National Toxicology Program Toxicity Report Series Number 27 NTP Technical Report on Toxicity Studies of Riddelliine (CAS No. 23246-96-0) Administered by Gavage to F344/N Rats and B6C3F1 Mice Po C. Chan, PhD, Study Scientist National Toxicology Program Post Office Box 12233 Research Triangle Park, NC 27709 NIH Publication 94-3350 December 1993 United States Department of Health and Human Services Public Health Service National Institutes of Health Note to the Reader The National Toxicology Program (NTP) is made up of four charter agencies of the United States Department of Health and Human Services (DHHS): • the National Cancer Institute (NCI) of the National Institutes of Health; • the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health; • the National Center for Toxicological Research (NCTR) of the Food and Drug Administration; and • the National Institute for Occupational Safety and Health (NIOSH) of the Centers for Disease Control. In July 1981, the Carcinogenesis Bioassay Testing Program was transferred from NCI to NIEHS. NTP coordinates the relevant Public Health Service programs, staff, and resources that are concerned with basic and applied research and with biological assay development and validation. 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. To carry out its mission, NTP designs and conducts studies to characterize and evaluate the toxicologic potential of selected chemicals in laboratory animals (usually two species, rats and mice). Chemicals selected for NTP toxicology studies are chosen primarily on the bases of human exposure, level of production, and chemical structure. Selection per se is not an indicator of a chemical's toxic potential. The studies described in this toxicity study report were performed under the direction of NIEHS and were conducted in compliance with NTP chemical health and safety requirements. These studies met or exceeded all applicable federal, state, and local health and safety regulations. Animal care and use were in accord and compliance with the Public Health Service Policy on Humane Care and Use of Animals. Single copies of this report are available without charge, while supplies last, from the NTP Public Information Office (telephone number 919/541-3991). NTP Public Information Office NIEHS Post Office Box 12233 Research Triangle Park, NC 27709 National Toxicology Program Toxicity Report Series Number 27 NTP Technical Report on Toxicity Studies of Riddelliine (CAS No. 23246-96-0) Administered by Gavage to F344/N Rats and B6C3F1 Mice Po C. Chan, PhD, Study Scientist National Toxicology Program Post Office Box 12233 Research Triangle Park, NC 27709 NIH Publication 94-3350 December 1993 United States Department of Health and Human Services Public Health Service National Institutes of Health RIDDELLIINE, NTP TOXICITY REPORT NUMBER 27 2 CONTRIBUTORS This NTP report on the toxicity studies of riddelliine was based primarily on 2-week studies that took place in November 1986 and 13-week studies that began in March 1987 and ended in September 1987 at SRI International, Menlo Park, CA. National Toxicology Program SRI International Evaluated experiment, interpreted results, and Principal contributors reported findings James R. Hill, PhD Po C. Chan, PhD, Study Scientist Jon B. Reid, PhD John R. Bucher, PhD Principal Investigators Leo T. Burka, PhD Richard Becker Rajendra S. Chhabra, PhD R. Long Michael P. Dieter, PhD Earl Meierhenry Michael R. Elwell, DVM, PhD Jon Mirsalis Joel Mahler, DVM Ron Spanggord Robert R. Maronpot, DVM H. B. Matthews, PhD Experimental Pathology Laboratories, Morrow B. Thompson, DVM, PhD Inc Gregory S. Travlos, PhD Provided pathology quality assessment Errol Zeiger, PhD John Peckham, DVM, MS, PhD Gary Riley, MVSc, PhD Coordinated report preparation Jane M. Lambert, BS Environmental Health Research and Edison McIntyre, BS Testing, Inc Kristine L. Witt, MS Provided sperm morphology and vaginal cytology Oak Ridge Associated Universities evaluation Teresa Cocanougher, BA NTP Pathology Working Group Dushant K. Gulati, PhD Evaluated slides and prepared pathology report Susan Russell, BA John C. Seely, DVM, Chair PATHCO, Inc. Analytical Sciences, Inc John Cullen, VMD, PhD Provided statistical analyses North Carolina State University Steven Seilkop, MS Michael P. Elwell, DVM, PhD Janet Teague, MS National Toxicology Program Seiichi Imoto, DVM, PhD (Observer) Biotechnical Services, Inc Shin Nippon Biomedical Laboratories, Provided toxicity report preparation Japan Janet L. Elledge, BA, Principal Investigator Margarita M. McDonald, DVM, PhD Chad J. Fitz, MA National Toxicology Program Paula C. Higginson, BA William F. MacKenzie, DVM, MS Theresa King-Hunter, BS Experimental Pathology Laboratories, Jennifer Rector, MAP Inc A.W. Macklin, DVM, PhD Burroughs Wellcome Research Laboratories 3 RIDDELLIINE, NTP TOXICITY REPORT NUMBER 27 TABLE OF CONTENTS ABSTRACT.......................................................................................................................... 5 PEER REVIEW PANEL........................................................................................................ 10 SUMMARY OF PEER REVIEW COMMENTS .......................................................................... 11 INTRODUCTION................................................................................................................. 13 Physical Properties, Occurrence, and Exposure .......................................................... 13 Disposition and Metabolism ....................................................................................... 14 Toxicity....................................................................................................................... 16 Study Rationale and Design........................................................................................ 21 MATERIALS AND METHODS .............................................................................................. 23 Procurement and Characterization of Riddelliine ........................................................ 23 Dose Formulations ..................................................................................................... 24 Toxicity Study Designs ............................................................................................... 24 Supplemental Evaluations.......................................................................................... 26 Genetic Toxicity Studies ............................................................................................. 32 Statistical Methods ..................................................................................................... 35 Quality Assurance ...................................................................................................... 38 RESULTS .......................................................................................................................... 39 2-Week Gavage Study in F344/N Rats........................................................................ 39 13-Week Gavage Study in F344/N Rats...................................................................... 43 2-Week Gavage Study in B6C3F1 Mice ....................................................................... 65 13-Week Gavage Study in B6C3F1 Mice ..................................................................... 67 Mating Trial Results ................................................................................................... 79 Genetic Toxicity .......................................................................................................... 80 DISCUSSION ..................................................................................................................... 87 REFERENCES ................................................................................................................... 95 APPENDIXES Appendix A Organ Weights and Organ-Weight-to-Body-Weight Ratios ................... A-1 Appendix B Hematology and Clinical Chemistry Results ........................................B-1 Appendix C Reproductive Tissue Evaluations and Estrous Cycle Characterization ...................................................C-1 Appendix D Genetic Toxicology .............................................................................D-1 RIDDELLIINE, NTP TOXICITY REPORT NUMBER 27 4 5 RIDDELLIINE, NTP TOXICITY REPORT NUMBER 27 Riddelliine Molecular Formula C18H23NO6 CAS Number 23246-96-0 Molecular Weight 349.4 Synonyms 13,19-didehydro-12,18-dihydroxy senecionan-11,16-dione; trans-15-ethylidine-12b-hydroxy-12a-hydroxymethyl-13- methylenesenec-1-enine; 3-ethylidine-3,4,5,6,9,11,13,14,14a,14b-decahydro-6-hydroxy-6- (hydroxymethyl)-5-methylene(1,6)di-oxacyclododecino (2,3,4-gh)-pyrrolizidine-2,7-dione ABSTRACT Riddelliine is a naturally occurring pyrrolizidine alkaloid, a class of compounds occurring in rangeland plants of the genera Crotalaria, Amsinckia, and Senecio. Two- week and 13-week rodent toxicity studies of riddelliine were conducted because riddelliine can be a contaminant of foodstuffs, such as meat, grains, seeds, milk, herbal tea, and honey. In addition to histopathology, evaluations included clinical pathology and reproductive toxicity. In vitro genetic toxicity studies included assessments of mutagenicity in Salmonella typhimurium and