N-ethyl-4-[[4-(ethylamino)-m-tolylphenyl][4-(ethylimino)-3-methylcyclohexa-2,5-dien-1-ylidene]methyl]-m-toluidine monoacetate

Endpoint summary

Additional information

No studies are available for the test substance, therefore read across was performed with ethyl red violet chloride containing the same active ingredient as the test substance. Ethyl red violet chloride is composed of ca. 84 % active ingredient, sodium chloride and water. The test substance includes acetic acid instead of sodium chloride. Both constituents (acetic acid and sodium chloride) are not classified and labelled as mutagenic. Both substances are used in food industry and belong to substances that are normally used for food preparation. Therefore it is assumed that the toxicological relevant part of the substance is the active ingredient. Thus, using ethyl red violet chloride as read across substance is scientifically justified.

Ames Test

In a GLP compliant reverse gene mutation assay in bacteria according to OECD guideline 471, strains of Salmonella typhimurium ( TA 1535, TA 100, TA 1537, TA 98) were exposed to the test substance in a standard plate incorporation (SPT) and in a pre-incubation assay (PIT) (BASF AG, 1995). The following concentrations were used:

0; 5; 10; 15; 20; 40 and 80 µg/plate (1st experiment, SPT)

0; 200; 400; 600; 800 and 1000 µg/plate (2nd experiment, SPT)

0; 5; 10; 15; 20; 40 and 80 µg/plate (PIT)

All experiments were conducted in the presence and absence of metabolic activation. Each concentration was tested in triplicate.

The positive controls induced the appropriate responses in the corresponding strains. A bacteriotoxic effect was observed at doses above 200 µg/plate (SPT) or from about 5 µg - 40 µg/plate (PIT) onward. There was no evidence or a concentration related positive response of induced mutant colonies over background. The test substance was not mutagenic in the S. typhimurium strains TA1535, TA1537, TA100 and TA98 under the experimental conditions with or without metabolic activation.

Chromosome Aberration test

A GLP compliant chromosome aberration assay was performed according to OECD 473 and EU method B.10 (BASF AG, 1995). The possible clastogenicity of the test substance was tested in two independent experiments with cultured V79 cells both with and without metabolic activation. According to pretests for the determination of the experimental doses and in consideration of the cytotoxicity actually found in the main experiment, 0.4 µg/mL, 0.6 µg/mL and 0.8 µg/mL culture medium (18 hours sampling time) and 0.8 µg/mL culture medium (28 hours sampling time) without metabolic activation or 3.0 µg/mL, 4.0 µg/mL and 5.0 µg/mL culture medium (18 hours sampling time) and 5.0 µg/mL culture medium (28 hours sampling time) in the experiment with metabolic activation were evaluated.

Crystal violet (= Basic violet 3) was tested in parallel as a positive reference compound. Chromosomes were prepared 18 hours (low, intermediate and top dose) and 28 hours (top dose only) after test substance treatment, which lasted for about 4 hours both in the experiment with S9 mix or without metabolic activation. Duplicate cultures were used for all experimental groups.

The negative controls (untreated and vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals, i.e. EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations indicating the sensitivity of the test system.

According to the results of the present study, the test substance did not cause any significant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system in two experiments independent of each other. In contrast, the positive reference compound crystal violet exhibited, as expected, a very clear clastogenic activity with a high proportion of exchanges. Thus, under the experimental conditions chosen here, the read across substance is considered not to be a chromosome-damaging (clastogenic) agent under in vitro conditions in V79 cells.

Justification for selection of genetic toxicity endpoint

GLP and guideline compliant studies for mutagenicity in bacteria and mammalian cell culture.

Short description of key information:

In a reverse mutation assay in bacteria (Ames Test) and in a chromosome aberration test with mammalian cells with a strcutrally similar compound, no mutagenicity was observed with or without metabolic activation in both tests. (BASF AG, 1995)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available experimental in vitro and in vivo test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled for mutagenic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation EC No 605/2014.