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The mutagenic potential of 2-Hexyl-2 ‘-hydroxy-5 ‘methyldecananilide (AF-654) was assessed in three in vitro tests.

A bacterial reverse mutation assay (Ames test) was performed using AF-654, according to OECD Guideline 471 (Buskens, 2003). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coliWP2 uvrA were exposed to the test substance at concentrations of 3, 10, 33, 100 and 333 µg/plate, using the plate incorporation assay.

At 333 µg/plate the test substance precipated on the plates. Due to the limited solubility of the test substance, 333 µg/plate was selected as highest concentration. Both experiments were performed in the absence or presence of a liver microsomal activation system (S9 mix). The S9 mix contained 5% S9 fraction in the first experiment and 10% S9 fraction in the second experiment. The test substance did not induce mutations in any of the S. typhimurium strains or in E. coli WP2 uvrA, with or without metabolic activation. No cytotoxic effects were observed up to and including the highest concentration tested. The positive controls included in the assay showed the expected results and verified the validity of the assay.

AF-654 was tested in a mammalian cell gene mutation assay according to OECD Guideline 476 (Lazová, 2013). Based on the results of a cytotoxicity range-finding study 2 main mutation experiments were conducted with duplicate cultures in the absence and presence of metabolic activation. Chinese hamster lung V79 cells were exposed to AF-654 at concentrations ranging from 18.75 to 400 µg/mL in the absence of metabolic activation, and from 12.5 to 300 µg/mL in the presence of metabolic activation, respectively. The upper dose limit in the absence of S9 mix was limited by precipitation, whereas the highest concentration tested in the presence of S9 mix was limited by toxicity. The treatment period for all the cultures was 3 hours. In one of the cultures tested without metabolic activation, a statistically significant increase (p<0.01) in mean mutant frequency was observed at 300 µg/mL. Since this increase in mean mutant frequency was not greater than 3-fold above that of the concurrent vehicle control, there was no dose-response relationship, and it was not duplicated in the second culture, the statistical significance at 300 µg/mL was considered to be biologically irrelevant. No increase in mean mutant frequency was noted following exposure to AF-654 at any dose level in the experiments with metabolic activation. Cytotoxicity was observed in the experiments with metabolic activation at concentrations ≥ 75 µg/mL, which yielded in relative plating efficiencies of 4 - 15%. The positive controls significantly increased mutant frequency, demonstrating the validity of the test system. In conclusion, AF-654 did not induce gene mutations in vitro at the HPRT locus of V79 Chinese hamster lung cells when tested under the conditions of this study.

The potential of AF-654 to induce chromosomal aberrations was tested in cultured human peripheral lymphocytes, according to OECD Guideline 473 (Buskens, 2004). The possible clastogenicity of AF-654 was determined in two independent experiments. The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h exposure time) or on toxicity (24 and 48 h continuous exposure time), inhibition of the mitotic index of approximately 50% or greater.

In the first experiment AF-654 was tested at concentrations of 10, 33 and 100 µg/mL for a 3 h exposure time with a 24 h fixation time with and without metabolic activation. Precipitation of the test substance in the culture medium was observed at 100 µg/mL.

In the second experiment the cultures were treated with the test substance at concentrations of 19, 20 and 25 µg/mL for 24 h (fixation time 24 h) and with 3, 10 and 15 µg/mL for 48 h (fixation time 48 h) in the absence of S9 mix, respectively. Appropriate toxicity (indicated by a mitotic index of about 50%) was reached at the highest dose levels used for the continuous exposure experiments. In the presence of S9 mix AF-654 was tested at concentrations of 10, 33 and 100 µg/mL for a 3 h exposure time with a 48 h fixation time. At 100 µg/mL the substance precipitated in the culture medium.

AF-654 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations compared with the solvent control. The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay.

Thus, based on these results AF-654 is considered not to be clastogenic in human lymphocytes.

2-Hexyl-2 ‘-hydroxy-5 ‘methyldecananilide (AF-654) showed no evidence of clastogenic and mutagenic potential with and without metabolic activation in 3 in vitro test systems.

Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
In vitro:
Bacterial reverse mutation assay, Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in E. coli WP2uvrA
Mammalian cytogenetic test, chromosome aberrations (OECD 473): negative with and without metabolic activation in human peripheral lymphocytes
Mammalian cell gene mutation test / HPRT test (OECD 476): negative with and without metabolic activation in V79 cells

In vivo: no further testing required

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test 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.