Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic nor induced micronuclei in an OECD 471, 476 and 487 guideline study up to 9000 µg/plate and 29,000 µg/mL, respectively.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a GLP-compliant study that was conducted in accordance with OECD 471 and EU method B.13/15, the test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The standard plate test and the preincubation test were performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. These tests were repeated later on with new selected concentrations, due to an update on the purity of the sample. The doses used were 33, 100, 333, 1000, 3333 and 6000 µg/plate in the first two experiments and 906.3, 1812.5, 3625, 7250, 14500 and 9000 µg/plate in the final two experiments. A weak bacteriotoxic effect was occasionally observed on the strain and test conditions from about 7250 µg/plate onward. None of the tested concentrations (up to 9000 µg/plate) of the test substance led to a meaningful increase in the incidence of either histidine- (Salmonella typhimurium) or tryptophan- (E. coli) prototrophic mutants by comparison with the negative control.

Also, a GLP-compliant in vitro study was performed according to OECD guideline 476 and EU method B.17, where the test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested that were evaluated for gene mutations in the presence and absence of S9 mix: 0; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL and 0; 5000.0; 10000.0; 20000.0; 29000.0 μg/mL. Two ranges were tested, due to an update on the purity of the compound after experiments with the first mentioned concentration range. The cells were treated with the test substance for 4 hours and were subsequently cultured for 6 - 8 days after which selection occurred in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle- and both positive control substances performed as expected. No cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in all valid experiments performed independently of each other. However, in one experiment in the presence of S9 mix, there was a slight but statistically significant and dose-dependent increase in the mutant frequencies. The corrected mutation frequencies were well within the concurrent negative control data and the historical negative control data range, however. Therefore, this finding had to be regarded as biologically irrelevant. Thus, the test substance 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated was not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Finally, an in vitro mammalian cell micronucleus test was performed according to OECD guideline 487 and EU method B.49 under GLP conditions. Here, the test substance was assessed for its potential to induce micronuclei in V79 cells (clastogenic or aneugenic activity). Four independent experiments were carried out, all with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, although extra experiments had to be performed, due to an update on the purity of the compound after the first experiments. The following concentrations and incubation/harvest times were tested that were evaluated for cytogenetic damage:

- 4 hours exposure, 24 hours harvest time, with and without S9 mix: 0;7250.0; 14500.0; 29000.0 μg/mL

- 24 hours exposure, 24 hours harvest time, without S9 mix: 0;7250.0; 14500.0; 29000.0 μg/mL

- 4 hours exposure, 44 hours harvest time, with S9 mix: 0; 906.3;7250.0; 14500.0; 29000.0 μg/mL

At least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. Negative controls and positive controls (ethyl methanesulfonate (EMS) and cyclophosphamide (CPP)) were in line with historical control data. No relevant cytotoxicity indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest applied test substance concentration under all experimental conditions. The test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test substance 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 metabolic activation.

Justification for classification or non-classification

Based on the available information the test substance classification for genetic toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.