Toxicity and carcinogenicity studies of methylene blue trihydrate in F344N rats and B6C3F1 mice

Abstract

Methylene blue trihydrate has a variety of biomedical and biologically therapeutic applications. Groups of 50 male and 50 female rats and mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 5, 25, or 50 mg/kg bw/day (rats) or 0, 2.5, 12.5, and 25 mg/kg bw/day (mice), 5 days per week for 2 years. In rats survival of all dosed groups was similar to that of the vehicle controls, whereas mice exhibited a dose-dependent increase in survival. Rats receiving 25 and 50 mg/kg bw/day and mice receiving 25 mg/kg bw/day developed mild anemia. The incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined) were increased in all dosed groups of male rats, but increases were statistically significant in 25 mg/kg bw/day males only and the dose-response was non-linear. There was a corresponding increase in the incidence of pancreatic islet cell hyperplasia but statistically significant only in the 50 mg/kg bw/day male rats. There were no significant increases in neoplastic transformation observed in the mice; however, positive trends were noted for adenoma or carcinoma (combined) of the small intestine and malignant lymphoma.

1. Introduction

Methylene blue trihydrate (Figure 1) occurs as odorless dark green crystals or crystalline powder with a bronze luster (Merck, 2001). It is one of a group of thiazinium halides, or phenothiazin-5-ium chloride compounds, that have a wide variety of uses, including biomedical applications and biological activity (Moura and Cordeiro 2003). A search of Pubmed using the term methylene blue identified over 10,000 abstracts dating back to 1914, highlighting its role in biomedical science over many years (NLM). Currently, methylene blue is used in human and veterinary medicine for a number of therapeutic and diagnostic procedures including use as a stain in bacteriology, as a redox coloring agent, as a targeting agent for melanoma, as an anti-methemoglobinemic, as an antiseptic and disinfectant, as a treatment for ifosfamide-induced encephalopathy, as a treatment of vasoplegia associated with septic shock, and as a tracer in parathyroid surgery (Kwok and Howes 2006; O’Neill 2001; Patel 2006). Of particular note is its potential use as a disease-modifying Alzheimer’s therapeutic agent (Scheindlin 2008). Unlike other treatment protocols that require short-term exposure to methylene blue, treatment of Alzheimer’s will likely require chronic therapy.

Figure 1.

The structure of methylene blue trihydrate

Disposition studies in adult Sprague-Dawley rats indicate that methylene blue is rapidly distributed out of the blood into a number of tissues including heart, lung, liver, and kidneys (DiSanto and Wagner 1972a). In humans methylene blue is absorbed well, reduced and excreted largely in urine as the reduced leuco (colorless) form (DiSanto and Wagner 1972b).

Genotoxicity studies of methylene blue performed by the NTP revealed consistent mutagenic activity in the presence and absence of S9 in Salmonella typhimurium TA98 and TA100 and Escherichia coli WPM uvrA/pKM101. CHO cell assays demonstrated methylene blue is capable of inducing sister chromatid exchange and chromosomal aberrations. Notably, however methylene blue failed to induce micronucleated erythrocytes in mouse bone marrow or peripheral blood after in vivo exposure (NTP 2008).

Acute toxic effects that have been described in animals exposed to methylene blue include hemoconcentration, hypothermia, acidosis, hypercapnia, hypoxia, increases in blood pressure, changes in respiratory frequency and amplitude, corneal injury, conjunctival damage, and Heinz body formation (Christiansen 1980). In a 90-day NTP rodent toxicity study, as little as 25 mg/kg bw/day elicited a toxic response in the hematopoietic system, as evidenced by methemoglobinemia (Hejtmancik et al. 2002). In humans, large doses of methylene blue (approximately 500 mg) administered intravenously have been reported to cause nausea, abdominal and chest pain, cyanosis, methemoglobinemia (note: at therapeutic doses it acts as a anti-methemoglobinemic), sweating, dizziness, headache, and confusion (Clifton and Leikin 2003). Numerous reports have demonstrated toxicity in infants exposed to methylene blue trihydrate during prenatal or perinatal diagnostic or therapeutic procedures, including hyperbilirubinemia, anemia, Heinz bodies, erythrocytic blister cells, skin discoloration, and photosensitization (Porat et al. 1996; Sills and Zinkham 1994).

Despite its use as a therapeutic, methylene blue trihydrate has not been adequately tested for carcinogenicity. Furthermore, there is a paucity of epidemiological studies related to the carcinogenicity of methylene blue. To identify the potential carcinogenic hazard to human health, the NTP designed and performed a rodent carcinogenicity bioassay. Prior to the 2-year bioassay described here, the NTP performed 3 month toxicity studies at doses of 0, 25, 50, 100 or 200 mg/kg bw/day. A dose-related regenerative Heinz body anemia was observed at 50 mg/kg bw/day and higher in rats and 25 mg/kg bw/day and higher in mice. The hematological toxicities documented in the 3 month study served as the basis for selecting the doses for the chronic bioassay, which were 0, 5, 25, and 50 mg/kg bw/day for rats and 0, 2.5, 12.5, and 25 mg/kg bw/day for mice. The lowest doses were selected to approximate the human therapeutic dose used to treat methemoglobinemia (1 to 2 mg/kg intravenously) (Camp 2007). Described here are the results of the 2-year bioassay.

2. Materials and Methods

For a detailed discussion of the materials and methods used to perform these studies the reader is referred to the NTP technical report 540 (NTP 2008).

2.1. Chemical.

Methylene blue trihydrate (CAS No. 7220-79-3) was obtained from Aldrich Chemical Company (Milwaukee, WI) in one lot (68H3728). Identity and purity analyses were conducted using high performance liquid chromatography (HPLC) by the study laboratory, Southern Research Institute (Birmingham, AL). HPLC indicated one major peak and three impurities with relative peak areas of 0.16%, 0.21%, and 6.55%.

2.2. Preparation and Analysis of Dose Formulations.

The vehicle was prepared by mixing methylcellulose (Aldrich Chemical Company) with heated, deionized water. The dose formulations were prepared every four weeks. The dose formulations were stored in sealed amber glass bottles at room temperature (25° C) for up to 35 days. Homogeneity and stability studies were performed using HPLC. Dose formulations were analyzed every 3 months; animal room samples were also analyzed. All dose formulations analyzed were within 10% of the target concentrations; all rat and mice animal room samples were within 10% of target concentrations.

2.3. Animals.

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms, Inc. (Germantown, NY), for use in the 2-year studies. Rats and mice were quarantined for 12 days before the beginning of the studies. Five male and five female rats and mice were randomly selected for parasite evaluation and gross observation of disease. Rats and mice were 6 weeks old at the beginning of the studies.. Rats were housed three (males) or five (females) per cage, and mice were housed individually (males) or five per cage (females). Feed (NTP2000) and tap water were available ad libitum. Cages were changed once (male mice) or twice weekly, and racks were rotated every 2 weeks.

2.5. Study Design.

Groups of 50 male and 50 female rats and mice were administered methylene blue trihydrate in a 0.5% aqueous methylcellulose solution by gavage at doses of 0, 5, 25, or 50 mg/kg bw/day (rats) or 0, 2.5, 12.5, or 25 mg/kg bw/day (mice) once daily, 5 days per week for 2 years. Vehicle control animals were gavaged with 0.5% aqueous methylcellulose. These animals were considered to be the core study. Additional groups of 10 male and 10 female rats and 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, and 18 months for use as satellite hematology study groups.

2.6. Clinical Examinations.

Animals were observed twice daily. Clinical findings for core study animals were recorded every 4 weeks beginning with week 5. Body weights for core study animals were recorded on day 1, weekly for the first 13 weeks, at 4-week intervals thereafter, and at terminal sacrifice.

2.7. Hematology.

Blood was taken from the retroorbital sinus of the 10 male and 10 female hematology study rats/mice per group at 2 weeks and 3, 12, and 18 months. At each collection interval, methemoglobin analyses were completed within 30 minutes of blood collection; the remaining hematology analyses, including reticulocyte counts, were performed within approximately 6 hours of blood collection. At 2 weeks and 3 months, the automated hematology analyses, excluding the methemoglobin and reticulocyte assays, were conducted using a Technicon H·1TM hematology analyzer (Technicon Corporation, Tarrytown, NY) with reagents manufactured by R&D Systems, Inc. (Minneapolis, MN), Bayer, Inc. (Tustin, CA), and Fisher Scientific (Norcross, GA); the reticulocyte analyses were conducted at 2 weeks and 3 months using a Coulter Model EPICS XL Flow Cytometer (Coulter Corporation, Miami, FL) with reagents manufactured by Coulter Corporation and Molecular Probes (Eugene, OR). At 12 and 18 months, hematology analyses, including the reticulocyte analyses, were conducted using an ADVIA 120 Hematology System Analyzer (Bayer Diagnostics, Tarrytown, NY) with reagents manufactured and/or supplied by Bayer, Inc., and Fisher Scientific. At all four intervals, the methemoglobin analyses were conducted using a Beckman DU spectrophotometer with reagents manufactured by Baker Chemical Company (Phillipsburg, NJ) and Fisher Scientific. Blood smears were prepared within approximately 2 hours of sample collection for Heinz body enumeration and for evaluation of platelet and erythrocyte morphology by light microscopy. The parameters measured were as follows: automated and manual hematocrit; hemoglobin concentration; erythrocyte, nucleated erythrocytes, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differentials; methemoglobin; and Heinz bodies.

2.8. Necropsy and Histology.

Complete necropsies and microscopic examinations were performed on all core study rats and mice. At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined. A complete list all tissues that underwent microscopic examination can be found in the technical report (NTP 2008). In addition, all gross lesions observed at necropsy and all tissues masses were examined microscopically.

2.9. Statistical Methods.

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (Kaplan and Meier 1958). Statistical analyses for possible dose-related effects on survival used the Cox (Cox 1972) method for testing two groups for equality and the Tarone (Tarone 1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Average severity values were analyzed for significance with the Mann–Whitney U test (Hollander and Wolfe 1973). The poly-k test (Bailer and Portier 1988; Portier and Bailer 1989) was used to assess neoplasm and nonneoplastic lesion prevalence. Unless otherwise specified, a value of k = 3 was used in the analysis of site-specific lesions. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided.

Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (Dunnett 1955) and Williams (Williams 1971, 1972). Hematology data which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) as modified by Williams (Williams 1986) and Dunn (Dunn 1964).

3. Results

3.1. Rats

Survival of all dosed groups of rats was similar to that of the vehicle controls. Mean body weights of 25 and 50 mg/kg bw/day male rats were less than those of the vehicle controls after weeks 29 and 21, respectively; mean body weights of these groups at the end of the study were 91% and 87% that of the vehicle controls, respectively. In the 25 and 50 mg/kg bw/day females, mean body weights were lower than control values after weeks 73 and 53 and the final mean body weights were 91% and 88% that of the vehicle controls. For a more detailed evaluation of the body weight effects the reader is referred to the NTP technical report (NTP 2008).

Hematology profiles for the 2 year rat studies are shown in table 1. Similar to what occurred in the short-term studies, the primary response to chemical administration was the development of a methemoglobinemia, Heinz body formation, and a macrocytic responsive anemia. A dose-related increase in methemoglobin concentrations occurred in the 50 mg/kg bw/day male and female dose groups at month 3. By month 6, the 25 mg/kg bw/day males and females were also affected; both groups remained affected at month 18. Significantly increased numbers of Heinz bodies only occurred in the 25 and 50 mg/kg bw/day female groups at month 18. Small decreases in the hematocrit, hemoglobin and erythrocyte count values occurred fairly consistently in the 25 and 50 mg/kg bw/day female and male groups as early as month 3, persisting through month 18. Evidence of a hematopoietic response was indicated by increased numbers of circulating nucleated erythrocytes and/or reticulocytes. The small increase in mean cell volume probably reflects the increased presence of the larger immature erythrocytes.

Table 1.

Select hematology from rats

	Males				Females
	Vehicle Control	5 mg/kg	25 mg/kg	50 mg/kg	Vehicle Control	5 mg/kg	25 mg/kg	50 mg/kg
Animal numbers
Week 2	10	10	10	10	10	10	10	10
Month 3	10	10	10	10	10	10	10	10
Month 12	10	10	8	10	10	10	9	9
Month 18	9	10	8	9	9	9	8	9
Hematocrit (spun) (%)
Week 2	42.9±0.6	43.9±0.4	42.9±0.6	42.2±0.5	46.1±0.6	45.2±0.6	45.4±0.4	45.3±0.5
Month 3	45.6±0.4	46.2±0.5	44.9±0.4	43.8±0.6*	44.8±0.4	45±0.4	44.5±0.3	42.8±0.4**
Month 12	45.4±0.3	45.4±0.4	43.5±0.4**	43.5±0.4**	44.9±0.3	44.5±0.4	43.4±0.4*	41.4±0.5**
Month 18	45.6±0.6	47.5±1	46.3±0.8	44.6±0.3	45.1±0.3	44.9±0.4	43.9±0.4	43.1±0.3*
Hematocrit (auto) (%)
Week 2	43.1±0.6	44.1±0.5	42.8±0.5	43±0.5	46.9±0.8	46±0.6	46±0.5	46.3±0.6
Month 3	46±0.3	46.3±0.5	45.2±0.4	43.9±0.5**	44.8±0.4	45±0.4	44.4±0.4	43.1±0.3*
Month 12	44.9±0.3	45±0.4	42.8±0.4**	42.6±0.3**	46±0.3	45.6±0.3	44.1±0.2**	41.5±0.4**
Month 18	46±0.6	48.3±1.1	47.2±0.9	45.1±0.5	43.7±0.4	43.4±0.5	42.3±0.4*	41.2±0.3**
Hemoglobin (g/dL)
Week 2	14.5±0.2	14.7±0.1	14.2±0.2	14.3±0.2	14.8±0.2	14.5±0.2	14.6±0.2	14.6±0.2
Month 3	15.5±0.1	15.5±0.1	15±0.1*	14.4±0.2**	15.2±0.1	15.3±0.1	14.9±0.1	14.2±0.1**
Month 12	15.1±0.1	15.2±0.1	14.4±0.1**	14.2±0.1**	15.5±0.1	15.4±0.1	14.8±0.1**	13.8±0.1**
Month 18	15.5±0.2	16.2±0.4	15.5±0.3	14.7±0.2*	14.9±0.1	14.7±0.2	14.2±0.1**	13.6±0.1**
Erythrocytes (106/μL)
Week 2	7.49±0.12	7.63±0.08	7.32±0.1	7.4±0.1	8.22±0.14	8.03±0.1	8.11±0.09	8.1±0.1
Month 3	8.79±0.06	8.82±0.1	8.5±0.08*	7.87±0.07**	8.21±0.06	8.27±0.05	7.99±0.06*	7.44±0.06**
Month 12	8.82±0.05	8.87±0.09	8.31±0.08**	8.02±0.03**	8.27±0.04	8.23±0.06	7.67±0.04**	7.04±0.06**
Month 18	8.22±0.11	8.66±0.19	8.3±0.13	7.68±0.11*	7.92±0.07	7.91±0.09	7.46±0.04**	7.01±0.06**
Reticulocytes (105/μL)
Week 2	4.64±0.13	4.94±0.25	4.93±0.27	4.77±0.27	3.29±0.24	3.33±0.17	3.46±0.15	3.63±0.23
Month 3	3.44±0.11	3.29±0.13	3.76±0.07	4.86±0.14**	3.1±0.07	2.98±0.12	3.48±0.11*	4.55±0.15**
Month 12	2.44±0.08	2.42±0.06	3.05±0.11**	3.95±0.09**	2.21±0.09	2.21±0.09	3.13±0.15**	4.86±0.12**
Month 18	2.84±0.15	2.87±0.18	3.77±0.13**	4.87±0.08**	2.64±0.1	2.46±0.06	3.52±0.11**	4.87±0.15**
Nucleated erythrocytes (103/μL)
Week 2	0.3±0.15	0.2±0.13	0.4±0.22	0.3±0.21	0.1±0.1	0.2±0.13	0.2±0.13	0.1±0.1
Month 3	0.4±0.16	0±0	0.4±0.16	0.9±0.28	0±0	0±0	0±0	0±0
Month 12	0.1±0.1	0.3±0.15	0.88±0.30*	1.7±0.42**	1.1±0.43	0.8±0.25	3.22±1	3.56±0.88*
Month 18	0±0	0.2±0.13	1±0.38*	0.56±0.34	1.33±0.37	0.89±0.39	1.25±0.56	1.11±0.26
Mean cell volume (fL)
Week 2	57.6±0.3	57.8±0.2	58.5±0.3	58.1±0.3	57.1±0.4	57.3±0.3	56.7±0.3	57.2±0.3
Month 3	52.4±0.1	52.4±0.3	53.2±0.2**	55.8±0.2**	54.5±0.1	54.5±0.2	55.6±0.1**	57.9±0.2**
Month 12	50.9±0.1	50.7±0.2	51.4±0.2	53.2±0.3**	55.6±0.2	55.5±0.1	57.6±0.1**	58.9±0.3**
Month 18	56±0.5	55.7±0.4	56.9±0.3	58.7±0.4**	55.2±0.2	55±0.1	56.8±0.2**	58.7±0.2**
Mean cell hemoglobin (pg)
Week 2	19.3±0.1	19.2±0.1	19.4±0.1	19.3±0.1	18±0.1	18.1±0.1	18±0.1	18±0.1
Month 3	17.6±0.1	17.6±0.1	17.7±0.1	18.4±0.2**	18.5±0.1	18.6±0.1	18.6±0.1	19.1±0.1**
Month 12	17.1±0.1	17.2±0.1	17.3±0.1	17.7±0.1**	18.8±0.1	18.7±0.1	19.3±0.1**	19.6±0.1**
Month 18	18.9±0.2	18.6±0.2	18.7±0.1	19.2±0.2	18.8±0.1	18.6±0.1	19.1±0.1	19.5±0.1**
Mean cell hemoglobin concentration (g/dl)
Week 2	33.5±0.1	33.2±0.2	33.1±0.1	33.2±0.2	31.6±0.3	31.6±0.2	31.8±0.1	31.5±0.1
Month 3	33.6±0.1	33.6±0.1	33.2±0.1*	32.9±0.2**	34±0.1	34±0.1	33.5±0.1*	33.1±0.2**
Month 12	33.7±0.1	33.8±0.2	33.6±0.1	33.3±0.1	33.7±0.2	33.7±0.1	33.6±0.2	33.3±0.1
Month 18	33.7±0.1	33.4±0.2	32.9±0.2**	32.7±0.1**	34.1±0.2	33.8±0.1	33.6±0.1*	33.1±0.1**
Methemoglobin (g/dL)
Week 2	0.15±0.02	0.15±0.02	0.21±0.05	0.19±0.01	0.19±0.02	0.2±0.02	0.23±0.02	0.43±0.22
Month 3	0.21±0.01	0.19±0.01	0.23±0.02	0.29±0.02**	0.16±0.02	0.16±0.02	0.2±0.03	0.27±0.02**
Month 12	0.16±0.04	0.14±0.02	0.25±0.02**	0.32±0.02**	0.19±0.01	0.19±0.01	0.28±0.02**	0.32±0.02**
Month 18	0.12±0.02	0.16±0.02	0.24±0.03**	0.33±0.02**	0.14±0.02	0.18±0.02	0.29±0.01**	0.32±0.02**
Heinz bodies (%)
Week 2	0.1±0	0.1±0	0.1±0	0.2±0.1	0.6±0.1	0.6±0.1	0.5±0	0.7±0.1
Month 3	0.4±0.1	0.7±0.1	0.7±0.1	0.5±0.1	0.2±0	0.1±0	0.2±0	0.3±0.2
Month 12	0.1±0	0.2±0.1	0.1±0	0.2±0.1	0.1±0	0.1±0	0.1±0	0.1±0.1
Month 18	0.1±0	0.1±0	0.1±0	0.2±0.1	0.1±0	0±0	0.4±0.1**	7.1±1.7**

The incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined) were increased in male rats dosed at 25 mg/kg bw/day (table 2). Pancreatic islet cell adenomas were characterized by discrete, well-demarcated, single nodules, 1 to 10 mm in diameter that often compressed the adjacent acinar tissue and were composed of a monomorphic population of cuboidal to polygonal cells with central round nuclei and vacuolated amphophilic cytoplasm. Islet cell carcinomas were similar to the adenomas with additional features of invasion, cellular anaplasia, and pleomorphism. The incidence of islet cell hyperplasia was significantly increased in the 50 mg/kg bw/day males (table 3). Affected hyperplastic islets were enlarged with round to oval outlines that could attain a diameter of 500 μm and consisted of either enlarged islet cells or retained normal cytologic appearances and arrangements. Islet cell hyperplasia, adenoma, and carcinoma are thought to constitute a morphological and biological continuum in the progression of islet cell proliferation. As in this study, islet cell proliferative lesions are observed to occur more frequently in males than females (Riley et al. 1990).

Table 2.

Incidence of Neoplasms exhibiting significant change in rats

	Effect	Vehicle Control	5 mg/kg	25 mg/kg	50 mg/kg
Male
Pancreatic Islet Adenoma (includes multiple)a	↑	4/50 (8%)	9/50 (18%)	12/50 (24%)*	8/50 (16%)
Mononuclear Cell Leukemiab	↓	23/50 (46%)	10/50(20%)	2/50 (4%)**	2/50 (4%)**
Adrenal Medulla, Benign Pheochromocytoma	↓	9/50 (18%)	13/50 (26%)	2/50 (4%)*	3/50 (6%)
Female
Mononuclear Cell Leukemiac	↓	12/50 (24%)	6/49 (12%0	3/50 (6%)*	2/50 (4%)**
Mammary Gland Fibroadenoma	↓	28/50 (56%)	30/49 (61%)	28/50 (56%)	17/50 (34%)*

^*Significantly different (P≤0.05) from vehicle control by the Poly−3 test

^**(P≤0.01)

^aHistorical incidence for 2−year studies, all routes, all vehicles (mean ± standard deviation): 66/1,448 (4.8% ± 3.1%), range 0%−10%

^bHistorical incidence for 2−year studies, all routes, all vehicles (mean ± standard deviation): 622/1,459 (41.4% ± 12.3%), range 22%−68%

^cHistorical incidence: 383/1,459 (26.7% ± 10.5%), range 12%−52%

Table 3.

Incidence of nonneoplastic lesions exhibiting significant change in rats

	Effect	Vehicle Control	5 mg/kg	25 mg/kg	50 mg/kg
Male
Pancreatic Islet Hyperplasia	↑	13/50 (1.2)a	13/50 (1.7)	17/50 (1.8)	26/50** (1.4)
Pancreatic Acinar Cell Hyperplasia	↑	4/50 (2.0)	6/50 (1.7)	15/50** (1.9)	12/50 * (1.8)
Pancreatic Acinar Atrophy	↓	43/50 (2.1)	31/50 **(1.8)	35/50* (2.0)	32/50* (1.9)
Splenic Hematopoietic Cell Proliferation	↑	11/50 (1.5)	12/50 (2.0)	17/50 (1.5)	20/50* (1.7)
Splenic Capsular Fibrosis	↑	1/50 (1.0)	7/50* (1.3)	12/50** (1.5)	30/50** (1.8)
Female
Splenic Capsular Fibrosis	↑	8/49 (1.0)	17/48* (1.1)	12/49** (1.1)	20/49** (1.0)
Mammary Gland Hyperplasia	↓	18/50 (2.3)	19/49 (2.1)	9/50* (2.0)	7/50* (2.6)

^*Significantly different (P≤0.05) from vehicle control by the Poly−3 test

^**(P≤0.01)

^aAverage severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

The incidences of pancreatic acinar cell focal hyperplasia were significantly increased in the 25 and 50 mg/kg bw/day males (table 3). Microscopically, focal acinar hyperplasia consisted of focal areas, less than 3 mm in overall diameter, of large, hypertrophic acini having increased cytoplasmic zymogen granules and slightly enlarged nuclei with prominent nucleoli. The increase in the incidences of acinar hyperplasia was not accompanied by significant increases in the incidence of acinar adenomas. Acinar atrophy occurred with significantly decreased incidences in all dosed groups of males and consisted of small, focal areas of acinar cell loss with relative increases in ducts and interstitial connective tissue often containing a mixed inflammatory cell infiltrate. Acinar atrophy is a common background lesion in older rats.

The incidences of splenic hematopoietic cell proliferation in dosed rats were greater than those in the vehicle controls, and the incidence in 50 mg/kg bw/day males was significantly increased (table 3). The incidences of capsular fibrosis were significantly increased in all dosed groups of males and in 5 and 50 mg/kg bw/day females (table 3). Splenic capsule fibrosis consisted of one or more small areas of slight thickening of the capsule by mature fibrous connective tissue with collagen deposition that often extended outward from the surface of the spleen; occasionally, this area contained hematopoietic tissue or hemosiderin-laden macrophages.

Significantly decreased incidences of mononuclear cell leukemia occurred in all dosed groups of males and in 25 and 50 mg/kg bw/day females (table 2). Mononuclear cell leukemia consisted of widely dispersed infiltrations of monomorphic populations of small, round to polygonal cells with central, round nuclei and scant cytoplasm in a variety of organs. Mononuclear cell leukemia was most often present in the bone marrow, spleen, and liver.

The incidence of mammary gland fibroadenoma was significantly decreased in 50 mg/kg bw/day female rats (table 2); incidences of hyperplasia were significantly decreased in the 25 and 50 mg/kg bw/day females (table 3).

Significant decreases in the incidences of benign pheochromocytoma and benign, complex, or malignant pheochromocytoma (combined) of the adrenal gland occurred in 25 mg/kg bw/day males (table 2).

3.2. Mice

Estimates of 2-year survival probabilities for male and female mice are shown in the Kaplan-Meier survival curves (NTP 2008). Survival of dosed male and female groups exceeded that of the vehicle controls in a generally dose-related manner.

Mean body weights of male mice in all dosed groups were similar to those of the vehicle control group (data not shown). 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 controls for the 2.5, 12.5, and 25 mg/kg bw/day groups, respectively. For a more detailed evaluation of the body weight effects the reader is referred to the NTP technical report (NTP 2008). There were no chemical-related clinical findings in males or females (NTP 2008).

Hematology profiles for the 2 year mouse studies are shown in table 4. Similar to what occurred in the short-term studies, the primary response to methylene blue trihydrate administration was the development of a methemoglobinemia and Heinz body formation. The highest dose in this study was 25 mg/kg bw/day, and the changes observed at this dose level were not dramatic or consistent. A dose-related Heinz body formation occurred in the 12.5 and 25 mg/kg bw/day male and female mice at essentially all time points. However, the percentage of erythrocytes with Heinz bodies varied widely from <1% to >20%, depending on the sex and time point. Increased concentrations of methemoglobin occurred in the 12.5 and 25 mg/kg bw/day female mice at month 18; the 25 mg/kg bw/day males also may have been affected. Also, it appeared that the macrocytic, hyperchromic, responsive anemia that had been apparent in the short-term studies was no longer macrocytic or hyperchromic and, at most, was minimal and transient.

Table 4.

Select hematology from mice

	Males				Females
	Vehicle Control	2.5 mg/kg	12.5 mg/kg	25 mg/kg	Vehicle Control	2.5 mg/kg	12.5 mg/kg	25 mg/kg
Animal numbers
Week 2	10	10	10	10	10	10	10	10
Month 3	9	10	9	10	10	10	10	9
Month 12	9	9	8	9	10	9	10	9
Month 18	10	9	8	8	9	9	10	10
Hematocrit (spun) (%)
Week 2	49.9±0.8	50.3±0.7	49.6±1	49.4±0.9	48.6±0.9	48.8±0.9	49.1±0.6	48.6±0.8
Month 3	49.6±0.7	48.6±0.8	48.5±0.8	46.3±0.5**	47.2±0.5	48.3±0.7	47±0.8	45.6±0.7
Month 12	46.7±0.6	46.9±0.4	46.7±0.8	45±0.8	47±0.4	47.1±0.3	45.2±1	45.7±0.5*
Month 18	41.6±1	45.5±1.8	42.8±1.4	42.2±1.2	42.2±0.5	42±1.3	42.1±0.8	43.1±0.5
Hematocrit (auto) (%)
Week 2	50.6±0.7	50.6±0.9	50.4±1	49.9±1	48.5±0.8	48.5±0.9	48.9±0.6	48.1±0.7
Month 3	50.4±0.8	49.5±0.8	49.2±0.8	46.9±0.7**	48.1±0.8	49±0.7	47.7±0.7	46±0.8
Month 12	46.3±0.5	46.7±0.4	46.5±0.9	44.2±0.7	47.6±0.4	48±0.3	45.8±1.1	46.1±0.6
Month 18	41.4±1	44.6±1.8	42.4±1.2	41.3±1	41.9±0.5	42±1.3	41.5±0.7	42.5±0.5
Hemoglobin (g/dL)
Week 2	16.9±0.3	16.9±0.3	16.8±0.4	16.6±0.4	15.8±0.2	15.8±0.3	15.9±0.2	15.6±0.2
Month 3	17±0.2	16.6±0.3	16.4±0.3	15.4±0.2**	16.3±0.3	16.5±0.2	15.8±0.3	15.8±0.2
Month 12	15.5±0.3	15.5±0.2	15.3±0.3	14.6±0.2	15.8±0.1	16±0.1	15.1±0.3	15.2±0.2
Month 18	14±0.3	15±0.6	14.5±0.4	13.7±0.3	14.2±0.2	14.1±0.6	13.9±0.2	14.1±0.2
Erythrocytes (106/μL)
Week 2	10.68±0.17	10.65±0.21	10.64±0.24	10.64±0.26	10.56±0.14	10.67±0.21	10.73±0.13	10.47±0.18
Month 3	10.98±0.19	10.7±0.17	10.63±0.15	10.11±0.13**	10.35±0.17	10.5±0.17	10.34±0.14	9.86±0.16
Month 12	10.02±0.11	10.19±0.16	10.13±0.2	9.43±0.13*	9.82±0.1	9.96±0.07	9.41±0.23	9.52±0.11
Month 18	9.2±0.26	10.29±0.58	9.91±0.52	9.07±0.34	9.04±0.14	9.03±0.33	8.74±0.23	9.12±0.14
Reticulocytes (105/μL)
Week 2	4.13±0.15	4.62±0.10*	4.19±0.11	3.93±0.17	4.26±0.2	4.6±0.24	4.39±0.19	4.43±0.24
Month 3	4.37±0.13	4.08±0.12	4.06±0.15	4.99±0.15	4.53±0.35	4.86±0.18*	4.89±0.18*	5.39±0.36**
Month 12	2.75±0.1	3.07±0.23	3.39±0.11**	4.23±0.12**	2.79±0.13	3.02±0.11	4.04±0.23**	4.6±0.41**
Month 18	2.72±0.17	3.25±0.41	2.79±0.25	3.41±0.35	2.93±0.2	4.1±0.91	3.03±0.23	3.21±0.2
Nucleated erythrocytes (103/μL)
Week 2	0.3±0.15	0.5±0.22	0.4±0.22	0.2±0.13	0±0	0±0	0±0	0±0
Month 3	0±0	0.1±0.1	0±0	0±0	0±0	0±0	0±0	0±0
Month 12	0±0.00	0±0	0±0	0.22±0.22	0±0	0±0.00b	0±0	0±0.00
Month 18	0.1±0.1	0±0	0±0	0±0	0.11±0.11	0.11±0.11	0±0	0.1±0.1
Mean cell volume (fL)
Week 2	47.4±0.1	47.6±0.2	47.4±0.3	47±0.3	45.9±0.2	45.5±0.1	45.6±0.2	46±0.2
Month 3	45.9±0.2	46.2±0.1	46.2±0.2	46.4±0.2	46.5±0.2	46.6±0.2	46.1±0.2	46.7±0.3
Month 12	46.2±0.3	45.9±0.4	45.9±0.2	46.8±0.3	48.4±0.2	48.2±0.2	48.7±0.2	48.4±0.3
Month 18	45.1±0.5	43.7±0.7	43.3±1.3	45.7±0.5	46.4±0.3	46.6±0.4	47.6±0.6	46.7±0.5
Mean cell hemoglobin (pg)
Week 2	15.8±0.1	15.9±0.1	15.8±0.1	15.6±0.1	14.9±0.1	14.8±0.1	14.8±0.1	14.9±0.1
Month 3	15.5±0.1	15.5±0.1	15.4±0.1	15.3±0.2	15.7±0.1	15.7±0.1	15.3±0.1**	16±0.2
Month 12	15.4±0.1	15.2±0.2	15.1±0.1	15.5±0.1	16.1±0.1	16.1±0.1	16.1±0.1	16±0.1
Month 18	15.3±0.2	14.7±0.2	14.7±0.3	15.1±0.2	15.7±0.1	15.6±0.2	16±0.2	15.5±0.1
Mean cell hemoglobin concentration (g/dl)
Week 2	33.4±0.2	33.4±0.2	33.4±0.2	33.3±0.2	32.6±0.2	32.6±0.2	32.5±0.2	32.3±0.2
Month 3	33.8±0.2	33.5±0.3	33.3±0.3	32.9±0.4**	33.8±0.1	33.7±0.1	33.1±0.2**	34.3±0.4
Month 12	33.4±0.4	33.1±0.2	33±0.1	33±0.1	33.2±0.1	33.4±0.1	33±0.1	33.1±0.2
Month 18	33.8±0.3	33.6±0.3	34.1±0.5	33±0.2	33.8±0.1	33.5±0.5	33.5±0.2	33.1±0.2*
Methemoglobin (g/dL)
Week 2	0.62±0.1	0.59±0.05	0.45±0.07	0.58±0.11	0.47±0.09	0.58±0.11	0.56±0.1	0.61±0.08
Month 3	0.33±0.04	0.38±0.03	0.44±0.07	0.52±0.05*	0.41±0.05	0.46±0.08	0.5±0.1	0.51±0.1
Month 12	0.38±0.04	0.38±0.04	0.4±0.07	0.51±0.04	0.34±0.04	0.33±0.05	0.41±0.04	0.63±0.05**
Month 18	0.22±0.04	0.18±0.05	0.3±0.04	0.44±0.1	0.22±0.04	0.31±0.04	0.34±0.03*	0.52±0.03**
Heinz bodies (%)
Week 2	0.2±0	0.2±0	0.3±0.1	1.2±0.1**	0.1±0	0.1±0	0.3±0.1*	5.1±0.9**
Month 3	0±0	0±0	2.2±0.4**	23.3±2.9**	0.1±0.1	0.1±0.1	1.5±0.2**	18.6±1.5**
Month 12	0±0.0	0±0	2.3±0.6**	13.3±1.6**	0.1±0	0±0.0	1±0.3**	6.2±1.7**
Month 18	0.1±0	0.2±0.1	5±0.5**	19.8±1.1**	0.2±0.1	0.1±0	4.9±1.0**	22±3.6**

The incidences of carcinoma of the small intestine occurred with a positive trend in males (table 5). The incidences were within the historical control range for all routes of administration; however, the incidence in the 25 mg/kg bw/day group was at the upper end of the historical control range for all gavage studies. In addition, the incidence of adenoma or carcinoma (combined) in the 25 mg/kg bw/day group exceeded the historical control range for all study routes. One vehicle control female had a carcinoma and one 2.5 mg/kg bw/day female had an adenoma (table 5). The carcinomas were invasive tumors arising from the mucosal epithelium. The neoplastic epithelium had cellular atypia, basophilia, and frequent mitotic figures. They were usually pedunculated with papillary growth into the intestinal lumen.

Table 5.

Incidence of Neoplasms exhibiting significant change in mice

	Effect	Vehicle Control	2.5 mg/kg	12.5 mg/kg	25 mg/kg
Male
Small Intestine, Carcinomaa	↑	0/50 (0%)$	1/50(2%)	2/50 (4%)	4/50 (8%)
Small Intestine, Adenoma or Carcinomab	↑	1/50 (2%)$	2/50 (4%)	4/50 (8%)	6/50 (12%)
Alveolar/bronchiolar Carcinoma (includes multiple)c	↑	1/50 (2%)$	4/50 (8%)	5/50 (10%)	7/50 (14%)*
Female
Alveolar/bronchiolar Carcinoma (includes multiple)	↓	5/50 (10%)	0/50 (0%)*	0/50 (0%)*	1/50 (2%)
Malignant lymphomad	↑	6/50(4%)$	2/50(8%)	9/50 (18%)	12/50 (24%)

^*Significantly different (P≤0.05) from vehicle control by the Poly−3 test

^**(P≤0.01)

^$Significant by Poly−3 trend test (P≤0.05)

^aHistorical incidence for 2−year studies, all routes, all vehicles (mean ± standard deviation): 33/1,508 (2.2% ± 2.7%), range 0%−10%

^bHistorical incidence: 39/1,508 (2.6% ± 2.8%), range 0%−10%

^cHistorical incidence: 151/1,507 (9.9% ± 5.0%), range 4%−24%

^dHistorical incidence: 308/1,508 (19.7% ± 13.3%), range 6%−58%

In the lung there was a positive trend in the incidences of alveolar/bronchiolar carcinoma in males, and the incidence in 25 mg/kg bw/day males was significantly greater than that in the vehicle control group (table 5). The incidences of alveolar/bronchiolar carcinoma in the methylene blue treated groups of males were within the historical control range for all routes of administration and historical vehicle control range for all gavage studies. The incidence in vehicle controls in the current study was below both historical control ranges. Incidences of focal hyperplasia of the alveolar epithelium were similar among all groups of males. In females, the incidences of alveolar/bronchiolar carcinoma were decreased, and the decreases were significant in the 2.5 and 12.5 mg/kg bw/day groups (table 5). The incidences of alveolar/bronchiolar adenoma and focal hyperplasia of the alveolar epithelium were similar among all female groups (data not shown). Microscopically, alveolar/bronchiolar carcinomas usually consisted of large growths that were well demarcated from the surrounding lung tissues. However, the cellular margins were often irregular with invasion of adjacent tissues, lymphatics, blood vessels, the pleural cavity, and mediastinum. The tumor cells ranged from round to oval and from cuboidal to tall columnar. They were pleomorphic, had nuclear atypism, and were arranged in one to multiple layers around prominent fibrovascular cores. They had heterogeneous growth patterns that included alveolar, papillary, and tubular structures or mixtures of these structures.

The incidences of malignant lymphoma occurred with a positive trend in females, and the incidences in the 12.5 and 25 mg/kg bw/day groups were significantly greater than that in the vehicle controls (table 5). The incidences, including that in the vehicle controls, were within the historical control ranges for all routes and gavage studies. In 25 mg/kg bw/day males, the incidence of malignant lymphoma was slightly increased (table 5) and exceeded the historical control ranges for all routes and gavage studies. Microscopically, lymphomas are a group of related neoplasms composed of relatively homogenous populations of lymphocytic cells that replace the normal structures of the spleen, thymus, various lymph nodes, and bone marrow and may infiltrate the portal areas of the liver.

The incidences of splenic hematopoietic cell proliferation were significantly increased in the 12.5 and 25 mg/kg bw/day males and in 25 mg/kg bw/day females (table 6). Hematopoietic cell proliferation was characterized by the presence of random foci of dense, basophilic, round nuclei consistent with red blood cell precursors in the splenic parenchyma.

Table 6.

Incidence of nonneoplastic lesions exhibiting significant change in mice

	Effect	Vehicle Control	2.5 mg/kg	12.5 mg/kg	25 mg/kg
Male
Splenic Hematopoietic Cell Proliferation	↑	14/49 (2.6)	16/50 (2.7)	25/49* (2.8)	29/48** (2.5)
Female
Splenic Hematopoietic Cell Proliferation	↑	23/47 (2.7)	21/47 (2.5)	31/49 (1.1)	40/50** (1.0)
Nose Inflammation	↑	0/50	3/50 (2.0)	7/50* (2.0)	11/50** (1.9)

^*Significantly different (P≤0.05) from vehicle control by the Poly−3 test

^**(P≤0.01)

^aAverage severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Dose-related increases in inflammation of the nose occurred in all dosed groups with significant increases in the 12.5 and 25 mg/kg bw/day females (table 6). Inflammation consisted of proteinaceous fluid and inflammatory cells, primarily neutrophils, in the nasal cavities.

4. Discussion

Methylene blue trihydrate has a variety of therapeutic and diagnostic uses in human and veterinary medicine. The primary goal of these studies was to identify the toxicity and carcinogenicity of methylene blue trihydrate in rodents. Negative effects on survival were not observed in animals administered methylene blue. Although there were no effects on body weight in the mouse study, the two top dose groups in the rat study exhibited mild, yet significant decreases in body weight. There was toxicity observed in the hematological system of both mice and rats. Dose-related toxicity and/or carcinogenicity were observed in the male rat pancreas, the male mouse small intestine and the mouse lymphatic system.

Because methylene blue may be an effective therapy for Alzheimer’s disease, its pharmacological and toxicological effects in central nervous system are of particular interest (Scheindlin 2008). Kinetic studies indicate methylene blue rapidly accumulates in the central nervous system, suggesting the potential for effects on neurological function (Peter et al. 2000). Clinical observations indicative of adverse neurological effects have been observed in humans following intravenous doses of 7.5 to 10 mg/kg bw/day of methylene blue (Martindale and Stedeford 2003). Consistent with the clinical observations, studies in rats indicate methylene blue produces neuronal apoptosis when administered intravenously at doses as low as 5 mg/kg bw/day (Vutskits et al. 2008). In our studies there were no treatment related effects in the central nervous system, however further histopathological evaluation is underway and will be the subject of a future manuscript.

A comparable decrease in the erythron with a concomitant increase in reticulocyte counts, methemoglobin concentration, and Heinz body formation, similar to that observed in male and female rats in the 25 and 50 mg/kg bw/day groups in the 3-month studies (Hejtmancik et al. 2002), was apparent at least to 18 months. The 2-year mouse study demonstrated a similar effect in the 25 mg/kg bw/day group (compared to the 3-month study (Hejtmancik et al. 2002)), except the erythron and reticulocyte responses were not apparent at 18 months; the increased methemoglobin (females) and Heinz body formation (males and females) continued. This suggests that the oxidative effect of methylene blue on erythrocytes (Hejtmancik et al. 2002) was present and continued throughout the 2-year administration period in rats and mice.

In general, all hematological and probably clinical changes observed for this series of toxicity studies were related to the mild hemolytic anemia induced by the methylene blue administration. The term “hemolytic anemia” refers to an anemia that is a result of increased destruction of circulating erythrocytes. This can occur within the vasculature or extravascularly within the mononuclear phagocyte system (e.g., the spleen). In most instances, the bone marrow compensates for the erythrocyte loss and increased marrow erythropoiesis is evidenced by a reticulocytosis (except in the mouse where a large percentage of the hematopoietic response also comes from the spleen) as observed in this study. Morphologically, the reticulocytosis is evidenced by an increase in RBC polychromasia and nucleated red blood cells. The mean cell volume is increased due to the large size of the reticulocytes. The mean cell hemoglobin concentration is increased due to the increased release of free hemoglobin into the circulation.

The increased incidences of pancreatic islet cell neoplasms in male rats, accompanied by hyperplasia, were considered to be associated with methylene blue trihydrate administration. The incidences of islet cell adenoma, as well as adenoma or carcinoma (combined), were increased in all dosed groups, and the increase was statistically significant in 25 mg/kg bw/day males, which had an incidence double the highest rate observed in historical controls. Out of approximately 540 chemicals tested in NTP rodent cancer studies prior to methylene blue, pancreatic islet cell neoplasms have been observed in only eight other studies. The incidence of this rather uncommon neoplasm peaked at an intermediate dose in five of the nine studies where it occurred. The reasons for this are unknown, but the high incidence of hyperplasia in the 50 mg/kg bw/day group of male rats in the present study suggests that proliferative lesions in these animals were somehow inhibited from progressing to adenomas.

In the nine studies with chemical-induced pancreatic islet cell neoplasia, there was no correlation with chemical structure; indeed, the structures of the chemicals where it has been observed are quite different. Furthermore, while promethazine and methylene blue are built on the phenothiazine ring structure, the NTP study of promethazine provided no evidence of carcinogenic activity in male and female rats or mice (NTP 1993).

Several proliferative lesions occurred at reduced incidences in dosed rats treated with methylene blue. These included mononuclear cell leukemia in males and females, mammary gland fibroadenoma in females, and adrenal medulla pheochromocytoma in males. The reduced incidence of leukemia has been observed in rats treated with other test article that exhibit toxicity to the spleen (Elwell et al. 1996). Mononuclear cell leukemia is thought to have its origins in the spleen, and chemicals that are directly toxic to the spleen or damage the spleen secondary to hematotoxicity, as with methylene blue trihydrate, probably act to inhibit the spontaneous development of mononuclear cell leukemia through this mechanism. It is hypothesized that precursor cells that give rise to MCL reside in the spleen and/or require the splenic microenvironment for differentiation/neoplastic transformation (Thomas et al. 2007).

The mechanism resulting in inhibition of mononuclear cell leukemia is not the same as that of hyperplasia in the bone marrow and hematopoietic cell proliferation in the spleen and liver. Bone marrow hyperplasia and hematopoietic cell proliferation occur in response to anemia. Inhibition of mononuclear cell leukemia appears to be associated with toxicity of methylene blue metabolites or products from damaged red blood cells in the spleen. Capsular fibrosis is additional evidence of spleen injury caused by damaged red blood cells (Stefanski et al. 1990).

Mammary gland fibroadenoma development is negatively correlated with body weight of female rats, and expected rates of fibroadenoma can be calculated for dosed groups that have reductions in body weight in relation to controls (Haseman et al. 1997). In the methylene blue trihydrate study, the expected rates of mammary gland fibroadenoma were consistent with the observed body weights only in the 50 mg/kg bw/day group. The other dosed groups and vehicle controls had rates higher than anticipated. The reasons for this are not known, and consequently, the relationship between methylene blue trihydrate administration and reduced mammary gland fibroadenoma, if any, is uncertain.

In the 2-year study in mice, survival of dosed male and female groups exceeded that of vehicle controls in a generally dose-related manner. Hematological effects observed at 3, 12, and 18 months were similar in male and female mice. The incidences of hematopoietic cell proliferation in the spleen were significantly increased in both sexes.

The incidences of malignant lymphoma occurred with a positive trend in female mice. The incidence in the 25 mg/kg bw/day group (24%) was well within the historical control range (6% to 58%) for this highly variable neoplasm, and thus, the response in this study was considered equivocal. In males, the incidence in the 25 mg/kg bw/day group was numerically elevated, though not statistically significant, and exceeded the historical control range and was also considered an equivocal response.

Although not identified as a target organ in the 3-month study, male mice at 2 years exhibited a significant positive trend in the incidence of carcinoma and adenoma or carcinoma (combined) of the small intestine (site unspecified). Although the incidences in the dosed groups were not significant by pairwise comparison, the rate of adenoma or carcinoma (combined) in the 25 mg/kg bw/day group exceeded the historical control range for these combined neoplasms, and the rate in vehicle controls was consistent with the historical mean. Thus, the small intestine neoplasms observed in male mice were considered to have some carcinogenic activity related to methylene blue trihydrate administration.

The incidence of alveolar/bronchiolar carcinoma of the lung in male mice was low but exhibited a positive trend and was also significant only in the 25 mg/kg bw/day group. However, incidences of alveolar/bronchiolar adenoma alone were decreased in dosed groups, and the incidence of focal hyperplasia of the alveolar epithelium was low and similar across all groups. The incidences of alveolar/bronchiolar carcinoma in dosed groups were well within the range observed for historical controls. For these reasons, the observed alveolar/bronchiolar carcinomas were not considered related to methylene blue trihydrate administration.

Overall, there were few treatment-related increases in tumor incidence in the rodents and the significant increases that were noted were marginal. Increased incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined) were observed in male rats, however no clear dose-response relationship was observed. There were no treatment-related increases in neoplasia in female rats. Male mice exhibited increased incidences of carcinoma and of adenoma or carcinoma (combined) in the small intestine; however the increases only occurred at the highest treatment level. Both male and female mice exhibited increases in malignant lymphoma that may or may not have been related to the administration of methylene blue trihydrate.