NATIONAL TOXICOLOGY PROGRAM Toxicity Report Series Number 62 NTP Technical Report on the Toxicity Studies of Wy-14,643 (CAS No. 50892-23-4) Administered in Feed to Sprague-Dawley Rats, B6C3F Mice, 1 and Syrian Hamsters October, 2007 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 Toxicity Study Report series began in 1991. The studies described in the Toxicity Study Report series are designed and conducted to characterize and evaluate the toxicologic potential of selected substances in laboratory animals (usually two species, rats and mice). Substances selected for NTP toxicity studies are chosen primarily on the basis of human exposure, level of production, and chemical structure. The interpretive conclusions presented in the Toxicity Study 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 toxic 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 Toxicity Study 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. NATIONAL TOXICOLOGY PROGRAM Toxicity Report Series Number 62 NTP Technical Report on the Toxicity Studies of Wy-14,643 (CAS No. 50892-23-4) Administered in Feed to Sprague-Dawley Rats, B6C3F Mice, 1 and Syrian Hamsters Michael L. Cunningham, Ph.D., Study Scientist National Toxicology Program Post Office Box 12233 Research Triangle Park, NC 27709 NIH Publication No. 08-4419 National Institutes of Health Public Health Service U.S. Department of Health and Human Services 2 CONTRIBUTORS National Toxicology Program NTP Pathology Working Group Evaluated and interpreted results and reported findings Evaluated slides and prepared pathology report (September 24, 1998) M.L. Cunningham, Ph.D., Study Scientist J.R. Bucher, Ph.D. M.P. Jokinen, D.V.M., Chairperson L.T. Burka, Ph.D. Pathology Associates International R.S. Chhabra, Ph.D. K.J. Brenneman, D.V.M., Observer A.P. King-Herbert, D.V.M. North Carolina State University G.E. Kissling, Ph.D. S. Botts, M.S., D.V.M., Ph.D. D.E. Malarkey, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. J. Mahler, D.V.M. J. Everitt, D.V.M. D.P. Orzech, M.S. Chemical Industry Institute of Toxicology C.S. Smith, Ph.D. R.A. Herbert, D.V.M., Ph.D. G.S. Travlos, D.V.M. National Toxicology Program M.K. Vallant, B.S., M.T. J. Mahler, D.V.M. K.L. Witt, M.S. National Toxicology Program A. Nyska, D.V.M. Battelle Columbus Laboratories National Toxicology Program Conducted studies and evaluated pathology findings Evaluated slides and prepared special pathology report M.R. Hejtmancik, Ph.D., Principal Investigator (January 25, 2005) P.J. Kurtz, Ph.D., Principal Investigator L.R. Goodchild, D.V.M. J.C. Turnier, V.M.D., Chairperson A.G. Manus Pathology Associates, A Charles River Company P.W. Mellick, D.V.M., Ph.D. J.R. Hailey, D.V.M. T.A. Peace, D.V.M. National Toxicology Program A.W. Singer, D.V.M. R.A. Herbert, D.V.M., Ph.D. J.T. Yarrington, D.V.M., Ph.D. National Toxicology Program D.E. Malarkey, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. National Toxicology Program Provided pathology review R.R. Maronpot, D.V.M. J.F. Hardisty, D.V.M., Principal Investigator National Toxicology Program G. Pearse, B.V.M. & S. S. Botts, M.S., D.V.M., Ph.D. G.E. Marrs, Jr., D.V.M., M.S. Experimental Pathology Laboratories, Inc. G. Pearse, B.V.M. & S. Dynamac Corporation Environmental Health Research Prepared quality assurance audits and Testing, Inc. S. Brecher, Ph.D., Principal Investigator Provided sperm motility evaluations Biotechnical Services, Inc. T. Cocanougher, B.A. Prepared Toxicity Study Report D.K. Gulati, Ph.D. S. Russell, B.A. S.R. Gunnels, M.A., Principal Investigator M.P. Barker, B.A. Constella Group, Inc. B.F. Hall, M.S. Provided statistical analyses L.M. Harper, B.S. D.C. Serbus, Ph.D. P.W. Crockett, Ph.D., Principal Investigator W.D. Sharp, B.A., B.S. L.J. Betz, M.S. R.A. Willis, B.A., B.S. K.P. McGowan, M.B.A. J.T. Scott, M.S. 3 PEER REVIEW The draft report on the toxicity studies of Wy-14,643 was evaluated by the reviewers listed below. These reviewers serve as independent scientists, not as representatives of any institution, company, or governmental agency. In this capacity, reviewers determine if the design and conditions of these NTP studies are appropriate and ensure that this Toxicity Study Report presents the experimental results and conclusions fully and clearly. Prescott L. Deininger, Ph.D. Jack Vanden Heuvel, B.S., Ph.D. Tulane University Medical Center Department of Veterinary and Biomedical Sciences New Orleans, LA Center for Molecular Toxicology and Carcinogenesis Pennsylvania State University University Park, PA 4 CONTENTS ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Genetic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 NIEHS Extramural Mechanistic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Study Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Procurement and Characterization of Wy-14,643 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Preparation and Analysis of Dose Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2-Week Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3-Month Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Statistical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Quality Assurance Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Hamsters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Toxicokinetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 APPENDIXES Appendix A Summary of Nonneoplastic Lesions in Rats, Mice, and Hamsters . . . . . . . . . . . . . . . . . A-1 Appendix B Clinical Pathology Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1 Appendix C Determinations of Wy-14,643 in Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1 Appendix D Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . . . . . . . . . . . D-1 Appendix E Reproductive Tissue Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1 Appendix F Cell Proliferation Indices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1 Appendix G Peroxisomal Enzyme Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1 Appendix H Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H-1 Appendix I Chemical Characterization and Dose Formulation Studies . . . . . . . . . . . . . . . . . . . . . . . I-1 Appendix J Toxicokinetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-1 Appendix K NIEHS Extramural Mechanistic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-1 Wy-14,643, NTP TOX 62 5 SUMMARY Background Wyeth-14,643 is a chemical that was developed by the pharmaceutical industry to lower serum cholesterol. It is not used in clinical applications. We studied the effects of Wyeth-14,643 on rats, mice, and hamsters because it was known that this chemical promotes the production of peroxisomes, organelles that contain a variety of enzymes involved in metabolism of lipids and cholesterol. Methods We gave groups of male rats, mice, and hamsters Wyeth-14,643 mixed in their food for three months. In each species, groups of 25 animals received either 5, 10, 50, 100, or 500 parts per million (ppm) of Wyeth-14,643 in feed. Other groups receiving undosed feed served as the control groups. Tissues from 35 sites were examined for each animal and measures of sperm motility were performed. Results All the animals survived until the end of the studies. All of the animal groups exposed to the chemical, except the groups receiving 5 ppm and the mice receiving 10 ppm, had lower body weights than their control groups although the feed consumption was generally similar in the various groups of each species. However, the liver weights of rats, mice, and hamsters fed Wyeth-14,643 were generally greater than those of the controls and liver foci were observed in three 100 ppm mice and one 500 ppm mouse. In all groups of animals exposed to Wyeth-14,643, there were significant increases in cytoplasmic alteration of the liver. In the examination of sperm motility, the weights of the cauda epididymis were decreased in all three species of rodents receiving 500 ppm, and in hamsters the testis weights and spermatid counts were decreased in all dosed groups. Conclusions Exposure to Wyeth-14,643 caused several changes in the livers of male rats, mice, and hamsters, including increased liver weights, increases in cytoplasmic alteration of the liver, and some liver foci. Wyeth-14,643 also had effects on the testes of exposed male rodents, decreasing the spermatid counts and the weights of the cauda epididymis. 6 Wy-14,643, NTP TOX 62 7 ABSTRACT Cl H N N N CH CH S CH COOH 3 3 2 WY-14,643 CAS No. 50892-23-4 Chemical Formula: C H ClN OS Molecular Weight: 323.79 14 14 3 2 Synonym: [4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid Wy-14,643 was selected for inclusion in a series of studies on peroxisome proliferators because it is known to produce considerable peroxisome proliferation and hepatocarcinogenicity in rats. Male Sprague-Dawley rats were exposed to Wy-14,643 (greater than 98% pure) in feed for up to 3 months; male B6C3F mice and male Syrian 1 hamsters were exposed to Wy-14,643 in feed for 2 weeks or up to 3 months. Animals were evaluated for clinical pathology, plasma concentrations of Wy-14,643, reproductive system effects, cell proliferation and peroxisomal enzyme analyses, and histopathology. Single and multiple-dose toxicokinetic studies of Wy-14,643 were conducted in additional groups of male Sprague-Dawley and Wistar Furth rats, B6C3F mice, and Syrian hamsters. Genetic 1 toxicology studies were conducted in vivo in Tg.AC mouse peripheral blood erythrocytes. In the 2-week studies, groups of five mice were fed diets containing 0, 10, 50, 100, 500, or 1,000 ppm Wy-14,643 (equivalent to average daily doses of approximately 2 to 184 mg Wy-14,643/kg body weight). Groups of five hamsters were fed diets containing 0, 10, 100, 500, 1,000, or 5,000 ppm Wy-14,643 (equivalent to average daily doses of approximately 1 to 550 mg/kg). All animals survived to the end of the studies. The mean body weight gain of 500 ppm mice was significantly less than that of the controls; hamsters exposed to 100 ppm or greater lost weight during the study. Feed consumption by 500 ppm mice was greater than that by the controls. Liver weights of all exposed groups of mice and hamsters were generally significantly increased. 8 Wy-14,643, NTP TOX 62 In the 2-week studies, an increase in peroxisomal enzyme activity occurred in 10 ppm mice; increases in peroxisomal $-oxidation, carnitine acetyltransferase, catalase, and acyl CoA oxidase occurred in all exposed mice compared to controls. Significantly increased BrdU-labeled hepatocyte percentages occurred in 100 and 1,000 ppm mice and 500 and 5,000 ppm hamsters; peroxisomal $-oxidation of lipids was increased in all exposed groups of mice and hamsters. Gross lesions in the 2-week studies included liver foci in one 500 ppm mouse and one 1,000 ppm hamster and enlarged livers in one hamster in each of the 100 and 500 ppm groups and two 5,000 ppm hamsters. All 500 and 1,000 ppm mice had hepatocyte hypertrophy of the liver, and 1,000 ppm mice also had widespread individual cell necrosis. Minimal to mild multifocal vacuolation of the liver occurred in hamsters exposed to 500 ppm or greater. In the 3-month core studies, groups of 10 male rats, mice, or hamsters were fed diets containing 0, 5, 10, 50, 100, or 500 ppm Wy-14,643 (equivalent to average daily doses of approximately 0.3 to 34 mg/kg for rats, 0.9 to 135 mg/kg for mice, and 0.4 to 42 mg/kg for hamsters). Groups of 15 male rats, mice, or hamsters designated for special studies received the same concentrations of Wy-14,643 for up to 13 weeks. Groups of six male rats, 36 male mice, or 12 male hamsters designated for plasma concentration studies were fed diets containing 50, 100, or 500 ppm Wy-14,643 for up to 9 weeks. All core study animals survived to the end of the studies. Mean body weights were significantly decreased in all exposed groups except the 5 ppm groups and 10 ppm mice; hamsters in the 100 and 500 ppm groups lost weight during the study. Feed consumption by exposed rats and mice was generally similar to that by the controls; during week 14, hamsters exposed to 50 ppm or greater consumed slightly less feed than did the controls. The only clinical finding of toxicity was thinness of two 50 ppm and five 500 ppm hamsters. At all time points, the liver weights of exposed groups of core and special study rats, mice, and hamsters were generally significantly greater than those of the controls. Testis weights were significantly decreased in 500 ppm hamsters on day 34, in hamsters exposed to 5 ppm or greater at week 13 (special study), and in 100 and 500 ppm core study hamsters at the end of the study. In the sperm motility evaluation, the cauda epididymis weight of 500 ppm rats, epididymis weights of 100 and 500 ppm rats and mice, and the testis weight of 500 ppm mice were significantly less than those of the controls. For hamsters, cauda epididymis, epididymis, and testis weights; spermatid heads per testis; and spermatid counts were significantly decreased in all exposed groups evaluated for sperm motility. Epididymal spermatozoal motility and concentration in the 100 and 500 ppm groups and spermatid heads per gram testis in the 500 ppm group were also significantly decreased. Serum concentrations of estradiol were significantly decreased in all exposed groups of hamsters, and concentrations of testosterone and luteinizing hormone were decreased in groups exposed to 50 ppm or greater.