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IN VITRO BACTERIAL GENE MUTATION

In the key study (Hargitai, 2012) the mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with standardised guidelines.

S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA were exposed to the test material at concentrations of 5000, 2500, 1250, 625, 312.5, 156.25 and 78.125 µg/plate using the preincubation method. Positive and solvent controls were run concurrently for comparison.

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), both in the presence and in the absence of metabolic activation.

IN VITRO MAMMALIAN CELL CYTOGENICITY

In the key study (Hargitai, 2013a) the clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with standardised guidelines.

Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. The experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.

Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication ofpolyploidy or endoreplication.

The test material is therefore considered not to be clastogenic in this test system.

IN VITRO MAMMALIAN CELL GENE MUTATION

In the key study (Hargitai, 2013b) the mutagenic potential of the test material was determined in an in vitro mouse lymphoma assay, conducted under GLP conditions and in line with standardised guidelines.

Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations of 5000, 1666.7, 555.6, 185.2, 61.73 and 20.58 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed as two experiments, both with a three hour exposure period; however the first was performed in the presence of metabolic activation and the second without. Assay 2 was again performed as two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine used as the selection agent.

Treatment with the test material did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of the study.


Justification for selection of genetic toxicity endpoint
One single study could not be selected at key, since all three studies are necessary to address this endpoint. The three studies assess different types of in vitro genetic toxicity; bacterial gene mutation, mammalian chromosome aberrations and mammalian gene mutation. All three studies were performed under GLP conditions and in accordance with standardised guidelines. The studies have been assigned a reliability score of 1 in line with Klimisch (1997).

Short description of key information:
Ames test: Negative, OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100, Hargitai (2012).
Chromosome aberration: Negative, OECD 473, EU Method B.10 and EPA OPPTS 870.5375, Hargitai (2013a).
Mouse lymphoma assay: Negative, OECD 476 and EU Method B.17, Hargitai (2013b).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the submitted in vitro data, testing does not indicate any evidence of genetic toxicity of the test material. In accordance with criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for genetic toxicity.