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An Ames test was conducted with the following tester strains: Salmonella typhimurium: TA 98, TA 100, TA 102, TA 1535 and TA 1537. In two independent experiments, several concentrations of NPEO were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls (negative and/or solvent as well as positive control), were tested in triplicateup to 2,500 µg/plate. Precipitation of the test substance was observed in all tester strains used in experiments 1 and 2 (with and without metabolic activation). Toxic effects were noted in all tester strains used in the experiments. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiments 1 and 2. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. Under the experimental conditions, NPEO did not cause gene mutations by base pair changes or frame shifts in the genome of the tester strains used. NPEO was considered not mutagenic in this bacterial mutation assay (Wallner B, 2010).

A mammalian cell gene mutation assay was conducted to investigate the potential of NPEO to induce mutations at the TK (thymidine kinase) locus in L5178Y mouse lymphoma cells. The study was performed according to OECD Guideline 473. The selection of the concentrations was based on data from pre-experiments. Two independent experiments, with and without a metabolic activation system, were conducted at the following concentrations:

Experiment 1:

5, 10, 15, 18, 21, 24 and 27 μg/mL (without S9 mix)

5, 10, 15, 20, 25, 30, 35 and 40 μg/ mL (with S9 mix)

Experiment 2:

0.5, 2, 10, 15, 21, 23, 27 and 30 μg/ mL (without S9 mix)

30, 38, 42, 46, 50, 54, 58 and 62 μg/ mL (with S9 mix)

Experiment 2 without metabolic activation was performed as a 24 h long-term exposure assay.

Growth inhibition was observed in both experiments, with and without metabolic activation. In experiments 1 and 2 (with metabolic activation), relative total growth (RTG) was 4.94 and 10.68%, respectively at the highest concentration (40 and 62 μg/ mL respectively). The highest concentration evaluated without metabolic activation was 27 and 30 μg/ mL in experiments 1 and 2, with an RTG of 11.56 and 12.12%, respectively. Colony sizing showed no clastogenic effects in either experiment (with and without metabolic activation). There was no biologically relevant increase of mutants. Under the test conditions, NPEO did not cause a relevant increase of mutants at the TK locus in L5178Y mouse lymphoma cells and was considered to be non mutagenic in this assay (Trenz K, 2010).

A chromosome aberration test was conducted to investigate whether NPEO has the ability to induce structural chromosome aberrations in human lymphocytes. The study was performed according to OECD Guideline 473 in compliance with GLP. The chromosomes were prepared 24 h after start of treatment with the test substance. The treatment interval was 4 h with and without metabolic activation (experiment 1) and 4 h with and 24 h without metabolic activation (experiment 2). Two parallel cultures were set up. Per culture, 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were used in the main experiment:

Experiment 1:

8, 15, 30, 50, 60, 70, 80, 100 and 125 μg/ mL, 4 h treatment and 24 h preparation interval (with S9)

4, 8, 15, 25, 30, 35, 40, 50 and 60 μg/ mL, 4 h treatment, 24 h preparation interval (without S9)

Experiment 2:

15, 30, 40, 50, 60, 65, 70, 75 and 80 μg/ mL, 4 h treatment, 24 h preparation interval (with S9)

5, 10, 15, 20, 25, 30, 35 and 40 μg/ mL, 24 h treatment, 24 h preparation interval (without S9)

In experiment 1, precipitation of the test item was observed at all concentrations evaluated with and without metabolic activation. In experiment 2, precipitation of the test item was observed at all concentrations evaluated with metabolic activation. Without metabolic activation, precipitation was observed at the concentration of 30 μg/ mL and higher. In experiment 1, toxic effects of the test item were noted with metabolic activation at the concentration of 70 μg/ mL and without metabolic activation at concentration of 35 μg/mL. In experiment 2 with metabolic activation, toxic effects of the test item were observed at concentration of 65 μg/ mL and higher and without metabolic activation were observed at concentration of 30 μg/mL and higher. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test substance with and without metabolic activation. The aberration rates of all dose groups were within the historical control data of the negative control. In the experiments 1 and 2 with and without metabolic activation, no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the controls. EMS (400 and 600 μg/ mL) and CPA (5 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. Under the test conditions, NPEO did not induce structural chromosomal aberrations in human lymphocyte cells and was considered to be non clastogenic this assay (Singh S, 2010).

Short description of key information:

Based on the results of in vitro studies, NPEO is considered to be non-genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

NPEO was negative for genotoxicity in in vitro studies. Therefore no classification is therefore required for this endpoint according to CLP (EC 1272/2008) and DSD (67/548/EEC) criteria.