National Toxicology Program

National Toxicology Program
https://ntp.niehs.nih.gov/go/32989

Abstract for TR-540 - Methylene Blue Trihydrate (CASRN 7220-79-3)

Toxicology and Carcinogenesis Studies of Methylene Blue Trihydrate (CAS No. 7220-79-3) in F344/N Rats and B6C3F1 Mice (Gavage Study)

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Chemical Formula: C16H24ClN3O3S - Molecular Weight: 373.9

Methylene blue trihydrate has a variety of biomedical and biologically therapeutic applications. Methylene blue trihydrate was nominated by the National Cancer Institute (NCI) for carcinogenicity testing based on the numerous uses of this compound and the lack of long-term toxicity data, including epidemiological studies of methylene blue trihydrate, as well as the inadequate animal data on this compound. Male and female F344/N rats and B6C3F1 mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose by gavage for 1 month, 3 months, or 2 years. Genetic toxicology studies were conducted using Salmonella typhimurium, Escherichia coli, cultured Chinese hamster ovary cells, mouse bone marrow cells, and mouse peripheral blood erythrocytes.

1-MONTH STUDY IN RATS

Groups of 10 male and 10 female core study rats and groups of 10 male and 10 female clinical pathology study rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000; mg/kg, 5; days per week for 5 weeks. In the 500 mg/kg groups, one male died the first week of the study and one male and four females died the second week of the study. All rats in the 1,000 ;mg/kg group died by study day 10, and all rats in the 2,000 mg/kg group died by study day 6. Final mean body weights of male and female rats in the 250 and 500 mg/kg groups were significantly less than those of the vehicle controls. Dosed rats developed methemoglobinemia and a regenerative Heinz body anemia.

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 males and 125 and 250 mg/kg females. Spleen lesions associated with methylene blue trihydrate administration included hematopoietic cell proliferation, pigmentation, lymphoid depletion of the lymphoid follicles, and capsular fibrosis. Hyperplasia of the bone marrow occurred in all dosed groups of rats. Liver lesions associated with methylene blue exposure included centrilobular necrosis in rats dying early, hematopoietic cell proliferation, and Kupffer cell pigmentation with erythrophagocytosis.

1-MONTH STUDY IN MICE

Groups of 10 male and 10 female core study mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 125, 250, 500, 1,000, or 2,000 mg/kg, 5 days per week for 5 weeks. None of the mice in the 500, 1,000, and 2,000 mg/kg groups survived to the end of the study. In the 250 mg/kg groups, two females died on days 16 and 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 respiration, hypothermia, lethargy, ataxia, and ruffled fur were observed in a few surviving animals in the 250 mg/kg groups. Hypothermia and abnormal posture were observed in mice in the 500, 1,000, and 2,000 mg/kg groups. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia. Significant increases in spleen weights occurred in all surviving dosed groups of mice compared to vehicle controls. Significant decreases occurred in the thymus weights of 250 mg/kg males and females. The heart weights of 125 and 250 mg/kg females were significantly increased. Lesions in the spleen associated with methylene blue trihydrate administration included hematopoietic cell proliferation, pigmentation, and congestion. Liver lesions associated with methylene blue trihydrate administration included periportal degeneration, hematopoietic cell proliferation, and Kupffer cell pigmentation with erythrophagocytosis. The incidences of bone marrow pigmentation were significantly increased in all dosed groups of mice. Forestomach lesions that were related to methylene blue trihydrate administration included focal ulcer, inflammation, and squamous hyperplasia.

3-MONTH STUDY IN RATS

Groups of 10 male and 10 female core study rats and groups of 20 male and 20 female clinical pathology study rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 25, 50, 100 or 200 mg/kg, 5 days per week for 14 weeks. Mean body weights of males in the 200 mg/kg group were significantly less than those of the vehicle controls. Dosed rats developed methemoglobinemia and a regenerative Heinz body anemia. Significant increases in spleen weights occurred in males and females administered 50 mg/kg or greater. Thymus and lung weights 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.

Spleen lesions in dosed rats included hematopoietic cell proliferation, congestion, lymphoid depletion of the lymphoid follicles, and capsular fibrosis. The incidences of bone marrow hyperplasia were significantly increased in groups administered 50 mg/kg or greater. There were no consistent effects of methylene blue trihydrate administration on reproductive system measures in male or female rats.

3-MONTH STUDY IN MICE

Groups of 10 male and 10 female core study mice and groups of 20 male and 20 female clinical pathology study mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 25, 50, 100, or 200 mg/kg, 5 days per week for 14 weeks. Mean body weights of all dosed groups were similar to or only slightly less than those of the vehicle control groups. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia. Spleen weights of 100 and 200 mg/kg males and 50 mg/kg or greater females were significantly greater than those of the vehicle control groups. Heart weights were significantly increased in 200 mg/kg males. In females, there were significant decreases in thymus weights at 50 mg/kg or greater. Males had decreased sperm motility and increased epididymal sperm counts at 200 mg/kg.

In all dosed groups, the incidences of hematopoietic cell proliferation and pigmentation in the spleen were significantly greater than those in the vehicle controls. In the liver, the incidences of hematopoietic cell proliferation were significantly increased in males and females in the 100 and 200 mg/kg groups, and the incidences of Kupffer cell pigmentation were significantly increased in groups administered 50 mg/kg or greater. The incidences of bone marrow pigmentation were significantly increased in all dosed groups of mice except 25 mg/kg females.

2-YEAR STUDY IN RATS

Groups of 50 male and 50 female rats were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 5, 25, or 50 mg/kg, 5 days per week for 2 years. Additional groups of 10 male and 10 female rats were administered the same doses for up to 18 months and were evaluated at 2 weeks and 3, 12, and 18 months for hematology. Survival of all dosed groups of rats was similar to that of the vehicle controls. Mean body weights of 25 and 50 mg/kg male rats were less than those of the vehicle controls after weeks 29 and 21, respectively. In the 25 and 50 mg/kg females, mean body weights were less after weeks 73 and 53. Dosed male and female rats developed methemoglobinemia, and females developed a regenerative Heinz body anemia.

The incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined) were increased in all dosed groups of males, were significantly increased in 25 mg/kg males, and exceeded the historical range in controls (all routes). The incidence of pancreatic islet cell hyperplasia was significantly increased in the 50 mg/kg males.

In the spleen, the incidence of hematopoietic cell proliferation in 50 mg/kg males was significantly increased; the incidences of capsular fibrosis were significantly increased in all dosed groups of males and in 5 and 50 mg/kg females.

2-YEAR STUDY IN MICE

Groups of 50 male and 50 female mice were administered methylene blue trihydrate in a 0.5% aqueous methylcellulose solution by gavage at doses of 0, 2.5, 12.5, or 25 mg/kg, 5 days per week for 2 years. Additional groups of 30 male and 30 female mice were administered the same doses for up to 18 months and were evaluated at 2 weeks and 3, 12, or 18 months for hematology. Survival of dosed male and female groups exceeded that of the vehicle controls in a generally dose-related manner. Mean body weights of dosed female mice began to increase after weeks 29, 61, and 85, reaching final values that were 113%, 111%, and 106% of vehicle controls for the 2.5, 12.5, and 25 mg/kg groups, respectively. Dosed mice developed methemoglobinemia and a regenerative Heinz body anemia.

The incidences of carcinoma and of adenoma or carcinoma (combined) of the small intestine occurred with a positive trend in males. The incidences of malignant lymphoma occurred with a positive trend in females, and the incidence in 25 mg/kg males exceeded the historical control range.

The incidences of hematopoietic cell proliferation of the spleen were significantly increased in 12.5 and 25 mg/kg males and in 25 mg/kg females. The incidences of inflammation of the nose were significantly increased in 12.5 and 25 mg/kg females.

GENETIC TOXICOLOGY

Methylene blue trihydrate was mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without rat or hamster liver S9 activation enzymes; mutagenicity was also observed in Escherichia coli strain WP2 uvrA/pKM101 with and without rat liver S9. In cytogenetic tests with cultured Chinese hamster ovary cells, methylene blue trihydrate induced sister chromatid exchanges and chromosomal aberrations with and without S9. However, in contrast to the positive results in the in vitro tests, no increase in the frequency of micronucleated erythrocytes was observed in bone marrow or blood samples collected from male mice and analyzed 48 hours after a single intraperitoneal injection of methylene blue trihydrate or in peripheral blood of male and female mice administered methylene blue trihydrate by gavage for 3 months. In the 3-month micronucleus tests, a dose-related increase in the percentage of reticulocytes among the total erythrocyte population was observed in both male and female mice.

Adjunct studies were conducted with three metabolites of methylene blue trihydrate: Azure A, Azure B, and Azure C. All three compounds were tested in the Ames assay, and all were positive, with and without rat liver S9 activation enzymes, in Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2 uvrA/pKM101.

CONCLUSIONS

Under the conditions of these 2-year gavage studies, there was some evidence of carcinogenic activity of methylene blue trihydrate in male F344/N rats based on increased incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined). There was no evidence of carcinogenic activity in female F344/N rats administered 5, 25, or 50 mg/kg. There was some evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of carcinoma and of adenoma or carcinoma (combined) in the small intestine. The increased incidence of malignant lymphoma in males receiving 25 mg/kg may have been related to the administration of methylene blue trihydrate. There was equivocal evidence of carcinogenic activity in female B6C3F1 mice based on marginally increased incidences of malignant lymphoma.

Methylene blue trihydrate administration caused methemoglobinemia and a regenerative Heinz body anemia with secondary injury to other organs in rats and mice.

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


Summary of the 2-Year Carcinogenesis Studies of Methylene Blue Trihydrate

 
Male
F344/N Rats
 
Female
F344/N Rats
 
Male
B6C3F1 Mice
 
Female
B6C3F1 Mice
 
Doses in 0.5% aqueous methylcellulose solution administered by gavage 0, 5, 25, or 50 mg/kg 0, 5, 25, or 50 mg/kg 0, 2.5, 12.5, or 25 mg/kg 0, 2.5, 12.5, or 25 mg/kg
Body weights 25 and 50 mg/kg groups less than the vehicle control group 25 and 50 mg/kg groups less than the vehicle control group Dosed groups similar to the vehicle control group 12.5 and 25 mg/kg groups greater than the vehicle control group
Survival rates 31/50, 33/50, 39/50, 31/50 35/50, 32/50, 36/50, 35/50 35/50, 38/50, 38/50, 41/50 33/50, 40/50, 42/50, 43/50
Hematologic effects Blood: Methemoglobinemia Blood: Methemoglobinemia and Heinz body formation Blood: Methemoglobinemia and Heinz body formation Blood: Methemoglobinemia and Heinz body formation
Nonneoplastic effects Pancreatic islets: hyperplasia (13/50, 13/50, 17/50, 26/50);

Spleen: hematopoietic cell proliferation (11/50, 12/50, 17/50, 20/50); capsule fibrosis (1/50, 7/50, 12/50, 30/50)
Spleen: capsule fibrosis (8/49, 17/48, 12/49, 20/49) Spleen: hematopoietic cell proliferation (14/49, 16/50, 25/49, 29/48) Spleen: hematopoietic cell proliferation (23/47, 21/47, 31/49, 40/50)
Neoplastic effects Pancreatic islets: adenoma (4/50, 9/50, 12/50, 8/50); adenoma or carcinoma (4/50, 9/50, 14/50, 8/50) None Small intestine: carcinoma (0/50, 1/50, 2/50, 4/50); adenoma or carcinoma (1/50, 2/50, 4/50, 6/50) None
Equivocal findings None None Malignant lymphoma: (2/50, 2/50, 2/50, 5/50) Malignant lymphoma: (6/50, 4/50, 9/50, 12/50)
Level of evidence of carcinogenic activity Some evidence No evidence Some evidence Equivocal evidence
Genetic Toxicology of Methylene Blue Trihydrate
Assay Test System Results
Bacterial Mutagenicity Salmonella typhimurium gene mutations: Methylene blue trihydrate Positive in strains TA98 and TA100 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations: Methylene blue trihydrate (lot 68H3728) Positive in strains TA98 and TA100 with and without S9 and in Escherichia coli WPM uvrA/pKM101 with and without S9
Bacterial Mutagenicity Salmonella typhimurium gene mutations: Azure A, Azure B, and Azure C Positive in strains TA98 and TA100 with and without S9 and in Escherichia coli WPM uvrA/pKM101 with and without S9
Sister chromatid exchanges Cultured Chinese hamster ovary cells in vitro: Positive with and without S9
Chromosomal aberrations Cultured Chinese hamster ovary cells in vitro: Positive with and without S9
Micronucleated erythrocytes Mouse bone marrow in vivo: Negative
Micronucleated erythrocytes Mouse peripheral blood in vivo: Negative in single dose and 3-month studies

Report Date: May 2008

Pathology Tables, Survival and Growth Curves from NTP 2-year Studies

Target Organs & Incidences from 2-year Studies

NTP is located at the National Institute of Environmental Health Sciences, part of the National Institutes of Health.