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Description of key information

Repeated dose toxicity via oral route - systemic effects (target organ): digestive: liver; glandular: thyroids

In the 2-year feeding study with rats LOAELs of 13 and 50 mg/kg bw/day were established for male and female rats, respectively, based on an increased incidence of thyroid gland follicular cell hyperplasia at all dose levels. In the 2-year study with mice, a LOAEL of 75 mg/kg bw/day was established for males based on increased incidences of hematopoietic cell proliferation and the increased incidence of hepatocellular adenoma or carcinoma in all exposed groups. For females, a NOAEL of 80 mg/kg bw/day was established for toxicity, based on an increased incidence of thyroid gland follicular cell hypertrophy and decreases in automated and manual hematocrit values, hemoglobin concentrations and erythrocyte counts at the next dose levels.
In the 14-week feeding study with rats, based on the increased incidences of diffuse thyroid follicular cell hyperplasia at 2500 ppm (160 mg/kg bw/d), as well as kidney and erythropoietic effects, a NOAEL of 1250 ppm (80 mg/kg bw/d) was established. In the 14-week feeding study with mice, a NOAEL of 625 ppm (100 mg/kg bw/d) was established, based on the effects observed in liver, kidney and the erythropoietic system.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
13 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a 28 -day repeated dose study (according to OECD guideline 407), Sprague-Dawley rats (10/sex/dose) were administered 2-methylimidazoleat 100, 200, 400 and 800 mg/kg bw/day (in water) by gavage (BASF AG, 1975) for a total of 20 days. One group of animals (10 per sex) was used as a control. None of the animals died during the test. Normal body weight gain was observed in all exposure groups, except for males at 800 mg/kg bw/day. No abnormal clinical observations were observed for the 100 and 200 mg/kg bw/day test groups, at 400 and 800 mg/kg bw/day bright yellow urine, ruffled fur and increased salivation was observed. No changes in heamatology were observed. With regard to clinical chemistry decreases in total protein in males and females in all dose groups (not dose related in males) and increased creatinine at 800 mg/kg bw/day in males were observed. Changes in organ weights were observed for the liver (females in 800 mg/kg bw/day exposure group – absolute and relative weight, females in the 400 mg/kg bw/day exposure group – relative weight), thyroid glands (males of the 200 to 800 mg/kg bw/day exposure groups and females of the 800 mg/kg bw/day exposure group – absolute and relative weight), kidneys (females of the 800 mg/kg bw/day exposure group - relative weight). Macroscopic and microscopic examination did not reveal substance treatment related abnormalities. A LOAEL of 100 mg/kg bw/day based on decreases in total protein in males and females in all dose groups (not dose related in males) was derived for 2-methylimidazole.

A number of feeding studies in rats and mice with 2-methylimidazole were conducted (NTP TR67, 2004; Chan et al., 2008; Chan et al., 2006, Tani et al., 2005), including 15 days, 14 weeks and 2 years exposure duration. The studies were conducted under GLP and according to protocols generally similar to OECD guidelines.

In the 15-day study, which was used as a dose-range finding study for the 14 weeks studies, groups of 5 male and 5 female Fischer 344 rats and B6C3F1mice were administered the test substance via feed at 0, 1200, 3000 or 10000 ppm (NTP, 2004; Chan et al., 2006). The animals were checked for clinical signs, body weights and food consumption. After the sacrifice at 15 days animals were subjected to complete necropsy and histopathological examinations. The consumed amount of substance was calculated according to Paulussen et al. (TNO report 98.390, 1998) based on theoretical average food consumption and animal body weight in subacute studies, and resulted in average daily doses of 0, 108, 297 and 900 mg/kg bw/day for rats and 0, 232, 638 and 1933 mg/kg bw/day for mice.

In the rat study, all animals survived until termination and no clinical signs were noted. Body weight gains were significantly reduced compared to the controls in the 900 mg/kg bw/day male and female rats. In rats of the 900 mg/kg bw/day group food consumption was significantly reduced for males and females. At necropsy enlarged thyroid glands were observed in mid- and high dose males and females. Increased incidences of hypertrophy of thyroid-stimulating hormone cells, evidenced by increase in basophilic cells containing cytoplasmic granules occurred in the pituitary pars distalis in all males receiving 297 and 900 mg/kg bw/day groups and in all females in the 900 mg/kg bw group. Thyroid gland follicular cell hyperplasia was observed in all males and females in the 297 and 900 mg/kg bw/day groups. Based on these data, the NOAEL for toxicity was set to 108 mg/kg bw/day.

In the 15-day study with mice, all animals survived until termination and no clinical signs were noted. Body weight gains were significantly reduced compared to the controls at 1933 mg/kg bw/day in female mice. The incidence of thyroid follicular cell hypertrophy was increased in all dosed groups of males and females. The severity of the lesions increased with increased doses. Hematopoietic-cell proliferation in the spleen occurred in controls and all dosed groups in both sexes; the severity was dose related. Based on increased incidence of thyroid follicular cell hypertrophy, the lowest dose level of 232 mg/kg bw/day was considered a LOAEL for mice.

In the 14 weeks study with rats (NTP, 2004; Chan et al., 2006), groups of 10 male and female rats were administered the test substance in diet at dose levels of 0, 625, 1250, 2500, 5000 and 10000 ppm, which resulted in average daily consumptions of 0, 40, 80, 160, 300 and 560 mg/kg bw/day test substance. Animals were observed for clinical signs and mortality, body weight and body weight gain and food consumption. Neurobehavioral examination was conducted on days 29 and 82. Sperm morphology and vaginal cytology were evaluated in 0, 160, 300 and 560 mg/kg bw/day groups 12 days prior to terminal sacrifice. Animals were sacrificed and subjected to histopathological examinations at the end of the exposure period. Additional 10 male and 10 female rats in each dose group were bled via the retroorbital sinus at days 8, 29 and 86 for evaluations of hematology, thyroid hormones (T3,T4), thyroid-stimulating hormone (TSH) and clinical chemistry.

All animals survived till termination. Body weight gains were significantly reduced in males after exposure to 2500 ppm (160 mg/kg bw/d) and above; in females after exposure to 10000 ppm (560 mg/kg bw). Reduced food consumption was observed in males and females of the high dose group.. Functional observational battery tests offered no evidence of neurobehavioral abnormalities related to exposure to 2-methylimidazole.

Relative increases in kidney and testicular weight were observed in all dosed male groups. Dose-related incidences of diffuse thyroid follicular cell hyperplasia was observed in males at 80 mg/kg bw/day and above, and in females at 160 mg/kg bw/day and above. Thyroid follicular-cell adenoma was diagnosed in two high-dose males.

The incidence of testicular degeneration was significantly increased in the high-dose males compared to controls. Spermatid heads per testis and the mean spermatid count were significantly decreased in high dose males (11.1 x 107per testis and 55.7/10-4mL suspension, respectively), compared to controls (13.0 x 107per testis and 65.1/10-4mL suspension, respectively). In females, the length of estrous cycle of the high-dose females was significantly increased (5.6 vs. 4.3 days).

Hematological evaluations at days 8, 29, and 90 showed minimal to mild anaemia characterized by slight decreases in red blood cell counts, hematocrit, and hemoglobin in male and female rats at 300 and 560 mg/kg bw/day. Female rats at 160 mg/kg bw/day showed mild anemia at terminal sacrifice. At day 8, statistically significant decreases in T3 and T4 and an increase in TSH were observed in males and females treated with 160 mg/kg bw/day and above. The decreasing trends in T3 and T4 and increasing trends in TSH levels could still be seen at days 29 and 86.

Based on the increased incidences of diffuse thyroid follicular cell hyperplasia at 2500 ppm (160 mg/kg bw/d), on the changes in absolute kidney weight in males plus the changes in creatinine concentrations in both sexes as well as the changes in absolute spleen weights plus the changes in hematology in both sexes, suggesting a treatment related erythropoietic effect, a NOAEL of 1250 ppm (80 mg/kg bw/d) was established.

In the 14-week study with mice (NTP, 2004; Chan et al., 2006), performed by the same protocol as therat study, the average test substance intake was 0, 100, 165, 360, 780 or 1740 mg/kg bw/day for males and 0, 90, 190, 400, 800 and 1860 mg/kg bw/day for females for the 0, 625, 1250, 2500, 5000 and 10000 ppm groups, respectively. All mice survived till termination. No chemical-related clinical signs of toxicity were observed.Significant reduction of body weight gain occurred in males and females of the 5000 and 10000 ppm groups. No differences were noted in food consumptions.

Increases in absolute and relative weights were observed in the liver and heart of all dosed males and the spleen of all dosed females. Compound-related and dose-dependent lesions occurred in the thyroid gland, spleen and kidney. Incidences of thyroid gland follicular-cell hypertrophy were increased in males and females treated with 2500 ppm and higher. The affected follicular epithelium showed increased in height and width. Increased incidences of hematopoietic cell proliferation in the spleen were observed in males treated with 1250 ppm and higher, and females treated at 2500 ppm and higher. Increased incidences of renal-tubule pigmentation were observed in males at 1250 ppm and higher and females at 2500 ppm and higher. Iron staining showed that the pigment granules consisted of hemosiderin.

Hematological evaluations conducted at termination of the study revealed mild to marked anemia characterized by a decrease in the number of red blood cells, hematocrit, and hemoglobin, and an increase in reticulocyte counts suggesting a hemolytic process at doses of 1250 ppm and above in male and female mice. The severity was dose-related. Serum T4 levels were decreased at days 8, 29 and 86, and T3 levels were increased at days 29 and 86 in the high-dose females. Levels of TSH were increased in the high-dose males at day 29 and high-dose females at day 86. No significant differences between exposed and control mice were found in the parameters of sperm motility or vaginal cytology.

Based on the increased absolute liver weights and the histopathological findings in spleen and kidney in males at 1250 ppm and above, as well as the hematological findings in both sexes at 1250 ppm and above, a NOAEL of 625 ppm (100 mg/kg bw/d) was established.

 

In the 2-year study with rats (NTP, 2004; Chan et al., 2008; Tani et al., 2005), groups of 60 male and female rats received the test substance in feed at concentrations 0, 300, 1000 and 3000 ppm for males and 0, 1000, 2500 and 5000 ppm for females. This corresponded to average daily intakes of 0, 13, 40 and 130 mg/kg bw for males and 0, 50, 120 and 230 mg/kg bw for females. Lower dose levels were selected for males because they appeared to be more sensitive to the effects of 2-methylimidazole in the 14-week study. The highest dose level selected for the 2-year study provided internal doses lower than the 100 mg/kg bolus dose used in toxicokinetic studies, which demonstrated evidence of saturation. When translated to concentrations in dosed feed, the highest dose levels were insufficient to reach this level.

In addition, groups of 20 male and female animals were placed in satellite groups exposed to the same dose levels for 8 days and 14 weeks. 10 animals from each group were designated for interim evaluation and were sacrificed at 6 months.

Animals were observed for mortality, clinical signs, body weights and food consumption. Blood for haematological and clinical chemistry evaluation was collected from 10 males and 10 females per each dose level at 8 days, 14 weeks and 6 months. Liver samples were collected from 10 male and 10 female satellite study rats on day 8 and at week 14 and 10 male and 10 female main study rats at 6 months for determination of total cytochrome P450 and UDP-glucuronosyltransferase activity. Gross pathological and histopathological examinations were conducted on all animals.

Survival of 2500 ppm females was significantly less than that of the controls. Mean body weights of high dose males and intermediate and high dose females were generally less than those of the controls during most of the study.Feed consumption by high dose males was less than that by the controls from week 4 through week 28, and that by high dose females was less throughout the study.

Exposure-related increases in absolute and relative thyroid gland weight were observed at 8 days, 14 weeks and 6 months in male and female rats. The pituitary gland weights of high-dose females were significantly decreased on day 8 and 14 weeks and 6 months. Relative liver weights were increased in mid- and high-dose males at 8 days and in high dose males at 6 months. In exposed groups of female rats, there were trends of increased relative liver weights at 8 days, 14 weeks, and 6 months. At 6 months, significant increases in absolute liver weight were observed in the low- and mid-dose groups of females.

The incidences of thyroid gland follicular cell hyperplasia in all exposed groups were significantly greater than in controls at 6 months and 2 years. Follicular cell hyperplasia is considered a precursor lesion to follicular cell neoplasms. The incidence of follicle mineralization in mid- and high-dose males and all exposed groups of females were significantly greater than those in the controls at 6 months. The incidences of follicle mineralization in low- and high-dose females were slightly increased at 2 years. The incidences of mixed cell foci in mid- and high-dose males and high dose females were significantly greater than in the controls at 2 years. The incidences of basophilic foci were decreased in mid- and high-dose males when compared to the controls. Foci are usually observed in some exposed F344/N rats at 6 to 12 months and in almost all exposed rats at 2 years. The incidences of bile duct hyperplasia in low- and high-dose females were significantly increased at 2 years. The low incidence of bile duct hyperplasia in mid-dose females was due to decreased survival in this group. Generally, bile duct hyperplasia developed in rats that survived for 600 days. Bile duct hyperplasia was regarded as a normal aging change that was accentuated by exposure to 2-methylimidazole. The incidence of granulomatous inflammation in high-dose females was significantly increased at 2 years. Focal granulomatous inflammation is considered to be a normal background lesion accentuated by 2-methylimidazole exposure. The incidence of granulomatous inflammation of the spleen in high dose females was significantly increased.  

Two high dose rats had thyroid gland follicular cell adenomas at 6 months. The incidences of thyroid gland follicular cell adenoma, follicular cell carcinoma and adenoma or carcinoma (combined) in high dose females were significantly greater than those in the controls at 2 years, and the incidences exceeded the historical range in controls. The incidences of follicular cell adenoma or carcinoma (combined) occurred with a positive trend in males, and the incidence in high dose male exceeded the historical control range.

In the liver, the incidences of hepatocellular adenoma or carcinoma (combined) in the mid- and high-dose exposure groups of males and females exceeded the historical ranges for controls, and the incidences of hepatocellular adenoma in females occurred with a positive trend.

The incidence of preputial gland adenoma or carcinoma (combined) in mid-dose males was significantly increased, but was not considered to be exposure-related since the incidence was within the historical range. Furthermore, there was no exposure concentration-response for these neoplasms and no supportive increases of hyperplastic lesions.

The incidence of fibroadenoma in the mammary gland of high-dose females was significantly lower than in controls at 2 years and was below the historical range in controls. The incidence of clitoral gland adenoma in high dose females was significantly lower than in controls at 2 years and was below the historical range.

The incidence of pituitary gland pars distalis adenoma of high-dose females was significantly lower than in controls at 2 years and was at the lower end of the historical range.

A minimal (less than 10%) decrease in the erythron characterized by a lack of a reticulocyte response and minimal (ca. 6%) decreases in mean cell volume and mean cell hemoglobin values was observed in male and female rats after 6 months of exposure. No Heinz body was found. Rats that were exposed to 2-methylimidazole for up to 6 months had alterations in thyroid hormone concentrations. On day 8, the thyroxine and triiodothyronine concentrations were decreased and thyroid stimulating hormone increased in exposed groups of males and females. By week 14, the effects of the thyroid hormones had ameliorated and the only evidence of an effect was increased thyroid stimulating hormone levels in the high dose females. At 6 months, the increase in thyroid stimulating hormone persisted in the females and was accompanied by a light decrease in triiodothyronine concentrations.Exposure of rats to 2-methylimidazole induced an increase in total hepatic UDP-glucuronosyltransferase at all time points evaluated through 6 months.

Based on the increased incidence of thyroid gland follicular cell hyperplasia at all dose levels, the LOAEL for toxicity was set at 13 mg/kg bw/day for males and 50 mg/kg bw/day for females. Based on an increased incidence of hepatocellular adenoma or carcinoma (combined) in the mid- and high dose groups, the NOAEL for carcinogenicity was 13 mg/kg bw/day for males and 50 mg/kg bw/day for females.

 

In the 2-year study with mice (NTP, 2004; Chan et al., 2008; Tani et al., 2005), performed according to the same protocol as the rat study, the concentrations of the test substance in diet were 0, 625, 1250 and 2500 ppm, which corresponded to the average daily intake of 0, 75, 150 and 315 mg/kg bw/day for males and 0, 80, 150 and 325 mg/kg bw/day for females. No clinical signs or mortality were observed. No effects were noted on food consumption. Mean body weights of mid- and high-dose males and high dose females were less than those of the controls during most of the study.

There were exposure-related changes in the absolute and relative weights of the thyroid gland in high dose males and females at 14 weeks and 6 months. In addition, the relative weights of the pituitary gland in mid- and high-dose males and mid-dose females were significantly greater than those in the controls at 6 months.

Absolute and relative liver weights increased in mid- and high dose males at 14 weeks and 6 months. Absolute and relative liver weights were generally increased in all groups of exposed females at all time points.

Spleen weights were significantly increased in mid- and high-dose males and high-dose females at 14 weeks and 6 months.

The incidences of thyroid gland follicular cell hypertrophy were significantly increased in all exposed groups of males and females at 6 months. The incidences of follicular cell hypertrophy in mid- and high-dose males and females were significantly greater than those in the control groups at 2 years. There was no evidence linking follicular cell hypertrophy to neoplastic development. The incidences of follicular cell hyperplasia in high-dose males and females were significantly greater than those in the control groups at 2 years. Follicular cell hyperplasia is considered a precursor lesion to follicular cell neoplasia.

The incidence of hepatocyte karyomegaly was significantly increased in high-dose males at 6 months, and the incidences were significantly increased in mid- and high-dose males at 2 years. The incidences of hepatocyte cytoplasmic vacuolization were significantly increased in high-dose males and females at 6 months. The incidences of cytoplasmic alteration in mid- and high-dose males were significantly greater than that in controls at 2 years. The incidence of Kupffer cell pigmentation in high-dose males was significantly increased at 2 years.

The incidences of hematopoietic cell proliferation in all exposed groups of males and in high-dose females were significantly greater than those in the controls at 6 months and 2 years. Hematopoietic cell proliferation is a typical regenerative response to the anemia observed in exposed mice. The incidences of hemosiderin pigmentation within macrophages in the red pulp of the spleen were significantly increased in mid- and high-dose males and females at 6 months and all groups of exposed males and mid- and high-dose group females at 2 years. The incidence of lymphoid follicular atrophy were significantly increased in mid- and high dose females at 6 months.

The incidences of bone marrow hyperplasia were significantly increased in mid- and high-dose males at 2 years.

The incidences of pigmentation in the proximal convoluted tubules were increased in high-dose males at 6 months and 2 years.

The incidences and severities of chronic active inflammation of the epididymis were increased in mid- and high-dose males at 2 years, and the incidence of sperm granuloma was increased in high-dose males. The incidences of germinal epithelial atrophy of the testis were increased in mid- and high-dose males at 2 years.

The incidence of thyroid gland follicular cell adenoma in high-dose males was significantly greater than that in the control group at 2 years and exceeded the historical range in controls. Follicular cell adenomas were also observed in one low-dose male and one high-dose female.

The incidences of hepatocellular adenoma occurred with positive trends in males and females; and the incidences in high-dose males and females were significantly increased at 2 years. The incidence of hepatocellular carcinoma was significantly increased in mid-dose males. The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in all exposed groups of males. The incidences of hepatocellular adenoma in high-dose males and females and the incidence of hepatocellular carcinoma in high-dose males exceeded the historical ranges in controls. The incidences of hepatocellular adenoma or carcinoma (combined) in exposed groups of males were at the upper end of the historical control range.

2-methylimidazole induced a mild to moderate, dose-related, macrocytic, responsive anemia in mice. The anemia was characterized by decreases in automated and manual hematocrit values, hemoglobin concentrations, and erythrocyte counts in mid- and high-dose males and females. An erythropoietic response was indicated by a mild to marked increase in reticulocyte count. The increase in reticulocyte numbers would also account for the erythrocyte macrocytosis and was indicated by increases in mean cell volume and mean cell hemoglobin values. The erythron effects were usually more severe for males.

There were alterations in thyroxine concentrations in the 2-year study, but the findings were not consistent in males and females. In high dose females, thyroxine concentrations were slightly decreased (< 20%) at 14 weeks and 6 months. Thyroxine concentrations were slightly increased (< 20%) in the high-dose males at the same time points.

In mice exposed to 2-methylimidazole for up to 6 months, hepatic UDP-glucuronosyltransferase activity (calculated as total activity per liter) was significantly increased in a few groups. The only significant effect on total hepatic cytochrome P450 in mice was a decrease at 14 weeks associated with a high control value. In exposed females, liver cytochrome P450 activities were decreased at 8 days and 14 weeks, but the depression in P450 levels was ameliorated at 6 months. A slight increase in UDP-glucuronosyltransferase activity per gram of liver was observed after 8 days in all groups of exposed female mice.

Based on the increased incidences of hematopoietic cell proliferation and the increased incidence of hepatocellular adenoma or carcinoma in all exposed groups of males, the lowest dose level of 75 mg/kg bw/day was considered a LOAEL for toxicity and carcinogenicity for male mice.

For females, a NOAEL of 80 mg/kg bw/day was established for toxicity, based on an increased incidence of thyroid gland follicular cell hypertrophy and decreases in automated and manual hematocrit values, hemoglobin concentrations, and erythrocyte counts at the next dose levels. For carcinogenicity, a NOAEL of 150 mg/kg bw/day was set for females, based on the increased incidence of hepatocellular adenoma at the next dose level.

Justification for classification or non-classification

Based on increased incidences of thyroid gland follicular cell neoplasms in the 2 -year NTP study (TR 516), there was some evidence of carcinogenic activity of 2-methylimidazole in male F344/N rats, and clear evidence of carcinogenic activity in female F344/N rats. There was some evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of thyroid gland follicular cell adenoma. There was some evidence of carcinogenic activity in male and female B6C3F1 mice based on increased incidences of hepatocellular adenoma.

For 2-methylimidazole, the relevance of the thyroid gland tumours for humans is questionable; while the induction of hepatocellular neoplasms was marginal. As the substance is not a directly acting mutagen in vivo, a threshold for the tumour induction exists, below which no tumourigenicity occurs. Based on these results, it is concluded that the evidence of carcinogenicity of 2-methylimidazole in humans is not sufficient for placing the substance into Category 1B, and the classification of the substance as Category 2, H351 (suspected of causing cancer) is warranted according to the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

A supplementary classification for repeated dose toxicity for the thyroid gland and the liver is not considered necessary, as the specific organ toxicity is already covered by the carcinogenicity classification. Furthermore, a classification for repeated dose toxicity observed in kidney and the spleen/erythopoietic system is not necessary because the effects were observed at high doses, not sufficient for classification.