Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An incresase in the number of his+ or trp+ revertants was not observed in the standard plate and preincubation tests either with or without S-9 mix. Therefore, the test substance is not mutagenic in the Ames tests under the experimental conditions chosen.
The test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, 2-ethyl-4-methylimidazole is considered to be non-mutagenic in this HPRT assay.
2-Ethyl-4-methylimidazole is considered to be non-mutagenic in the in vitro Micronucleus test system, when tested up to precipitating or the highest evaluable concentrations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reverse mutation assays:

In the first test (according to OECD guideline 471, no data on GLP), the substance was tested usingSalmonella typhimuriumstrains TA98, TA100, TA1535 and TA1537 in a standard plate test and a preincubation test (1989). In both experiments, 20, 100, 500, 2500 and 5000 µg/plate were tested in all strains. All test were performed both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). No precipitation of the test substance was found and no bacteriotoxic effect was observed. An increase in the number his+revertants was not observed both in the standard plate test and in the preincubation test either with or without S-9 mix. According to the results of the present study, the test substance,2 -ethyl-4-methylimidazole is not mutagenic in the Ames test under the experimental conditions chosen here.

In the second test (according to OECD guideline 471, GLP compliant), the substance was tested using Salmonella typhimuriumstrains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain E. coli WP2 uvrA in a standard plate test and a preincubation test (2012). In both experiments, 33, 100, 333, 1000, 2500 and 5000 µg/plate were tested in all strains. All test were performed both with and without metabolic activation (phenobarbital/ß-naphthoflavone-induced rat liver S-9 mix). No precipitation of the test substance was found and a bacteriotoxic effect was occasionally observed in the preincubation test depending on the strain and test conditions at 5000 µg/plate. An increase in the number his+or trp+revertants was not observed both in the standard plate test and in the preincubation test either with or without S-9 mix. Thus, under the experimental conditions of this study, the test substance2-ethyl-4-methylimidazole is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and presence of metabolic activation.

 

HPRT assay:

In a GLP-compliant study performed according to OECD guideline 476, the potential of 2-ethyl-4-methylimidazole to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated (2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.The cell cultures in experiment I and II were evaluated at the following concentrations 75, 150, 300, 600 and 1200 µg/mL. The maximum concentration of 1200 μg/mL was equal to a molar concentration of about 10 mM. The test item was dissolved in deionised water.Precipitation was observed in the second experiment with metabolic activation at 600 µg/mL and above following a 4 hours treatment period. Cytotoxic effects in both parallel cultures were observed in the second experiment without metabolic activation at 600μg/mL and above following 24 hours treatment. The recommended cytotoxic range of approximately 10 - 20% relative cloning efficiency I or relative cell density was covered without metabolic activation in experiment II. No relevant cytotoxicity was noted after 4 hours treatment with and without metabolic activation.No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2-ethyl-4-methylimidazole is considered to be non-mutagenic in this HPRT assay.

Micronucleus test (in vitro):

In a GLP compliant study performed according OECD guideline 487 the substance 2 -ethyl-4 -methylimidazole was assessed for its potential to induce micronuclei in V79 cells of the Chinese hamster in vitro (clastogenic or aneugenic activity). (2013) Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested. In the first experiment with 4 hours exposure and 24 hours harvest time; with and without S9 mix: the concentrations 0; 34.38; 68.75; 137.5; 275; 550 and 1100 μg/mL were tested. The concentrations 275, 500, and 1100 µg/mL were evaluated for cytogenetic damage. In the second experiment without metabolic activation, 24 hours exposure, and 24 hours harvest time concentrations of 0, 34.38; 68.75; 137.5; 275; 550 and 1100 μg/mL were tested and the concentrations 68.75, 137.50, and 275 µg/mL were evaluated for cytogenetic damage. In the second experiment with metabolic activation, 4 hours exposure, and 24 hours harvest time, the concentrations 0; 103.13; 206.25; 412.5; 825 and 1100 μg/mL were tested and the concentrations 412.5, 825, and 1100 µg/mL were evaluated. A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2 000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In this study, cytotoxicity indicated by reduced relative increase in cell count (RICC) was observed in both experimental parts without S9 mix. In the experimental parts with S9 mix no cytotoxicity indicated by reduced RICC was observed. In none of the experimental parts the proliferation index (PI) was adversely influenced. On the basis of the results of the present study, the test substance did not cause any increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. Thus, under the conditions of this in vitro test in V79 cells, 2-ethyl-4-methylimidazole is considered not to have a chromosome-damaging (clastogenic) effect and not to induce numerical chromosomal aberrations (aneugenic activity) in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the results of the Ames tests, the in vitro micronucleus test, and the HPRT assay, 2 -ethyl-4 -methylimidazole does not need to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.