[2-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxoallyl]oxy]ethyl][3-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxoallyl]oxy]propyl]dimethylammonium chloride

Endpoint summary

Additional information

Bacterial reverse mutation assay (Ames test)

In a reverse gene mutation assay in bacteria (BASF AG, 1981, OECD 471), strains TA 1538, TA1537, TA 1535, TA100, TA 98 of Salmonella typhimurium were exposed to a dye preparation containing 50% dye stuff in doses up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The study was performed according to the standard plate test method. Cytotoxicity was observed at doses > 2500 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Therefore, the test item was considered to be non-mutagenic in this bacterial reverse mutation assay.

In a supporting gene mutation assay in bacteria (BASF AG, 1982, OECD 471) the Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 and the strain E. coli WP2 uvr A were exposed to a dye preparation containing 50% dye stuff in the presence and absence of mammalian metabolic activation (rat liver S9 -mix) in doses up to 10000 µg/plate. In two additional test series the test item was tested only after metabolic activation using the two frameshift strains TA 1538 and TA 98 and using two different solvents (DMSO and acetone). The study was performed according to the standard plate test method. Without metabolic activation the test item was not mutagenic. The number of revertant colonies was always in the same range as that of a metabolizing system. In the test stains TA 98 and TA 1538 of Salmonella typhimurium in the presence of metabolic activation weakly positive and solvent dependent (only with DMSO) reactions were observed at high dose levels. The authors concluded that an impurity can be regarded as the cause of this effect.

Since the key gene mutation assay (BASF AG, 1981) revealed that a dye preparation containing 50% dye stuff was not mutagenic it can be considered that the registered dye itself has no mutagenic potential. This assumption is in line with the results obtained in the supporting gene mutation assay in bacteria (BASF AG, 1982). Thereby another dye preparation containing 50% dye stuff showed a weak mutagenicity only under sporadic conditions (i.e. DMSO used as solvent, with metabolic activation, only in the Salmonella typhimurium strains TA 98 and TA 1538). Thus, an impuritiy is regarded as the cause of the mutagenic effects and the registered dye is consequently considered as non-mutagenic.

 

In vitro mammalian cell gene mutation test (HPRT test)

The registered dye was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD guideline 476 (BASF SE, 2013). The assay was performed in three independent experiments with identical experimental procedures. The first and second experiment were performed with and without liver microsomal activation and a treatment period of 4 hours. The third experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The cell cultures were evaluated at the following concentrations:

 

Experiment I:

without S9 mix: 3.9; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0 and 500.0 µg/mL

with S9 mix: 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 and 1000.0 µg/mL

Experiment II:

without S9 mix: 3.9; 7.8; 15.6; 31.3; 62.5; 125.0 and 250.0 µg/mL 

with S9 mix: 15.6; 31.3; 62.5; 125.0; 250.0; 500.0 and 1000.0 µg/mL

Experiment III:

without S9 mix: 3.9; 7.8; 15.6; 31.3; 62.5; 125.0 and 250.0 µg/mL 

with S9 mix: 25.0; 50.0; 100.0; 200.0; 400.0 and 800.0 µg/mL

 

Osmolality and pH values were not influenced by the test item treatment. In the absence of metabolic activation test substance precipitation in culture medium at the end of treatment was observed at 125 µg/mL and above in the 3rd Experiment only. In the presence of metabolic activation, test substance precipitation was observed at 500 µg/mL and above in the 2nd Experiment and at 100 µg/mL and above in the 3rd Experiment, respectively. In this study, no increase in the number of mutant colonies was observed either without metabolic activation or after the addition of a metabolizing system. In all experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies were close to the respective vehicle control values and clearly within the range of the laboratory´s historical negative control data. The positive control substances induced clearly increased mutant frequencies as expected. Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in all experiments at least in the highest applied concentrations. In the absence and presence of metabolic activation, after 4 hours and 24 hours treatment in all experimental parts scored for gene mutations the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration. All together under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

In vitro micronucleus assay in V79 cells

The registered dye was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) according to OECD guideline 487 in compliance with GLP in the absence and the presence of metabolic activation. The assay was performed in two independent experiments with identical experimental procedures and a 24 hours harvest time. In experiment I exposure time was 4 hours with and without metabolic activation. In experiment II the exposure time was 24 hours without metabolic activation and 4 hours with metabolic activation. For the determination of the experimental doses an initial range-finding cytotoxicity test was performed resulting in the following doses for the main study:

1st Experiment

4 hours exposure; 24 hours harvest time; without S9 mix:

7.8; 15.6; 31.3; 62.5; 125.0 and 250.0.µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

15.6; 31.3; 62.5; 125.0; 250.0 and 500.0µg/mL

 

2nd Experiment

24 hours exposure, 24 hours harvest time, without S9 mix:

6.3; 12.5;25.0;50.0;100.0and 200.0 µg/mL

4 hours exposure, 24 hours harvest time, with S9 mix:

50.0;100.0;200.0and 400.0 µg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. For the test item no increased number of cells containing micronuclei was observed either with or without metabolic activation. The vehicle controls showed frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. In this study, cytotoxicity indicated by reduced relative increase in cell count (RICC) was at least observed in the highest dose group in all experimental parts. Besides, no cytotoxicity indicated by reduced proliferation index (PI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the regeistered dye is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of mammalian metabolic activation.

Justification for selection of genetic toxicity endpoint
No study was selected because all in vitro studies were negative.

Short description of key information:
The absence of a mutagenic potential was demonstrated for the registered dye in vitro in bacteria (two Ames tests, both conducted with 50% dye stuff) and in mammalian cells (HPRT assay). An in vitro micronucleus assay in V79 cells with the registered dye demonstrated that the test sustance has no potential to induce chromosome aberrations.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results the regeistered dye is not classified and labelled for genotoxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No. 1272/2008 (CLP).