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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce an increase in mutation frequency in the Ames test, did not induce gene mutations at the HPRT locus in V79 cells in the HPRT assay and did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, and therefore, it can be concluded that the test substance does not exert genotoxic effects.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There are three in vitro studies available that assessed the possible genotoxic potential of the test substance. They were performed according to GLP and internationally accepted guidelines.

Ames assay:

This key study was performed according to OECD 471. The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test substance was tested in concentrations of 15 to 1500 µg per plate. Both in the absence and presence of S9-mix the test item was bacteriotoxic towards various strains at different concentrations, however precipitation of the test compound was not observed.

The positive controls confirmed the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency both in the presence and absence of a metabolic activation system.

HPRT assay:

In this study the potential of the test item to induce gene mutations was assessed in an in vitro mammalian cell gene mutation test according to OECD 476.

The HPRT locus in the Chinese hamster cell line V79 was the target gene.

The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (1762 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity and phase separation caused by the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

In vitro micronucleus assay:

In the current study the potential of the test item to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487.

The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of structural chromosomal damage and aneugenicity in cells that have undergone cell division during or after exposure to the test substance. Micronuclei arise from chromosomal fragments or whole chromosomes and are inducible by clastogens or agents affecting the spindle apparatus.

The induction of cytogenetic damage in human lymphocytes was assessed in two independent experiments and in each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The test item was dissolved in DMSO. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix.

The cells were stimulated for 48 hour with phytohemeagglutinine (PHA) to activate the proliferation, before exposure. After exposure, the cells were washed and left to recover for 16 hours before the cytokinesis was blocked by cytochalasin B for 20 minutes. The cells were prepared 40 hours after start of treatment with the test item.

The highest applied concentration in this study (1007 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. The chosen treatment concentrations are in Experiment I (4 hours with and without S9 mix): 107, 188, 329, 575 and 1007 μg/mL and in Experiment II (20 hours without S9 mix): 107, 187 and 327 μg/mL.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleate cells was observed after treatment with the test item. However, in Experiment IB in the absence of S9 mix statistically significant increases in micronucleate cells were observed after treatment with 107 and 329 μg/mL (0.85 and 1.00 %). Since these values are clearly within the range of the historical solvent control data (0.08 – 1.12 % micronucleate cells), they are considered as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test.

Conclusions:

Taken together, the test substance did not induce an increase in mutation frequency in the Ames test, did not induce gene mutations at the HPRT locus in V79 cells in the HPRT assay and did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, and therefore, it can be concluded that the test substance does not exert genotoxic effects in the three key studies performed and under the conditions of testing.

Justification for selection of genetic toxicity endpoint

All three key studies are well documented and according to GLP and internationally accepted guidelines.

Justification for classification or non-classification

As no genotoxic effects are observed in the available studies addressing genetic toxicity, classification for genetic toxicity under EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) is not required.