronchiolar carcinoma, multiple Carcinoma, metastatic, harderian gland Carcinoma, metastatic, uncertain primary site Hepatocellular carcinoma, metastatic, liver Hepatocholangiocarcinoma, metastatic, liver Mediastinum, alveolar/bronchiolar carcinoma, metastatic, lung Nose Carcinoma, metastatic, harderian gland Hemangioma Hemangiosarcoma Respiratory epithelium, adenoma (50) (50) (50) 1 (2%) 3 (6%) 3 (6%) 1 (2%) 4 (8%) (50) 1 (2%) (50) (49) (50) 13 (26%) 13 (26%) 10 (20%) 6 (12%) 5 (10%) 1 (2%) (50) 1 (2%) (50) (50) (50) 16 (32%) 20 (40%) 13 (26%) 7 (14%) 2 (4%) (50) 1 (2%) 500 ppm (48) 1 (2%) (50) 1 (2%) (50) 1 (2%) 2 (4%) (50) (50) 1 (2%) (50) 1 (2%) (50) 8 (16%) 30 (60%) 15 (30%) 19 (38%) 1 (2%) 3 (6%) 1 (2%) (50) 1 (2%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm Respiratory System (continued) Pleura Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocholangiocarcinoma, metastatic, liver Trachea (50) (1) 1 (100%) (50) Special Senses System Eye Harderian gland Adenoma Carcinoma (49) (50) 8 (16%) 5 (10%) (50) (49) 4 (8%) 1 (2%) Urinary System Kidney Alveolar/bronchiolar carcinoma, metastatic, lung Carcinoma, metastatic, uncertain primary site Hepatocholangiocarcinoma, metastatic, liver Urinary bladder Transitional epithelium, carcinoma (49) 1 (2%) (48) (50) 1 (2%) (48) Systemic Lesions Multiple organsb Histiocytic sarcoma Lymphoma malignant (50) 7 (14%) (50) 1 (2%) 15 (30%) Neoplasm Summary Total animals with primary neoplasmsc Total primary neoplasms Total animals with benign neoplasms Total benign neoplasms Total animals with malignant neoplasms Total malignant neoplasms Total animals with metastatic neoplasms Total metastatic neoplasms Total animals with malignant neoplasms- uncertain primary site 42 44 81 120 34 39 48 68 26 33 33 52 76 16 14 1 a b c Number of animals examined microscopically at the site and the number of animals with neoplasm Number of animals with any tissue examined microscopically Primary neoplasms: all neoplasms except metastatic neoplasms (1) 1 (100%) (50) (50) (50) 1 (2%) (50) (49) 1 (2%) (50) 2 (4%) 6 (12%) 50 131 47 76 37 55 3 6 D-5 500 ppm (2) 1 (50%) 1 (50%) (50) (49) (50) 2 (4%) 5 (10%) (50) 1 (2%) 1 (2%) (48) (50) 12 (24%) 50 163 47 94 43 69 7 19 1 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-6 Cumene, NTP TR 542 TABLE D2 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm Harderian Gland: Adenoma OATevdrejmurasintleladrlarrtaaettaeecb PFoirlsyt-i3ncteidstednce (days) Harderian Gland: Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Harderian Gland: Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Liver: Hepatocellular Carcinoma or Hepatoblastoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Lung: Alveolar/bronchiolar Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 8/50 (16%) 18.0% 7/37 (19%) 656 P=0.012N 5/50 (10%) 11.2% 3/37 (8%) 683 P=0.441 12/50 (24%) 26.8% 9/37 (24%) 656 P=0.056N 18/50 (36%) 40.5% 17/37 (46%) 654 P=0.040 10/50 (20%) 22.2% 7/37 (19%) 607 P=0.311 25/50 (50%) 55.6% 22/37 (60%) 607 P=0.024 10/50 (20%) 22.2% 7/37 (19%) 607 P=0.305 1/50 (2%) 2.3% 1/37 (3%) 731 (T) P<0.001 4/50 (8%) 8.9% 3/36 (8%) 726 P=0.171N 1/50 (2%) 2.2% 1/36 (3%) 731 (T) P=0.099N 5/50 (10%) 11.2% 4/36 (11%) 726 P=0.050N 23/50 (46%) 50.0% 17/36 (47%) 609 P=0.243 7/50 (14%) 15.5% 5/36 (14%) 623 P=0.291N 26/50 (52%) 56.5% 20/36 (56%) 609 P=0.549 7/50 (14%) 15.5% 5/36 (14%) 623 P=0.291N 26/50 (52%) 56.3% 21/36 (58%) 555 P<0.001 0/50 (0%) 0.0% 0--/3e9 (0%) P=0.003N 1/50 (2%) 2.1% 1/39 (3%) 731 (T) P=0.090N 1/50 (2%) 2.1% 1/39 (3%) 731 (T) P<0.001N 27/50 (54%)f 56.4% 22/39 (56%) 618 P=0.091 6/50 (12%) 12.7% 4/39 (10%) 651 P=0.177N 29/50 (58%)f 60.4% 23/39 (59%) 618 P=0.395 7/50 (14%) 14.7% 4/39 (10%) 618 P=0.255N 36/50 (72%) 74.5% 31/39 (80%) 495 P<0.001 500 ppm 2/50 (4%) 4.3% 2/35 (6%) 731 (T) P=0.036N 5/50 (10%) 10.6% 3/35 (9%) 672 P=0.591N 6/50 (12%) 12.7% 4/35 (11%) 672 P=0.073N 29/50 (58%) 59.8% 19/35 (54%) 662 P=0.046 12/50 (24%) 25.4% 10/35 (29%) 702 P=0.455 36/50 (72%) 74.1% 25/35 (71%) 662 P=0.043 12/50 (24%) 25.4% 10/35 (29%) 702 P=0.455 38/50 (76%) 77.9% 29/35 (83%) 565 P<0.001 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE D2 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm Lung: Alveolar/bronchiolar Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 6.7% 2/37 (5%) 533 P<0.001 Lung: Alveolar/bronchiolar Adenoma or Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 4/50 (8%) 8.9% 3/37 (8%) 533 P<0.001 Ovary: Cystadenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 5/48 (10%) 11.5% 5/37 (14%) 731 (T) P=0.274N Pituitary Gland (Pars Distalis): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 8/50 (16%) 18.1% 8/37 (22%) 731 (T) P=0.299 Spleen: Hemangiosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 0/49 (0%) 0.0% 0/37 (0%) -- P=0.271 Thyroid Gland (Follicular Cell): Adenoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 1/50 (2%) 2.3% 1/37 (3%) 731 (T) P=0.432 Thyroid Gland (Follicular Cell): Adenoma or Carcinoma Overall rate 2/50 (4%) Adjusted rate 4.5% Terminal rate 1/37 (3%) First incidence (days) 683 Poly-3 test P=0.519N 16/50 (32%) 35.3% 13/36 (36%) 646 P<0.001 31/50 (62%) 66.8% 25/36 (69%) 555 P<0.001 2/50 (4%) 4.5% 2/36 (6%) 731 (T) P=0.204N 6/49 (12%) 13.6% 6/36 (17%) 731 (T) P=0.385N 0/50 (0%) 0.0% 0/36 (0%) ----g 4/50 (8%) 8.9% 4/36 (11%) 731 (T) P=0.183 5/50 (10%) 11.2% 5/36 (14%) 731 (T) P=0.220 20/50 (40%) 41.9% 15/39 (39%) 618 P<0.001 42/50 (84%) 86.0% 34/39 (87%) 495 P<0.001 0/50 (0%) 0.0% 0/39 (0%) -- P=0.025N 6/50 (12%) 12.8% 5/39 (13%) 709 P=0.342N 3/50 (6%) 6.4% 2/39 (5%) 673 P=0.130 0/50 (0%) 0.0% 0/39 (0%) -- P=0.489N 0/50 (0%) 0.0% 0/39 (0%) -- P=0.226N D-7 500 ppm 34/50 (68%) 69.5% 24/35 (69%) 513 P<0.001 46/50 (92%) 92.4% 33/35 (94%) 513 P<0.001 3/49 (6%) 6.5% 3/34 (9%) 731 (T) P=0.326N 10/48 (21%) 21.8% 8/34 (24%) 565 P=0.433 1/50 (2%) 2.1% 1/35 (3%) 731 (T) P=0.513 3/50 (6%) 6.4% 3/35 (9%) 731 (T) P=0.329 3/50 (6%) 6.4% 3/35 (9%) 731 (T) P=0.527 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-8 Cumene, NTP TR 542 TABLE D2 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm Uterus: Carcinoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test Uterus: Stromal Polyp Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Hemangiosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Hemangioma or Hemangiosarcoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Malignant Lymphoma Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Benign Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test All Organs: Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 3/50 (6%) 6.8% 3/37 (8%) 731 (T) P=0.081N 3/50 (6%) 6.8% 2/37 (5%) 693 P=0.560N 1/50 (2%) 2.3% 1/37 (3%) 731 (T) P=0.518N 1/50 (2%) 2.3% 1/37 (3%) 731 (T) P=0.304 7/50 (14%) 15.4% 5/37 (14%) 292 P=0.372 34/50 (68%) 73.3% 29/37 (78%) 193 P<0.001 26/50 (52%) 55.3% 19/37 (51%) 292 P<0.001 0/50 (0%) 0.0% 0/36 (0%) -- P=0.116N 0/50 (0%) 0.0% 0/36 (0%) -- P=0.116N 3/50 (6%) 6.6% 1/36 (3%) 555 P=0.318 3/50 (6%) 6.6% 1/36 (3%) 555 P=0.318 15/50 (30%) 33.0% 12/36 (33%) 645 P=0.041 39/50 (78%) 82.6% 30/36 (83%) 555 P=0.192 33/50 (66%) 67.6% 21/36 (58%) 481 P=0.149 1/50 (2%) 2.1% 1/39 (3%) 731 (T) P=0.285N 2/50 (4%) 4.3% 2/39 (5%) 731 (T) P=0.476N 6/50 (12%) 12.8% 5/39 (13%) 673 P=0.066 6/50 (12%) 12.8% 5/39 (13%) 673 P=0.066 6/50 (12%) 12.7% 4/39 (10%) 639 P=0.471N 47/50 (94%) 95.7% 38/39 (97%) 495 P<0.001 37/50 (74%) 74.0% 26/39 (67%) 495 P=0.040 500 ppm 0/50 (0%) 0.0% 0/35 (0%) -- P=0.108N 2/50 (4%) 4.2% 1/35 (3%) 677 P=0.471N 1/50 (2%) 2.1% 1/35 (3%) 731 (T) P=0.746N 3/50 (6%) 6.4% 3/35 (9%) 731 (T) P=0.329 12/50 (24%) 25.0% 7/35 (20%) 565 P=0.187 47/50 (94%) 95.3% 34/35 (97%) 565 P=0.002 43/50 (86%) 86.0% 28/35 (80%) 513 P<0.001 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 D-9 TABLE D2 Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm 500 ppm All Organs: Benign or Malignant Neoplasms Overall rate Adjusted rate Terminal rate First incidence (days) Poly-3 test 42/50 (84%) 85.9% 32/37 (87%) 193 P<0.001 44/50 (88%) 90.2% 32/36 (89%) 481 P=0.366 50/50 (100%) 100% 39/39 (100%) 495 P=0.007 50/50 (100%) 100% 35/35 (100%) 513 P=0.007 (aT) Terminal sacrifice Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, ovary, b c d pituitary gland, spleen, and thyroid gland; for other tissues, denominator is number of animals necropsied. Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality Observed incidence at terminal kill Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not e f g reach terminal sacrifice. A negative trend or a lower incidence in an exposed group is indicated by N. Not applicable; no neoplasms in animal group One animal with adenoma also had hepatoblastoma. Value of statistic cannot be computed. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-10 Cumene, NTP TR 542 TABLE D3a Historical Incidence of Alveolar/bronchiolar Neoplasms in Control Female B6C3F1 Mice Study Adenoma Incidence in Controls Carcinoma Adenoma or Carcinoma Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide 1/50 1/49 4/50 4/50 1/50 3/50 2/50 2/50 1/50 Overall Historical Incidence: Inhalation Studies Total (%) Mean standard deviation Range 19/449 (4.2%) 4.2% 2.5% 2%-8% Overall Historical Incidence: All Routes Total (%) Mean standard deviation Range 77/1,596 (4.8%) 4.9% 2.7% 0%-12% a Data as of March 2, 2007 3/50 6/49 2/50 0/50 1/50 2/50 1/50 0/50 0/50 15/449 (3.3%) 3.4% 3.9% 0%-12% 57/1,596 (3.6%) 3.6% 3.1% 0%-12% 4/50 7/49 6/50 4/50 2/50 5/50 3/50 2/50 1/50 34/449 (7.6%) 7.6% 4.0% 2%-14% 129/1,596 (8.1%) 8.2% 3.9% 2%-18% NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 D-11 TABLE D3b Historical Incidence of Hepatocellular Neoplasms in Control Female B6C3F1 Micea Study Adenoma Incidence in Controls Carcinoma Historical Incidence: Inhalation Studies Cumene Decalin Divinylbenzene Methyl isobutyl ketone "-Methylstyrene Propargyl alcohol Propylene glycol mono-t-butyl ether Stoddard solvent IIC Vanadium pentoxide Overall Historical Incidence: Inhalation Studies Total (%) Mean standard deviation Range Overall Historical Incidence: All Routes Total (%) Mean standard deviation Range a Data as of March 2, 2007 18/50 7/49 17/49 13/50 10/50 15/50 14/49 9/50 6/50 109/447 (24.4%) 24.4% 8.7% 12%-36% 402/1,593 (25.2%) 25.8% 15.8% 2%-62% 10/50 4/49 5/49 6/50 3/50 4/50 4/49 6/50 6/50 48/447 (10.7%) 10.7% 4.1% 6%-20% 159/1,593 (10.0%) 10.2% 6.6% 0%-28% Adenoma or Carcinoma 25/50 11/49 19/49 17/50 13/50 17/50 18/49 13/50 12/50 145/447 (32.4%) 32.4% 8.8% 22%-50% 505/1,593 (31.7%) 32.4% 17.5% 8%-64% Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-12 Cumene, NTP TR 542 TABLE D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Cumenea Chamber Control 125 ppm 250 ppm 500 ppm Disposition Summary Animals initially in study Early deaths Moribund Natural deaths Survivors Died last week of study Terminal sacrifice Animals examined microscopically 50 50 8 10 54 37 36 50 50 Alimentary System Esophagus Gallbladder Intestine large, cecum Intestine large, colon Intestine small, duodenum Necrosis Intestine small, ileum Intestine small, jejunum Hyperplasia, lymphoid Liver Angiectasis Basophilic focus Clear cell focus Cyst Eosinophilic focus Erythrophagocytosis Fatty change Hematopoietic cell proliferation Inflammation, granulomatous Mixed cell focus Necrosis Tension lipidosis Centrilobular, necrosis Mesentery Inflammation, chronic active Fat, hemorrhage Fat, necrosis Pancreas Atrophy Inflammation, chronic active Salivary glands Stomach, forestomach Hemorrhage Inflammation Ulcer Epithelium, erosion Epithelium, hyperplasia Stomach, glandular Mineralization Necrosis (50) (38) (46) (47) (46) 1 (2%) (46) (46) (50) 3 (6%) 4 (8%) 1 (2%) 8 (16%) 1 (2%) 1 (2%) 4 (8%) 3 (6%) 2 (4%) (11) 11 (100%) (49) 1 (2%) (50) (49) 1 (2%) 1 (2%) (49) 1 (2%) (50) (41) (47) (50) (47) (47) (47) (50) 1 (2%) 3 (6%) 6 (12%) 11 (22%) 1 (2%) 1 (2%) 3 (6%) 3 (6%) 1 (2%) (12) 1 (8%) 10 (83%) (50) (50) (50) 2 (4%) (50) a Number of animals examined microscopically at the site and the number of animals with lesion 50 8 3 39 50 (50) (44) (49) (50) (49) (49) (49) 1 (2%) (50) 1 (2%) 4 (8%) 1 (2%) 7 (14%) 3 (6%) 4 (8%) (11) 1 (9%) 10 (91%) (50) 1 (2%) (50) (50) 1 (2%) 1 (2%) 2 (4%) (50) 1 (2%) 50 12 3 1 34 50 (50) (37) (47) (48) (48) (47) (47) (50) 1 (2%) 5 (10%) 1 (2%) 14 (28%) 1 (2%) 1 (2%) 2 (4%) 4 (8%) 1 (2%) (9) 9 (100%) (50) 3 (6%) (50) (50) 2 (4%) 1 (2%) 3 (6%) (49) 1 (2%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 D-13 TABLE D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm 500 ppm Cardiovascular System Heart Cardiomyopathy Inflammation, chronic Mineralization Thrombosis (50) 12 (24%) (50) 17 (34%) 1 (2%) 1 (2%) (50) 6 (12%) 1 (2%) 1 (2%) (50) 7 (14%) Endocrine System Adrenal cortex Hyperplasia Hypertrophy Adrenal medulla Hyperplasia Islets, pancreatic Hyperplasia Pituitary gland Pars distalis, angiectasis Pars distalis, hyperplasia Thyroid gland Follicular cell, hyperplasia General Body System None (50) 5 (10%) 2 (4%) (50) 1 (2%) (49) 1 (2%) (50) 3 (6%) 11 (22%) (50) 15 (30%) (50) 7 (14%) 1 (2%) (49) 4 (8%) (50) 1 (2%) (49) 1 (2%) 19 (39%) (50) 10 (20%) (50) 7 (14%) 1 (2%) (50) 2 (4%) (50) 2 (4%) (50) 7 (14%) 10 (20%) (50) 16 (32%) (49) 5 (10%) 6 (12%) (49) 2 (4%) (50) 3 (6%) (48) 1 (2%) 6 (13%) (50) 10 (20%) Genital System Ovary Angiectasis Cyst Thrombosis Uterus Angiectasis Decidual reaction Inflammation, suppurative Thrombosis Endometrium, hyperplasia, cystic (48) 1 (2%) 9 (19%) 1 (2%) (50) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 8 (16%) (50) 12 (24%) 1 (2%) (50) 2 (4%) 2 (4%) 11 (22%) (50) 14 (28%) (50) 1 (2%) 18 (36%) (49) 11 (22%) 1 (2%) (50) 3 (6%) 1 (2%) 2 (4%) 8 (16%) Hematopoietic System Bone marrow Lymph node Iliac, ectasia Lumbar, angiectasis Lumbar, ectasia Lumbar, hyperplasia, lymphoid Renal, ectasia Lymph node, bronchial Lymph node, mandibular Inflammation, granulomatous Lymph node, mediastinal Hyperplasia, lymphoid Lymph node, mesenteric (49) (4) 1 (25%) 2 (50%) 1 (25%) (39) (35) 1 (3%) (44) (46) (50) (9) 1 (11%) 1 (11%) 1 (11%) 2 (22%) (36) (42) (40) (46) (50) (5) 1 (20%) (40) (38) (42) (48) (50) (5) 1 (20%) (46) (42) (40) 1 (3%) (48) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-14 Cumene, NTP TR 542 TABLE D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm 500 ppm Hematopoietic System (continued) Spleen Hematopoietic cell proliferation Hyperplasia, lymphoid Thymus (49) 1 (2%) 1 (2%) (49) Integumentary System Mammary gland Hyperplasia Skin Inflammation, chronic active (50) 1 (2%) (50) Musculoskeletal System Bone Skeletal muscle (50) (1) Nervous System Brain Necrosis Meninges, infiltration cellular, mononuclear cell (50) Respiratory System Larynx Inflammation, suppurative Metaplasia, squamous Lung Foreign body Hemorrhage Inflammation, suppurative Thrombosis Alveolar epithelium, bronchiole, hyperplasia Alveolar epithelium, bronchiole, metaplasia Alveolar epithelium, hyperplasia Alveolar epithelium, metaplasia, squamous Alveolus, infiltration cellular, histiocyte Bronchiole, hyperplasia Nose Inflammation, suppurative Glands, olfactory epithelium, cyst Glands, olfactory epithelium, hyperplasia Olfactory epithelium, atrophy Olfactory epithelium, hyperplasia, atypical Olfactory epithelium, hyperplasia, basal cell Olfactory epithelium, necrosis Respiratory epithelium, accumulation, hyaline droplet Respiratory epithelium, metaplasia, squamous Respiratory epithelium, necrosis Turbinate, necrosis Pleura Trachea Inflammation, suppurative (50) 1 (2%) (50) 2 (4%) 1 (2%) (50) 1 (2%) 4 (8%) (50) (50) 6 (12%) (50) (50) (50) 1 (2%) (50) (1) (50) (49) (50) 2 (4%) 2 (4%) 42 (84%) 3 (6%) 1 (2%) 17 (34%) (50) 1 (2%) 4 (8%) 11 (22%) 1 (2%) 2 (4%) 1 (2%) (1) (50) (50) 2 (4%) (48) (50) (50) 1 (2%) (50) (1) (50) 1 (2%) (50) (50) 1 (2%) 49 (98%) 6 (12%) 1 (2%) 10 (20%) (50) 3 (6%) 1 (2%) 4 (8%) 9 (18%) 2 (4%) 11 (22%) 2 (4%) 1 (2%) 1 (2%) 1 (2%) (1) (50) (50) 2 (4%) 1 (2%) (48) (50) 1 (2%) (50) (50) (50) 1 (2%) (50) 1 (2%) (50) 1 (2%) 1 (2%) 47 (94%) 6 (12%) 3 (6%) 14 (28%) (50) 7 (14%) 11 (22%) 18 (36%) 10 (20%) 25 (50%) 3 (6%) 6 (12%) 2 (4%) (2) (50) 2 (4%) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 D-15 TABLE D4 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Inhalation Study of Cumene Chamber Control 125 ppm 250 ppm 500 ppm Special Senses System Eye Cataract Degeneration Cornea, epithelium, hyperplasia Cornea, inflammation, acute Cornea, inflammation, chronic active Cornea, mineralization Harderian gland Atrophy Hyperplasia (49) 2 (4%) 1 (2%) 4 (8%) (50) 1 (2%) 6 (12%) (50) 1 (2%) (49) 2 (4%) (50) 1 (2%) 1 (2%) 1 (2%) (50) 1 (2%) (49) 2 (4%) 1 (2%) 1 (2%) 2 (4%) 1 (2%) (50) 1 (2%) Urinary System Kidney Cyst Infarct Inflammation, suppurative Metaplasia, osseous Nephropathy Capsule, fibrosis Renal tubule, karyomegaly Renal tubule, necrosis Urinary bladder Inflammation, chronic active Artery, inflammation, chronic active (49) 1 (2%) 2 (4%) 36 (73%) 1 (2%) 1 (2%) 3 (6%) (48) (50) 1 (2%) 3 (6%) 3 (6%) 34 (68%) 1 (2%) (48) 1 (2%) (50) 2 (4%) 1 (2%) 34 (68%) 1 (2%) (49) 1 (2%) (50) 1 (2%) 1 (2%) 34 (68%) 1 (2%) (48) Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION D-16 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft E-1 APPENDIX E GENETIC TOXICOLOGY SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RAT BONE MARROW MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EVALUATION PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE E1 Mutagenicity of Cumene in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE E2 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated with Cumene by Intraperitoneal Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE E3 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with Cumene by Inhalation for 3 Months . . . . . . . . . . . . . . . . . . . . . . E-2 E-2 E-3 E-3 E-3 E-4 E-5 E-6 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION E-2 Cumene, NTP TR 542 GENETIC TOXICOLOGY SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL Testing was performed as reported by Zeiger et al. (1988). Cumene was sent to the laboratory as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella typhimurium tester strains TA97, TA98, TA100, and TA1535 either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37 C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37 C. Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of cumene. The high dose was limited by toxicity. All trials were repeated; negative trials conducted with S9 were repeated with a higher S9 concentration. In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive. RAT BONE MARROW MICRONUCLEUS TEST PROTOCOL Factors affecting dose selection included chemical solubility and toxicity and the extent of cell cycle delay induced by cumene exposure. A high dose of 2,500 mg/kg was selected based on toxicity. The standard three-exposure protocol is described in detail by Shelby et al. (1993). Male F344/N rats were injected intraperitoneally (three times at 24-hour intervals) with cumene dissolved in corn oil. Vehicle control animals were injected with corn oil only. The positive control animals received injections of cyclophosphamide (25 mg/kg). The animals were killed 24 hours after the third injection, and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained; 2,000 polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in up to five rats per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of cumene-induced bone marrow toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among PCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted previously). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 E-3 MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female B6C3F1 mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronucleated cells in 1,000 normochromatic erythrocytes (NCEs) in each of 9 or 10 mice per exposure group. In addition, the percentage of PCEs in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity. The results were tabulated as described for PCEs in the rat bone marrow micronucleus test. Results of the 3-month study were accepted without repeat tests because additional data could not be obtained. EVALUATION PROTOCOL These are the basic guidelines for arriving at an overall assay result for assays performed by the National Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical procedures for data analysis have been described in the preceding protocols. There have been instances, however, in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation. Results obtained in the absence of activation are not combined with results obtained in the presence of activation; each testing condition is evaluated separately. The summary table in the Abstract of this Technical Report presents a result that represents a scientific judgement of the overall evidence for activity of the chemical in an assay. RESULTS Cumene (1 to 333 g/plate) was not mutagenic in S. typhimurium strain TA97, TA98, TA100, or TA1535 when tested with and without induced rat or hamster liver S9 activation enzymes (Table E1). In vivo, cumene induced small but significant increases in micronucleated PCEs in bone marrow of male rats treated by intraperitoneal injection (Table E2). Two trials were performed in rats. In the first trial, doses ranging from 78.13 to 2,500 mg/kg were administered three times at 24-hour intervals, and results were positive, based both on the trend (P=0.011) and the response at the 1,250 mg/kg dose. The data from the 2,500 mg/kg dose were excluded from analysis because only two animals survived and a minimum of three animals is required for a valid dose point. The second confirmatory trial also produced a positive response, although the trend test was not significant (P=0.085). Micronucleated erythrocytes were elevated at all four doses in trial 2; the responses at the 312 and 1,250 mg/kg levels were statistically significant (P<0.006). The percentage of PCEs in the bone marrow fluctuated unrelated to dose and likely represented variation within the normal range of 40% to 60% PCEs among the total erythrocyte population in the bone marrow. In contrast to the results in male rats, no increase in micronucleated erythrocytes was observed in peripheral blood of male or female mice exposed to cumene by inhalation (62.5 to 1,000 ppm) for 3 months (Table E3). For both male and female mice, no significant changes in the percentage of PCEs were observed over the exposure range tested, indicating an absence of treatment-related toxicity to the bone marrow. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION E-4 Cumene, NTP TR 542 TABLE E1 Mutagenicity of Cumene in Salmonella typhimuriuma Strain Dose S9 (g/plate) Trial 1 Trial 2 Revertants/Plateb +hamster S9 10% 30% +rat S9 10% 30% TA100 0 1 3 10 33 100 166 333 TPorisailtisvuemcmonatrryold TA1535 0 1 3 10 33 100 166 Trial summary Positive control TA97 0 1 3 10 33 100 166 Trial summary Positive control TA98 0 1 3 10 33 100 166 333 Trial summary Positive control 99 5.0 103 3.0 93 6.0 96 5.0 100 47 11.40.0c Negative 914 8.0 15 1.0 10 2.0 17 0.0 14 1.0 13 1.0 11 2.0 Negative 851 8.0 133 12.0 128 1.0 144 5.0 148 12.0 136 13.0 99 8.0 Negative 426 13.0 9 1.0 9 0.0 9 1.0 14 1.0 8 4 00..00c Negative 328 28.0 105 1.0 100 8.0 104 4.0 116 7.0 93 6.0 111 8.0 121 11.0 107 4.0 112 8.0 102 7.0 102 3.0 106 6.0 Negative 875 26.0 14 0.0 13 2.0 13 1.0 13 3.0 12 1.0 16 5.0 Negative 399 17.0 156 7.0 177 3.0 174 2.0 172 2.0 158 53 41.70.0c Negative 515 13.0 20 1.0 22 4.0 16 3.0 20 2.0 18 5.0 16 2.0 Negative 1,420 121.0 15 2.0 12 2.0 17 1.0 11 1.0 10 2.0 9 1.0 Negative 198 12.0 188 2.0 181 6.0 185 3.0 195 9.0 153 125 91..00c Negative 545 22.0 22 3.0 22 2.0 23 1.0 22 3.0 23 2.0 21 3.0 Negative 414 24.0 Negative 1,227 35.0 126 20.0 119 6.0 127 8.0 105 7.0 112 7.0 79 4.0c Negative 603 25.0 14 1.0 12 1.0 9 2.0 14 3.0 15 1.0 10 2.0 Negative 321 15.0 159 1.0 165 6.0 171 4.0 185 4.0 186 7.0 153 4.0 Negative 481 18.0 14 3.0 14 2.0 13 3.0 13 3.0 18 2.0 10 1.0c Negative 291 13.0 125 7.0 112 8.0 116 10.0 113 5.0 105 3.0 95 5.0 Negative 1,111 30.0 21 2.0 17 2.0 17 1.0 15 2.0 12 2.0 11 1.0 Negative 140 2.0 192 4.0 172 8.0 176 7.0 190 4.0 149 115 91.50.c0 Negative 478 12.0 19 1.0 25 5.0 24 3.0 16 1.0 14 1.0 23 1.0 Negative 890 63.0 125 11.0 137 11.0 129 3.0 122 4.0 138 5.0 91 9.0 Negative 792 16.0 13 3.0 14 0.0 14 2.0 12 2.0 12 2.0 11 4.0 Negative 222 7.0 170 11.0 189 5.0 191 3.0 188 2.0 168 15.0 163 2.0 Negative 435 23.0 19 1.0 11 2.0 14 3.0 13 1.0 11 2.0 12 3.0 Negative 669 17.0 a b c d Study performed at SRI International. The detailed protocol is presented by Zeiger et al. (1988). 0 g/plate was the solvent control. Revertants are presented as mean standard error from three plates. Slight toxicity The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA97), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 E-5 TABLE E2 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated with Cumene by Intraperitoneal Injectiona Compound Dose (mg/kg) Number of Male Rats Micronucleated PCEs/ with Erythrocytes Scored 1,000 PCEsb Pairwise P Valuec PCEb (%) Trial 1 Corn oild Cumene 0 78.13 156.25 312.5 625 1,250 2,500 Cyclophosphamideg 25 5 5 5 5 5 52e 5 0.50 0.16 1.20 0.25 1.20 0.34 1.30 0.54 0.80 0.41 2.60 0.29 1.25 0.25 P=0.011f 17.30 2.32 0.0447 0.0447 0.0296 0.2026 0.0001 0.0000 50.2 2.9 59.4 5.1 64.8 4.2 54.6 3.1 45.1 1.7 46.6 4.8 49.3 2.8 50.3 4.3 Trial 2 Corn oil Cumene 0 312 625 1,250 2,500 Cyclophosphamide 25 5 5 5 5 3 5 0.50 0.27 1.70 0.20 1.40 0.33 1.80 0.34 1.50 1.00 P=0.085 7.80 1.63 0.0052 0.0194 0.0033 0.0192 0.0000 53.2 3.8 50.2 1.0 47.6 3.1 44.5 3.0 54.3 2.1 38.7 2.7 a Study was performed at ILS, Inc. The detailed protocol is presented by Shelby et al. (1993). b c PCE=polychromatic erythrocyte Mean standard error Pairwise comparison with the vehicle control; dosed group values are significant at P#0.005 (trial 1) or P#0.006 (trial 2); positive control d e f g values are significant at P#0.05 (ILS, 1990) Vehicle control Statistical tests not performed due to high mortality Significance of micronucleated PCEs/1,000 PCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990) Positive control Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION E-6 Cumene, NTP TR 542 TABLE E3 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with Cumene by Inhalation for 3 Monthsa Compound Concentration (ppm) Number of Mice with Erythrocytes Scored Micronucleated NCEs/ 1,000 NCEsb P-Valuec PCEsb (%) Male Aird 0 10 2.40 0.69 2.7 0.1 Cumene 62.5 10 2.20 0.66 0.6161 2.6 0.1 125 10 2.10 0.48 0.6728 2.6 0.1 250 10 1.80 0.36 0.8230 2.8 0.1 500 10 2.00 0.26 0.7270 2.9 0.1 1,000 10 2.20 0.42 0.6161 2.9 0.2 P=0.553e Female Air 0 10 2.30 0.40 3.3 0.1 Cumene 62.5 9 1.33 0.37 0.9396 2.3 0.1 125 10 1.70 0.30 0.8289 3.1 0.2 250 10 2.10 0.53 0.6186 3.3 0.2 500 10 2.10 0.35 0.6186 3.4 0.1 P=0.329 a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990). b c d e NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte Mean standard error Pairwise comparison with the chamber controls, significant at P#0.025 (PCEs) or P#0.005 (NCEs) (ILS, 1990) Chamber control Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990) NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft F-1 APPENDIX F CLINICAL PATHOLOGY RESULTS TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-2 TABLE F2 Hematology Data for Mice in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . F-8 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION F-2 Cumene, NTP TR 542 TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Male Hematology n Day 3 Day 23 Week 14 10 10 10 Hematocrit (%) Day 3 43.6 0.7 Day 23 49.0 0.5 Week 14 45.5 0.4 Packed cell volume (mL/dL) Day 3 41.3 0.6 Day 23 48.0 0.7 Week 14 45.5 0.3 Hemoglobin (g/dL) Day 3 13.4 0.2 Day 23 15.2 0.2 Week 14 15.1 0.1 Erythrocytes (106/L) Day 3 6.47 0.09 Day 23 7.87 0.13 Week 14 8.20 0.05 Reticulocytes (106/L) Day 3 0.25 0.04 Day 23 0.20 0.02 Week 14 0.18 0.02 Nucleated erythrocytes/100 leukocytes Day 3 1.00 0.33 Day 23 0.10 0.10 Week 14 0.30 0.15 Mean cell volume (fL) Day 3 64.0 0.4 Day 23 61.0 0.5 Week 14 55.6 0.2 Mean cell hemoglobin (pg) Day 3 20.7 0.2 Day 23 19.3 0.2 Week 14 18.5 0.1 Mean cell hemoglobin concentration (g/dL) Day 3 32.5 0.4 Day 23 31.7 0.2 Week 14 33.3 0.2 Platelets (103/L) Day 3 868.3 23.8 Day 23 857.7 19.8 Week 14 671.3 8.4 10 10 10 42.1 0.4 48.4 0.4 45.4 0.3 40.1 0.5 47.9 0.5 45.4 0.5 13.1 0.2 15.1 0.1 14.9 0.1 6.29 0.09 7.68 0.10 8.19 0.08 0.32 0.03 0.24 0.03 0.18 0.01 1.40 0.40 0.20 0.13 0.30 0.15 63.8 0.3 62.5 0.3 55.3 0.2 20.9 0.2 19.7 0.1 18.3 0.1 32.8 0.2 31.5 0.1 33.0 0.2 859.6 15.7 858.7 22.0 677.6 13.9 10 10 10 42.7 0.4 48.1 0.5 45.1 0.4 40.5 0.5 47.2 0.5 44.8 0.4 12.8 0.2 15.0 0.2 14.9 0.1 6.23 0.09 7.56 0.10 8.11 0.06 0.35 0.02* 0.30 0.02* 0.19 0.01 2.20 0.39* 0.20 0.13 0.50 0.17 64.7 0.3 62.7 0.4 55.4 0.2 20.5 0.2 19.9 0.2 18.4 0.1 31.6 0.3 31.8 0.2 33.3 0.2 941.7 28.7 924.7 30.8 644.8 10.1 10 10 10 10 10 10 10 10 9 42.9 0.5 47.7 0.4 44.3 0.3 40.8 0.5 46.8 0.5 44.4 0.5 13.0 0.1 14.9 0.1 14.8 0.1 6.28 0.09 7.48 0.10 7.98 0.09* 0.41 0.03** 0.27 0.02 0.19 0.01 2.50 0.54* 0.30 0.21 0.30 0.15 64.9 0.4 62.6 0.4 55.4 0.2 20.7 0.2 19.9 0.2 18.5 0.1 31.9 0.4 31.9 0.2 33.3 0.2 924.3 34.4 880.3 32.6 679.0 14.1 42.9 0.5 48.4 0.3 44.3 0.3 40.7 0.5 46.9 0.4 44.1 0.4 12.9 0.1 15.1 0.1 14.7 0.1* 6.33 0.09 7.53 0.08 7.95 0.06* 0.43 0.04** 0.23 0.03 0.19 0.02 2.30 0.37* 0.10 0.10 0.10 0.10 64.4 0.5 62.3 0.4 55.5 0.2 20.4 0.3 20.0 0.2 18.5 0.1 31.7 0.4 32.1 0.2 33.4 0.3 951.3 24.1* 996.8 34.9** 676.7 7.6 42.5 0.4 48.9 0.5 44.4 0.2 39.9 0.4 48.8 0.5 44.7 0.3 12.9 0.1 15.1 0.2 14.8 0.1 6.32 0.09 7.67 0.10 8.06 0.05* 0.42 0.04** 0.26 0.02 0.18 0.02 2.80 0.33** 0.80 0.29* 0.44 0.34 63.1 0.7 63.7 0.4** 55.4 0.2 20.5 0.3 19.7 0.1 18.4 0.1 32.4 0.2 30.9 0.2 33.2 0.2 958.2 32.1* 1,075.7 40.9** 757.6 10.5** NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 F-3 TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Male (continued) Hematology (continued) n Day 3 Day 23 Week 14 10 10 10 Leukocytes (103/L) Day 3 7.74 0.35 Day 23 7.64 0.44 Week 14 5.54 0.45 Segmented neutrophils (103/L) Day 3 0.80 0.07 Day 23 0.78 0.12 Week 14 0.95 0.08 Bands (103/L) Day 3 0.01 0.01 Day 23 0.00 0.00 Week 14 0.00 0.00 Lymphocytes (103/L) Day 3 6.68 0.34 Day 23 6.64 0.45 Week 14 4.48 0.40 Monocytes (103/L) Day 3 0.22 0.05 Day 23 0.21 0.06 Week 14 0.07 0.01 Basophils (103/L) Day 3 0.015 0.015 Day 23 0.000 0.000 Week 14 0.000 0.000 Eosinophils (103/L) Day 3 0.02 0.01 Day 23 0.03 0.01 Week 14 0.04 0.03 Clinical Chemistry n Urea nitrogen (mg/dL) Day 3 Day 23 Week 14 Creatinine (mg/dL) Day 3 Day 23 Week 14 Total protein (g/dL) Day 3 Day 23 Week 14 10 9.3 0.8 10.0 0.7 13.9 0.4 0.71 0.02 0.72 0.01 1.05 0.03 5.6 0.1 6.0 0.0 6.7 0.1 10 10 10 8.07 0.24 8.98 0.36 5.35 0.40 0.87 0.10 0.71 0.08 0.87 0.14 0.00 0.00 0.00 0.00 0.00 0.00 6.93 0.22 8.08 0.34 4.35 0.27 0.21 0.04 0.19 0.05 0.11 0.02 0.007 0.007 0.000 0.000 0.000 0.000 0.07 0.03 0.01 0.01 0.03 0.01 10 7.5 0.3 10.2 0.5 13.5 0.4 0.73 0.02 0.74 0.02 1.08 0.04 5.5 0.1 6.1 0.1 6.8 0.1 10 10 10 8.24 0.26 8.30 0.38 6.07 0.60 1.02 0.08 0.90 0.12 1.05 0.09 0.02 0.01 0.00 0.00 0.00 0.00 6.96 0.23 7.28 0.29 4.88 0.52 0.19 0.04 0.10 0.05 0.09 0.02 0.025 0.013 0.000 0.000 0.000 0.000 0.03 0.02 0.02 0.01 0.05 0.02 10 7.2 0.2 9.4 0.3 14.7 0.3 0.70 0.02 0.73 0.02 1.05 0.02 5.4 0.1 6.1 0.0 6.7 0.0 10 10 10 7.95 0.30 8.88 0.37* 6.21 0.41 1.00 0.12 1.02 0.08 0.93 0.09 0.01 0.01 0.00 0.00 0.00 0.00 6.69 0.23 7.67 0.29 5.13 0.36 0.21 0.04 0.17 0.06 0.05 0.02 0.006 0.006 0.000 0.000 0.000 0.000 0.04 0.01 0.04 0.02 0.11 0.03* 10 6.7 0.4 8.6 0.6 14.0 0.3 0.71 0.01 0.71 0.01 1.11 0.03 5.3 0.1 6.1 0.0 6.8 0.1 10 10 10 8.00 0.33 9.68 0.28** 7.91 0.39** 1.00 0.05 1.29 0.44 1.12 0.14 0.01 0.01 0.01 0.01 0.00 0.00 6.85 0.33 8.18 0.39** 6.67 0.38** 0.12 0.03 0.13 0.05 0.10 0.02 0.000 0.000 0.000 0.000 0.000 0.000 0.03 0.01 0.07 0.03 0.03 0.01 10 10 9 5.68 0.58 9.76 0.30** 6.11 0.59* 0.72 0.09 0.98 0.08 0.96 0.09 0.00 0.00 0.00 0.00 0.00 0.00 4.82 0.49* 8.50 0.31** 5.06 0.55* 0.09 0.02* 0.22 0.05 0.05 0.01 0.005 0.005 0.011 0.011 0.000 0.000 0.05 0.01 0.05 0.02 0.04 0.02 10 8.4 0.4 10.1 0.3 14.3 0.4 0.72 0.01 0.71 0.01 1.11 0.03 5.4 0.1 6.2 0.1 6.8 0.2 10 12.5 0.6 13.6 0.7* 14.2 0.3 0.69 0.01 0.74 0.02 1.06 0.03 5.4 0.1 6.4 0.1** 7.0 0.0** Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION F-4 Cumene, NTP TR 542 TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm Male (continued) Clinical Chemistry (continued) n 10 Albumin (g/dL) Day 3 4.0 0.1 Day 23 3.8 0.1 Week 14 3.8 0.1 Globulin (g/dL) Day 3 1.6 0.1 Day 23 2.2 0.1 Week 14 2.9 0.0 Albumin/globulin ratio Day 3 2.5 0.2 Day 23 1.8 0.1 Week 14 1.3 0.0 Alanine aminotransferase (IU/L) Day 3 64 2 Day 23 44 1 Week 14 113 6 Alkaline phosphatase (IU/L) Day 3 733 14 Day 23 496 15 Week 14 309 7 Creatine kinase (IU/L) Day 3 376 21 Day 23 301 29 Week 14 126 20 Sorbitol dehydrogenase (IU/L) Day 3 13 1 Day 23 13 0 Week 14 26 1 Bile acids (mol/L) Day 3 33.8 1.1 Day 23 32.1 2.9 Week 14 28.6 1.7 Female n Hematology Hematocrit (%) Day 3 Day 23 Week 14 Packed cell volume (mL/dL) Day 3 Day 23 Week 14 10 45.5 0.5 48.4 0.4 42.1 0.5 43.3 0.5 46.6 0.6 41.9 0.3 10 3.8 0.1 3.7 0.1 3.9 0.0 1.7 0.1 2.4 0.1 2.8 0.1 2.4 0.1 1.6 0.1 1.4 0.0 61 1 39 1* 113 11 759 16 490 11 294 4 422 50 273 36 125 21 12 1 13 0 24 2 39.9 0.9** 33.2 1.4 31.2 1.3 10 3.6 0.1* 3.8 0.1 3.8 0.0 1.7 0.0 2.3 0.0 2.9 0.0 2.1 0.1 1.7 0.1 1.3 0.0 61 1 39 1** 110 12 779 14 506 15 293 10 476 75 210 25* 144 20 12 1 13 0 22 1 47.1 0.7** 37.7 1.4* 32.7 1.5 10 3.6 0.1** 3.8 0.1 3.9 0.0 1.7 0.1 2.3 0.1 3.0 0.1 2.1 0.1 1.7 0.1 1.3 0.0 64 2 38 1** 70 4** 794 24 485 15 283 9* 619 231 2966b 129 20 11 1 12 1 20 1** 50.9 1.7** 41.1 1.0** 30.9 0.9 10 3.8 0.1 3.9 0.1 4.1 0.1** 1.7 0.1 2.3 0.1 2.7 0.2 2.3 0.1 1.8 0.1 1.6 0.2** 60 2 35 1** 61 3** 771 24 456 27 275 7** 470 56 220 15 179 13 12 0 12 0 17 1** 50.4 2.0** 43.4 1.7** 32.4 0.9* 10 10 10 10 45.1 0.4 48.9 0.6 41.8 0.4 43.3 0.5 48.3 0.7 41.9 0.4 45.3 0.7 48.1 0.3 42.0 0.5 43.2 0.6 45.9 0.4 41.0 0.4 44.4 0.7 48.6 0.7 42.4 0.5 42.3 0.7 47.6 0.6 42.5 0.5 44.0 0.5 48.4 0.5 42.5 0.4 41.7 0.5 47.6 0.7 42.6 0.4 1,000 ppm 10 3.8 0.1 3.9 0.0 4.2 0.0** 1.6 0.1 2.4 0.1 2.9 0.0 2.5 0.1 1.6 0.1 1.5 0.0** 56 1** 35 0** 50 2** 674 15 470 12 250 6** 436 62 278 29 118 13 11 1 12 0 17 1** 55.6 4.1** 44.2 1.4** 35.9 2.2** 10 44.3 0.5 48.5 0.6 43.3 0.4 42.5 0.5 47.9 0.7 43.3 0.3* NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 F-5 TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Female (continued) n 10 Hematology (continued) Hemoglobin (g/dL) Day 3 13.9 0.2 Day 23 15.5 0.2 Week 14 14.4 0.1 Erythrocytes (106/L) Day 3 6.65 0.09 Day 23 7.42 0.11 Week 14 6.96 0.03 Reticulocytes (106/L) Day 3 0.33 0.03 Day 23 0.14 0.01 Week 14 0.15 0.01 Nucleated erythrocytes/100 leukocytes Day 3 0.40 0.22 Day 23 0.30 0.15 Week 14 0.70 0.26 Mean cell volume (fL) Day 3 65.3 0.3 Day 23 62.8 0.3 Week 14 60.2 0.3 Mean cell hemoglobin (pg) Day 3 20.9 0.1 Day 23 20.9 0.2 Week 14 20.7 0.1 Mean cell hemoglobin concentration (g/dL) Day 3 32.2 0.1 Day 23 33.3 0.3 Week 14 34.4 0.2 Platelets (103/L) Day 3 964.0 24.6 Day 23 1,026.6 35.1 Week 14 620.3 10.6 Leukocytes (103/L) Day 3 10.24 0.46 Day 23 7.99 0.47 Week 14 4.12 0.46 Segmented neutrophils (103/L) Day 3 0.98 0.07 Day 23 0.79 0.07 Week 14 0.67 0.11 Bands (103/L) Day 3 0.00 0.00 Day 23 0.00 0.00 Week 14 0.00 0.00 10 10 13.8 0.2 15.7 0.2 14.4 0.1 6.67 0.07 7.75 0.13 6.99 0.07 0.34 0.03 0.14 0.01 0.15 0.01 1.00 0.21 0.20 0.20 1.30 0.40 64.9 0.4 62.4 0.4 60.1 0.2 20.7 0.2 20.2 0.2 20.5 0.2 31.8 0.4 32.4 0.2 34.2 0.3 860.5 940.7 3314..35b 605.8 7.7 9.72 0.53 9.53 0.57 2.93 0.28 1.13 0.14 0.83 0.12 0.48 0.07 0.00 0.00 0.00 0.00 0.00 0.00 13.9 0.2 15.4 0.1 14.3 0.1 6.70 0.10 7.35 0.08 6.83 0.07 0.33 0.03 0.17 0.01 0.12 0.01 0.20 0.13 0.20 0.13 0.40 0.22 64.4 0.4 62.5 0.4 60.0 0.2 20.8 0.1 20.9 0.3 20.9 0.2 32.2 0.1 33.4 0.3 34.8 0.3 922.1 46.2 996.6 37.6 615.8 12.9 9.77 0.53 8.78 0.54 3.18 0.27 0.93 0.09 0.84 0.14 0.52 0.04 0.00 0.00 0.00 0.00 0.00 0.00 10 10 10 13.5 0.3 15.5 0.2 14.5 0.1 6.52 0.13 7.58 0.12 7.07 0.08 0.27 0.02 0.16 0.01 0.14 0.01 0.60 0.16 0.30 0.21 0.50 0.22 64.9 0.3 62.7 0.3 60.1 0.3 20.7 0.2 20.5 0.2 20.5 0.2 32.0 0.2 32.6 0.3 34.1 0.3 907.5 26.0 963.8 24.0 618.3 14.2 8.57 0.40* 8.45 0.51 3.57 0.40 0.84 0.11 0.78 0.09 0.62 0.10 0.00 0.00 0.00 0.00 0.00 0.00 13.3 0.1 15.6 0.2 14.5 0.1 6.50 0.08 7.65 0.14 7.07 0.07 0.38 0.02 0.15 0.01 0.15 0.01 0.70 0.34 0.10 0.10 0.30 0.21 64.3 0.4 62.4 0.5 60.3 0.3 20.6 0.1 20.4 0.3 20.5 0.2 32.0 0.2 32.8 0.3 33.9 0.3 787.4 26.9** 990.8 51.8 640.3 13.9 8.79 0.41* 9.96 0.52* 4.38 0.70 1.09 0.10 0.94 0.19 0.77 0.29 0.00 0.00 0.00 0.00 0.00 0.00 13.7 0.1 15.6 0.2 14.6 0.1 6.62 0.10 7.71 0.15 7.17 0.04 0.28 0.03 0.17 0.02 0.12 0.01 0.70 0.26 0.30 0.15 0.60 0.22 64.4 0.3 62.2 0.3 60.3 0.2 20.7 0.2 20.2 0.3 20.4 0.1 32.3 0.2 32.5 0.4 33.8 0.3 904.3 41.7 1,042.0 30.1 677.7 8.5** 8.38 0.67* 10.60 0.57** 4.60 0.61 1.09 0.15 0.83 0.09 0.73 0.12 0.00 0.00 0.00 0.00 0.00 0.00 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION F-6 Cumene, NTP TR 542 TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Female (continued) n Hematology (continued) Lymphocytes (103/L) Day 3 Day 23 Week 14 Monocytes (103/L) Day 3 Day 23 Week 14 Basophils (103/L) Day 3 Day 23 Week 14 Eosinophils (103/L) Day 3 Day 23 Week 14 10 8.99 0.46 7.03 0.51 3.39 0.38 0.21 0.04 0.11 0.03 0.02 0.01 0.000 0.000 0.010 0.010 0.000 0.000 0.06 0.03 0.05 0.02 0.04 0.01 Clinical Chemistry Urea nitrogen (mg/dL) Day 3 9.0 0.5 Day 23 12.9 0.6 Week 14 14.4 0.7 Creatinine (mg/dL) Day 3 0.69 0.02 Day 23 0.72 0.01 Week 14 0.97 0.02 Total protein (g/dL) Day 3 5.7 0.1 Day 23 6.1 0.1 Week 14 6.6 0.1 Albumin (g/dL) Day 3 3.6 0.1 Day 23 3.8 0.0 Week 14 4.2 0.1 Globulin (g/dL) Day 3 2.1 0.0 Day 23 2.4 0.1 Week 14 2.5 0.1 Albumin/globulin ratio Day 3 1.7 0.1 Day 23 1.6 0.0 Week 14 1.7 0.1 Alanine aminotransferase (IU/L) Day 3 50 1 Day 23 37 1 Week 14 80 8 10 8.30 0.54 8.54 0.51 2.43 0.23 0.22 0.07 0.13 0.03 0.01 0.01 0.000 0.000 0.000 0.000 0.000 0.000 0.08 0.02 0.03 0.01 0.02 0.01 8.4 0.5 12.7 0.7 14.5 0.3 0.69 0.02 0.71 0.01 1.05 0.02 5.7 0.1 6.1 0.0 6.7 0.1 3.6 0.1 3.8 0.0 4.0 0.1 2.1 0.0 2.4 0.0 2.7 0.1 1.8 0.1 1.6 0.0 1.5 0.1 51 2 37 1 64 6 10 8.55 0.48 7.83 0.57 2.62 0.25 0.25 0.06 0.05 0.03 0.03 0.01 0.018 0.012 0.017 0.017 0.000 0.000 0.04 0.03 0.05 0.02 0.01 0.01 7.7 0.3 12.3 0.5 14.6 0.5 0.64 0.02 0.70 0.00 1.01 0.02 5.6 0.0 6.0 0.0 6.9 0.1 3.6 0.1 3.7 0.0 4.3 0.1 2.0 0.0 2.3 0.0 2.5 0.1 1.8 0.1 1.6 0.0 1.7 0.1 48 2 34 1 63 4 10 10 10 7.55 0.38 7.52 0.52 2.89 0.30 0.13 0.04 0.10 0.04 0.03 0.01 0.000 0.000 0.000 0.000 0.000 0.000 0.06 0.03 0.05 0.02 0.03 0.02 7.59 0.40 8.82 0.44* 3.57 0.47 0.05 0.01** 0.17 0.05 0.01 0.01 0.018 0.012 0.000 0.000 0.000 0.000 0.05 0.02 0.03 0.02 0.02 0.01 7.06 0.54** 9.51 0.52** 3.83 0.50 0.20 0.04 0.17 0.04 0.00 0.00 0.008 0.008 0.009 0.009 0.000 0.000 0.03 0.01 0.09 0.03 0.04 0.02 7.8 0.5 12.1 0.3 15.1 0.4 0.62 0.01** 0.72 0.01 1.07 0.02* 5.5 0.1 6.1 0.1 6.9 0.1 3.5 0.1 3.9 0.1 4.2 0.1 2.1 0.1 2.2 0.0* 2.7 0.1 1.7 0.1 1.8 0.0 1.6 0.1 48 2 35 1 53 4** 8.8 0.4 11.7 0.5 14.4 0.5 0.65 0.02* 0.73 0.02 1.03 0.03 5.6 0.1 6.2 0.1 6.9 0.1* 3.6 0.0 3.8 0.1 4.3 0.1 2.0 0.1 2.4 0.1 2.6 0.1 1.8 0.1 1.6 0.1 1.7 0.1 47 2 34 1 53 3** 10.5 0.5 11.2 0.5 13.5 0.6 0.62 0.01** 0.71 0.01 1.04 0.03 5.7 0.0 6.2 0.1 7.1 0.1** 3.7 0.1 3.8 0.1 4.6 0.1** 2.0 0.1 2.4 0.0 2.4 0.1 1.9 0.1 1.6 0.1 2.0 0.1 42 2** 33 1 33 1** NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 F-7 TABLE F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Inhalation Study of Cumene Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm Female (continued) n 10 Clinical Chemistry (continued) Alkaline phosphatase (IU/L) Day 3 Day 23 Week 14 Creatine kinase (IU/L) Day 3 Day 23 Week 14 Sorbitol dehydrogenase (IU/L) Day 3 Day 23 Week 14 Bile acids (mol/L) Day 3 Day 23 Week 14 642 18 372 6 272 9 303 26 242 28 149 22 13 1 12 1 22 1 27.2 1.3 23.0 0.7 29.7 2.6 10 10 10 10 636 23 388 11 275 8 291 28b 233 27 123 15 13 0 11 0 21 1 32.6 1.9* 27.6 1.5** 24.3 1.3 618 19 370 8 262 12 326 26 209 15 120 14 13 0 11 0 19 0 34.4 1.8** 29.1 0.9** 26.2 1.6 608 12 362 9 267 7 314 189 1374b 140 17 12 0 12 0 19 1 38.5 1.8** 31.5 1.1** 32.1 4.3 585 15* 348 7* 243 7* 367 25 229 24 125 20 12 0 11 0 18 1** 42.5 0.9** 38.5 2.9** 25.7 2.3 * Significantly different (P#0.05) from the chamber control group by Dunn's or Shirley's test *a * b P#0.01 Data are given as mean standard error. n=9 Ratios were calculated and statistical tests were performed on unrounded data. 1,000 ppm 10 474 10** 319 7** 207 6** 320 49 245 24 104 9 12 0 12 0 15 1** 38.0 1.7** 38.4 3.6** 23.8 0.9 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION F-8 Cumene, NTP TR 542 TABLE F2 Hematology Data for Mice in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm Male n 10 Hematocrit (%) 49.5 0.7 Packed cell volume (mL/dL) 49.8 0.6 Hemoglobin (g/dL) Erythrocytes (106/L) Reticulocytes (106/L) 15.6 0.2 9.74 0.10 0.17 0.02 Nucleated erythrocytes/ 100 leukocytes 0.00 0.00 Howell-Jolly bodies (% erythrocytes) 0.1 0.0 Mean cell volume (fL) 51.2 0.2 Mean cell hemoglobin (pg) 16.0 0.1 Mean cell hemoglobin concentration (g/dL) 31.3 0.2 Platelets (103/L) 724.2 14.7 Leukocytes (103/L) 2.65 0.29 Segmented neutrophils (103/L) 0.32 0.05 Bands (103/L) 0.00 0.00 Lymphocytes (103/L) 2.31 0.25 Monocytes (103/L) 0.00 0.00 Basophils (103/L) 0.000 0.000 Eosinophils (103/L) 0.02 0.01 10 49.7 0.4 50.2 0.6 15.8 0.2 9.84 0.10 0.16 0.02 0.10 0.10 0.1 0.0 51.0 0.2 16.1 0.1 31.6 0.2 733.8 9.0 2.92 0.30 0.34 0.06 0.00 0.00 2.52 0.26 0.03 0.01* 0.000 0.000 0.03 0.01 10 49.3 0.5 49.2 0.5 15.6 0.2 9.64 0.08 0.14 0.01 0.00 0.00 0.1 0.1 50.8 0.2 16.2 0.1 31.8 0.2 732.1 9.0 3.18 0.43 0.34 0.05 0.00 0.00 2.82 0.39 0.01 0.01 0.000 0.000 0.02 0.01 10 50.4 0.4 50.6 0.4 15.9 0.1 9.91 0.08 0.16 0.02 0.00 0.00 0.1 0.0 51.0 0.2 16.1 0.1 31.5 0.2 736.7 10.5 3.38 0.46 0.43 0.09 0.00 0.00 2.91 0.40 0.01 0.01 0.000 0.000 0.03 0.01 10 49.9 0.5 50.5 0.3 15.8 0.1 9.90 0.06 0.15 0.02 0.10 0.10 0.1 0.0 51.0 0.2 16.0 0.1 31.4 0.1 760.6 13.8* 3.04 0.43 0.50 0.21 0.00 0.00 2.49 0.31 0.02 0.01 0.002 0.002 0.03 0.01 Female n 10 Hematocrit (%) 49.2 0.8 Packed cell volume (mL/dL) 49.6 0.7 Hemoglobin (g/dL) Erythrocytes (106/L) Reticulocytes (106/L) 15.6 0.2 9.56 0.11 0.21 0.02 Nucleated erythrocytes/ 100 leukocytes 0.00 0.00 Howell-Jolly bodies (% erythrocytes) 0.1 0.0 Mean cell volume (fL) 51.9 0.2 Mean cell hemoglobin (pg) 16.4 0.1 Mean cell hemoglobin concentration (g/dL) 31.5 0.1 Platelets (103/L) 715.2 13.1 Leukocytes (103/L) 2.79 0.41 Segmented neutrophils (103/L) 0.26 0.03 Bands (103/L) 0.00 0.00 Lymphocytes (103/L) 2.50 0.39 Monocytes (103/L) 0.01 0.01 Basophils (103/L) 0.000 0.000 Eosinophils (103/L) 0.02 0.01 10 50.5 0.5 50.7 0.5 15.9 0.2 9.73 0.09 0.18 0.02 0.00 0.00 0.1 0.0 52.0 0.0 16.4 0.1 31.4 0.1 694.7 20.6 3.07 0.15 0.36 0.09 0.00 0.00 2.68 0.12 0.01 0.00 0.000 0.000 0.03 0.01 10 50.2 0.3 50.5 0.5 15.9 0.2 9.71 0.09 0.22 0.02 0.00 0.00 0.1 0.0 52.2 0.1 16.3 0.1 31.4 718.4 103.1.4b 2.95 0.17 0.28 0.05 0.00 0.00 2.62 0.14 0.02 0.01 0.000 0.000 0.03 0.01 10 50.1 0.5 50.6 0.5 16.0 0.2 9.74 0.11 0.20 0.02 0.30 0.21 0.1 0.0 52.0 0.3 16.4 0.1 31.5 0.1 730.5 10.6 3.18 0.24 0.30 0.04 0.00 0.00 2.83 0.21 0.03 0.01 0.000 0.000 0.03 0.01 10 50.6 0.6 51.0 0.6 15.9 0.2 9.75 0.10 0.24 0.02 0.10 0.10 0.1 0.0 52.3 0.2 16.3 0.1 31.3 754.2 206.2.5b 3.49 0.24 0.39 0.07 0.00 0.00 3.02 0.20 0.02 0.01 0.000 0.000 0.06 0.02 * Significantly different (P#0.05) from the chamber control group by Dunn's or Shirley's test a b Data are given as mean standard error. Ratios were calculated and statistical tests were performed on unrounded data. n=9 1,000 ppm 10 49.4 0.6 48.7 0.5 15.5 0.2 9.45 0.09 0.15 0.02 0.00 0.00 0.1 0.0 51.7 0.2 16.4 0.1 31.9 0.3 765.4 7.1* 3.02 0.35 0.41 0.06 0.00 0.00 2.51 0.31 0.04 0.01* 0.000 0.000 0.05 0.02 2 49.0 1.0 49.3 0.5 15.6 0.4 9.42 0.11 0.21 0.06 0.00 0.00 0.1 0.1 52.0 0.0 16.6 0.2 31.6 0.4 778.5 1.5 2.35 0.25 0.34 0.00 0.00 0.00 1.98 0.24 0.02 0.02 0.000 0.000 0.03 0.01 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft G-1 APPENDIX G ORGAN WEIGHTS AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS TABLE G1 TABLE G2 TABLE G3 TABLE G4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 2-Week Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 2-Week Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-2 G-3 G-4 G-5 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION G-2 Cumene, NTP TR 542 TABLE G1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 2-Week Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm 2,000 ppm Male n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative R. Testis Absolute Relative Thymus Absolute Relative 5 146 4 0.612 0.033 4.208 0.258 0.568 0.017 3.896 0.071 6.282 0.228 43.052 0.696 1.068 0.063 7.324 0.403 0.918 0.012 6.310 0.171 0.454 0.013 3.123 0.116 5 149 4 0.566 0.015 3.788 0.036 0.660 0.022* 4.413 0.047** 7.442 0.329* 49.706 1.030** 1.244 0.110 8.344 0.782 0.913 0.069 6.081 0.360 0.459 0.007 3.080 0.104 5 148 7 5 148 5 3 102 3** 0.576 0.029 3.893 0.110 0.648 0.026 4.380 0.033** 7.544 0.481* 50.775 1.242** 1.088 0.101 7.320 0.456 0.868 0.104 5.780 0.537 0.434 0.023 2.932 0.064 0.558 0.022 3.778 0.046 0.690 0.028** 4.672 0.080** 9.214 0.411** 62.329 0.830** 1.262 0.082 8.582 0.614 0.947 0.026 6.421 0.092 0.452 0.026 3.061 0.130 0.457 0.017** 4.471 0.101 0.563 0.015 5.526 0.231** 8.207 0.247** 80.343 0.194** 1.070 0.142 10.425 1.121* 0.630 0.089* 6.135 0.716 0.170 0.011** 1.662 0.103** Female n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative Thymus Absolute Relative 5 120 3 0.494 0.010 4.132 0.137 0.486 0.011 4.060 0.102 5.004 0.134 41.800 1.128 0.896 0.021 7.488 0.216 0.385 0.017 3.223 0.164 5 121 3 0.516 0.031 4.245 0.205 0.572 0.023** 4.706 0.116** 5.632 0.163* 46.390 0.869* 1.180 0.129 9.787 1.219 0.399 0.022 3.301 0.230 5 125 1 5 122 3 2 93 3** 0.510 0.011 4.095 0.098 0.594 0.006** 4.768 0.022** 6.204 0.183** 49.764 1.089** 0.978 0.071 7.835 0.505 0.383 0.008 3.074 0.034 0.498 0.015 4.069 0.071 0.582 0.015** 4.761 0.119** 7.022 0.251** 57.371 1.450** 1.056 0.140 8.592 1.036 0.357 0.030 2.903 0.193 0.440 0.030 4.718 0.178 0.550 0.030 5.900 0.142** 7.030 0.010** 75.544 2.419** 0.790 0.050 8.505 0.797 0.187 0.007** 2.007 0.014** * Significantly different (P#0.05) from the chamber control group by Williams' or Dunnett's test a** P#0.01 Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean standard error). No data are reported for the 4,000 ppm groups due to 100% mortality. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 G-3 TABLE G2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm n Male Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative R. Testis Absolute Relative Thymus Absolute Relative 10 10 10 10 10 10 312 8 313 6 322 5 331 4 314 5 323 5 0.850 0.024 2.726 0.022 0.923 0.024 2.962 0.032 9.518 0.327 30.481 0.348 1.431 0.049 4.586 0.078 1.349 0.034 4.333 0.067 0.316 0.011 1.016 0.038 0.881 0.019 2.812 0.021 0.980 0.025 3.128 0.051** 10.123 0.267 32.279 0.278* 1.520 0.044 4.851 0.103 1.364 0.022 4.360 0.069 0.326 0.014 1.042 0.046 0.888 0.018 2.754 0.028 1.010 0.031*b 3.131 0.056**b 0.893 0.015 2.695 0.030 1.059 0.021** 3.194 0.036** 10.260 0.264 31.792 0.430* 11.170 0.302** 33.660 0.558** 1.523 0.038 4.724 0.090 1.376 0.034b 4.279 0.059b 1.578 0.027 4.765 0.078 1.390 0.015 4.198 0.051 0.300 0.020 0.930 0.056 0.337 0.013 1.017 0.034 0.885 0.015 2.820 0.035 1.070 0.017** 3.411 0.045** 11.589 0.282** 36.895 0.563** 1.548 0.051 4.925 0.116 1.343 0.027 4.281 0.077 0.323 0.012 1.028 0.028 0.915 0.015 2.832 0.037 1.152 0.023** 3.561 0.029** 12.637 0.288** 39.068 0.549** 1.681 0.081** 5.210 0.272* 1.399 0.017 4.331 0.054 0.316 0.011 0.978 0.038 Female Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative Thymus Absolute Relative 195 2 190 3 194 4 190 3 185 3 187 4 0.646 0.011 3.312 0.034 0.637 0.016 3.263 0.061 5.553 0.130 28.442 0.389 1.093 0.019 5.606 0.089 0.288 0.015 1.476 0.066 0.634 0.011 3.347 0.065 0.636 0.010 3.355 0.049 5.669 0.148 29.858 0.458 1.100 0.030 5.807 0.168 0.289 0.007 1.527 0.041 0.633 0.007 3.248 0.045 0.649 0.018 3.322 0.057 5.885 0.204 30.094 0.634* 1.169 0.027 6.003 0.168 0.280 0.014 1.432 0.059 0.655 0.016 3.438 0.045 0.655 0.017 3.439 0.057* 5.959 0.137 31.289 0.412** 1.186 0.028 6.232 0.107** 0.277 0.011 1.450 0.043 0.625 0.012 3.380 0.058 0.645 0.011 3.486 0.044** 5.979 0.133 32.286 0.386** 1.098 0.041 5.920 0.163 0.270 0.016 1.453 0.072 0.662 0.016 3.540 0.058** 0.675 0.011 3.612 0.040** 6.923 0.227** 36.958 0.724** 1.104 0.023 5.908 0.100 0.273 0.013 1.456 0.050 * Significantly different (P#0.05) from the chamber control group by Williams' or Dunnett's test a** P#0.01 Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean standard error). b n=9 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION G-4 Cumene, NTP TR 542 TABLE G3 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 2-Week Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm Male n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative R. Testis Absolute Relative Thymus Absolute Relative 5 27.0 0.5 0.142 0.002 5.268 0.167 0.234 0.005 8.677 0.290 1.324 0.051 48.933 0.989 0.200 0.014 7.394 0.489 0.099 0.005 3.657 0.223 0.059 0.003 2.201 0.147 5 27.6 0.6 0.140 0.008 5.066 0.282 0.270 0.007** 9.769 0.152** 1.520 0.044** 54.966 0.752** 0.268 0.026* 9.749 1.067 0.103 0.003 3.741 0.104 0.051 0.003 1.845 0.132 5 26.7 0.5 0.132 0.002 4.946 0.154 0.250 0.004 9.355 0.181 1.570 0.022** 58.763 1.077** 0.198 0.005 7.412 0.213 0.102 0.002 3.834 0.114 0.056 0.001 2.107 0.087 5 27.0 0.9 0.126 0.007 4.665 0.160 0.246 0.007 9.122 0.099 1.808 0.056** 67.040 0.739** 0.206 0.019 7.640 0.651 0.103 0.004 3.823 0.209 0.044 0.004** 1.643 0.109* Female n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative Thymus Absolute Relative 5 22.5 0.3 0.130 0.003 5.768 0.126 0.172 0.004 7.641 0.230 1.150 0.035 50.985 0.981 0.194 0.022 8.599 0.965 0.076 0.006 3.379 0.238 5 22.6 0.7 0.126 0.005 5.567 0.174 0.216 0.027 9.608 1.357 1.296 0.057 57.172 1.306* 0.216 0.017 9.543 0.701 0.073 0.003 3.251 0.192 5 23.3 0.7 0.122 0.007 5.234 0.181 0.188 0.007 8.089 0.263 1.458 0.084** 62.501 2.211** 0.216 0.015 9.271 0.525 0.080 0.005 3.423 0.190 1 23.8 0.130 5.462 0.210 8.824 1.560 65.546 0.200 8.403 0.062 2.605 * Significantly different (P#0.05) from the chamber control group by Williams' or Dunnett's test a** P#0.01 Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean standard error). No data is reported for the 2,000 and 4,000 ppm groups due to 100% mortality. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 G-5 TABLE G4 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 3-Month Inhalation Study of Cumenea Chamber Control 62.5 ppm 125 ppm 250 ppm 500 ppm 1,000 ppm Male n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative R. Testis Absolute Relative Thymus Absolute Relative 10 38.3 0.7 10 37.7 0.9 10 37.0 0.8 10 36.1 0.8 10 35.8 0.9* 10 34.7 0.6** 0.166 0.003 4.344 0.075 0.333 0.007 8.707 0.119 1.531 0.027 40.048 0.559 0.242 0.006 6.327 0.138 0.117 0.003 3.058 0.088 0.043 0.002 1.117 0.069 0.167 0.006 4.437 0.128 0.332 0.007 8.828 0.154 1.601 0.049 42.490 0.894 0.237 0.010 6.289 0.208 0.122 0.001 3.248 0.079 0.041 0.002 1.096 0.053 0.175 0.005 4.745 0.136 0.337 0.007 9.140 0.204 1.607 0.041 43.485 0.551* 0.230 0.007 6.218 0.111 0.125 0.003 3.390 0.130 0.043 0.002 1.160 0.062 0.162 0.004 4.501 0.119 0.305 0.010* 8.460 0.234 1.591 0.048 44.052 0.746** 0.219 0.006 6.090 0.196 0.120 0.002 3.340 0.083 0.043 0.003 1.182 0.076 0.164 0.006 4.591 0.135 0.311 0.006* 8.712 0.144 1.705 0.048* 47.668 0.795** 0.225 0.010 6.280 0.178 0.117 0.003 3.293 0.099 0.040 0.003 1.132 0.084 0.152 0.005 4.387 0.125 0.285 0.007** 8.229 0.184 1.913 0.070** 55.103 1.501** 0.214 0.005* 6.177 0.124 0.116 0.002 3.348 0.054 0.039 0.001 1.124 0.039 Female n Necropsy body wt Heart Absolute Relative R. Kidney Absolute Relative Liver Absolute Relative Lung Absolute Relative Thymus Absolute Relative 10 32.4 1.1 10 31.0 1.2 10 31.4 1.1 10 31.5 1.1 10 29.8 0.7 2 30.8 1.3 0.140 0.004 4.335 0.094 0.209 0.006 6.469 0.134 1.453 0.037 45.016 1.043 0.223 0.006 6.909 0.159 0.059 0.002 1.831 0.038 0.139 0.002 4.527 0.154 0.202 0.004 6.563 0.179 1.430 0.047 46.200 0.778 0.238 0.009 7.718 0.302 0.050 0.003* 1.592 0.074 0.140 0.003 4.484 0.117 0.210 0.003 6.740 0.181 1.495 0.053 47.622 0.576* 0.235 0.008 7.501 0.185 0.056 0.002 1.797 0.055 0.144 0.003 4.605 0.132 0.208 0.004 6.640 0.133 1.552 0.045 49.380 0.521** 0.229 0.006 7.316 0.230 0.057 0.003 1.810 0.077 0.144 0.003 4.848 0.114* 0.218 0.006 7.329 0.130** 1.593 0.042* 53.510 0.663** 0.240 0.008 8.079 0.265** 0.053 0.002 1.770 0.072 0.135 0.005 4.404 0.342 0.230 0.000 7.492 0.305** 1.910 0.110** 62.071 1.054** 0.240 0.000 7.818 0.318 0.052 0.007 1.669 0.144 * Significantly different (P#0.05) from the chamber control group by Williams' or Dunnett's test a** P#0.01 Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean standard error). Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION G-6 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft H-1 APPENDIX H REPRODUCTIVE TISSUE EVALUATIONS AND ESTROUS CYCLE CHARACTERIZATION TABLE H1 TABLE H2 TABLE H3 TABLE H4 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrous Cycle Characterization for Female Rats in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary of Reproductive Tissue Evaluations for Male Mice in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Estrous Cycle Characterization for Female Mice in the 3-Month Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H-2 H-2 H-3 H-3 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION H-2 Cumene, NTP TR 542 TABLE H1 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm n Weights (g) Necropsy body wt L. Cauda epididymis L. Epididymis L. Testis Spermatid measurements Spermatid heads (103/mg testis) Spermatid heads (106/testis) Spermatid count (106/cauda epididymis) Epididymal spermatozoal measurements Motility (%) Concentration (103/mg cauda epididymal tissue) 10 312 8 0.1805 0.0072 0.4781 0.0200 1.4148 0.0325 127.6 4.7 166.8 4.9 100.28 5.52 85.45 3.10 562.4 33.2 10 331 4* 0.1877 0.0050 0.4754 0.0072 1.4627 0.0148 131.8 4.1b 179.7 5.6 88.53 4.55 81.28 2.83 475.6 28.7 10 314 5 0.1948 0.0034 0.4843 0.0079 1.4299 0.0288 128.8 4.4 170.1 6.3 95.54 3.36 84.10 2.03 492.4 22.0 10 323 5 0.1738 0.0040 0.4502 0.0103 1.4546 0.0201 129.5 5.4 172.3 8.2 90.53 2.32 87.62 1.30 523.2 18.2 *a Significantly different (P#0.05) from the chamber control group by Dunnett's test Data are presented as mean standard error. Differences from the chamber control group are not significant by Dunnett's test (tissue b weights) or Dunn's test (spermatid and epididymal spermatozoal measurements). n=9 TABLE H2 Estrous Cycle Characterization for Female Rats in the 3-Month Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm n Necropsy body wt (g) Estrous cycle length (days) Estrous stages (% of cycle) Diestrus Proestrus Estrus Metestrus 10 195 5.06 02.13b 49.2 19.2 15.8 15.8 10 190 3 4.85 0.11 41.7 14.2 25.8 18.3 10 185 3 4.80 0.11 41.7 9.2 28.3 20.8 10 187 4 4.90 0.07 44.2 11.7 25.0 19.2 a Necropsy body weight and estrous cycle length data are presented as mean standard error. Differences from the chamber control group are not significant by Dunnett's test (body weight) or Dunn's test (estrous cycle length). Evidence shows that exposed female groups differ significantly (Wilk's Criterion, P#0.05) from the chamber control females in the relative length of time spent in the estrous stages. Exposed b females spent more time in estrus and less time in proestrus than chamber control females. Estrous cycle was longer than 12 days or unclear in one animal. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 H-3 TABLE H3 Summary of Reproductive Tissue Evaluations for Male Mice in the 3-Month Inhalation Study of Cumenea Chamber Control 250 ppm 500 ppm 1,000 ppm n Weights (g) Necropsy body wt L. Cauda epididymis L. Epididymis L. Testis Spermatid measurements Spermatid heads (103/mg testis) Spermatid heads (106/testis) Spermatid count (106/cauda epididymis) Epididymal spermatozoal measurements Motility (%) Concentration (103/mg cauda epididymal tissue) 10 38.3 0.7 0.0196 0.0010 0.0497 0.0013 0.1119 0.0027 184.4 7.6 18.80 0.77 18.05 0.95 85.44 1.96 931.4 52.1 10 36.1 0.8 0.0190 0.0007 0.0514 0.0022 0.1160 0.0024 191.3 7.2 19.77 0.79 17.62 1.11 82.75 2.41 928.6 46.1 10 36.3 0.8 0.0173 0.0006 0.0493 0.0010 0.1116 0.0026 204.5 7.7 20.67 0.70 17.53 1.04 79.95 2.13 1,017.4 56.7 10 34.7 0.6** 0.0171 0.0006* 0.0463 0.0015 0.1112 0.0022 202.2 7.3 20.77 0.88 14.70 0.87* 83.65 2.43 870.9 61.5 * Significantly different (P#0.05) from the chamber control group by Williams' (body weights), Dunnett's (tissue weights), or Dunn's (spermatid measurements) test a** P#0.01 Data are presented as mean standard error. Differences in epididymal spermatozoal measurements between exposed groups and the chamber control group are not significant by Dunn's test. TABLE H4 Estrous Cycle Characterization for Female Mice in the 3-Month Inhalation Study of Cumenea Chamber Control 125 ppm 250 ppm 500 ppm n Necropsy body wt (g) Estrous cycle length (days) Estrous stages (% of cycle) Diestrus Proestrus Estrus Metestrus 10 32.4 1.1 3.96 0.07 24.2 0.0 51.7 24.2 10 31.4 1.1 3.93 0.08 25.0 0.0 51.7 23.3 10 31.5 1.1 3.88 0.05 25.0 0.0 52.5 22.5 10 29.8 0.7 4.01 0.12 26.7 0.0 53.3 20.0 a Necropsy body weight and estrous cycle length data are presented as mean standard error. Differences from the chamber control group are not significant by Dunnett's test (body weight) or Dunn's test (estrous cycle length). By multivariate analysis of variance, exposed females do not differ significantly from the chamber control females in the relative length of time spent in the estrous stages. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION H-4 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft I-1 APPENDIX I CHEMICAL CHARACTERIZATION AND GENERATION OF CHAMBER CONCENTRATIONS PROCUREMENT AND CHARACTERIZATION OF CUMENE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VAPOR GENERATION AND EXPOSURE SYSTEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VAPOR CONCENTRATION MONITORING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHAMBER ATMOSPHERE CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FIGURE I1 1H-Nuclear Magnetic Resonance Spectrum of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FIGURE I2 Mass Spectrum of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FIGURE I3 Infrared Absorption Spectrum of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE I1 Gas Chromatography Systems Used in the Inhalation Studies of Cumene . . . . . . . . . . . . . FIGURE I4 Schematic of the Vapor Generation and Delivery System in the Inhalation Studies of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE I2 Summary of Chamber Concentrations in the 2-Week Inhalation Studies of Cumene . . . . . TABLE I3 Summary of Chamber Concentrations in the 3-Month Inhalation Studies of Cumene . . . . TABLE I4 Summary of Chamber Concentrations in the 2-Year Inhalation Studies of Cumene . . . . . I-2 I-2 I-3 I-3 I-5 I-6 I-7 I-8 I-9 I-10 I-10 I-11 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION I-2 Cumene, NTP TR 542 CHEMICAL CHARACTERIZATION AND GENERATION OF CHAMBER CONCENTRATIONS PROCUREMENT AND CHARACTERIZATION OF CUMENE Cumene was obtained from Sunoco, Inc. (Philadelphia, PA), in one lot (200556852) that was used in the 2-week, 3-month, and 2-year studies. Identity and purity analyses were conducted by the study laboratory at Battelle Toxicology Northwest (Richland, WA), by the analytical chemistry laboratory at Midwest Research Institute (Kansas City, MO), and by Chemir/Polytech Laboratories, Inc. (Maryland Heights, MO). Reports on analyses performed in support of the cumene studies are on file at the National Institute of Environmental Health Sciences. Lot 200556852 of the chemical, a colorless liquid with a sharp, penetrating, aromatic odor, was identified as cumene by the analytical chemistry laboratory using 1H-nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC/MS) and by Chemir/Polytech Laboratories, Inc., using infrared (IR) and 1H-NMR spectroscopy. All spectra were consistent with the literature spectra (Aldrich, 1993, 1997; NIST/EPA/NIH, 1994) of cumene. The 1H-NMR, GC/MS, and IR spectra are presented in Figures I1, I2, and I3, respectively. The purity of lot 200556852 was determined by the analytical chemistry laboratory using gas chromatography (GC) by system A and by the study laboratory using GC by system B, C, or D (Table I1). In addition, Chemir/Polytech Laboratories, Inc., determined the moisture content of this lot by Karl Fischer titration and measured its purity by elemental analysis. For lot 200556852, Karl Fischer titration indicated a water content ranging from approximately 50 to 220 ppm; elemental analyses for carbon and hydrogen were in agreement with the theoretical values for cumene. GC by system A detected no impurities greater than 0.05%, and the purity was determined to be approximately 100%. Using GC by system B, the area percent purity for the major cumene peak was 99.9%, and no peaks were detected with an area percent greater than 0.1%. The overall purity of lot 200556852 was determined to be greater than 99.9%. To ensure stability, the bulk chemical was stored at controlled room temperature in the original shipping containers (55-gallon metal drums). Stability was monitored by the study laboratory during the 2-week, 3-month, and 2-year studies with GC by system B, C, or D. No degradation of the bulk chemical was detected. VAPOR GENERATION AND EXPOSURE SYSTEM A diagram of the vapor generation and delivery system used in the studies is shown in Figure I4. The design of the system was influenced by the relatively high boiling point of cumene (approximately 152 C) and the need to reach relatively high concentrations. Therefore, with the exception of individual chamber inlets, all vapor transport lines and dilution air were heated to the minimum temperature needed to move vapor to the chambers without condensation. A bulk supply of cumene was held in an 8-gallon stainless steel chemical reservoir and pumped through a preheater into the top of a heated glass column filled with glass beads to increase the surface area for evaporation. Heated nitrogen entering the column from below vaporized the chemical as it conveyed it out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Because the cumene vapor leaving the generator was above room temperature, it was transported to the exposure room at an elevated temperature to prevent condensation. In the distribution manifold cabinet, the vapor was mixed with additional heated air before it entered a short vapor distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate, all of which NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 I-3 were monitored by the exposure operator. The pressure in the distribution manifold was fixed to ensure constant flow through the manifold and into the chambers as the flow of vapor to each chamber was adjusted. Electronically actuated metering valves controlled the flow to each chamber. In addition, an exposure-shutoff valve, mounted in series with each chamber-metering valve, controlled vapor delivery to each chamber. Vapor was diverted to the exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To start the exposure, the valves were opened to allow the flow of vapor to reach the chamber-metering valves and move into individual temperature-controlled delivery lines to each chamber. The vapor was then injected into the chamber inlet duct where it was diluted with conditioned chamber air to achieve the desired exposure concentration. The study laboratory designed the inhalation exposure chamber (Harford Systems Division of Lab Products, Inc., Aberdeen, MD) so that uniform vapor concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3. A condensation particle counter (Model 3022A, TSI, Inc., St. Paul, MN) was used to count the particles in all chambers before and during generation to ensure that cumene vapor, and not aerosol, was produced. No particle counts greater than 200 particles/cm3 were detected. VAPOR CONCENTRATION MONITORING Summaries of the chamber vapor concentrations are given in Tables I2 through I4. Concentrations of cumene in the exposure chambers were monitored by an on-line gas chromatograph using system E (Table I1). Samples were drawn from each exposure chamber approximately every 20 (2-week and 3-month studies) or 26 (2-year studies) minutes during each 6-hour exposure period using Hasteloy-C stream-select and gas-sampling valves (Valco Instruments Co., Houston, TX) in a separate, heated valve oven. The sample lines composing each sample loop were made from Teflon tubing and were connected to the exposure chamber relative humidity sampling lines at a location close to the gas chromatograph. A vacuum regulator maintained a constant vacuum in the sample loop to compensate for variations in sample line pressure. An in-line flow meter between the vacuum regulator and the gas chromatograph allowed digital measurement of sample flow. The on-line gas chromatograph was checked throughout the day for instrument drift against an on-line standard of cumene in nitrogen supplied by a permeation tube standard generator (Kin-Tek Model 491, Kin-Tek Laboratories, Inc., La Marque, TX). The on-line gas chromatograph was calibrated prior to the start of each study, three times during the 2-week studies, and monthly during the 3-month and 2-year studies by a comparison of chamber concentration data to data from grab samples that were collected with charcoal sampling tubes (ORBOTM-101, Supelco, Bellefonte, PA), extracted with toluene containing 1,2,4-trimethylbenzene as an internal standard, and analyzed by an off-line gas chromatograph using system F. The volumes of gas were sampled from each chamber at a constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standard solutions of cumene and the internal standard (1,2,4-trimethylbenzene) in toluene. CHAMBER ATMOSPHERE CHARACTERIZATION Buildup and decay rates for chamber vapor concentrations were determined with (all studies) and without (3-month and 2-year studies) animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 12.5 minutes. For the 2-week studies in rats and mice with animals present, T90 values ranged from 10 to 12 minutes, and T10 values ranged from 10 to 11 minutes. For rats and mice in the 3-month studies, T90 values ranged from 9 to 10 minutes without animals present and averaged 11 minutes with animals present; T10 values ranged from 9 to 10 minutes without animals present and from 10 to 12 minutes Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION I-4 Cumene, NTP TR 542 with animals present. For rats and mice in the 2-year studies, T90 values ranged from 9 to 10 minutes without animals present and from 10 to 15 minutes with animals present; T10 values ranged from 8 to 9 minutes without animals present and from 9 to 13 minutes with animals present. A T90 value of 12 minutes was selected for all studies. The uniformity of cumene vapor concentration in the inhalation exposure chambers without animals present was evaluated before the 3-month and 2-year studies began; concentration uniformity with animals present in the chambers was measured once during the 2-week studies, once during the 3-month studies, and approximately quarterly in the 2-year studies. The vapor concentration was measured using the on-line gas chromatograph (system E, Table I1) with the stream-selection valve fixed in one position to allow continuous monitoring from a single input line. During the 2-week study and prior to the 3-month and 2-year studies, concentrations were measured at 12 chamber positions, one in front and one in back for each of the six possible animal cage unit positions per chamber. During the 3-month and 2-year studies, concentrations were measured at the regular monitoring port and from sample ports at levels where animals were present. Chamber concentration uniformity was maintained throughout the studies. The persistence of cumene in the chambers after vapor delivery ended was determined by monitoring the postexposure vapor concentration in the 4,000 ppm chambers in the 2-week studies and the 1,000 ppm chambers in the 3-month and 2-year studies, with (all studies) and without (3-month and 2-year studies) animals present in the chambers. In the 2-week studies, the concentration decreased to 1% of the target concentration within 77 minutes. In the 3-month studies, the concentration decreased to 1% of the target concentration within 25 minutes without animals present and within 28 minutes with animals present. In the 2-year studies, the concentration decreased to 1% of the target concentration within 25 (rats) and 24 (mice) minutes without animals present and within 35 (rats) and 27 (mice) minutes with animals present. Samples of the test atmosphere from the distribution lines and low and high exposure concentration chambers were collected prior to the 3-month and 2-year studies and also at the beginning and end of one generation day during the 2-week, 3-month, and 2-year studies; the atmosphere samples were collected with sorbent tubes (ORBOTM-101, Supelco) and extracted with methylene chloride. Additional samples were collected from the generator reservoir and vapor trap, and all of the samples were analyzed using GC by system B to measure the stability and purity of cumene in the generation and delivery system. To assess whether impurities or degradation products co-eluted with cumene or the solvent, a second GC analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities (system G). The relative purity of cumene in the generator reservoir and vapor trap was measured using GC by systems C or D in conjunction with the stability and purity measurements described above by major peak comparison to the bulk test chemical. No evidence of degradation of cumene was noted in any part of the exposure system. With the exception of one peak noted in the distribution line samples taken during the 3-month studies, no impurity peaks were resolved with an area >0.1% of the total peak area, and no additional impurities were detected with the polar GC analyses. The identity of the spurious peak was established by GC/mass spectrometry and by standard addition as 2-phenyl-2-propanol. The spectra for this peak (accounting for 0.14% and 0.10% of the total peak areas for the samples taken at the beginning and end, respectively, of the sampled generation day) closely matched a library reference spectrum for 2-phenyl-2-propanol (NIST/EPA/NIH, 1994) and the spectrum of a purchased standard of this chemical. The relative purity of all generator reservoir and vapor trap samples exceeded 99% compared to the bulk chemical, and these samples were 99.97% pure by area percent. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 I-5 FIGURE I1 1H-Nuclear Magnetic Resonance Spectrum of Cumene Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION I-6 Cumene, NTP TR 542 FIGURE I2 Mass Spectrum of Cumene NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 I-7 Board Draft FIGURE I3 Infrared Absorption Spectrum of Cumene NOT FOR DISTRIBUTION OR ATTRIBUTION I-8 Cumene, NTP TR 542 TABLE I1 Gas Chromatography Systems Used in the Inhalation Studies of Cumenea Detection System Column Carrier Gas Oven Temperature Program System A Flame ionization System B Flame ionization System C Flame ionization System D Flame ionization System E Flame ionization System F Flame ionization System G Flame ionization DBWax, 30 m 0.53 mm, 1.0-m film (J&W Scientific, Folsom, CA) Helium at at 10 mL/minute 35 C for 6 minutes, then 10 C/minute to 205 C, held for 5 minutes Rtx-5, 30 m 0.25 mm, 1.0-m film (Restek) or DB-5, 30 m 0.25 mm, 1.0-m film (J&W Scientific) Helium at 24 psi head pressure 45 C for 1 minute, then 5 C/minute to 250 C Rtx-5, 30 m 0.25 mm, 1.0-m Helium at 24 psi head pressure film (Restek, Bellefonte, PA) 60 C for 1 minute, then 10 C/minute to 200 C Rtx-5, 30 m 0.25 mm, 1.0-m Helium at 24 psi head pressure film (Restek) 80 C for 0.5 minutes, then 8 C/minute to 135 C, then 30 C/minute to 200 C DB-5, 15 m 0.53 mm, 1.5-m Nitrogen at 20 mL/minute film (J&W Scientific) Isothermal at 85 C DB-5, 30 m 0.53 mm, 1.5-m Helium at 6 psi head pressure film (J&W Scientific) 90 C for 1 minute, then 16 C/minute to 150 C, held for 1 minute DBWax-Etr, 30 m 0.25 mm, 0.5-m film (J&W Scientific) Helium at 24 psi head pressure 45 C for 1 minute, then 5 C/minute to 250 C a The gas chromatographs were manufactured by Hewlett-Packard (Palo Alto, CA). NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 I-9 FIGURE I4 Schematic of the Vapor Generation and Delivery System in the Inhalation Studies of Cumene Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION I-10 Cumene, NTP TR 542 TABLE I2 Summary of Chamber Concentrations in the 2-Week Inhalation Studies of Cumene Target Concentration (ppm) Total Number of Readings Average Concentrationa (ppm) Rat Chambers Mouse Chambers 250 500 1,000 2,000 4,000b 250 500 1,000 24,,000000bb a b Mean standard deviation Includes data only from the first day of exposure 212 215 216 221 20 231 234 235 20 20 254 4 504 10 986 30 2,047 65 4,002 58 254 4 504 9 990 31 2,076 49 4,002 58 TABLE I3 Summary of Chamber Concentrations in the 3-Month Inhalation Studies of Cumene Target Concentration (ppm) Total Number of Readings Average Concentrationa (ppm) Rat Chambers Mouse Chambers 62.5 125 250 500 1,000 62.5 125 250 500 1,000 a Mean standard deviation 1,291 1,298 1,305 1,309 1,312 1,331 1,338 1,345 1,349 1,352 62.9 1 125 3 251 5 504 10 1,009 16 62.9 1 125 3 252 5 503 10 1,009 16 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 TABLE I4 Summary of Chamber Concentrations in the 2-Year Inhalation Studies of Cumene Target Concentration (ppm) Total Number of Readings Average Concentrationa (ppm) Rat Chambers Mouse Chambers 250 500 1,000 125 250 500 1,000 a Mean standard deviation 7,386 7,440 7,497 7,804 7,708 7,418 7,480 251 5 502 11 1,005 23 125 2 250 6 501 13 1,007 24 I-11 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION I-12 Cumene, NTP TR 542 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft J-1 APPENDIX J INGREDIENTS, NUTRIENT COMPOSITION, AND CONTAMINANT LEVELS IN NTP-2000 RAT AND MOUSE RATION TABLE J1 TABLE J2 TABLE J3 TABLE J4 Ingredients of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vitamins and Minerals in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . Nutrient Composition of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . J-2 J-2 J-3 J-4 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION J-2 TABLE J1 Ingredients of NTP-2000 Rat and Mouse Ration Ingredients Ground hard winter wheat Ground #2 yellow shelled corn Wheat middlings Oat hulls Alfalfa meal (dehydrated, 17% protein) Purified cellulose Soybean meal (49% protein) Fish meal (60% protein) Corn oil (without preservatives) Soy oil (without preservatives) Dried brewer's yeast MCViaitlnacmeiruiamnl ppcrraeermmboiixxnbaate (USP) Calcium phosphate, dibasic (USP) Sodium chloride Choline chloride (70% choline) Methionine a Wheat middlings as carrier b Calcium carbonate as carrier Percent by Weight 22.26 22.18 15.0 8.5 7.5 5.5 5.0 4.0 3.0 3.0 1.0 0.9 0.5 0.5 0.4 0.3 0.26 0.2 Cumene, NTP TR 542 TABLE J2 Vitamins and Minerals in NTP-2000 Rat and Mouse Rationa Amount Vitamins A D K "-Tocopherol acetate Niacin Folic acid d-Pantothenic acid Riboflavin Thiamine B12 Pyridoxine Biotin Minerals Magnesium Iron Zinc Manganese Copper Iodine Chromium a Per kg of finished product 4,000 IU 1,000 IU 1.0 mg 100 IU 23 mg 1.1 mg 10 mg 3.3 mg 4 mg 52 g 6.3 mg 0.2 mg 514 mg 35 mg 12 mg 10 mg 2.0 mg 0.2 mg 0.2 mg Source Stabilized vitamin A palmitate or acetate D-activated animal sterol Menadione sodium bisulfite complex d-Calcium pantothenate Thiamine mononitrate Pyridoxine hydrochloride d-Biotin Magnesium oxide Iron sulfate Zinc oxide Manganese oxide Copper sulfate Calcium iodate Chromium acetate NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 J-3 TABLE J3 Nutrient Composition of NTP-2000 Rat and Mouse Ration Nutrient Mean Standard Deviation Range Number of Samples Protein (% by weight) Crude Fat (% by weight) Crude Fiber (% by weight) Ash (% by weight) Amino Acids (% of total diet) Arginine Cystine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine Essential Fatty Acids (% of total diet) Linoleic Linolenic Vitamins Vitamin A (IU/kg) Vitamin D (IU/kg) "-Tocopherol (ppm) Thiamine (ppm)b Riboflavin (ppm) Niacin (ppm) Pantothenic Acid (ppm) Pyridoxine (ppm)b Folic Acid (ppm) Biotin (ppm) Vitamin Choline (Bp1p2m(p)bpb) Minerals Calcium (%) Phosphorus (%) Potassium (%) Chloride (%) Sodium (%) Magnesium (%) Sulfur (%) Iron (ppm) Manganese (ppm) Zinc (ppm) Copper (ppm) Iodine (ppm) Chromium (ppm) Cobalt (ppm) 14.6 0.63 8.1 0.27 9.0 0.45 5.2 0.27 0.750 0.048 0.225 0.025 0.701 0.039 0.365 0.090 0.533 0.038 1.077 0.059 0.703 0.125 0.402 0.049 0.615 0.035 0.492 0.040 0.135 0.018 0.378 0.048 0.658 0.043 3.90 0.256 0.30 0.035 14,,070904a 759 84.2 16.60 7.3 1.04 6.8 2.11 79.0 10.50 23.9 3.73 9.21 2.20 1.75 0.54 0.332 0.12 60.5 46.5 3,064 270 1.024 0.054 0.613 0.035 0.665 0.023 0.376 0.041 0.191 0.017 0.201 0.009 0.170 0.029 182 46.7 54.1 7.89 55.0 9.55 6.65 1.790 0.512 0.221 0.604 0.253 0.25 0.074 a b From formulation As hydrochloride (thiamine and pyridoxine) or chloride (choline) 13.3 15.7 7.6 8.6 8.0 9.9 4.8 5.8 0.670 0.850 0.150 0.250 0.620 0.750 0.310 0.680 0.430 0.590 0.960 1.150 0.310 0.830 0.260 0.460 0.540 0.660 0.430 0.590 0.110 0.160 0.280 0.460 0.550 0.710 3.49 4.54 0.21 0.35 3,060 6,460 52.0 110.0 5.9 9.2 4.20 11.20 66.4 98.2 17.4 29.8 6.4 13.7 1.20 3.27 0.225 0.704 18.3 174.0 2,700 3,790 0.873 1.140 0.556 0.701 0.626 0.694 0.300 0.474 0.160 0.222 0.185 0.217 0.116 0.209 135 311 42.1 73.1 43.3 78.5 3.21 10.50 0.233 0.972 0.330 1.380 0.20 0.47 23 23 23 23 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 23 15 23 15 15 15 15 15 15 15 15 23 23 15 15 15 15 15 15 15 15 15 15 14 14 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION J-4 Cumene, NTP TR 542 TABLE J4 Contaminant Levels in NTP-2000 Rat and Mouse Rationa Nutrient Mean Standard Deviationb Range Number of Samples Contaminants Arsenic (ppm) Cadmium (ppm) Lead (ppm) Mercury (ppm Selenium (ppm) Aflatoxins (ppb) Nitrate nitrogen (ppm)c Nitrite nitrogen (ppm)c BHA (ppm)d BHT (ppm)d Aerobic plate count (CFU/g) Coliform (MPN/g) Escherichia coli (MPN/g) Salmonella (MPN/g) Total nitrosoamines (ppb)e N-Nitrosodimethylamine (ppb)e N-Nitrosopyrrolidine (ppb)e Pesticides (ppm) "-BHC $-BHC (-BHC *-BHC Heptachlor Aldrin Heptachlor epoxide DDE DDD DDT HCB Mirex Methoxychlor Dieldrin Endrin Telodrin Chlordane Toxaphene Estimated PCB's Ronnel Ethion Trithion Diazinon Methyl chlorpyrifos Methyl parathion Ethyl parathion Malathion Endosulfan 1 Endosulfan 2 Endosulfane Sulfate 0.32 0.151 0.04 0.009 0.07 0.026 <0.02 0.23 0.052 <5.00 14.2 4.13 <0.61 <1.0 <1.0 15 15 3.1 0.2 <10 Negative 4.7 1.45 2.9 1.37 1.8 0.78 <0.01 <0.02 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.05 <0.01 <0.01 <0.01 <0.05 <0.10 <0.20 <0.01 <0.02 <0.05 <0.10 0.117 0.070 <0.02 <0.02 0.343 0.486 <0.01 <0.01 <0.03 0.17 0.50 0.04 0.07 0.05 0.17 0.14 0.36 6.85 23.2 10 70c 3.0 3.6 2.4 8.4 1.2 6.9 0.9 3.1 0.020 0.259 0.020 1.850 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 23 a All samples were irradiated. CFU=colony-forming units; MPN=most probable number; BHC=hexachlorocyclohexane or benzene b c d e hexachloride For values less than the limit of detection, the detection limit is given as the mean. Sources of contamination: alfalfa, grains, and fish meal Sources of contamination: soy oil and fish meal All values were corrected for percent recovery. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft K-1 APPENDIX K SENTINEL ANIMAL PROGRAM METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-2 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-4 Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION K-2 Cumene, NTP TR 542 SENTINEL ANIMAL PROGRAM METHODS Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally clean facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel animals come from the same production source and weanling groups as the animals used for the studies of chemical compounds. Serum samples were collected from five male and five female chamber control rats and mice at the end of the 2-week and 3-month studies, five male and five female sentinel rats and mice 1 week after the start of the 3-month and 2-year studies, five male and five female sentinel rats and mice at 6, 12, and 18 months in the 2-year studies, and five males and five females from the 1,000 ppm rats and 500 ppm mice at the end of the 2-year studies. Blood from each animal was collected and allowed to clot, and the serum was separated; fecal samples were collected from five male and five female mice. Samples were processed appropriately and sent to BioReliance Corporation (Rockville, MD) for determination of antibody titers. The laboratory serology methods and viral agents for which testing was performed are tabulated below; the times at which blood was collected during the studies are also listed. Method and Test RATS 2-Week Study ELISA H-1 (Toolan's H-1 virus) KRV (Kilham rat virus) Mycoplasma pulmonis PVM (pneumonia virus of mice) RCV/SDA (rat coronavirus/sialodacryoadenitis virus) Sendai 3-Month Study ELISA H-1 KRV M. arthritidis M. pulmonis PVM RCV/SDA Sendai Immunofluorescence Assay Parvovirus Time of Analysis Study termination Study termination Study termination Study termination Study termination Study termination 1 week, study termination 1 week Study termination 1 week, study termination 1 week, study termination 1 week, study termination 1 week, study termination Study termination NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 Method and Test RATS (continued) 2-Year Study ELISA H-1 KRV M. arthritidis M. pulmonis PVM RCV/SDA Sendai Immunofluorescence Assay Parvovirus MICE 2-Week Study ELISA GDVII (mouse encephalomyelitis virus) MHV (mouse hepatitis virus) MVM (minute virus of mice) M. pulmonis PVM Sendai 3-Month Study ELISA Ectromelia virus EDIM (epizootic diarrhea of infant mice) GDVII LCM (lymphocytic choriomeningitis virus) mouse adenoma virus-FL MCMV (mouse cytomegalovirus) MHV MVM M. arthritidis M. pulmonis PVM Reovirus 3 Sendai Immunofluorescence Assay Parvovirus Time of Analysis K-3 1 week 1 week Study termination 1 week and study termination 1 week, 6, 12, and 18 months, study termination 1 week, 6, 12, and 18 months, study termination 1 week, 6, 12, and 18 months, study termination 6, 12, and 18 months, study termination Study termination Study termination Study termination Study termination Study termination Study termination Study termination Study termination 1 week, study termination Study termination Study termination Study termination 1 week, study termination 1 week Study termination 1 week, study termination 1 week, study termination Study termination 1 week, study termination Study termination Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION K-4 Method and Test MICE (continued) 2-Year Study ELISA Ectromelia virus EDIM GDVII LCM Mouse adenoma virus MCMV MHV MVM M. arthritidis M. pulmonis PVM Reovirus 3 Sendai Immunofluorescence Assay Helicobacter billis Helicobacter hepatica Parvovirus RESULTS All results were negative. Time of Analysis Cumene, NTP TR 542 6, 12, and 18 months, study termination 6, 12, and 18 months, study termination 1 week, 6, 12, and 18 months, study termination 6, 12, and 18 months, study termination 6, 12, and 18 months, study termination Study termination 1 week, 6, 12, and 18 months, study termination 1 week Study termination 1 week, study termination 1 week, 6, 12, and 18 months, study termination 6, 12, and 18 months, study termination 1 week, 6, 12, and 18 months, study termination 18 months 18 months 6, 12, and 18 months, study termination NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft L-1 APPENDIX L CHARACTERIZATION OF K-ras AND p53 MUTATIONS IN LUNG NEOPLASMS OF MICE IN THE 2-YEAR INHALATION STUDY OF CUMENE Hue-Hua L. Hong, Thai-Vu T. Ton, Yongbaek Kim, Nobuko Wakamatsu, and Robert C. Sills National Institute of Environmental Health Sciences Research Triangle Park, North Carolina INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE L1 K-ras Mutations in Lung Neoplasms of B6C3F1 Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TABLE L2 p53 Mutations in Lung Neoplasms of B6C3F1 Mice in the 2-Year Inhalation Study of Cumene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L-2 L-2 L-3 L-3 L-4 L-8 L-8 L-2 Cumene, NTP TR 542 CHARACTERIZATION OF K-ras AND p53 MUTATIONS IN LUNG NEOPLASMS OF MICE IN THE 2-YEAR INHALATION STUDY OF CUMENE INTRODUCTION Following exposure of male and female B6C3F1 mice by inhalation to 125 (females only), 250, 500, or 1,000 (males only) ppm cumene for 2 years, there were increased incidences of lung neoplasms in all groups of exposed males and females, specifically alveolar/bronchiolar adenomas and carcinomas. Cumene was not genotoxic in several studies involving bacterial and mammalian cells in culture and in in vivo studies involving mice and rats (Appendix E; USEPA, 1997; HSDB, 2003). In vitro cell transformation assays using BALB/3T3 mouse embryo cells and unscheduled DNA synthesis assays using rat primary hepatocytes yielded conflicting results regarding a cumene effect that were not reproducible. Cumene was weakly positive with no clear dose response for the induction of micronuclei in male rat bone marrow at intraperitoneal doses ranging from 78 to 2,500 mg/kg body weight (Appendix E). Mouse alveolar/bronchiolar adenomas, which are the most common spontaneous and chemical-induced lung tumors in mice, are similar in histomorphology and molecular characteristics, including activation of the K-ras gene, to human adenocarcinomas (Meuwissen and Berns, 2005). The patterns of mutations in cancer genes, such as ras and p53, have been found to aid in the understanding of tumorigenesis (Harris, 1993; Maronpot et al., 1995; Osada and Takahashi, 2002; Le Calvez et al., 2005). For example, in some neoplasms, the profile of activating mutations in ras genes or inactivating mutations in the p53 gene are specific for particular chemicals and differ from those detected in spontaneous neoplasms (Sills et al., 1999, 2004). In the present study, 52 alveolar/bronchiolar neoplasms from B6C3F1 mice exposed to cumene for 2 years were examined for mutations in exons 1 and 2 of K-ras, for overexpression of mutant p53 protein using immunohistochemistry, and for mutations in exons 5 through 8 of p53 gene. Other studies to assess changes in the K-ras/MAP kinase signaling pathway are in progress. MATERIALS AND METHODS Lung Neoplasms Male and female B6C3F1 mice were exposed to 0, 125 (females only), 250, 500, or 1,000 (males only) ppm cumene (50 animals per group per sex) by inhalation for 6 hours per day, 5 days per week for 2 years. At necropsy, tissues were fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin, sectioned to a thickness of 5 m, and stained with hematoxylin and eosin. Subsequently, five unstained serial sections, 10 m thick, were prepared from paraffin blocks containing alveolar/bronchiolar adenomas or carcinomas. In order to isolate adequate amounts of DNA, neoplasms greater than 1 mm in diameter were identified for analysis. Fifty-two cumene-induced alveolar/bronchiolar neoplasms (six adenomas and 46 carcinomas), seven spontaneously occurring carcinomas, and six normal lung tissues were evaluated for K-ras mutations in exons 1 and 2 (codons 12, 13, and 61) and p53 mutations in exons 5 through 8. DNA Isolation, Amplification, and Cycle Sequencing DNA was isolated and extracted from paraffin-embedded sections containing lung neoplasms and normal lung tissue and amplified by polymerase chain reaction (PCR). Details of the use of nested primers for K-ras and p53 genes have been described previously (Sills et al., 1995; Lambertini et al., 2005). Positive DNA controls for K-ras and p53 mutations and controls lacking DNA were run with all sets of reactions. PCR products were NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 L-3 purified using a QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, CA). The purified samples were sequenced utilizing a cycle sequencing kit (USB Corporation, Cleveland, OH), which incorporated "-33p-dideoxynucleotide triphosphate (ddNTP) terminators (A, C, G, T) into the sequencing products. Detected mutations were confirmed by repeat analysis, starting from amplification of the original DNA extract. Immunohistochemistry for p53 Protein Alveolar/bronchiolar adenomas and carcinomas were examined for p53 protein expression by immunohistochemical analysis using an avidin-biotin-peroxidase detection system [VECTASTAIN Elite ABC Kit (Rabbit IgG), Vector Laboratories, Burlingame, CA]. The immunohistochemical staining for expression of mutant p53 protein was performed, as previously described (Hong et al., 2000; Sills et al., 2004). RESULTS A higher frequency of K-ras mutations (45/52, 87%) was observed in the cumene-induced lung neoplasms, as compared to spontaneous lung neoplasms from control animals (historical controls, 33/117, 28%; current controls, 1/7, 14%) (Table L1). The predominant K-ras mutations were codon 12 G to T transversions (GTT) and codon 61 A to G transitions (CGA). These two mutations were found at frequencies of 21% (11/52) and 25% (13/52), respectively, in the neoplasms from exposed mice, compared to 0.008% (1/124) and 2% (3/124), respectively, in lung neoplasms from control mice in the historical database. Three codon 12 CGT mutations and one codon 61 CTA mutation were found in exposed groups but none in spontaneous lung neoplasms (0/124) (Table L1). p53 mutations were identified in 52% (27/52) of the cumene-induced lung neoplasms; none were identified in seven spontaneous carcinomas, and six were identified in normal lung tissue (Table L2). The predominant p53 mutations were identified in exon 5 (24/27, 89%) (Table L2). There were dose-related increases in the incidences of K-ras and p53 mutations; however, a similar spectrum of both mutations was detected in cumene-induced neoplasms regardless of whether the neoplasms were adenomas or carcinomas. The p53 protein expression was detected in 56% (29/52) of the cumene-induced lung neoplasms, mostly corresponding to p53 mutations and was localized to the nucleus, compared to 14% (1/7) in the spontaneously occurring neoplasms without p53 mutation. DISCUSSION A high frequency (87%) of K-ras mutations was identified in cumene-induced alveolar/bronchiolar neoplasms compared to that in spontaneous alveolar/bronchiolar neoplasms from control B6C3F1 mice (28% historical database; 14% current study). The predominant mutations were K-ras codon 12 G to T transversions (GGT to GTT, 21%) and codon 61 A to G transitions (CAA to CGA, 25%), which clearly differed from those identified in control mice (0.008% and 2%, respectively). Point mutations at codon 12 of the K-ras gene are activating mutations, rendering ras insensitive to the down-regulatory action of GTPase activating proteins, thereby locking the protein in the active state and promoting cellular transformation (Ellis and Clark, 2000). G to T transversions are commonly detected DNA base changes associated with active oxygen species and are consistent with 8-OH-G adducts produced during oxidative damage to DNA (Shigenaga and Ames, 1991; Tchou et al., 1991; Janssen et al., 1993). Exposure of B6C3F1 mice to ozone or vanadium pentoxide may have resulted in the generation of hydroxyl radicals, which could have induced G to T transversions at codon 12 of the K-ras gene (Sills et al., 1995; Devereux et al., 2002). Interestingly, G to T transversions in K-ras codon 12 are the most common mutations detected in human adenocarcinomas (Rodenhuis et al., 1987). In human lung tumors, K-ras codon 12 G to T mutations appear to correlate with DNA adducts of benzo(a)pyrene and are associated with smoking (Reynolds et al., 1987). It is possible that smoking in combination with cumene exposure in humans may have an additive effect on K-ras mutations. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION L-4 Cumene, NTP TR 542 The high frequency and pattern of K-ras mutations in mouse lung tumors may directly depend on the nature of the chemical carcinogen or its metabolites. In the present study, the development of lung neoplasms in the B6C3F1 mouse exposed to cumene may involve multiple carcinogenic processes, including direct DNA damage and/or indirect DNA damage such as oxidative stress. There were neoplasms without mutations of the K-ras gene, suggesting that other genetic events should be considered (Anderson et al., 1992; Loeb, 2001). Previous studies showed that benzene is carcinogenic and genotoxic (Gut et al., 1996; Snyder and Hedli, 1996; Valentine et al., 1996; Abernethy et al., 2004). Side-chain oxidation of cumene is rapid and extensive and occurs in both hepatic and extrahepatic tissues, including the lung (Sato and Nakajima, 1987), with the secondary alcohol 2-phenyl-2-propanol being the principal metabolite in rats (RTI, 1989; USEPA, 1997) and humans (Lee, 1987; USEPA, 1997). The C-isopropyl bonds are readily cleaved, and the remaining electrophilic carbon moiety may form DNA adducts and cause subsequent DNA damage. Interestingly, the in vivo tumor response in the present study did not show an exposure concentration-related response, suggesting that K-ras mutational analysis may be a more sensitive method for identifying dose response with cumene. A high frequency (52%) of p53 mutations were detected in cumene-induced alveolar/bronchiolar neoplasms that were correlated to p53 protein expression (56%) by immunohistochemistry. The presence of p53 protein expression without p53 mutation could be due to p53 mutations outside the regions exons 5 through 8 examined or possibly to alterations of other proteins downstream of p53 (Greenblatt et al., 1994). The predominance of cumene-induced alveolar/bronchiolar neoplasms containing p53 mutations is consistent with these mutations, providing a selective advantage for unregulated growth and the avoidance of apoptosis (Greenblatt et al., 1994; Harris, 1996; Osada and Takahashi, 2002; Rodin and Rodin, 2005). A study of aflatoxin-B1 (AFB1)-induced mouse lung tumors also found a high proportion (>70%) of tumors with p53 accumulation and mutations (Tam et al., 1999). In addition, 84% p53 mutations were detected in lung tumors of mice exposed transplacentally to AZT (Hong et al., 2007), while other studies have found a low frequency of p53 mutations in methylene chlorideinduced mouse lung tumors (Hegi et al., 1993), and no p53 mutation was present in vanadium pentoxide-induced mouse lung tumors (Devereux et al., 2002). Unlike the mostly random mutation pattern for the AFB1-induced tumors (Tam et al., 1999), the cumene-induced tumors had specific p53 mutations. Only exon 5 (24/27, 89%) and exon 7 (11%) appeared to account for the p53 mutations. The data on K-ras and p53 mutations provide evidence that these genetic alterations play an important role in cumene-induced mouse lung carcinogenesis. Additional evidence (Santillo et al., 2001) showed that K-ras activation may play a major role in the formation of these neoplasms. Other microarray studies to investigate the role of MAP kinase signaling pathway in these neoplasms are in progress. In conclusion, the patterns of mutations identified in the lung tumors suggest that DNA damage and genomic instability may be the contributing factors to the mutation profile and development of lung cancer in these mice. The molecular alterations identified in the cumene-induced lung neoplasms affect the same pathways as those reported in human lung cancer, suggesting that the response in the mouse may be of relevance to humans. REFERENCES Abernethy, D.J., Kleymenova, E.V., Rose, J., Recio, L., and Faiola, B. (2004). Human CD34+ hematopoietic progenitor cells are sensitive targets for toxicity induced by 1,4-benzoquinone. Toxicol. Sci. 79, 82-89. Anderson, M.W., Reynolds, S.H., You, M., and Maronpot, R.M. (1992). Role of proto-oncogene activation in carcinogenesis. Environ. Health Perspect. 98, 13-24. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 L-5 Devereux, T.R., Holliday, W., Anna, C., Ress, N., Roycroft, J., and Sills, R.C. (2002). 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(1996). p53 tumor suppressor gene: From the basic research laboratory to the clinic--an abridged historical perspective. Carcinogenesis 17, 1187-1198. Hazardous Substances Data Bank (HSDB) (2003). Cumene. National Institute for Occupational Safety and Health, HSDB database available through the National Library of Medicine MEDLARS System. Hegi, M.E., Soderkvist, P., Foley, J.F., Schoonhoven, R., Swenberg, J.A., Kari, F., Maronpot, R., Anderson, M.W., and Wiseman, R.W. (1993). Characterization of p53 mutations in methylene chloride-induced lung tumors from B6C3F1 mice. Carcinogenesis 14, 803-810. Hong, -H.H.L., Devereux, T.R., Melnick, R.L., Moomaw, C.R., Boorman, G.A., and Sills, R.C. (2000). Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years. Toxicol. Pathol. 28, 529-534. Hong, H.H., Dunnick, J., Herbert, R., Devereux, T.R., Kim, Y., and Sills, R.C. (2007). Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from Swiss (CD-1) male mice exposed transplacentally to AZT. Environ. Mol. Mutagen. Submitted for publication. Janssen, Y.M., Van Houten, B., Borm, P.J., and Mossman, B.T. (1993). Cell and tissue responses to oxidative damage. Lab. Invest. 69, 261-274. Lambertini, L., Surin, K., Ton, T.V., Clayton, N., Dunnick, J.K., Kim, Y., Hong, H.H., Devereux, T.R., and Sills, R.C. (2005). Analysis of p53 tumor suppressor gene, H-ras protooncogene and profilerating cell nuclear antigen (PCNA) in squamous cell carcinomas of HRA/Skh mice following exposure to 8-methoxypsoralen (8-MOP) and UVA radiation (PUVA therapy). Toxicol. Pathol. 33, 292-299. Le Calvez, F., Mukeria, A., Hunt, J.D., Kelm, O., Hung, R.J., Taniere, P., Brennan, P., Boffetta, P., Zaridze, D.G., and Hainaut, P. (2005). TP53 and KRAS mutation load and types in lung cancers in relation to tobacco smoke: Distinct patterns in never, former, and current smokers. Cancer Res. 65, 5076-5083. Lee, E.W. (1987). Cumene. In Ethel Browning's Toxicity and Metabolism of Industrial Solvents, 2nd ed. (R. Snyder, Ed.), Vol. 1, Hydrocarbons, Section 1.5. Elsevier Science Publishers, New York. Loeb, L.A. (2001). A mutator phenotype in cancer. Cancer Res. 61, 3230-3239. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION L-6 Cumene, NTP TR 542 Maronpot, R.R., Fox, T., Malarkey, D.E., and Goldsworthy, T.L. (1995). Mutations in the ras proto-oncogene: Clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101, 125-156. Meuwissen, R., and Berns, A. (2005). Mouse models for human lung cancer. Genes Dev. 19, 643-664. Osada, H., and Takahashi, T. (2002). Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 21, 7421-7434. Research Triangle Institute (RTI) (1989). Metabolism, Disposition and Pharmacokinetics of Cumene in F 344 Rats Following Oral, IV Administration or Nose-Only Inhalation Exposure. RTI/4353-01F. Research Triangle Institute, Research Triangle Park, NC. Reynolds, S.H., Stowers, S.J., Patterson, R.M., Maronpot, R.R., Aaronson, S.A., and Anderson, M.W. (1987). Activated oncogenes in B6C3F1 mouse liver tumors: Implications for risk assessment. Science 237, 1309-1316. Rodenhuis, S., van de Wetering, M.L., Mooi, W.J., Evers, S.G., van Zandwijk, N., and Bos, J.L. (1987). Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung. N. Engl. J. Med. 317, 929-935. Rodin, S.N., and Rodin, A.S. (2005). Origins and selection of p53 mutations in lung carcinogenesis. Semin. Cancer Biol. 15, 103-112. Santillo, M., Mondola, P., Seru, R., Annella, T., Cassano, S., Ciullo, I., Tecce, M.F., Iacomino, G., Damiano, S., Cuda, G., Paterno, R., Martignetti, V., Mele, E., Feliciello, A., and Avvedimento, E.V. (2001). Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals. Curr. Biol. 11, 614-619. Sato, A., and Nakajima, T. (1987). Pharmacokinetics of organic solvent vapors in relation to their toxicity. Scand. J. Work Environ. Health 13, 81-93. Shigenaga, M.K., and Ames, B.N. (1991). Assays for 8-hydroxy-2N-deoxyguanosine: A biomarker of in vivo oxidative DNA damage. Free Radic. Biol. Med. 10, 211-216. Sills, R.C., Hong, H.L., Greenwell, A., Herbert, R.A., Boorman, G.A., and Devereux, T.R. (1995). Increased frequency of K-ras mutations in lung neoplasms from female B6C3F1 mice exposed to ozone for 24 or 30 months. Carcinogenesis 16, 1623-1628. Sills, R.C., Hong, H.L., Melnick, R.L., Boorman, G.A., and Devereux, T.R. (1999). High frequency of codon 61 K-ras A to T transversions in lung and harderian gland neoplasms of B6C3F1 mice exposed to chloroprene (2-chloro-1,3-butadiene) for 2 years, and comparisons with the structurally related chemicals isoprene and 1,3-butadiene. Carcinogenesis 20, 657-662. Sills, R.C., Hong, H.L., Flake, G., Moomaw, C., Clayton, N., Boorman, G.A., Dunnick, J., and Devereux, T.R. (2004). o-Nitrotoluene-induced large intestinal tumors in B6C3F1 mice model human colon cancer in their molecular pathogenesis. Carcinogenesis 25, 605-612. Snyder, R., and Hedli, C.C. (1996). An overview of benzene metabolism. Environ. Health Perspect. 104 (Suppl. 6), 1165-1171. Tam, A.S., Foley, J.F., Devereux, T.R., Maronpot, R.R., and Massey, T.E. (1999). High frequency and heterogenous distribution of p53 mutations in aflatoxin B1-induced mouse lung tumors. Cancer Res. 59, 3634-3640. NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft Cumene, NTP TR 542 L-7 Tchou, J., Kasai, H., Shibutani, S., Chung, M.H., Laval, J., Grollamn, A.P., and Nishimura, S. (1991). 8-oxoguanine (8-hydroxyguanine) DNA glycosylase and its substrate specificity. Proc. Natl. Acad. Sci. U.S.A. 88, 4690-4694. U.S. Environmental Protection Agency (USEPA) (1997). Toxicological Review of Cumene (CAS No. 98-82-8) in Support of Summary Information on the Integrated Risk Information System. Office of Research and Development, U.S. Environmental Protection Agency, Washington, DC. Valentine, J.L., Lee, S.S., Seaton, M.J., Asgharian, B., Farris, G., Corton, J.C., Gonzalez, F.J., and Medinsky, M.A. (1996). Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression. Toxicol. Appl. Pharmacol. 141, 205-213. Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION L-8 Cumene, NTP TR 542 TABLE L1 K-ras Mutations in Lung Neoplasms of B6C3F1 Mice in the 2-Year Inhalation Study of Cumenea Exposure Concentration (ppm) Activate K-ras Codon 12 Codon 13 Codon 61 (GGT) (GGC) (CAA) (GAT) (TGT) (GTT) (CGT) (CGA) (CAT) (CAC) (CTA) Historical controlsb 33/117 (28%) 14 5 10 Chamber controls 1/7 (14%) 00 00 Cumene total 45/52 (87%) 6 5 11 3 125 1/4 (25%) 01 00 250 10/13 (77%) 00 12 500 17/18 (94%) 41 60 1,000 17/17 (100%) 23 41 6 2410 0 1000 4 13 0 2 1 0 0000 0 5020 2 4000 2 4001 a b Only neoplasms greater than 1 mm in diameter were used; 125 ppm, females only; 1,000 ppm, males only. Historical incidences of spontaneous lung neoplasms in control B6C3F1 mice (Hong et al., 2007) TABLE L2 p53 Mutations in Lung Neoplasms of B6C3F1 Mice in the 2-Year Inhalation Study of Cumenea Exposure Concentration (ppm) Activate p53b IHC Positive Exon 5 Exon 7 Chamber controls Cumene total 125 250 500 1,000 0/7 27/52 (52%) 0/4 5/13 (38%) 11/18 (61%) 11/17 (65%) 1/7 (14%) 29/52 (56%) 1/4 (25%) 6/13 (46%) 8/18 (44%) 14/17 (82%) 0 24 0 4 10 10 a b Only neoplasms greater than 1 mm in diameter were used; 125 ppm, females only; 1,000 ppm, males only. No mutation detected for p53 at exons 6 and 8 in the samples examined 0 3 0 1 1 1 NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft