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TOXICOLOGY AND CARCINOGENESIS STUDIES OF METHYLENE BLUE TRIHYDRATE PDF

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NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF METHYLENE BLUE TRIHYDRATE (CAS NO. 7220-79-3) IN F344/N RATS AND B6C3F1 MICE (GAVAGE STUDIES) National Toxicology Program P.O. Box 12233 Research Triangle Park, NC 27709 May 2008 NTP TR 540 NIH Publication No. 08-4429 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 NTP TECHNICAL REPORT ON THE TOXICOLOGY AND CARCINOGENESIS STUDIES OF METHYLENE BLUE TRIHYDRATE (CAS NO. 7220-79-3) IN F344/N RATS AND B6C3F1 MICE (GAVAGE STUDIES) National Toxicology Program P.O. Box 12233 Research Triangle Park, NC 27709 May 2008 NTP TR 540 NIH Publication No. 08-4429 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 rats (December 8, 2004) D.W. Bristol, Ph.D., Study Scientist W.G. Lieuallen, D.V.M., Ph.D., Chairperson J.C. Peckham, D.V.M., M.S., Ph.D., Study Pathologist Pathology Associates International J.R. Bucher, Ph.D. M.F. Cesta, D.V.M., Observer R.S. Chhabra, Ph.D. National Toxicology Program B.J. Collins, M.S. K.J. Cimon, D.V.M., M.S. R.A. Herbert, D.V.M., Ph.D. Experimental Pathology Laboratories, Inc. A.P. King-Herbert, D.V.M. D. Dixon, D.V.M., Ph.D. G.E. Kissling, Ph.D. National Toxicology Program D.E. Malarkey, D.V.M., Ph.D. R.A. Herbert, D.V.M., Ph.D. National Toxicology Program R.R. Maronpot, D.V.M. D.E. Malarkey, D.V.M., Ph.D. S.D. Peddada, Ph.D. National Toxicology Program J.H. Roycroft, Ph.D. K. Mozzachio, D.V.M., Observer R.C. Sills, D.V.M., Ph.D. National Toxicology Program C.S. Smith, Ph.D. G. Pearse, B.V.M. & S. G.S. Travlos, D.V.M. National Toxicology Program K.L. Witt, M.S. J.C. Peckham, D.V.M., M.S., Ph.D. Experimental Pathology Laboratories, Inc. A. Suttie, B.V.Sc., Ph.D. Battelle Columbus Operations Conducted 1- and 3-month studies and evaluated pathology findings ILS, Inc. K. Yoshizawa, D.V.M., Ph.D., Observer P.J. Kurtz, Ph.D., Principal Investigator National Toxicology Program R.L. Persing, D.V.M. Evaluated slides and prepared pathology report on mice A.W. Singer, D.V.M. (January 26, 2005) J.D. Toff, D.V.M., M.S. W.G. Lieuallen, D.V.M., Ph.D., Chairperson Pathology Associates International Southern Research Institute Conducted 2-year studies and evaluated pathology findings M.F. Cesta, D.V.M., Observer National Toxicology Program W. Richter, D.V.M., Principal Investigator K.J. Cimon, D.V.M., M.S. Experimental Pathology Laboratories, Inc. C.D. Hébert, Ph.D., Principal Investigator D. Dixon, D.V.M., Ph.D. D.R. Farnell, D.V.M., Ph.D. National Toxicology Program J.E. Heath, D.V.M. G.P. Flake, M.D. National Toxicology Program Experimental Pathology Laboratories, Inc. R.A. Herbert, D.V.M., Ph.D. Provided pathology review National Toxicology Program D.E. Malarkey, D.V.M., Ph.D. J.F. Hardisty, D.V.M., Principal Investigator National Toxicology Program K.J. Cimon, D.V.M., M.S. A. Nyska, D.V.M. J.C. Peckham, D.V.M., M.S., Ph.D. National Toxicology Program G. Pearse, B.V.M. & S. National Toxicology Program Dynamac Corporation J.C. Peckham, D.V.M., M.S., Ph.D. Prepared quality assurance audits Experimental Pathology Laboratories, Inc. A. Suttie, B.V.Sc., Ph.D. S. Brecher, Ph.D., Principal Investigator ILS, Inc. Methylene Blue Trihydrate, NTP TR 540 3 Constella Group, Inc. Biotechnical Services, Inc. Provided statistical analyses Prepared Technical Report P.W. Crockett, Ph.D., Principal Investigator S.R. Gunnels, M.A., Principal Investigator L.J. Betz, M.S. B.F. Hall, M.S. K.P. McGowan, M.B.A. L.M. Harper, B.S. J.I. Powers, M.A.P. D.C. Serbus, Ph.D. 4 CONTENTS ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY. . . . . . . . . . . . . . . . . 12 TECHNICAL REPORTS REVIEW SUBCOMMITTEE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS. . . . . . . . . . . . . 14 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 DISCUSSION AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Appendix A Summary of Lesions in Male Rats in the 2-Year Gavage Study of Methylene Blue Trihydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Appendix B Summary of Lesions in Female Rats in the 2-Year Gavage Study of Methylene Blue Trihydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Appendix C Summary of Lesions in Male Mice in the 2-Year Gavage Study of Methylene Blue Trihydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Appendix d Summary of Lesions in Female Mice in the 2-Year Gavage Study of Methylene Blue Trihydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Appendix e Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Appendix F Clinical Pathology Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Appendix G Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . . . . . . . . . . . 193 Appendix H Reproductive Tissue Evaluations and Estrous Cycle Characterization . . . . . . . . . . . . . . 199 Appendix i Chemical Characterization and Dose Formulation Studies . . . . . . . . . . . . . . . . . . . . . . . . 203 Appendix J Ingredients, Nutrient Composition, and Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Appendix K Sentinel Animal Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Methylene Blue Trihydrate, NTP TR 540 5 SUMMARY Background Methylene blue trihydrate has a variety of medical uses, including the treatment of methemoglobinemia and psychiatric disorders and as a disinfectant and biological stain. We studied the effects of methylene blue trihydrate on male and female rats and mice to identify potential toxic or cancer-related hazards. Methods We deposited solutions containing methylene blue trihydrate in aqueous methylcellulose directly into the stomachs of male and female rats and mice. Groups of 50 male and female rats received 5, 25, or 50 milligrams of methylene blue trihydrate per kilogram body weight five days per week for two years; groups of 50 male and female mice received 2.5, 12.5, or 25 milligrams of methylene blue per kilogram of body weight for the same duration. Groups of animals receiving methylcellulose alone served as the control groups. At the end of the study, tissues from more than 40 sites were examined for every animal. Results The two highest dose groups of male and female rats weighed less than the control animals, while the two highest dose groups of female mice weighed more than their corresponding control group. In male and female rats and mice, the blood of the animals was affected, with animals receiving the highest doses experiencing methemoglobinemia and anemia. This caused secondary injury to the spleen in these animals. Cancer of the pancreatic islets was increased in male rats receiving methylene blue trihydrate, and some uncommon tumors of the small intestine were seen in exposed male mice. There was a slight increase in malignant lymphomas in exposed male and female mice. Conclusions We conclude that exposure to methylene blue caused pancreatic islet tumors in male rats and small intestine tumors in male mice. Malignant lymphomas in male and female mice were possibly associated with methylene blue trihydrate exposure. Methylene blue trihydrate caused blood abnormalities and anemia in male and female rats and mice. 6 Methylene Blue Trihydrate, NTP TR 540 7 ABSTRACT METHYLENE BLUE TRIHYDRATE CAS No. 7220-79-3 Chemical Formula: C16H24ClN3O3S Molecular Weight: 373.9 Synonyms: Aizen methylene blue; basic blue 9 (8CI); C.I. 52015; methylthionine chloride; methylthioninium chloride; phenothiazine-5-ium, 3,7-bis, (dimethylamino)-, chloride; swiss blue; tetramethylthionine chloride IUPAC Name: (7-dimethylaminophenothiazin-3-ylidene)-dimethyl-ammonium chloride trihydrate IUPAC International Chemical Identifier: InChI=1/C16H18N3S.CIH.3H2O/c1-18(2)11-5-7-13-15(9-11)20-16-10-12(19(3)4)6-8-14(16)17- 13;;;;/h5-10H,1-4H3;1H;3*1H2/q+1;;;;/p-1/fC16H18N3S.Cl.3H2O/h;1h;;;/qm;-1;;; Canonical SMILES: CN(C)C1=CC2=C(C=C1)N=C3C=CC(=[N+](C)C)C=C3S2.O.O.O.[Cl-] Trade Names: Desmoid piller, Desmoidpillen, Methylene Blue, Panatone, Urolene Blue, Vitableu Methylene blue trihydrate has a variety of biomedical study rats were administered methylene blue trihydrate and biologically therapeutic applications. Methylene in 0.5% aqueous methylcellulose solution by gavage at blue trihydrate was nominated by the National Cancer doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days Institute (NCI) for carcinogenicity testing based on the per week for 5 weeks. In the 500 mg/kg groups, one numerous uses of this compound and the lack of long- male died the first week of the study and one male and term toxicity data, including epidemiological studies four females died the second week of the study. All of methylene blue trihydrate, as well as the inadequate rats in the 1,000 mg/kg group died by study day 10, and animal data on this compound. Male and female F344/N all rats in the 2,000 mg/kg group died by study day 6. rats and B6C3F1 mice were administered methylene blue Final mean body weights of male and female rats in the trihydrate in 0.5% aqueous methylcellulose by gavage 250 and 500 mg/kg groups were significantly less than for 1 month, 3 months, or 2 years. Genetic toxicology those of the vehicle controls. Dosed rats developed studies were conducted using Salmonella typhimurium, met hemoglobinemia and a regenerative Heinz body Escherichia coli, cultured Chinese hamster ovary cells, anemia. mouse bone marrow cells, and mouse peripheral blood erythrocytes. Significant increases in spleen weights occurred in all surviving dosed groups. There were also significant decreases in the thymus weights of 250 and 500 mg/kg 1-MontH Study in RAtS males and 125 and 250 mg/kg females. Spleen lesions Groups of 10 male and 10 female core study rats and associated with methylene blue trihydrate administration groups of 10 male and 10 female clinical pathology included hematopoietic cell proliferation, pigmentation, 8 Methylene Blue Trihydrate, NTP TR 540 lymphoid depletion of the lymphoid follicles, and 14 weeks. Mean body weights of males in the 200 mg/ c apsular fibrosis. Hyperplasia of the bone marrow kg group were significantly less than those of the vehicle occurred in all dosed groups of rats. Liver lesions controls. Dosed rats developed methemoglobinemia and associated with methylene blue exposure included cen- a regenerative Heinz body anemia. Significant increases trilobular necrosis in rats dying early, hematopoietic in spleen weights occurred in males and females admin- cell proliferation, and Kupffer cell pigmentation with istered 50 mg/kg or greater. Thymus and lung weights erythrophagocytosis. of 50, 100, and 200 mg/kg males (except relative lung weight at 100 mg/kg) were significantly less than those of the vehicle controls. 1-MontH Study in MiCe Spleen lesions in dosed rats included hematopoietic Groups of 10 male and 10 female core study mice were cell proliferation, congestion, lymphoid depletion of administered methylene blue trihydrate in 0.5% aque- the lymphoid follicles, and capsular fibrosis. The inci- ous methylcellulose solution by gavage at doses of 0, dences of bone marrow hyperplasia were significantly 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week increased in groups administered 50 mg/kg or greater. for 5 weeks. None of the mice in the 500, 1,000, and There were no consistent effects of methylene blue tri- 2,000 mg/kg groups survived to the end of the study. In hydrate administration on reproductive system measures the 250 mg/kg groups, two females died on days 16 and in male or female rats. 18 and two males died on days 6 and 13. Mean body weights of surviving dosed mice were similar to those of the vehicle controls. Thinness, abnormal respira- tion, hypothermia, lethargy, ataxia, and ruffled fur were 3-MontH Study in MiCe observed in a few surviving animals in the 250 mg/ Groups of 10 male and 10 female core study mice and kg groups. Hypothermia and abnormal posture were groups of 20 male and 20 female clinical pathology observed in mice in the 500, 1,000, and 2,000 mg/kg study mice were administered methylene blue trihydrate groups. Dosed mice developed methemoglobinemia in 0.5% aqueous methylcellulose solution by gavage at and a regenerative Heinz body anemia. Significant doses of 0, 25, 50, 100, or 200 mg/kg, 5 days per week increases in spleen weights occurred in all surviving for 14 weeks. Mean body weights of all dosed groups dosed groups of mice compared to vehicle controls. were similar to or only slightly less than those of the Significant decreases occurred in the thymus weights of vehicle control groups. Dosed mice developed meth- 250 mg/kg males and females. The heart weights of 125 emoglobinemia and a regenerative Heinz body anemia. and 250 mg/kg females were significantly increased. Spleen weights of 100 and 200 mg/kg males and 50 mg/ Lesions in the spleen associated with methylene blue tri- kg or greater females were significantly greater than hydrate administration included hematopoietic cell pro- those of the vehicle control groups. Heart weights were liferation, pigmentation, and congestion. Liver lesions significantly increased in 200 mg/kg males. In females, associated with methylene blue trihydrate administra- there were significant decreases in thymus weights at tion included periportal degeneration, hematopoietic 50 mg/kg or greater. Males had decreased sperm motil- cell proliferation, and Kupffer cell pigmentation with ity and increased epididymal sperm counts at 200 mg/ eryth-rophagocytosis. The incidences of bone marrow kg. pigmentation were significantly increased in all dosed groups of mice. Forestomach lesions that were related to In all dosed groups, the incidences of hematopoietic methylene blue trihydrate administration included focal cell proliferation and pigmentation in the spleen were ulcer, inflammation, and squamous hyperplasia. s ignificantly greater than those in the vehicle controls. In the liver, the incidences of hematopoietic cell prolifer- ation were significantly increased in males and females 3-MontH Study in RAtS in the 100 and 200 mg/kg groups, and the incidences of Groups of 10 male and 10 female core study rats and Kupffer cell pigmentation were significantly increased groups of 20 male and 20 female clinical pathology in groups administered 50 mg/kg or greater. The inci- study rats were administered methylene blue trihydrate dences of bone marrow pigmentation were significantly in 0.5% aqueous methylcellulose solution by gavage at increased in all dosed groups of mice except 25 mg/kg doses of 0, 25, 50, 100 or 200 mg/kg, 5 days per week for females.

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