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Mutagenicity in bacterial reverse mutation assays (Ames test) was investigated in a complete study on the substance (Iwahara S. and Kanatani H., 1980). The test was to be carried out for the purpose of checking the back mutation from Histidine requisite to non-requisite in Salmonella Tryphimurium and back mutation from Tryptophane requisite to non-requisite in Caliform Bacilli. All the strains tested (Salmonella Typhinurium TA 1535, TA 100, TA 1537, TA 1538, TA 98; Escherichia coll WP2 uvrA) with and without Metabolic Activation Tests, did not shown remarkable increase in the mutation colony.

A further AMES test confirmed these outcomes (Shapiro R., 1988): the results show that the test strains are sensitive to the positive control mutagens, indicating that under the assay conditions, the test strains were sensitive to the detection of potentially genotoxic agents. The test article was found to be soluble in DMSO and showed no apparent cytotoxicity at the concentrations tested. No increase in numbers of revertant colonies was observed on plates containing the test article without and with metabolic activation compared to negative control plates.

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) were tested for bacteria reverse mutation and chromosomal aberration and none of the existing tests arisen any concern for mutagenicity or genotoxicity.

CAS 27344-06-5 was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test; V79 hamster fibroblast were used for testing. A preliminary cytotoxicity assay (3-hour treatment) was performed at first. First experiment, with 3 hours treatment by the test substance followed then. Four concentrations were used - the maximum concentration 5.0 mg/ml (in experiment with metabolic activation) and 3.0 mg/ml (without metabolic activation) and three lower concentrations, 1.5; 0.5 and 0.15 mg/ml. Each concentration was tested in two replicates.

Experiments were performed without as well as with of metabolic activation using the supernatant of rat liver and a mixture of cofactors.

Results of first experiment were negative, so the modification of experiment was performed with extended treatment time of 24 hours. Concentrations of 1.5; 1.0; 0.5 and 0.25 mg/ml were used according to results of cytotoxicity test. Additional dose 5.0 mg/ml was tested in parallel with 24 hours experiment. Also the second expreriment gives no evidence of the mutagenicity of test substance (Täublová E., 2015).

Further mammalian mutagenicity assays were performed on three representative members of the category (CAS 16090-02-1, CAS 68971-49-3 and CAS 67786-25-8), on the basis of three different levels of solubility and covering those groups which could be the most biologically reactive, based on chemical constitution and expected metabolism.

The In Vitro Mammalian Cell Gene Mutation assay performed on the similar substance CAS 67786-25-8 (the bis(2-hydroxypropyl)amino derivative), 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 (Täublová E., 2014).

The same test was also performed on the CAS 68971-49-3 (the dihydroxyethylamino hexasulphonated sodium salt) and on the CAS 16090-02-1 (the morpholino disulphonated, disodium salt). As well as in the first case, both the test substances were non-mutagenic for V79 cells with and without metabolic activation (Täublová E., 2014 and Täublová E., 2014).

 

The substance was tested for its potential to cause chromosome aberrations in cultured Chinese hamster ovary cells with and without an exogenous S-9 activation mixture. The Chromosome Aberration Assay was performed at five dose levels, 5000, 4000, 3000, 2000 and 1000 µg/ml, in the non-activated system and at four dose levels, 5000, 4000, 3000 and 2000 µg/ml, in the activated system. When compared to the solvent control data, the test doses did not cause a significant increase in the number of cells with chromosome aberrations in the non-activated or activated system. In the test was not reported a detectable dose response in the number of aberrations per cell or number of cells with aberrations in the activated or non-activated system (Thilagar A., 1988).

 

Furthermore, two in vivo test performed on the analogous substances (CAS 4193-55-9 and CAS 16470-24-9) were reported in order to confirm the outcomes from in vitro test.

A dominant lethal assay was performed on the similar substance CAS 4193-55-9 (Lorke D. and Machemer L., 1973). The compound was administered orally in single dose to NMRI mice by gavage at the concentration of 5000 mg/kg. Methylmethanesulfonate (MMS) and trimethyl phosphate (TMPO) were used as positive controls. Each of the 20 male mice in each group was mated with three untreated females directly after treatment. After insemination (determined by vaginal smear), or after a week, the females were isolated. This procedure was repeated weekly for eight weeks. On the fourteenth day of gestation the females were sacrificed and the numbers of fertile matings, implantations, resorptions, live foetuses, and corpora lutea were determined. The dominant lethal test investigation did not shown any evidence of mutagenicity caused by the test substance.

The chromosome aberration potential was investigated also for the analogous CAS 16470-24-9: the substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse (CCR - Cytotest Cell Research GmbH & Co KG, 1991).

In order to assess the mutagenicity potential on mammalian cells, an in vitro Ames test in combination with the micronucleous assay has been planned. This choice has been determined 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 attached to the section 13.

All substances of the category were modelled using the 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 also for the substance tunder registration that can then be considered as not genotoxic.

Read across within the same group is well justified in this case taking also into account the impurities of the involved substances: the identified organic impurities can have different substitution on the molecule, nevertheless 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 test (Iwahara and Kanatani, 1980), in vitro gene mutation on mammalian cells (Täublová, 2015), both in vitro and in vivo chromosomal aberration (Thilagar 1988, Bayer AG., 1973 and CCR - Cytotest Cell Research GmbH & Co KG, 1991).
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).