Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro

- Gene mutation in bacteria:

A bacterial gene mutation assay (Ames test) was conducted in compliance with OECD guideline 471 and under GLP conditions (Andres, 2012). The main study included two series of experiments, both performed in the absence and in the presence of S9 mix. In the first experiment, the direct plate incorporation procedure was conducted with Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 at concentrations ranging from 50 to 5001 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 313 to 5001 µg/plate were analysed using the same strains, but with modification of the study design (with pre-incubation period of 20 min). No signs of cytotoxicity were observed in both experiments. The positive and negative controls included showed the expected results in each experiment. Under the conditions of both experiments, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535).

The test substance was investigated in a second bacterial reverse mutation assay (Ames test) according to OECD guideline 471 and under GLP conditions using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence or absence of Aroclor-induced rat liver S9 (Dakoulas and Meena, 2010). The assay was performed in two phases using the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice. In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed in the presence or absence of S9 activation. Precipitate was observed beginning at 50, 150, 500, 1500 or at 5000 μg per plate. Toxicity was observed, as a reduction in revertant counts, at 5000 μg per plate with tester strains TA100, TA1537 and WP2 uvrA in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the concentrations tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed in the presence or absence of S9 activation. Precipitate was observed beginning at 500, 1500 or at 5000 μg per plate. Toxicity was observed, as a reduction in revertant counts, at 5000 μg per plate with tester strains TA100, TA1535 and WP2 uvrA in the presence of S9 activation. All criteria for a valid study were met as described in the protocol. The vehicle controls and positive controls in the initial toxicity-mutation and confirmatory mutagenicity assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay. Under the conditions of this study, test substance was concluded to be negative in the bacterial reverse mutation assay.

 

- Gene mutation in mammalian cells:

The test substance was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay according to OECD guideline 476 and under GLP conditions (Schisler, 2010). The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of metabolic activation (S9) system. The concentrations ranged from 10 to 200 μg/mL in the absence of S9 and from 2.5 to 200 μg/mL in the presence of S9. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The results of the in vitro CHO/HGPRT forward gene mutation assay with the test substance indicate that under the conditions of this study, the test substance was non-mutagenic when evaluated in the absence or presence of metabolic activation.

 

- Cytogenicity in mammalian cells:

The test substance was evaluated in an in vitro chromosomal aberration assay in rat lymphocytes according to OECD guideline 473 and under GLP conditions (Schisler, 2010). Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence (4 and 24 hour treatments) or presence of S9 (4 hour treatment) with concentrations ranging from 0 (solvent control) to 200 µg/mL of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. Based upon the mitotic indices, cultures treated for 4 hours with target concentrations of 0 (solvent control), 12.5, 25, and 50 µg/mL in the absence and 0 (solvent control), 25, 75, and 150 µg/mL in the presence of S9 were selected for determining the incidence of chromosomal aberrations. Cultures treated for 24 hours in the absence of S9 with 0 (solvent control), 25, 50, and 100 µg/mL were selected for determining the incidence of chromosomal aberrations. There were no significant increases in the frequencies of cells with aberrations in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells in all assays. Based on these results, the test substance was considered to be non-genotoxic in rat lymphocytes under the conditions of this in vitro chromosomal aberration assay.


Short description of key information:
Negative Ames tests with S. typhimurium TA 97a, TA 98, TA 100, TA 102, TA 1535, TA1537 and E. coli WP2 uvrA, with and without metabolic activation.
Negative HGPRT test in CHO cells in-vitro, with and without metabolic activation.
Negative chromosomal aberration test in rat lymphocytes in-vitro, with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.