NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDY OF ETHINYL ESTRADIOL (CAS NO. 57-63-6) IN SPRAGUE-DAWLEY RATS (FEED STUDY) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 July 2010 NTP TR 548 NIH Publication No. 10-5889 National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES FOREWORD The National Toxicology Program (NTP) is an interagency program within the Public Health Service (PHS) of the Department of Health and Human Services (HHS) and is headquartered at the National Institute of Environmental Health Sciences of the National Institutes of Health (NIEHS/NIH). Three agencies contribute resources to the program: NIEHS/NIH, the National Institute for Occupational Safety and Health of the Centers for Disease Control and Prevention (NIOSH/CDC), and the National Center for Toxicological Research of the Food and Drug Administration (NCTR/FDA). Established in 1978, the NTP is charged with coordinating toxicological testing activities, strengthening the science base in toxicology, developing and validating improved testing methods, and providing information about potentially toxic substances to health regulatory and research agencies, scientific and medical communities, and the public. The Technical Report series began in 1976 with carcinogenesis studies conducted by the National Cancer Institute. In 1981, this bioassay program was transferred to the NTP. The studies described in the Technical Report series are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected substances in laboratory animals (usually two species, rats and mice). Substances selected for NTP toxicity and carcinogenicity studies are chosen primarily on the basis of human exposure, level of production, and chemical structure. The interpretive conclusions presented in NTP Technical Reports are based only on the results of these NTP studies. Extrapolation of these results to other species, including characterization of hazards and risks to humans, requires analyses beyond the intent of these reports. Selection per se is not an indicator of a substance’s carcinogenic potential. The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public review. NTP Technical Reports are indexed in the NIH/NLM PubMed database and are available free of charge electronically on the NTP website (http://ntp.niehs.nih.gov) or in hardcopy upon request from the NTP Central Data Management group at [email protected] or (919) 541-3419. NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDY OF ETHINYL ESTRADIOL (CAS NO. 57-63-6) IN SPRAGUE-DAWLEY RATS (FEED STUDY) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 July 2010 NTP TR 548 NIH Publication No. 10-5889 National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 2 CONTRIBUTORS The study on ethinyl estradiol was conducted at the FDA’s National Center for Toxicological Research under an interagency agreement between the FDA and the NIEHS. The study was designed and monitored by a Toxicology Study Selection and Review Committee composed of representatives from the NCTR and other FDA product centers, NIEHS, and other ad hoc members from other government agencies and academia. The interagency agreement was designed to use the staff and facilities of the NCTR in the testing of FDA priority chemicals and to provide FDA scientists and regulatory policymakers information for hazard identification and risk assessment. Toxicology Study Selection National Center for Toxicological Research, and Review Committee Food and Drug Administration Conducted studies, evaluated and interpreted results D.A. Casciano, Ph.D., Chairperson and pathology findings, and reported findings National Center for Toxicological Research W.T. Allaben, Ph.D. K.B. Delclos, Ph.D., Study Scientist National Center for Toxicological Research R.R. Newbold, M.S., Co-Study Scientist F.A. Beland, Ph.D. National Institute of Environmental Health Sciences National Center for Toxicological Research S.A. Ferguson, Ph.D., Co-Study Scientist J.R. Bucher, Ph.D. A.C. Scallet, Ph.D., Co-Study Scientist National Institute of Environmental Health Sciences C.C. Weis, B.S., Study Coordinator K.J. Greenlees, Ph.D. W.T. Allaben, Ph.D. Center for Veterinary Medicine, J.R. Appleget, B.S. Food and Drug Administration F.A. Beland, Ph.D. R.J. Lorentzen, Ph.D. W. Campbell, B.S. Center for Food Safety and Applied Nutrition, M.I. Churchwell, B.S. Food and Drug Administration M. Miller, Ph.D. D.R. Doerge, Ph.D. Office of Women’s Health, K.M. Flynn, Ph.D. Food and Drug Administration J.M. Meredith, Ph.D. R.R. Newbold, M.S. J.M. Reed, M.S. National Institute of Environmental Health Sciences W.M. Witt, D.V.M., Ph.D. F.D. Sistare, Ph.D. Center for Drug Evaluation and Research, Conducted chemical analyses of the feed Food and Drug Administration and purity of the test chemical E. Yetley, Ph.D. Center for Food Safety and Applied Nutrition, S.M. Billedeau, B.S. Food and Drug Administration W.M. Cooper, B.S. F.E. Evans, Ph.D. Bionetics R.L. Evans, M.S. Prepared animal feed and cared for rats J.P. Freeman, Ph.D. T.M. Heinze, M.S. J. Carson, B.S. C.L. Holder, B.S. C. Hotchkiss, D.V.M., Ph.D. P.H. Siitonen, B.S. A. Matson, B.S. M. Vanlandingham Conducted quality assurance audits S.J. Culp, Ph.D. R.D. Smith, B.S. Y.E. Whiteside, B.S. Ethinyl Estradiol, NTP TR 548 3 Pathology Associates Z-Tech Corp. A Charles River Company Provided statistical analyses and software systems development Evaluated pathology findings K. Carroll T.J. Bucci, V.M.D., Ph.D. W.A. McCracken, M.S. P.W. Mellick, D.V.M., Ph.D. B.T. Thorn, M.S. G.R. Olson, D.V.M., Ph.D. J. Wulff, M.S. NTP Pathology Working Group Biotechnical Services, Inc. Evaluated slides and contributed to pathology report on rats Prepared Technical Report (April 20-21, 2005) S.R. Gunnels, M.A., Principal Investigator C.C. Shackelford, D.V.M., M.S., Ph.D., Coordinator B.F. Hall, M.S. Experimental Pathology Laboratories, Inc. L.M. Harper, B.S. C.H. Frith, D.V.M., Ph.D. E.S. Rathman, M.S. Tox-Path Associates (retired) J.R. Latendresse, D.V.M., Ph.D. P. Rathman, B.S. National Center for Toxicological Research D.C. Serbus, Ph.D. P.W. Mellick, D.V.M., Ph.D. National Center for Toxicological Research R.A. Miller, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. G.R. Olson, D.V.M., Ph.D. National Center for Toxicological Research G. Pearse, B.V.M.&S. National Institute of Environmental Health Sciences M.J. Van Zwieten, D.V.M., Ph.D. Merck (retired) G.A. Willson, B.V.M.S. Experimental Pathology Laboratories, Inc. 4 CONTENTS ABSTRACT . 7 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . 11 TECHNICAL REPORTS REVIEW SUBCOMMITTEE . 12 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . 13 OVERVIEW . 15 INTRODUCTION . 19 MATERIALS AND METHODS . 25 RESULTS . 35 DISCUSSION AND CONCLUSIONS . 53 REFERENCES . 57 APPENDIX A Summary of Lesions in Male Rats in the 2-Year Feed Study of Ethinyl Estradiol . 63 APPENDIX B Summary of Lesions in Female Rats in the 2-Year Feed Study of Ethinyl Estradiol . 109 APPENDIX C Chemical Characterization and Dose Formulation Studies . 145 APPENDIX D Body Weights . 157 APPENDIX E Feed Consumption . 171 APPENDIX F Onset of Aberrant Estrous Cycles . 185 APPENDIX G Organ Weights and Organ-Weight-to-Body-Weight Ratios . 189 APPENDIX H Ingredients, Nutrient Composition, and Contaminant Levels in Purina 5K96 Rat Ration . 205 APPENDIX I Sentinel Animal Program . 209 Ethinyl Estradiol, NTP TR 548 5 SUMMARY Background Ethinyl estradiol is a potent synthetic estrogen that is widely prescribed in oral contraceptives and is also used in the treatment of breast and prostate cancer. Ethinyl estradiol is one of a class of chemicals known as “environmental estrogens” that can affect the hormone activities and possibly reproductive function of wildlife and humans through exposure. The NTP conducted a series of studies on three such chemicals to detect if exposure over the course of multiple generations could have any cumulative effect on animals’ reproductive systems or development of cancers. This report describes the results of a set of studies in which rats were exposed to ethinyl estradiol for part or all of the study period and examined at the end of two years. Methods The study consisted of three separate study components; in each, animals were exposed to ethinyl estradiol from the time of conception and through weaning through their mothers, who were given ethinyl estradiol in their feed. In one study we gave feed containing 2, 10, or 50 parts per billion (ppb) of ethinyl estradiol to groups of 50 male and female rats from conception through two years. In the second study, groups of 50 male and female rats were given the same feed concentrations up to 20 weeks following birth, followed by untreated feed for the remainder of the two years. In the third study groups of 50 male and female rats were exposed from conception through weaning, and then given untreated feed for the duration of the study. Control animals received the same feed with no ethinyl estradiol added. Enthinyl estradiol is known to cause cancer at higher dose levels; the concentrations given in this study were below the levels of detection by chemical analysis, to determine the possible effects of trace amounts in the environment.