[2-[4-(dihexylamino)-2-methylbenzylidene]benzo[b]thien-3(2H)-ylidene]malononitrile S,S-dioxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the key study, the test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test, according to the OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test item was soluble in dimethyl sulphoxide and showed mild precipitation at 0.9 mg/plate. Based on these results, the initial cytotoxicity test was performed at 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 mg/plate. Initial cytotoxicity test was performed with TA100 both in the presence and absence of metabolic activation system. The tester strain, TA100 treated with test item, in the presence and absence of metabolic activation system, resulted in no cytotoxicity when compared to vehicle control.

On the basis of cytotoxicity results 0.9 mg/plate was considered as the highest test concentration for mutation assay.

The concentrations tested in the mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test. The two independent trials (trial 1 and 2) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of 0.009, 0.03, 0.09, 0.3 and 0.9 mg/plate Vehicle control (dimethyl sulphoxide) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

The tester strains used in the mutation assay wereSalmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.

The number of revertant colonies in the positive controls resulted in 2.2 to 17.0 fold increase under identical conditions.

Based on the results obtained from the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.9 mg/plate under the test conditions.

In the supporting study the potential mutagenic effect of the test item was investigated using Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 100 and TA 98 strains. The experiments were performed with and without metabolic activation. Dimethylsulphoxide was used as solvent. The substance was tested at the concentrations of 1.6, 8, 40, 200, 1000 and 5000 µg/plate. Adequate positive controls were also assayed.

No test item treatments of any of the test strains, either in the absence or in the presence of S9, induced a statistically signifrcant increase in revertant numbers, when the data were analysed at the 1 % level using Dunnett's test. The study was considered to have provided no evidence of any test item mutagenic activity. All maximum test dose plates were manually counted due to the dark nature of the test article, which affected the accuracy of the automated counter.

No clear evidence of toxicity, as would normally be indicated by a thinning of the background bacterial lawn, was observed following any of the test treatments.

Precipitation of the test article was observed following all treatments at 200 µl/plate and above.

Thus, test item did not induce mutation in five strains of Salmonella typhimurium under the conditions employed in this screening study, confirming the outcome of the key study. These conditions included treatment at concentrations up to 5000 µg/plate in the absence and in the presence of a rat liver metabolic activation system (S9).

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests asIn vitromutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

Under test conditions, the substance did not show any mutagenic effect on the bacteria strains tested, therefore it does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).