NTP TECHNICAL REPORT ON THE TOXICOLOGY STUDIES OF INDOLE-3-CARBINOL (CASRN 700-06-1) IN F344/N RATS AND B6C3F1/N MICE AND TOXICOLOGY AND CARCINOGENESIS STUDIES OF INDOLE-3-CARBINOL IN HARLAN SPRAGUE DAWLEY RATS AND B6C3F1/N MICE (GAVAGE STUDIES) NTP TR 584 JULY 2017 NTP Technical Report on the Toxicology Studies of Indole-3-carbinol (CASRN 700-06-1) in F344/N Rats and B6C3F1/N Mice and Toxicology and Carcinogenesis Studies of Indole-3-carbinol in Harlan Sprague Dawley Rats and B6C3F1/N Mice (Gavage Studies) Technical Report 584 July 2017 National Toxicology Program Public Health Service U.S. Department of Health and Human Services ISSN: 2378-8925 Research Triangle Park, North Carolina, USA Indole-3-carbinol, NTP TR 584 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, 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 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. 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. The NTP Technical Reports are available free of charge on the NTP website and cataloged in PubMed, a free resource developed and maintained by the National Library of Medicine (part of the National Institutes of Health). Data for these studies are included in NTP’s Chemical Effects in Biological Systems database. For questions about the reports and studies, please email NTP or call 984-287-3211. ii Indole-3-carbinol, NTP TR 584 Table of Contents Foreword.................................................................................................................................... ii Tables ........................................................................................................................................ vi Figures ......................................................................................................................................vii About This Report ...................................................................................................................... ix Explanation of Levels of Evidence of Carcinogenic Activity .....................................................xii Peer Review ............................................................................................................................. xiv Publication Details .................................................................................................................... xv Abstract ................................................................................................................................... xvi Three-month Study in F344/N Rats ..................................................................................... xvi Three-month Study in B6C3F1/N Mice ............................................................................... xvi Two-year Study in Sprague Dawley Rats ............................................................................ xvii Two-year Study in B6C3F1/N Mice ................................................................................... xvii Genetic Toxicology ...........................................................................................................xviii Conclusions .......................................................................................................................xviii Introduction ................................................................................................................................ 1 Chemical and Physical Properties ........................................................................................... 1 Production, Use, and Human Exposure ................................................................................... 1 Regulatory Status ................................................................................................................... 2 Absorption, Distribution, Metabolism, and Excretion ............................................................. 2 Experimental Animals ..................................................................................................... 2 Humans ........................................................................................................................... 3 Toxicity .................................................................................................................................. 3 Experimental Animals ..................................................................................................... 3 Humans ........................................................................................................................... 5 Reproductive and Developmental Toxicity ............................................................................. 6 Experimental Animals ..................................................................................................... 6 Humans ........................................................................................................................... 6 Carcinogenicity ...................................................................................................................... 6 Experimental Animals ..................................................................................................... 6 Humans ........................................................................................................................... 7 Genetic Toxicity ..................................................................................................................... 7 Study Rationale ...................................................................................................................... 8 Materials and Methods ................................................................................................................ 9 Procurement and Characterization .......................................................................................... 9 Indole-3-carbinol ............................................................................................................. 9 Corn Oil .......................................................................................................................... 9 Preparation and Analysis of Dose Formulations .................................................................... 10 Animal Source ...................................................................................................................... 10 Animal Welfare .................................................................................................................... 10 Three-month Studies ............................................................................................................ 11 iii Indole-3-carbinol, NTP TR 584 Two-year Studies .................................................................................................................. 13 Study Design ................................................................................................................. 13 Clinical Examinations and Pathology............................................................................. 13 Statistical Methods ............................................................................................................... 18 Survival Analyses .......................................................................................................... 18 Calculation of Incidence ................................................................................................ 18 Analysis of Neoplasm and Nonneoplastic Lesion