2-methylimidazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2-Methylimidazole did not cause gene mutations in Salmonella typhimurium (2 Ames tests). Further mutagenicity studies were performed in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

It gave negative result in one in vivo dominant lethal test with mice performed according to the OECD TG 478. This test was performed to clarify ambiguous results that had been obtained earlier when 2-methylimidazole was also tested in a mouse dominant lethal assay. However, the assay followed an early protocol established by Roehrborn and Vogel (1967) that differs from the OECD test guideline 478 in that only one dose level was tested in a low number of males. Therefore there were significant methodological deficiencies that invalidate this study and it can be concluded that the OECD 478 guideline study outweighs the ambiguous result of the early study.
The substance did not induce micronuclei induction in the two short-term tests with rat and mice, but caused statistically significant, dose-related increases in the frequency of micronucleated normochromatic erythrocytes in the peripheral blood of male and female mice in a 14-week study. The positive results observed in the 14-week micronucleus assay are likely to be caused by (an) indirect mechanism(s) rather than a direct interaction with DNA, for which a threshold is expected. The overall evidence strongly suggests that 2-methylimidazole is not a directly acting mutagen in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No further details.

Additional information

In vitro tests

2-Methylimidazole was tested in the reverse mutation assay in a study performed according to OECD guideline 471, usingSalmonella typhimuriumstrains TA98, TA100, TA1535 and TA1537 at 20 to 5000 µg/plate (standard plate test) with and without metabolic activation (BASF AG, 1985). 2-Methylimidazole was not cytotoxic nor mutagenic in any of theSalmonella typhimuriumstrains. Complete solubility of the test substance in the vehicle (water) was observed. Under the conditions tested, 2-methylimidazole is not considered to be genotoxic.

 

Another supporting test withSalmonella typhimuriumstrains TA97, TA98, TA100 and TA1535 (National Toxicology Program, 2004), both with and without metabolic activation, using rat and hamster S9 (10% and 30%) was also available for assessment. The concentration levels were 0, 100, 333, 1000, 3333 and 10000 μg/plate. Trials initially conducted with 10% S9 were repeated with 30% S9. Slight toxicity was observed at the highest concentration in TA100, without S9 and with hamster S9. No cytotoxicity was noted in other strains. The results were negative in all strains, both with and without metabolic activation.

 

In vivo tests

2-Methylimidazole was tested in a GLP-compliant rodent dominant lethal test, tested according to OECD guideline 478, with NMRI mice (RCC Umweltchemie, 1990). Male mice (40 per dose) were exposed to 90 or 180 mg/kg bw 2-methylimidazole administered via one intraperitoneal injection at the start of the test. As a positive control 80 mg/kg bw methyl methanesulfonate was administered. Each male was placed overnight in a cage with one sexually mature untreated virgin female, 14 days after successful mating (or after last mating) female mice were sacrificed by cervical dislocation, necropsied and the uteri examined for the number of live embryos and embryonic deaths. Each male mated for six consecutive periods of 4 days.

The single intraperitoneal injection of 2-methylimidazole to male mice caused ruffled fur during a period of fifteen days at the 180 mg/kg bw dose level. No further reaction to the treatment was observed. 2-Methylimidazole did not adversely affect the mating ratio of the paired mice or the pregnancy and implantation rates of the female mice. The results of the subsequent mating did not indicate dominant lethal mutations under the described conditions of this study. The results obtained from the positive control animals confirmed the suitability of the mouse strain used for this dominant lethal study.

 

In a earlier rodent dominant lethal test with NMRI mice (BASF AG, 1974), male mice (20 per dose) were exposed to 90 mg/kg bw 2-methylimidazole administered via one intraperitoneal injection at the start of the test. As a positive control 0.056 mg/kg Trenimon was administered. Each male was placed overnight in a cage with three sexually mature untreated virgin females, 18 days after successful mating (or after last mating) female mice were sacrificed and the uteri examined for the number of live embryos and embryonic deaths. Each male mated for two consecutive periods of 7 days (thus in total 20 * 3 * 2 = 120 females were used). In this study, 2-methylimidazole significantly reduced the conception rate in the first mating period. In the second mating period the conception rate was also reduced, although not significantly. In the second mating period, 2-methylimidazole also significantly increased the number of embryonic deaths (mutagenicity index). This test following an earlier protocol established by Roehrborn and Vogel in 1967 differs from the OECD test guideline 478 in that only one dose level was tested in a low number of males.

Therefore, the negative OECD 478 guideline study outweighs the ambiguous result of this early study.

 

Two GLP-compliant in vivo micronucleus tests with rats and mice according to a protocol similar to OECD guideline 474, using intraperitoneal route of administration were also available (National Toxicoly Program, 2004). Five males received 3 injections separated by 24 hours time interval, of 0, 25, 50, 100, 200 and 400 mg/kg bw (rats), or 200, 300, 400 and 500 mg/kg bw (mice) in phosphate-buffered saline. The dose levels were chosen based on the dose-range finding study, with the high dose limited by toxicity. Animals were sacrificed 24 hours after the last injection and bone marrow was obtained from femurs. 2000 polychromatic erythrocytes (PCEs) were scored per animal for frequency of micronucleated cells. Two hundred erythrocytes were counted to establish the percentage of PCEs.

In the rat study, the dose level of 400 mg/kg bw was lethal to all animals. One rat dosed with 200 mg/kg bw also died. In the study with mice, one animal of the high-dose group died. In rats, the substance did not induce an increased frequency of micronucleated PCEs in bone marrow. In mice, small increased frequencies of micronucleated PCEs were observed (ratio of 1.2 ± 0.3 for phosphate-buffered saline group, 2.5 ± 0.4 for 200 mg/kg bw group, 1.9 ± 0.3 for 300 mg/kg bw group; 2.2 ± 0.4 for 400 mg/kg bw group, 3.5 for 500 mg/kg bw group (determined for 1 animal only as the other animals died) and 23.8 ± 0.7 for positive control group), but as they were non-significant, the results of both studies were considered negative.

It can be concluded, that 2-methylimidazole failed to increase the number of micronucleated PCEs in rat and mouse bone marrow following 3 i.p. injections, a somewhat worst case testing scenario. Due to data on kinetics and the route of application, one can assume that 2-MI reached the target cells of the bone marrow.

 

Furthermore, a micronucleus assay was performed as a part of the 14-week feeding study with mice (National Toxicology Program, 2004; Chan et al., 2006). At the end of the 14-week exposure period peripheral blood samples were obtained from male and female mice. The dose levels were 0, 625, 1250, 2500, 5000 and 10000 ppm in diet, which corresponded to the average daily intakes of 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. The frequency of micronuclei in 2000 normochromatic erythrocytes was determined in each of five animals per exposure group. One thousand erythrocytes were examined to determine the percentage of polychromated erythrocytes. An exposure concentration-related increase in the percentage of PCEs (an increase in the rate of hematopoiesis) in peripheral blood was seen in males and females. Statistically significant exposure-related increases in the frequencies of micronucleated normochromatic erythrocytes (NCEs) were found in peripheral blood samples of males and females. The increases in the frequencies of micronuclei noted in female mice were greater than those observed in male mice but the overall magnitudes of the responses in males and females were similar.

 

A known mechanism which could explain the increase of MNs after 14 weeks is a responsive stimulation of erythropoiesis. The elevated peripheral blood frequencies of micronucleated erythrocytes in mice resulted from the increased splenic erythropoiesis observed in the animals treated with 2-methylimidazole. Rapidly dividing cells in the spleen may be more prone to chromosomal damage (aneuploidy and breakage) than the more slowly cycling bone marrow cells. Rapidly cell turnover may increase rate of mitotic error and the frequency of damaged cells. The authors of the study speculated that the positive effects migh have been a consequence of metabolic pathway overloading or that the metabolism to the proximate genotoxin requires time. However, for both assumptions there is no experimental evidence from the NTP studies, as there were no indications of hepatic cytochrome P450 induction after treatment for a period of 6 months nor are there any indications for accumulation of toxic metabolites over time. In addition, 2-methylimidazole is a water soluble compound which is rapily and completely absorbed after oral uptake and which is quickly excrected in the urine mainly in an unchanged form.

 

In summary, the positive results observed in the 14-week micronucleus assay are likely to be caused by (an) indirect mechanism(s) rather than a direct interaction with DNA, for which a threshold is expected. This means that the substance induces tumours by a non-stochastic mechanism. Taking into account the negative results in the in vitro tests, in the two standardized short-term intraperitoneal tests and the negative dominant lethal assay with mice, the overall evidence strongly suggests that 2-methylimidazole is not a directly acting mutagen in vivo.

Justification for classification or non-classification

Based on the results of the available studies, classification of 2-methylimidazole as genotoxic is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.