TOXICOLOGY AND CARCINOGENESIS STUDIES OF METHYLENE BLUE TRIHYDRATE

226 Pages·2008·10.7 MB·English

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
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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
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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
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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.