NTP REPORT ON THE TOXICOLOGY STUDIES OF DICHLOROACETIC ACID (CAS NO. 79-43-6) IN GENETICALLY MODIFIED (FVB Tg.AC HEMIZYGOUS) MICE (DERMAL AND DRINKING WATER STUDIES) AND CARCINOGENICITY STUDIES OF DICHLOROACETIC ACID IN GENETICALLY MODIFIED [B6.129-Trp53tm1Brd (N5) HAPLOINSUFFICIENT] MICE (DRINKING WATER STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 April 2007 NTP GMM 11 NIH Publication No. 07-4428 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 Genetically Modified Model (GMM) Report series began in 2005 with studies conducted by the NTP. The studies described in the GMM Report series are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity, of selected agents in laboratory animals that have been genetically modified. These genetic modifications may involve inactivation of selected tumor suppressor functions or activation of oncogenes that are commonly observed in human cancers. This may result in a rapid onset of cancer in the genetically modified animal when exposure is to agents that act directly or indirectly on the affected pathway. An absence of a carcinogenic response may reflect either an absence of carcinogenic potential of the agent or that the selected model does not harbor the appropriate genetic modification to reduce tumor latency and allow detection of carcinogenic activity under the conditions of these subchronic studies. 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 GMM 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 GMM 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 REPORT ON THE TOXICOLOGY STUDIES OF DICHLOROACETIC ACID (CAS NO. 79-43-6) IN GENETICALLY MODIFIED (FVB Tg.AC HEMIZYGOUS) MICE (DERMAL AND DRINKING WATER STUDIES) AND CARCINOGENICITY STUDIES OF DICHLOROACETIC ACID IN GENETICALLY MODIFIED [B6.129-Trp53tm1Brd (N5) HAPLOINSUFFICIENT] MICE (DRINKING WATER STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 April 2007 NTP GMM 11 NIH Publication No. 07-4428 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 on mice (July 16, 2002) G.A. Boorman, Ph.D., Study Scientist S.D. Rousselle, D.V.M., Chairperson R.A. Herbert, D.V.M., Ph.D., Study Pathologist Pathology Associates, A Charles River Company D.W. Bristol, Ph.D. K.J. Cimon, D.V.M., M.S. J.R. Bucher, Ph.D. Experimental Pathology Laboratories, Inc. L.T. Burka, Ph.D. R.A. Herbert, D.V.M., Ph.D. R.S. Chhabra, Ph.D. National Toxicology Program J.E. French, Ph.D. P.C. Howroyd, M.A., VetMB J.R. Hailey, D.V.M. Experimental Pathology Laboratories, Inc. G.E. Kissling, Ph.D. J. Mahler, D.V.M. A.P. King-Herbert, D.V.M. National Toxicology Program D.E. Malarkey, D.V.M., Ph.D. G. Pearse, B.V.M.&S. R.R. Maronpot, D.V.M. National Toxicology Program J.C. Peckham, D.V.M., M.S., Ph.D. M.K. Vallant, B.S., M.T. Experimental Pathology Laboratories, Inc. S.D. Peddada, Ph.D. D.C. Wolf, D.V.M., Ph.D. C.S. Smith, Ph.D. United States Environmental Protection Agency G.S. Travlos, D.V.M. K.L. Witt, M.S. Constella Group, Inc. Provided statistical analyses Battelle Columbus Operations Principal Investigator Conducted studies and evaluated pathology findings P.W. Crockett, Ph.D., L.J. Betz, M.S. M.R. Hejtmancik, Ph.D., Principal Investigator K.P. McGowan, M.B.A. J.D. Toff, II, D.V.M., M.S. Biotechnical Services, Inc. Experimental Pathology Laboratories, Inc. Prepared Report Provided pathology review S.R. Gunnels, M.A., Principal Investigator M.H. Hamlin, II, D.V.M., Principal Investigator L.M. Harper, B.S. K.J. Cimon, D.V.M., M.S. J.I. Powers, M.A.P. P.C. Howroyd, M.A., VetMB D.C. Serbus, Ph.D. J.C. Peckham, D.V.M., M.S., Ph.D. Dynamac Corporation Prepared quality assurance audits S. Brecher, Ph.D., Principal Investigator 3 CONTENTS ABSTRACT . 5 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . 11 TECHNICAL REPORTS REVIEW SUBCOMMITTEE . 12 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . 13 INTRODUCTION . 15 MATERIALS AND METHODS . 21 RESULTS . 31 DISCUSSION AND CONCLUSIONS . 67 REFERENCES . 71 APPENDIX A Summary of Lesions in Tg.AC Hemizygous Mice in the Dermal Studies of Dichloroacetic Acid . 77 APPENDIX B Summary of Lesions in Tg.AC Hemizygous Mice in the Drinking Water Studies of Dichloroacetic Acid . 95 APPENDIX C Summary of Lesions in p53 Haploinsufficient Mice in the Drinking Water Studies of Dichloroacetic Acid . 113 APPENDIX D Genetic Toxicology . 129 APPENDIX E Hematology Results . 137 APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios . 141 APPENDIX G Chemical Characterization and Dose Formulation Studies . 149 APPENDIX H Water and Compound Consumption in the 26- and 41-Week Drinking Water Studies of Dichloroacetic Acid . 159 4 Dichloroacetic Acid, NTP GMM 11 SUMMARY Background Dichloroacetic acid is a by-product of water disinfection. We tested if dichloroacetic acid could cause cancer in two different strains of genetically modified mice. Methods We applied solutions containing dichloroacetic acid to the backs of male and female Tg.AC mice for 6 or 9 months and gave dichloroacetic acid dissolved in drinking water to male and female Tg.AC hemizygous mice and to male and female p53 haploinsufficient mice for 6 or 10 months. Animals given plain water served as the control groups. Tissues from 18 sites were examined for every animal. Results Male and female Tg.AC mice with dichloroacetic acid applied to their backs had greatly increased rates of epidermal hyperkeratosis at the site where the chemical was applied, and Tg.AC mice receiving the highest concentrations (0.5 grams per kilogram of body weight each day) developed squamous cell papillomas. In Tg.AC mice receiving dichloroacetic acid in the drinking water, a few alveolar/bronchiolar tumors of the lung were seen in exposed animals in each of the four studies. There were no increases in cancers in the p53 haploinsufficient mice given dichloroacetic acid in the drinking water. Conclusions We conclude that dichloroacetic acid did not cause cancer in the genetically modified p53 haploinsufficient mice and gave only weakly positive responses when given to Tg.AC mice. Dichloroacetic acid did cause cancer in several other long-term studies with different rodents, and thus we conclude these genetically modified mice may not be as sensitive for detecting certain cancer-causing compounds. 5 ABSTRACT O Cl OH Cl DICHLOROACETIC ACID CAS No. 79-43-6 Chemical Formula: C2H2Cl2O2 Molecular Weight: 128.9426 Synonyms: Acetic acid, dichloro; bichloracetic acid; DCA; dichlorethanoic acid; 2,2-dichloroacetic acid; dichloroethanoic acid; kyselina dichloroctova; Urner’s liquid Dichloroacetic acid was nominated for study by the was tested for potential toxicity and carcinogenicity in United States Environmental Protection Agency and by two relatively well-studied models, the Tg.AC hemizy­ the National Institute of Environmental Health Sciences gous strain and the p53 haploinsufficient strain. Male because of its widespread occurrence in drinking water and female Tg.AC hemizygous and p53 haploinsuffi­ as a by-product of water disinfection using chlorination. cient mice were exposed to dichloroacetic acid in the It was also nominated because dichloroacetic acid is the drinking water (greater than 98% pure) for 26 or most studied representative of the class of haloacetic 41 weeks. In addition, male and female Tg.AC hemizy­ acids and has been shown to cause liver tumors in both gous mice were exposed by dermal application for 26 or rats and mice. Haloacetic acids are second only to tri­ 39 weeks. Genetic toxicology studies were conducted in halomethanes as a family of disinfection by-products Salmonella typhimurium strains TA98, TA100, and found in many drinking water supplies.