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Key value for chemical safety assessment

Additional information

In vitro data

Bacterial reverse mutation:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14. Furthermore, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF and the USA, EPA (TSCA) OPPTS harmonised guidelines. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation and pre-incubation methods, at five and seven dose levels, respectively, both with and without metabolic activation. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate using the plate incorporation method and 5, 15, 50, 150, 500, 1500 and 5000 µg/plate using the pre-incubation method. The test material caused a visible reduction in the growth of the bacterial background lawns of S. typhimurium strains TA100 (absence of S9-mix only) and TA1537 (absence and presence of S9-mix) at 5000 µg/plate employing both exposure methods. No signs of toxicity where displayed by any of the remaining bacterial strains, at any dose level with or without S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains. The vehicle controls gave revertant colony counts within the normal range. The positive controls gave the expected increases in revertants, validating the sensitivity of the assay and the efficacy of the S9-mix. Under the conditions of the test, the test material was considered to be non-mutagenic in bacteria.

Chromosome aberration test in vitro:

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 and EU Method B.10. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, including at least one precipitating concentration. Vehicle and positive controls were run concurrently. Four treatment conditions were used for the study. In Experiment 1, cultures were exposed for 4 hours with a 20 hour expression time, both in the presence and absence of metabolic activation (an induced rat liver homogenate at a final concentration of 2%). In Experiment 2, the 4 hour exposure period was repeated with metabolic activation (1 % final S9 concentration); whilst in the absence of metabolic activation cells were assayed with a continuous 24 hour exposure time. The frequencies of chromosome aberrations in both vehicle and positive controls were within the expected range and verified the sensitivity of the assay and the efficacy of the S9-mix.
Under the conditions of the test, exposure to the test material did not induce a dose-response reduction in mitotic indexes, or statistically significant increases in the frequency of structural chromosome aberrations, both in the presence and absence of metabolic activation. Furthermore, no polyploidy cells were recorded in any of the exposed cultures. In conclusion the test material was considered to be non-cytotoxic and non-clastogenic.

Gene mutation in mammalian cells:

A study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells in vitro in accordance with the standardised guidelines OECD 476, EU Method B.17, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the EPA OPPTS 870.5300. The study was assigned a reliability score of 1 according to the criteria of Klimisch (1997).

During the study CHO cells were treated with the test material at up to eight dose levels, in duplicate, together with negative and positive controls. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.

Four treatment conditions were used for the test. In Experiment 1, a 4 hour exposure in the presence of 2 % S9 (dose levels 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL) and a 4 hour exposure in the absence of metabolic activation (dose levels 0, 239.06, 478.13, 956.25, 1912.5, 2868.75 and 3825 µg/mL). In Experiment 2, the 4 hour exposure was repeated using a 1 % final S9 concentration dose levels 0, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL), whilst in the absence of metabolic activation the exposure time was increased to 24 hours (dose levels 0, 29.88, 59.77, 119.53, 239.06, 478.13, 956.25, 1912.5 and 3825 µg/mL).

In Experiment 1, a precipitate of the test material was seen at the end of exposure at and above 478.13 and 239.06 µg/mL in the absence and presence of S9, respectively. In Experiment 2, precipitate of the test material was seen at the end of the exposure period at and above 119.53 µg/mL in the presence of S9 and at and above 59.77 µg/mL in the absence of S9.

The 4 hour exposure group in the presence of S9 demonstrated no marked toxicity in any of the Experiments. The 4 hour exposure group in the absence of S9 demonstrated marked toxicity in Experiment 1 and the 24 hour exposure group also demonstrated a dose related increase in toxicity. The test material demonstrated no dose related increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The negative and positive controls gave mutant frequencies within the range expected, indicating the satisfactory performance of the test and of the metabolising system. Therefore, under the conditions of this study, the test material is considered to be non-mutagenic to CHO cells at the HPRT locus.


Justification for selection of genetic toxicity endpoint
Multiple studies have been provided to address the different endpoint of genetic toxicity, each addressing different types of genetic toxicity. Since all the studies showed negative results, a single study could not be selected as key over the others.

Short description of key information:
In vitro Gene Mutation Study in Bacteria:
Ames: Harlan (2013), OECD 471 and EU Method B.13/14; Negative (with and without metabolic activation).

In vitro Mammalian Cell Cytogenicity:
Chromosome Aberration: Harlan (2013), OECD 473, EU Method B.10; Negative (with and without metabolic activation).

In vitro Gene Mutation Study in Mammalian Cells:
Chinese hamster Ovary (CHO): Harlan (2013), OECD 476, EU Method B.17, and EPA OPPTS 870.5300; Negative (with and without metabolic activation).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.