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In vitro - Ames Test:

Evaluation of the mutagenic activity of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). The study procedures described in this report were based on the most recent OECD and EEC guidelines.

Batch 99/484 of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was a light yellow viscous liquid with a purity of 99%. The test substance was dissolved in Milli-Q water, In the dose range finding test, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA 1535, TA1537 and TA98. In the second mutation assay, 1-ETHYL-3-METHYL IMIDAZOLIUM

ETHYLSULFATE was tested at the same concentration range in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain·specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro - chromosomal aberration test:

Evaluation of the ability of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE to induce chromosome aberrations in cultured peripheral human lymphocytes.

The study report describes the effect of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). The possible clastogenicity of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EEC guidelines.

Batch 99/484 of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was a light yellow viscous liquid with a purity of 99.0%. The test substance was dissolved in RPMI 1640 medium.

In the first cytogenetic assay, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested up to 2363 µg/ml (= 0.01 M) for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This dose level is the highest recommended dose level for testing.

In the second cytogenetic assay, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested up to 2363 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 1500 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE was tested up to 2363 µg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix in two independently repeated experiments. No effects of 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE is not clastogenic in human lymphocytes under the experimental conditions described in this report.

HPRT gene mutation test in vitro:

In the GLP OECD476 guideline study (BASF SE, 2010) 1 -ethyl-3-methylimidazolium ethylsulphate (EMIM-ES) did not induce gene mutations at the HPRT locus in V79 cells. Cells were incubated with the test substance with and without metabolic activation (S9 mix) for 4 h and 24 h with concentrations up to 2420 µg/mL. Minor increases of the mutation frequency occasionally occurred. However, these increases were generally not dose dependent as indicated by the lacking statistical significance. The only significant increase was judged as irrelevant fluctuation since the mutation frequency did not exceed the range of historical solvent controls. Furthermore, no cytotoxicity occured.

Micronucleus test in vivo:

Micronucleus test in bone marrow cells of the mouse with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate.

1-Ethyl-3 Methyl Imidazolium Ethylsulfate was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines.

1-Ethyl-3 Methyl Imidazolium Ethylsulfate was a light yellow liquid with a purity of ± 99.5%. The test substance was dissolved in physiological saline. Five male and five female animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight (b.w.) of cyclophosphamide (CP), respectively. Animals were dosed with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate at 375 (two groups), 190 (one group), and 95 (one group) mg/kg b.w.. After dosing the animals of the dose level of 375 mg/kg b.w. showed the following toxic signs: quick breathing, convulsions, tremors, eyes closed, ataxia, hunched posture and rough coat. The animals of the dose levels of 190 and 95 mg/kg b.w. showed no abnormalities after dosing except for two male animals of the group treated with 190 mg/kg b.w. which were breathing quickly. Bone marrow of the groups treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical negative control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, both criteria for an acceptable assay were met. The groups that were treated with 1-Ethyl-3 Methyl Imidazolium Ethylsulfate showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the negative controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the negative controls, demonstrating toxic effects on erythropoiesis. It is concluded that 1-Ethyl-3 Methyl Imidazolium Ethylsulfate is not clastogenic in the micronucleus test under the experimental conditions described in this report.


Short description of key information:
1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Furthermore, 1-ETHYL-3-METHYL IMIDAZOLIUM ETHYLSULFATE (EMIM ES) is not clastogenic in vitro in human lymphocytes under the experimental conditions described, did not induce gene mutations at the HPRT locus in V79 cells in vitro, and showed no mutagenicity/genotoxicity in vivo in the micronucleus test in vivo under the experimental conditions described.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification