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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic on bacteria (OECD 471).

Non genotoxic in the micronucleus test (OECD 487).

No mutagenic in the mammalian cell gene mutation assay (OECD 476).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

IN VITRO GENE MUTATION IN BACTERIA

Test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay, in accordance to OECD guideline 471. The experiments were carried out using Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA,in the presence and absence of a post mitochondrial rat liver supernatant (S9) mix.

The study included a preliminary solubility tests, a preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

The results of the preliminary concentration range finding test allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, test item has no mutagenic activity on the applied bacterium tester strains under the test conditions.

IN VITRO MAMMALIAN CELL MICRONUCLEUS TEST

In the In Vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, the frequency of the cells with micronuclei did not show biologically and statistically significant increases compared to the concurrent and historical controls in the absence and in the presence of metabolic activation, up to the cytotoxic concentrations.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

Test concentrations were selected on the basis of cytotoxicity results in a preliminary study (with and without metabolic activation by S9 mix). In this experiment (both run in duplicate with concurrent negative and positive controls) at least 2000 cells were analyzed for micronucleus at concentrations and treatment (exposure)/sampling (expression) intervals given below, ranging from no or little to maximum (55 ± 5 % survival) toxicity.

4/24 h treatment/sampling time

without S9-mix:         78.2, 156.3, 312.5 and 625 g/ml test item

24/24 h treatment/sampling time

without S9-mix:         78.2, 156.3 and 312.5 g/ml test item

4/24 h treatment/sampling time

with S9-mix:              156.3, 312.5, 625 and 937.5 g/ml test item

Positive and negative controls were valid.

The test item, both with and without metabolic activation, did not induce breakage and /or chromosomal loss in Chinese Hamster lung cells under the test conditions. Therefore, test item is considered as non-genotoxic with the micronocucleus test.

IN VITRO MAMMALIAN GENE MUTATION TEST

Test item dissolved in Ham's F12 medium, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix:

5-hour treatment period without S9-mix: 800, 1000, 1200, 1400, 1500 and 1600 µg/ml

5-hour treatment period with S9-mix: 800, 1000, 1200, 1400, 1500 and 1600 µg/ml

In the absence and presence of metabolic activation clear cytotoxicity (survival between 19-20 %) of the test item was observed at the highest concentration applied.

Phenotypic expression was evaluated up to 8 days following exposure.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95 % control limit).

Negative and positive controls were valid.

Overall, the test item was not mutagenic in the in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).