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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Results after valid testing in in vitro test systems (ames test and UDS test) as well as in vivo test systems (MNT and SCE) are all negative. Therefore the substance is considered to be non genotoxic.

Additional information

Gene Mutation

In vitro studies

Studies in bacterial cells:

In a reverse gene mutation assay, bacterial strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium were exposed to the test substance (no data on substance purity) in acetone at concentrations of 0, 25, 75, 225, 675 and 2,025 µg/plate in the presence and absence of mammalian metabolic activation using the standard plate method. A confirmatory second experiment was performed only with bacteria strains TA 1535 and TA 1537 which gave equivocal responses in the first experiment. Each concentration was tested in triplicate cultures per experiment.

Cytotoxicty was absent at the tested concentrations but tests were performed up to precipitating concentrations. At the concentrations of 225 µg/plate and above, substance precipitation in soft agar was observed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study does not fully satisfy the requirements of OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data since strains capable of detecting cross-linkers and/or oxidizing agents are not included in the panel of tested strains. The study is nevertheless considered as acceptable for assessment as it is performed similarly to the protocol of OECD 471.

Studies in mammalian cells:

In a genotoxicity test (unscheduled DNA synthesis assay), primary rat hepatocyte cultures were exposed to the test substance (commercial grade) in acetone at concentrations of 0, 0.4, 2.0, 10.0 and 50.0 µg/mL (based on the results of a preliminary cytotoxicity test (3.125 µg/mL - 200 µg/mL)). Treatment duration was 5 hours. Each test concentration was applied in triplicates and 150 nuclei were evaluated per concentration.

Cytotoxicity was seen with the test substance in the preliminary tests at concentrations higher than 100 µg/mL. Substance precipitation was present from concentrations of 12.5 µg/mL and higher. The positive controls induced the appropriate response (14.8 silver grains vs. 1.04 in controls). There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [1.23 – 1.61 silver grains versus 1.04 silver counts in control cultures] was induced.

This study is classified as acceptable as it fully satisfies the requirements for Test Guideline OECD 482 for other genotoxic mutagenicity data.

In vivo Studies

The necessity for the evaluation of gene mutation in vivo is not indicated in available in vitro data.

Chromosomal Aberration

In vitro studies

In accordance with column two of REACH Annex VIII, an in vitro chromosomal aberration test is redundant as adequate in vivo data for the evaluation of chromosomal aberration is available.

In vivo studies

In a Cricetulus griseus Chinese Hamster bone marrow micronucleus assay, 3 animals per sex per dose were treated with the test substance (commercial grade) at doses of 1,250, 2,500, 5,000 mg/kg bw in 0,5% aqueous solution of sodium-carboxymethylcellulose (CMC). The control animals received the vehicle. Single dose applications were performed once daily for two consecutive days via gavage (20 mL/kg bw). Animals were sacrificed 24 hours after the second application and bone marrow cells were harvested. Following parameters were evaluated: single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploid cells. At least 2 animals per dose were evaluated.

There were no signs of overt systemic toxicity in the preliminary toxicity test as well as in the main study at doses up to 5,000 mg/kg bw. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated PCEs in bone marrow after treatment with the test substance.

This study does not fully satisfy the requirements of OECD 474 for in vivo mutagenicity (Mammalian Erythrocyte Micronucleus Test) data since only 1,000 PCEs instead of 2,000 PCEs were scored per animal per dose. In addition, the proportion of PCE to NCE was not studied in the test. Moreover, a maximum of only 2 animals were analysed in some doses. The study is nevertheless considered as acceptable for assessment as there are no other major deviations from the guideline standard.

In a Cricetulus griseus Chinese Hamster sister chromatid exchange assay (SCE), 4 animals per sex per dose were treated with the test substance (commercial grade) at doses of 1,250, 2,500, 5,000 mg/kg bw in 0,5% aqueous solution of sodium-carboxymethylcellulose (CMC). The control animals were treated with the vehicle. Doses were applied once via gavage (20 mL/kg bw) 2 hours after 5-bromodeoxyuridine (BUdR) had been administered (subcutaneous implantation). 2 hours before sacrifice, Colcemide (10 mg/kg bw) was administered intraperitoneally. Bone marrow cells were harvested upon sacrifice (24 hours after gavage), prepared and scored for SCE. Two animals per dose per sex were evaluated. Twenty five differently stained metaphases of the second cell-cycle with BUdR-substitution were analysed per animal.

There were no signs of overt systemic toxicity in the preliminary toxicity test as well as in the main study at doses up to 5,000 mg/kg bw. The positive control group showed a highly significant increase of SCE's per cell (5.96) in comparison with the negative control (3.15). No significant increase of the number of SCE's was found for the test substance in comparison with the negative control at any dose level.

This study does not fully satisfy the requirements of EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay) as only 4 animals per sex per dose were treated in the study. In addition only 2 animals per sex per dose were analysed. Plus, the sequence of test substance and BUdrR administration is reversed. The study is nevertheless considered as acceptable for assessment as there are no other major deviations from the guideline standard.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC 944/2013, classification as a mutagen is not warranted.