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Genetic toxicity in vitro

Description of key information

Three in vitro genetic toxicity studies were conducted on C4 methacrylate. The results of the studies were:

 

Chromosome aberration: Negative with and without metabolic activation when tested according to OECD 473 (2016).

 

Ames assay: Negative with and without metabolic activation when tested according to OECD 471 (1997).

 

Mouse Lymphoma assay: Negative with and without metabolic activation when tested according to OECD 490 (2016).

Additional information

The test substance was examined for its potential to induce structural chromosomal aberrations in cultured human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix). The study was conducted according to OECD 473 and was conducted in compliance with OECD GLP regulations. Dimethylsulfoxide (DMSO) was used as the solvent. Dose levels ranging from 3.13 to 500 μg/ml were tested. The maximum final concentration (500 μg/ml) was based on the solubility of the test substance in the culture medium. The purity of the test substance (99.3%) was taken into account while preparing the dosing solutions. In all instances, duplicate cultures were used. The mitotic index was used as measurement for cytotoxicity. In the first chromosomal aberration test, in both the absence and presence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment). In the second test, the treatment/harvesting times were 24/24 hours (continuous treatment). Solvent and positive controls were run in parallel. In the first test, in the pulse treatment groups both with and without S9-mix, the highest test substance concentration induced a moderate cytotoxicity to the cells. At the higher concentrations, cytotoxicity showed some fluctuation and did not exceed the maximal cytotoxicity of 55±5% as stated in the OECD guideline 473. In both pulse treatment groups, four test substance concentrations (with S9-mix: 50, 200, 400 and 500 μg/ml; without S9-mix: 50, 100, 300 and 500 μg/ml), together with the cultures of the solvent and the positive control (Cyclophosphamide) were analyzed. In the second test, a dose-related increase in cytotoxicity was observed. It should be noted that, at the four highest tests substance concentrations (200 – 500 μg/m) the observed cytotoxicity (as measured by mitotic index) was comparable between the concentrations. Four test substance concentrations (12.5, 50, 150 and 500 μg/ml), together with the cultures of the solvent and the positive control (Mitomycin C) were analyzed. In both chromosomal aberration tests, the numbers of cells with structural aberrations observed in the solvent control (1% DMSO) cultures were within the historical range. Treatment with the positive controls Cyclophosphamide and Mitomycin C resulted in statistically significant increases in the numbers of metaphases containing one or more chromosomal aberrations, when compared to the numbers observed in the cultures treated with the solvent. This demonstrates the validity of the study. In both chromosomal aberration tests, the test substance did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment conditions analyzed, when compared to the number of aberrant cells observed in the solvent control cultures. Based on the results of the study, the test article is not clastogenic to cultured human lymphocytes.

 

The test substance was examined for possible mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix). The study was performed according to OECD 471 (1997) and was conducted in compliance with OECD GLP. The test article was dissolved in DMSO. Two independent tests were performed. In both tests, all strains both in the absence and presence of S9-mix were tested. In the first test, five concentrations, ranging from 62 to 5000 μg/plate were tested and in the second test, six concentrations, ranging from 125 to 3000 μg/plate were tested. In the second test the maximum concentration tested was limited by the solubility of the test substance in the final treatment mix. In both tests, negative controls (solvent) and positive controls were run simultaneously with the test substance. In both tests, the mean numbers of his+ and trp+ revertant colonies of the negative controls used were within the acceptable range in all strains and the positive controls gave the expected increase in the mean numbers of revertant colonies. Therefore, both tests were considered valid. In both tests, no toxicity was observed in any strain, which was evidenced by an absence of a clearing of the background lawn of bacterial growth compared to the negative controls, absence of a decrease in the mean number of revertants was observed and absence of pinpoint colonies. In both tests, a precipitation of the test substance was observed in the final treatment mix and on the agar plates at and above 1667 μg/plate and at and above 1000 μg/plates in the first and second test, respectively. In the first test, the revertants could not be counted at a concentration of 5000 μg/plate due to the precipitation on the agar plates. In both tests, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control with strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA, in both the absence and presence of S9-mix. It is concluded that the results obtained in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions used in this study.

 

The test material was examined for its potential to induce gene mutations at the Tk-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). The study was conducted according to OECD 490 and was conducted in compliance with OECD GLP regulations. Prior to the main study, a dose-range finding study (DRF) was performed. A single test was conducted. In the test, ten single cultures were treated for 4 hours in the presence and absence of S9-mix. The test substance was dissolved in DMSO. The highest nominal concentration of evaluated for mutagenicity was 500 μg/ml both in the absence and presence of S9-mix, which was the maximum feasible concentration based on the solubility of the test substance in the culture medium. The purity of the test substance (99.3%) was taken into account while preparing the dosing solutions. 3-methylcholanthrene (MCA) was used as positive control substance in the presence of S9-mix; this single positive control response demonstrates both the activity of the metabolic activation system and the responsiveness of the test system. Negative control cultures were treated in duplicate with cell culture medium supplemented with 1% DMSO. Negative and positive controls met all the acceptance criteria and therefore the results were considered valid. Following 4 h exposure in the absence and presence of S9-mix, the test material was cytotoxic to the cells resulting in a reduction in the RTG. The mean RTG at the highest three concentrations evaluated (500, 400 and 320 μg/ml) was 39, 48 and 56% in the absence and 19, 39 and 60% in the presence of S9-mix, respectively. Following 4 h exposure in the absence and presence of S9-mix, no increase in the MF by more than 126 mutants per 1,000,000 clonable cells, i.e. no positive response, compared to the negative control was observed at any of the concentrations tested. Based on the results of the study, the test article is not mutagenic at the Tk-locus of mouse lymphoma L5178Y cells in the presence and absence of metabolic activation (S9 -mix).

Justification for classification or non-classification

Based on the results of the studies, C4 methacrylate does not meet the classification criteria for genotoxicity.