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Mutagenicity in bacterial reverse mutation assays (Ames test) was investigated in Salmonella T. strains TA 1530, TA 1532, TA 1535, TA153 with and without metabolic activation, with negative results (Kilbey B. J., 1975) and the results were confirmed in an analogous study, performed only on strains TA 1535 and TA 1538 (McGregor D. B., 1976). In order to confirm the outcomes of these studies and in order to evaluate all the recommended combination of strains, an in vitro bacteria reverse mutation assay on Escherichia Coli, according to the OECD guideline 471 was performed. The substance was tested for its mutagenic potential in standard plate test (SPT) and preincubation test (PIT), both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. Under the experimental conditions, the test item resulted to be not a mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF SE 2015).

Furthermore, Saccharomyces ceresiae was investigated conducting the following experiments:

- growth of Saccharomyces in presence of test item: the increase in cellular material of yeast strains W10 and D4 with the time in the presence of the FWA was evaluated measuring the changes in extinction at 620 nm when inoculated for overnight.

- petite induction and gene conversion: W10 was grown with test substance in the dark and the stationary phase populations assayed for petite individuals using the sodium tellurite method of Nagai et al. (J, Bateriol 90: 220 -222 (1965)).

- penetration: it is possible to obtain a crude estimate of the distribution of the material in the yeast cell by observing the patterns of fluorescence induced by UV under the microscope.

The results provide no indication that the substance produces mutagenic changes or any alteration in the gene material. This may be explained because the substance fails to reach the nucleus (Kilbey B.J. and Zetterberg L.B., 1975).

In order to assess the mutagenicity potential on mammalian cells, the In Vitro Mammalian Cell Gene Mutation assay performed on the similar substance CAS 67786-25-8 (the bis(2-hydroxypropyl)amino derivative) was considered. The test was conducted in V79 hamster fibroblast and experiments were performed without as well as with of metabolic activation using the supernatant of rat liver and a mixture of cofactors. The test substance was non-mutagenic (Research Institute for Organic Syntheses, Inc. CETA, 2014).

The chromosomal aberration potential was investigated in three in vivo studies.

Nucleus Anomaly Test on Somatic Interphase Nuclei was conducted in Chinese Hamster in order to evaluate the mutagenic effects on bone marrow cells. Doses of 1250, 2500, 5000 mg/kg were given by oral gavage. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 10.8, whereas the negative control yielded a percentage of 0.15. The difference is highly significant (p < 0.01) (Ciba-Geigy Ltd., 1974).

The Chromosome Study on Somatic Cells was conducted on the test substance in order to evaluate the mutagenic effects on bone marrow cells. Hamsters were dosed at 1250, 2500, 5000 mg/kg, by oral gavage.

In the lowest dosage group (1250 mg/kg) one chromatid-type aberration per 400 cells (0.25 %) was found in form of chromatid break. In the dosage groups 2500 and 5000 mg/kg there were no chromatid- type aberrations. Chromosome-type aberrations were not found at all. In all dosage groups the percentage did not significantly exceed the control value (Ciba-Geigy Ltd., 1974).

Dominant Lethal Study was conducted on the test substance in order to evaluate the potential for cytotoxic or mutagenic effects on male germinal cells. Mice were administered by oral gavage with a single dose of 1650 and 5000 mg/kg. The data on mating ratio, on the numbers of implantations and embryonic deaths are comparable for all groups. No signs of intolerability were noted in the males (Ciba-Geigy Ltd., 1974).

Mutagenicity is a non-threshold end-point, therefore mutagenicity potential is evaluated firstly based on the reactivity of the substance in itself, bound to chemical structure, functional groups and metabolism pathway, than on the bioavailability potential. Moreover the first screening in vitro is conservative regarding the end point, since the substance is put into the reaction plate even if potentially it will never been absorbed and will never express the mutagenic potential.

Within the whole category ten over fourteen registered substances covering at least one member per group (see data matrix in the Category Justification Report attached to the section 13 of the technical dossier) were tested for bacteria reverse mutation and chromosomal aberration and none of the existing tests arisen any concern for mutagenicity or genotoxicity.

Mutagenicity on mammalian cells was not tested for the subgroup 3b, but based on the position of The Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) that has a remit to provide UK Government Departments and Agencies with advice on the most suitable approaches to testing chemical substances for genotoxicity, the best strategy to assess genotoxicity in vitro is the combination of Ames test and micronucleous, because together with a better sensitivity, a better specificity is also demonstrated respect than testing mutagenicity in mammalian cells. More details are specified within the Category Justification Report in section 13 of the technical dossier.

Nevertheless, Mammalian mutagenicity test was performed on three representative members of the category (CAS 16090-02-1, CAS 68971-49-3 and CAS 67786-25-8) based on three different levels of solubility and covering those groups that could be the most biologically reactive based on chemical constitution and expected metabolic pathway.

All substances of the category were modelled with OECD Toolbox and the provisional results about mutagenicity alerts were calculated for all members and their metabolites. The same alert was reported based on the Hacceptor-path3-Hacceptor. This alert explores the possibility that a chemical interacts with DNA and/or proteins via non-covalent binding, such as DNA intercalation or groove-binding (Snyder et al. 2006). Among the descriptors potentially accounting for non-covalent interactions, the present molecular framework representing two bonded atoms connecting two H bond acceptors (calculated with software Leadscope Enteprise 2.4.15-6) resulted in an increased sensitivity/specificity for what concerns the Micronucleus training set. Experimental tests both in vivo and in vitro demonstrate that this alert is not expressed in none of the substances of the group. Based on all those considerations the available studies on the analogous substances are representative for the substance under registration also that can then be considered as not genotoxic.

Read across within the same group is well justified in this case also taking into account the impurities of the considered substances, since the identified organic impurities can have different substitution on the molecule, but the functional reactive groups are potentially the same, and molecules are of the same molecular size and polarity of the main component. As a consequence the systemic absorption and reactivity is practically the same than the main constituent and Read Across is justified.

REFERENCES

Snyder, R. D., Ewing, D. and Hendry, L. B. 2006. DNA intercalative potential of marketed drugs testing positive in in vitro cytogenetics assays.


Justification for selection of genetic toxicity endpoint
Evaluation of the endpoint has been performed with the integrated evaluation of the following studies: in vitro Ames tests (BASF SE, 2014; Kilbey and Zetterberg, 1975; McGregor and Ainsworth, 1976), in vitro gene mutation on mammalian cells (Research Institute for Organic Syntheses, Inc. CETA, 2014) and in vivo chromosomal aberration (Ciba-Geigy Ltd., 1974).
Detailed justification for Read Across is indicated within any endpoint and in the Category Justification Report attached to the Section 13.
Detailed justification for Read Across is indicated within any endpoint and in the Category Justification Report attached to the Section 13.

Short description of key information:
Non genotoxic

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- 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 or

- substances, which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.


On the basis of the results of the available studies, the substance can be considered as not having mutagenic or genotoxic properties.


In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).