NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF MILK THISTLE EXTRACT (CAS NO. 84604-20-6) IN F344/N RATS AND B6C3F1 MICE (FEED STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 May 2011 NTP TR 565 NIH Publication No. 11-5907 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 Technical Report series began in 1976 with carcinogenesis studies conducted by the National Cancer Institute. In 1981, this bioassay program was transferred to the 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. 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 Technical 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 TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF MILK THISTLE EXTRACT (CAS NO. 84604-20-6) IN F344/N RATS AND B6C3F1 MICE (FEED STUDIES) NATIONAL TOXICOLOGY PROGRAM P.O. Box 12233 Research Triangle Park, NC 27709 May 2011 NTP TR 565 NIH Publication No. 11-5907 National Institutes of Health Public Health Service U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES 2 CONTRIBUTORS National Toxicology Program Dynamac Corporation Evaluated and interpreted results and reported findings Prepared quality assessment audits J.K. Dunnick, Ph.D., Study Scientist S. Brecher, Ph.D., Principal Investigator A. Nyska, D.V.M., Study Pathologist S. Iyer, B.S. J.B. Bishop, Ph.D. V.S. Tharakan, D.V.M. J.R. Bucher, Ph.D. R.S. Chhabra, Ph.D. NTP Pathology Working Group P.M. Foster, Ph.D. Evaluated slides and contributed to pathology report R.A. Herbert, D.V.M., Ph.D. on 2-year rats (January 22 and 31, 2008) M.J. Hooth, Ph.D. A.P. King-Herbert, D.V.M. S. Newbigging, M.Sc., D.V.M., D.V.Sc., Coordinator G.E. Kissling, Ph.D. ILS, Inc. N. Allison, D.V.M. D.E. Malarkey, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. J.H. Roycroft, Ph.D. D. Dixon, D.V.M., Ph.D. J.M. Sanders, Ph.D. National Toxicology Program B.P. Singh, D.V.M. S.A. Elmore, D.V.M., M.S. C.S. Smith, Ph.D. National Toxicology Program G.S. Travlos, D.V.M. G.P. Flake, M.D. N.J. Walker, Ph.D. National Toxicology Program K.L. Witt, M.S. R.A. Herbert, D.V.M., Ph.D. National Toxicology Program D.E. Malarkey, D.V.M., Ph.D. Southern Research Institute National Toxicology Program Conducted studies and evaluated pathology findings A. Nyska, D.V.M. ILS, Inc. C.D. Hébert, Ph.D., Principal Investigator J.C. Peckham, D.V.M., M.S., Ph.D. J.E. Heath, D.V.M. Experimental Pathology Laboratories, Inc. J.F. Mann, B.S., D.V.M. B.P. Singh, B.V.Sc., M.S. National Toxicology Program Experimental Pathology Laboratories, Inc. Provided pathology review M.H. Hamlin, II, D.V.M., Principal Investigator N. Allison, D.V.M. H.M. Kolenda-Roberts, D.V.M., Ph.D. J.C. Peckham, D.V.M., M.S., Ph.D. TherImmune Research Corporation Provided SMVCE analysis G.W. Wolfe, Ph.D., Principal Investigator H.S. Seung, M.S. Milk Thistle Extract, NTP TR 565 3 NTP Pathology Working Group SRA International, Inc. Evaluated slides and contributed to pathology report Provided statistical analyses on 2-year mice (December 6 and 13, 2007) P.W. Crockett, Ph.D., Principal Investigator P.E. Blackshear, D.V.M., Ph.D., Coordinator L.J. Betz, M.S. ILS, Inc. K.P. McGowan, M.B.A. D. Dixon, D.V.M., Ph.D. National Toxicology Program Biotechnical Services, Inc. S.A. Elmore, D.V.M., M.S. Prepared Technical Report National Toxicology Program G.P. Flake, M.D. S.R. Gunnels, M.A., Principal Investigator National Toxicology Program B.F. Hall, M.S. R.A. Herbert, D.V.M., Ph.D. L.M. Harper, B.S. National Toxicology Program K. Hobbie, D.V.M. J.I. Powers, M.A.P. ILS, Inc. D.C. Serbus, Ph.D. H.M. Kolenda-Roberts, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. D.E. Malarkey, D.V.M., Ph.D. National Toxicology Program A. Nyska, D.V.M. ILS, Inc. J.B. Nold, D.V.M. GlaxoSmithKline J.C. Peckham, D.V.M., M.S., Ph.D. Experimental Pathology Laboratories, Inc. B.P. Singh, B.V.Sc., M.S. National Toxicology Program 4 CONTENTS ABSTRACT ................................................................................................................................................................. 7 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 .............................................................................................................................. 25 RESULTS ................................................................................................................................................................... 35 DISCUSSION AND CONCLUSIONS ..................................................................................................................... 59 REFERENCES .......................................................................................................................................................... 61 APPENDIX A Summary of Lesions in Male Rats in the 2-Year Feed Study of Milk Thistle Extract ................................................................................................................. 71 APPENDIX B Summary of Lesions in Female Rats in the 2-Year Feed Study of Milk Thistle Extract ................................................................................................................. 85 APPENDIX C Summary of Lesions in Male Mice in the 2-Year Feed Study of Milk Thistle Extract ................................................................................................................ 99 APPENDIX D Summary of Lesions in Female Mice in the 2-Year Feed Study of Milk Thistle Extract .............................................................................................................. 111 APPENDIX E Genetic Toxicology ...................................................................................................................... 123 APPENDIX F Clinical Pathology Results .......................................................................................................... 135 APPENDIX G Organ Weights and Organ-Weight-to-Body-Weight Ratios ................................................... 143 APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization ................................. 147 APPENDIX I Chemical Characterization and Dose Formulation Studies ................................................... 151 APPENDIX J Feed and Compound Consumption in the 2-Year Feed Studies of Milk Thistle Extract ............................................................................................................... 165 Milk Thistle Extract, NTP TR 565 5 APPENDIX K Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration .......................................................................................... 171 APPENDIX L Sentinel Animal Program ........................................................................................................... 175 6 Milk Thistle Extract, NTP TR 565 SUMMARY Background Milk thistle extracts have been used in herbal medicine for the treatment of liver cirrhosis, chronic hepatitis, and gallbladder disorders, in addition to a variety of other ailments. We studied the effects of milk thistle extract given to rats and mice in the feed to identify potential toxic or cancer-related hazards. Methods We gave feed containing 12,500, 25,000, or 50,000 parts per million (1.25%, 2.5%, or 5%) of milk thistle extract to groups of 50 male and female rats and mice for two years. Similar groups of animals were given feed with no chemical added and served as the control groups. At the end of the study, tissues from more than 40 sites were examined for every animal. Results Survival of all exposed groups of animals was similar to their controls. The body weights of male and female mice given milk thistle extract were less than for the control animals. There were no increases in the incidences of cancers at any sites, and the rates of mammary gland cancers in female rats and liver cancers in male mice were lower in animals given milk thistle extract than in their control groups. Conclusions We conclude that milk thistle extract did not cause cancer in male or female rats or mice. The incidences of mammary gland cancers in female rats and liver cancers in male mice were lower than the background rate in animals receiving milk thistle extract. 7 ABSTRACT Milk Thistle Extract (CAS No. 84604-20-6) (Milk thistle drawing obtained from AMR, 1999) Synonyms of milk thistle: Bull thistle; cardo blanco; cardui mariae fructus; cardui mariae herba; Cardum marianum L.; Carduus marianus L.; chardon-marie; emetic root; frauendistel; fructus silybi mariae; fruit de chardon marie; heal thistle; holy thistle; isosilibinin; kanger; kocakavkas; kuub; lady’s thistle; lady’s thistle extract; marian thistle; mariana mariana; mariendistel; Marienkrörner; Mary thistle; mild thistle; milk ipecac; pig leaves; royal thistle; Saint-Mary thistle; shui fei ji; silidianin; silybi mariae fructus; silybin; silybinin; Silybum marianum extract; silychristin; snake milk; sow thistle; St. Mary’s thistle; variegated thistle; venue thistle; wild artichoke Trade names for milk thistle extract (silymarin): Legalon®, Thisilyn® Milk thistle extracts have been used as medicinal herbs the despined leaves are added to salads. Roasted milk in the treatment of liver cirrhosis, chronic hepatitis thistle fruit has been used as a coffee substitute. Milk (liver inflammation), and gallbladder disorders. thistle extract was nominated for study by the National Treatment claims also include lowering cholesterol Institute of Environmental Health Sciences because it is levels; reducing insulin resistance; reducing the growth one of the most widely used herbs in the United States. of cancer cells in breast, cervical, and prostate gland Male and female F344/N rats and B6C3F1 mice were cancers; and antiviral activity. Other reported uses of exposed to an ethanol/water extract of milk thistle fruit milk thistle in folk medicine include as a treatment for (milk thistle extract) containing approximately malarial fever, bronchitis, gallstones, jaundice, peri- 65% silymarin in feed for 3 months or 2 years. Genetic tonitis, uterine congestion, varicose veins, and as a milk toxicology studies were conducted in Salmonella typhi- production stimulant for nursing mothers. The roots murium and Escherichia coli and mouse peripheral soaked in water overnight are used in food, and blood erythrocytes. 8 Milk Thistle Extract, NTP TR 565 3-MONTH STUDY IN RATS Significantly decreased incidences of mammary gland fibroadenoma, adenoma, or carcinoma (combined) Groups of 10 male and 10 female rats were fed diets occurred in females exposed to 25,000 or 50,000 ppm. containing 0, 3,125, 6,250, 12,500, 25,000, or 50,000 ppm milk thistle extract (equivalent to average Significantly increased incidences of clear cell and daily doses of approximately 260, 525, 1,050, 2,180, or mixed cell focus of the liver occurred in 25,000 and 4,500 mg milk thistle extract/kilogram body weight to 50,000 ppm females. The incidences of bile duct males and 260, 510, 1,050, 2,150, or 4,550 mg/kg to hyperplasia were significantly decreased in 50,000 ppm females) for 14 weeks. All rats survived to the end of males and in all exposed groups of females, and the the study. Mean body weights of exposed groups were incidence of mixed inflammatory cell infiltration was within 10% of those of the controls. Feed consumption significantly decreased in 50,000 ppm males. by exposed and control groups was similar. The sperm motility in 12,500, 25,000, and 50,000 ppm males was decreased by 5%, 11%, and 9%, respectively, relative to 2-YEAR STUDY IN MICE that of the controls; the total number of spermatid heads Groups of 50 male and 50 female mice were fed diets per testis decreased by 11%, 21%, and 9% in containing 0, 12,500, 25,000, or 50,000 ppm milk thistle 12,500, 25,000, and 50,000 ppm males. No significant extract (equivalent to average daily doses of approx- differences in estrous cyclicity were observed between imately 1,610, 3,530, or 7,770 mg/kg to males and exposed and control groups of female rats. No 1,500, 3,175, or 7,180 mg/kg to females) for 105 to exposure-related histopathologic lesions were observed. 106 weeks. Exposure to milk thistle extract had no effect on survival of male or female mice. The mean body weights of the 25,000 ppm groups were less than 3-MONTH STUDY IN MICE those of controls after week 25; mean body weights of Groups of 10 male and 10 female mice were fed diets 50,000 ppm groups were less than those of controls after containing 0, 3,125, 6,250, 12,500, 25,000, or week 12. Feed consumption by exposed groups of 50,000 ppm milk thistle extract (equivalent to average males and females was generally similar to that by the daily doses of approximately 640, 1,340, 2,500, 5,280, controls throughout the study. or 11,620 mg/kg to males and 580, 1,180, 2,335, 4,800, or 9,680 mg/kg to females) for 14 weeks. All mice Significantly decreased incidences of hepatocellular survived to the end of the study. Mean body weights adenoma and hepatocellular carcinoma occurred in and feed consumption of all exposed groups were sim- 50,000 ppm males, and decreased incidences of hepa- ilar to those of the controls. Absolute and relative tocellular adenoma or carcinoma (combined) occurred thymus weights were significantly decreased in 25,000 in 25,000 and 50,000 ppm males. and 50,000 ppm males. No significant differences were observed between exposed and control groups, for sperm parameters of male mice, for estrous cyclicity of GENETIC TOXICOLOGY female mice, or for reproductive organ weights of male Five milk thistle extracts were tested independently in or female mice, when mice were administered milk bacterial mutagenicity studies using a variety of thistle extract in feed at 12,500, 25,000, or 50,000 ppm. S. typhimurium tester strains and one E. coli strain. No exposure-related histopathologic lesions were Results were negative in three of the five studies, with observed. and without exogenous metabolic activation. In two studies, milk thistle extract was mutagenic in S. typhi- murium strain TA98 in the presence of exogenous 2-YEAR STUDY IN RATS metabolic activation enzymes. Silymarin, a major Groups of 50 male and 50 female rats were fed diets constituent of milk thistle extract, was positive in containing 0, 12,500, 25,000, or 50,000 ppm milk thistle S. typhimurium strains TA98 and TA100, when testing extract (equivalent to average daily doses of approx- occurred in the presence of exogenous metabolic acti- imately 570, 1,180, or 2,520 mg/kg to males and 630, vation enzymes. Silybin, another component of milk 1,300, or 2,750 mg/kg to females) for 105 to 106 weeks. thistle extract, was negative in a S. typhimurium gene Exposure to milk thistle extract had no effect on sur- mutation assay, with and without liver S9 activation vival of male or female rats. Mean body weights of all enzymes. exposed groups were similar to those of the controls throughout the study. Feed consumption by exposed Administration of milk thistle extract in feed for groups of males and females was generally similar to 3 months did not increase the frequencies of micro- that by the controls throughout the study. nucleated normochromatic erythrocytes, an indication of