NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF THEOPHYLLINE (CAS NO. 58-55-9) IN F344/N RATS AND B6C3F1 MICE (FEED AND GAVAGE STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 August 1998 NTP TR 473 NIH Publication No. 98-3963 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. 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. Listings of all published NTP reports and ongoing studies are available from NTP Central Data Management, NIEHS, P.O. Box 12233, MD E1-02, Research Triangle Park, NC 27709 (919-541-3419). The Abstracts and other study information for 2-year studies are also available at the NTP’s World Wide Web site: http://ntp-server.niehs.nih.gov. NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF THEOPHYLLINE (CAS NO. 58-55-9) IN F344/N RATS AND B6C3F1 MICE (FEED AND GAVAGE STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 August 1998 NTP TR 473 NIH Publication No. 98-3963 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health 2 Theophylline, NTP TR 473 CONTRIBUTORS National Toxicology Program NTP Pathology Working Group Evaluated and interpreted results and reported findings Evaluated slides, prepared pathology report on rats (28 March 1996) P.C. Chan, Ph.D., Study Scientist D.A. Bridge, B.S. P.K. Hildebrandt, D.V.M., Chairperson J.R. Bucher, Ph.D. PATHCO, Inc. E.T. Gaillard, M.S., D.V.M. R.E. Chapin, Ph.D. Experimental Pathology Laboratories, Inc. J.R. Hailey, D.V.M. J.R. Hailey, D.V.M. J.K. Haseman, Ph.D. National Toxicology Program R.R. Maronpot, D.V.M. R.A. Herbert, D.V.M., Ph.D. A. Nyska, D.V.M. National Toxicology Program G.N. Rao, D.V.M., Ph.D. E.E. McConnell, D.V.M. Experimental Pathology Laboratories, Inc. J.H. Roycroft, Ph.D. A. Nyska, D.V.M. C.S. Smith, Ph.D. National Toxicology Program G.S. Travlos, D.V.M. A. Radovsky, D.V.M., Ph.D. D.B. Walters, Ph.D. National Toxicology Program K.L. Witt, M.S., Oak Ridge Associated Universities Evaluated slides, prepared pathology report on mice (28 March 1996) Southern Research Institute Conducted studies, evaluated pathology findings P.K. Hildebrandt, D.V.M., Chairperson PATHCO, Inc. J.D. Prejean, Ph.D., Principal Investigator (all studies) E.T. Gaillard, M.S., D.V.M. D.G. Serota, Ph.D., Principal Investigator (2-year studies) Experimental Pathology Laboratories, Inc. D.R. Farnell, D.V.M., Ph.D. R.A. Herbert, D.V.M., Ph.D. J.E. Heath, D.V.M. National Toxicology Program E.E. McConnell, D.V.M. C. Lindamood III, Ph.D. Experimental Pathology Laboratories, Inc. T. Makovec, D.V.M. A. Nyska, D.V.M. A.G. Manus National Toxicology Program J. Page A. Radovsky, D.V.M., Ph.D. R.B. Thompson, D.V.M., Ph.D. National Toxicology Program Experimental Pathology Laboratories, Inc. Analytical Sciences, Inc. Provided pathology quality assurance Provided statistical analyses J.F. Hardisty, D.V.M., Principal Investigator R.W. Morris, M.S., Principal Investigator E.T. Gaillard, M.S., D.V.M. S. Lloyd, M.S. E.E. McConnell, D.V.M. N.G. Mintz, B.S. Dynamac Corporation Biotechnical Services, Inc. Prepared quality assurance audits Prepared Technical Report S. Brecher, Ph.D., Principal Investigator S.R. Gunnels, M.A., Principal Investigator J.R. Dias, M.S. L.M. Harper, B.S. A.M. Macri, M.A., M.F.A. 3 CONTENTS ABSTRACT ............................................................. 5 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ............. 10 TECHNICAL REPORTS REVIEW SUBCOMMITTEE ................................ 11 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS .......... 12 INTRODUCTION ......................................................... 13 MATERIALS AND METHODS ................................................ 21 RESULTS ............................................................... 39 DISCUSSION AND CONCLUSIONS ............................................ 75 REFERENCES ........................................................... 81 APPENDIX A Summary of Lesions in Male Rats in the 2-Year Gavage Study of Theophylline ................................................ 89 APPENDIX B Summary of Lesions in Female Rats in the 2-Year Gavage Study of Theophylline ................................................ 125 APPENDIX C Summary of Lesions in Male Mice in the 2-Year Gavage Study of Theophylline ................................................ 157 APPENDIX D Summary of Lesions in Female Mice in the 2-Year Gavage Study of Theophylline ................................................ 187 APPENDIX E Genetic Toxicology ............................................. 225 APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios .................. 237 APPENDIX G Hematology Results ............................................. 257 APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization ........... 269 APPENDIX I Chemical Characterization and Dose Formulation Studies ................... 275 APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels in NIH-07 Rat and Mouse Ration ................................... 295 APPENDIX K Sentinel Animal Program ......................................... 299 APPENDIX L Impact of Helicobacter hepaticus Infection in B6C3F1 Mice from 12 NTP 2-Year Carcinogenesis Studies ............................ 305 4 Theophylline, NTP TR 473 5 ABSTRACT O CH3 H N N O N N CH3 THEOPHYLLINE CAS No. 58-55-9 Chemical Formula: C7 H 8N 4O 2 Molecular Weight: 180.17 Synonyms: 3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione; 1,3-dimethylxanthine; 1H-purine-2,6-dione; NSC 2066; pseudotheophylline; theocin; theophylline, anhydrous Trade names: Accurbron; Aerobin; Aerolate III; Afonilum; Aminophylline; Aquaphyllin; Armophylline; Asmalix; Bilordyl; Bronchoretard; Bronkodyl; Cetraphylline; Constant-T; Diffumal; Duraphyl; Duraphyllin; Elixicon; Elixophyllin; Euphylline L.A.; Euphylong; LaBID; Labophylline; Lanophyllin; Lasma; Liquophylline; Optiphyllin; Parkophyllin; Phylocontin; Physpan; Pro-Vent; PulmiDur; Pulmo-Timelets; Quibron; Respbid; Rona-Phyllin; Sabidal; Slo-bid; Slo-Phyllin; Solosin; Sustaire; Tefamin; Teobid; Teofyllamin; Tesona; Theal tablets; Theo-24; Theobid; Theocap; Theochron; Theoclear; Theocontin; Theo-Dur; Theofol; Theograd; Theolair; Theolan; Theolix; Theophyl; Theoplus; Theo-Sav; Theosol; Theospan; Theostat; Theovent; TheoX; T-Phyl; Truphylline; Uni-Dur; Unifyl; Uniphyl; Uniphyllin; Xanthium Theophylline is an alkaloid found in tea (Thea the evaluation of theophylline. The oral route of sinensis) and chocolate and is structurally related to administration was selected because it is the primary caffeine and theobromine. Theophylline is used as a route of human exposure, and the gavage route was pharmaceutical agent. It stimulates the heart and cen­ selected because it mimics the pharmaceutical use of tral nervous system, relaxes the smooth muscles of the theophylline in humans. Male and female F344/N rats bronchi and blood vessels, and causes diuresis. The and B6C3F1 mice were given theophylline (greater drug is used mainly as a bronchodilator in obstructive than 99% pure) in feed or in corn oil by gavage for airway