reaction product of: C.I. Leuco Sulfur Black 1 with (3-chloro-2-hydroxypropyl)trimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

IN VITRO BACTERIAL CELL REVERSE MUTATION ASSAY

The potential of the test item to induce point mutations by base pair changes or frameshifts was evaluated in an experimental study according to the OECD Guideline 471 (1983) and the EU Method B.14 (1992).

8 concentrations of the test item (3.3, 10, 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate) were tested for toxicity in strains TA 98 and TA 100 in triplicate using the pre-incubation method in a pre-study. Based on the results obtained, concentrations of 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate were selected for the main study.

Two independent main experiments, utilsing the pre-incubation and plate-incoporation methods, were performed to Salmonella typhimurium strains TA 1535, TA 1537, TA 100 and TA 98, both with and without metabolic activation (S9 Mix). In each experiment, 3 agar plates were prepared for each strain and dose level, including controls.

In the pre-study the plates incubated with the test item showed normal background growth up to 5000 μg/plate, both with and without S9 mix, in all strains used. No toxic effects occurred at any of the dosages, any strain, with or without metabolic activation, in either the first or second experiment. No substantial increase in revertant colony numbers was observed in any S. typhimurium strain; with or without metabolic activation. The strain TA 98 showed an enhancement factor of 2.0 at 2500 µg/plate in the presence of metabolic activation in the first experiment, however this increase was judged as irrelevant since it only occurred at a single concentration in the first experiment and could not be reproduced at any concentration in the second experiment, and was not dose-related. Appropriate reference mutagens used as positive controls showed a distinct increase in induced revertant colonies.

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce point mutations by base pair changes and frameshifts in the genome of the strains used. Therefore, the substance is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

IN VITRO MAMMALIAN CELL MUTAGENESIS ASSAY

Structural chromosomal aberration potential was evaluated in an in vitro experimental study on mammalian cells, according to the OECD Guideline 473 (1983). Chinese hamster lung fibroblast V79 cells were exposed to the test item in two parallel experiments (1 and 2), with and without metabolic activation (S9 mix), and evaluated for percentage of cells demonstrating chromosomal aberration 18 and 28 hours after fixation. Per culture 100 metaphases were scored for structural chromosome aberrations.

The concentration range of the test item for cytogenetic experiment I was determined in a pre-test using the XTT-assay and a qualitative evaluation of cell density and morphology as indicator for toxicity response. Test concentrations between 3 µg/ml and 5000 µg/ml (with and without S9 mix) were chosen for evaluation of cytotoxicity. In the absence of S9 mix toxic effects could be observed after treatment with 1000 pg/ml - 5000 µg/ml, whereas in the presence of S9 mix no test item induced cytotoxicity could be observed. Visible test item precipitation occurred within a concentration range from 1000 µg/ml - 5000.0 pg/ml (with S9 mix) and 300 µg/ml – 5000 µg/ml (without S9 mix). In the absence of S9 mix, test concentrations within a range from 1 µg/ml - 100 µg/ml (experiment I) and from 10 µg/ml - 500 µg/ml (experiment II) were applied for the investigation of the potential to induce cytogenetic damage. In experiment I and II, in the presence of S9 mix concentrations within a range from 30 µg/ml - 5000 µg/ml were applied.

In the absence of S9 mix, in both experiments at the top evaluated experimental points, the mitotic index as a measure for cytotoxicity was reduced at the 18 h interval in experiment I and at both preparation intervals in experiment II. In the presence of S9 mix, no reduction of the mitotic index could be observed in the evaluated cultures except at the 18 h interval in experiment I after treatment with 5000 µg/ml.

In both independent experiments, there were neither biologically relevant nor statistically significant increases in cells carrying structural chromosome aberrations after treatment with the test item at fixation intervals 18 h and 28 h (with and without S9 mix).

In both experiments, no biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test item compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced a statistically significant increase (p < 0.05) of cells showing structural chromosome aberrations.

In conclusion, the test item did not induce structural chromosome aberrations in the V79 cells (Chinese hamster cell line), under the experimental conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), the term ‘mutation’ refers a permanent change in the amount or structure of the genetic material in a cell, both to heritable genetic changes that may be manifestedat the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The terms ‘mutagenic’ and ‘mutagen’ are used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms. For the purpose of the classification for germ cell mutagenicity, substances may be allocated to one of two categories:

-Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans. Further sub-classification can be made into the following:

-Sub-category 1A: in the presence of positive evidence from human epidemiological studies; or

-Sub-category 1B: in the presence of positive result(s) from (i) in vivo heritable germ cell mutagenicity tests in mammals; (ii) in vivo somatic cell mutagenicity tests in mammals, in combination with some evidence that the substance has the potentialto cause germ cells mutations (derived from mutagenicity/genotoxicity tests in germ cells in vivo, or by demonstrating the ability of the substance or its metabolite(s) to interact with the genetic material of germ cells); or (iii) tests showing mutagenic effects in the germ cells of humans, without demonstration of transmission to progeny; for example, an increase in the frequency of aneuploidy in sperm cells of exposed people.

-Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans. Classification in Category 2 is based on positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from either (i) somatic cell mutagenicity tests in vivo, in mammals; or (ii) other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Regarding the present test item, both in vitro gene mutation in bacterial cell and in vitro chromosomal aberration in mammalian cell demonstrated negative results. The test item is therefore not considered mutagenic according to the CLP Regulation (EC 1272/2008) and no further testing is warranted.