NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF trans-CINNAMALDEHYDE (MICROENCAPSULATED) (CAS NO. 14371-10-9) IN F344/N RATS AND B6C3F1 MICE (FEED STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 February 2004 NTP TR 514 NIH Publication No. 04-4448 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health FOREWORD The National Toxicology Program (NTP) is made up of four charter agencies of the U.S. Department of Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health; the National Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention. In July 1981, the Carcinogenesis Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the relevant programs, staff, and resources from these Public Health Service agencies relating to basic and applied research and to biological assay development and validation. The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous chemicals. This knowledge is used for protecting the health of the American people and for the primary prevention of disease. The studies described in this Technical Report were performed under the direction of the NIEHS and were conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use were in accordance with the Public Health Service Policy on Humane Care and Use of Animals. The prechronic and chronic studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice Regulations, and all aspects of the chronic studies were subjected to retrospective quality assurance audits before being presented for public review. These studies are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology and carcinogenesis studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. The interpretive conclusions presented in this Technical Report are based only on the results of these NTP studies. Extrapolation of these results to other species and quantitative risk analyses for humans require wider analyses beyond the purview of these studies. Selection per se is not an indicator of a chemical’s carcinogenic potential. Details about ongoing and completed NTP studies are available at the NTP’s World Wide Web site: http://ntp-server.niehs.nih.gov. Abstracts of all NTP Technical Reports and full versions of the most recent reports and other publications are available from the NIEHS’ Environmental Health Perspectives (EHP) http://ehp.niehs.nih.gov (866-541-3841 or 919-653-2590). In addition, printed copies of these reports are available from EHP as supplies last. A listing of all the NTP Technical Reports printed since 1982 appears at the end of this Technical Report. NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF trans-CINNAMALDEHYDE (MICROENCAPSULATED) (CAS NO. 14371-10-9) IN F344/N RATS AND B6C3F1 MICE (FEED STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 February 2004 NTP TR 514 NIH Publication No. 04-4448 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health 2 CONTRIBUTORS National Toxicology Program NTP Pathology Working Group Evaluated and interpreted results and reported findings Evaluated slides and prepared pathology report on rats and mice (March 1, 2001) M.J. Hooth, Ph.D., Study Scientist C. Picut, V.M.D., J.D., Chairperson R.C. Sills, D.V.M., Ph.D., Study Pathologist ILS, Inc. D.W. Bristol, Ph.D. A.E. Brix, D.V.M., Ph.D. J.R. Bucher, Ph.D. Experimental Pathology Laboratories, Inc. J.R. Hailey, D.V.M. R.A. Herbert, D.V.M., Ph.D. J.K. Haseman, Ph.D. National Toxicology Program R.A. Herbert, D.V.M., Ph.D. M.P. Jokinen, D.V.M. R.R. Maronpot, D.V.M. Pathology Associates International D.P. Orzech, M.S. G. Pearse, B.V.M.&S. S.D. Peddada, Ph.D. National Toxicology Program G.N. Rao, D.V.M., Ph.D. R.C. Sills, D.V.M., Ph.D. National Toxicology Program J.H. Roycroft, Ph.D. J.C. Wolf, D.V.M., Ph.D. C.S. Smith, Ph.D. Experimental Pathology Laboratories, Inc. G.S. Travlos, D.V.M. K.L. Witt, M.S., ILS, Inc. Analytical Sciences, Inc. Provided statistical analyses Battelle Columbus Operations Conducted studies and evaluated pathology findings P.W. Crockett, Ph.D., Principal Investigator L.J. Betz, M.S. M.R. Hejtmancik, Ph.D., Principal Investigator K.P. McGowan, M.B.A. G.B. Marit, D.V.M., 2-year mouse study J.T. Scott, M.S. J.D. Toft II, D.V.M., M.S., 3-month and 2-year rat studies J.T. Yarrington, D.V.M., Ph.D., 3-month mouse study Biotechnical Services, Inc. Prepared Technical Report Experimental Pathology Laboratories, Inc. Provided pathology quality assurance S.R. Gunnels, M.A., Principal Investigator M.P. Barker, B.A. J.F. Hardisty, D.V.M., Principal Investigator P.H. Carver, B.A. A.E. Brix, D.V.M., Ph.D. P.A. Gideon, B.A. J.C. Wolf, D.V.M., Ph.D. L.M. Harper, B.S. D.C. Serbus, Ph.D. Dynamac Corporation R.A. Willis, B.A., B.S. Prepared quality assurance audits S. Brecher, Ph.D., Principal Investigator 3 CONTENTS ABSTRACT . 5 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . 9 TECHNICAL REPORTS REVIEW SUBCOMMITTEE . 10 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . 11 INTRODUCTION . 13 MATERIALS AND METHODS . 25 RESULTS . 35 DISCUSSION AND CONCLUSIONS . 55 REFERENCES . 59 APPENDIX A Summary of Lesions in Male Rats in the 2-Year Feed Study of trans-Cinnamaldehyde . 67 APPENDIX B Summary of Lesions in Female Rats in the 2-Year Feed Study of trans-Cinnamaldehyde . 113 APPENDIX C Summary of Lesions in Male Mice in the 2-Year Feed Study of trans-Cinnamaldehyde . 151 APPENDIX D Summary of Lesions in Female Mice in the 2-Year Feed Study of trans-Cinnamaldehyde . 187 APPENDIX E Genetic Toxicology . 227 APPENDIX F Clinical Pathology Results . 239 APPENDIX G Hippuric Acid – Biomarker of Exposure . 247 APPENDIX H Organ Weights and Organ-Weight-to-Body-Weight Ratios . 251 APPENDIX I Chemical Characterization and Dose Formulation Studies . 255 APPENDIX J Feed and Compound Consumption in the 2-Year Feed Studies of trans-Cinnamaldehyde . 269 APPENDIX K Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration . 275 APPENDIX L Sentinel Animal Program . 279 4 trans-Cinnamaldehyde, NTP TR 514 SUMMARY Background trans-Cinnamaldehyde is used in foods, drinks, and cosmetics to give a cinnamon flavor and fragrance. We studied the effects of trans-cinnamaldehyde on male and female rats and mice to identify potential toxic or cancer­ related hazards to humans. Methods Because trans-cinnamaldehyde can evaporate easily, we enclosed it in starch microcapsules and placed them in the feed of rats and mice for two years. The doses given were 1,000, 2,100, or 4,100 parts per million (ppm) trans-cinnamaldehyde (equivalent to 0.1%, 0.21%, or 0.41%). Control animals received empty starch microcapsules in their feed. Tissues from more than 40 sites were examined for every animal. Results Rats receiving 4,100 ppm trans-cinnamaldehyde and mice receiving 2,100 or 4,100 ppm weighed less on average than the control animals, although they ate the same amount of feed. No more tumors or other toxic effects were observed in the groups of rats or mice given trans-cinnamaldehyde compared with the animals that were not. Mice receiving 4,100 ppm trans-cinnamaldehyde developed pigmentation of the olfactory epithelium of the nose. Conclusions We conclude that trans-cinnamaldehyde did not cause cancer in male or female rats or in male or female mice. 5 ABSTRACT O trans-CINNAMALDEHYDE CAS No. 14371-10-9 Chemical Formula: C9H8O Molecular Weight: 132.16 Synonyms: trans-Benzenepropenal; (E)-cinnamaldehyde; cinnamaldehyde; trans-cinnamic aldehyde; (E)-cinnamyl aldehyde; trans-cinnamylaldehyde; (E)-3-phenylacrolein; (E)-3-phenylpropenal; trans-3-phenyl-2-propenal; (E)-3-phenylprop-2-enal; (E)-3-phenyl-2-propenal; trans-3-phenylpropenal; 2-propenal, 3-phenyl-, (E)-; 2-propenal, 3-phenyl Cinnamaldehyde is used in foods, beverages, medical 3-MONTH STUDY IN RATS products, perfumes, cosmetics, soaps, detergents, Groups of 10 male and 10 female F344/N rats were fed creams, and lotions. Cinnamaldehyde has been used as diets containing 4,100, 8,200, 16,500, or 33,000 ppm a filtering agent and a rubber reinforcing agent and is microencapsulated trans-cinnamaldehyde (equivalent to used as a brightener in electroplating processes, as an average daily doses of approximately 275, 625, 1,300, or animal repellent, as an insect attractant, and as an anti­ 4,000 mg trans-cinnamaldehyde/kg body weight to fungal agent. trans-Cinnamaldehyde was nominated for males and 300, 570, 1,090, or 3,100 mg/kg to females) study by the Food and Drug Administration based on its for 3 months. Additional groups of 10 male and widespread use as a flavor and fragrance ingredient and 10 female rats received untreated feed (untreated con­ its structural similarity to cinnamyl anthranilate and trols) or feed containing placebo microcapsules (vehicle 3,4,5-trimethoxy cinnamaldehyde, two known rodent controls). All rats survived to the end of the study.