N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)hexane-1,6-diamine

Endpoint summary

Additional information

The mutagenic potential of the substance was tested in a fully guideline (OECD TG 471, EU Method B.13/14) and GLP compliant bacterial reverse mutation assay (Ames test). The test material was dissolved in acetone and both the plate incorporation method (first main experiment) and the pre-incubation method (second main experiment were performed with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA strain up to a test concentration of 5000 µg/plate. Both tests were conducted in the absence and presence of metabolic activation (phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver S9 mix). Cytotoxicity occurred in the Initial Mutation Test in some S. typhimurium strains at 5,000 μg/plate with and without metabolic activation and in Escherichia coli WP2 uvrA strain at 5,000 μg/plate without metabolic activation as well as in the Confirmatory Mutation Test in all examined bacterial strains at 5,000 and 1,581 μg/plate with and without metabolic activation; and in S. typhimurium TA 98, TA100 and TA1535 strains at 500 μg/plate without metabolic activation. Under the experimental conditions applied in the study, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used (Study director 2015).

The negative outcome in the key study is supported by another bacterial reverse mutation assay (Ames test). The test material was dissolved in acetone and both a direct plate incorporation procedure (main test) and a preincubation test were performed with Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations up to 5,000 µg/plate in the main test, and up to 2000 µg/plate in the preincubation test. Both tests were carried out in the absence and in the presence of a metabolic activator (Aroclor 1254-induced rat liver S9). During the test, signs of toxicity occurred from 1,000 µg/plate. The test substance did not induce mutations in both bacterial mutation tests in the absence or presence of metabolic activator in any strain tested. The positive and negative controls included in the experiment showed the expected results (Study director 1993d).

A further Ames test available is considered not reliable. In that study, no genotoxic effects were observed after exposure of S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 to the test substance at concentrations up to 320 µg/plate in the presence of metabolic activation. At this concentration, cytotoxic effects were reported only for strain TA 100 (IFREB).

 

The test substance was assessed for mutagenic potential in a Mammalian Cell Gene Mutation Test following OECD guideline 476 and in compliance with GLP. Cultured V79 cells (genetic marker HPRT) were co-incubated with the test substance at up to 5,000 µg/mL medium for 24 h in the absence, and for 4 h in the presence of a metabolic activator (Aroclor 1254-induced rat S9 fraction). After incubation of the cells in culture medium for about 6-7 days to allow genotype expression, the selection of mutants was carried out by co-incubating the cells with 6-thioguanine (10 µg/mL) for 12 days. The test was repeated in an independent experiment.

Cytotoxicity was observed at 5,000 µg/mL in a preliminary test and in both main tests in the absence of metabolic activation, while no cytotoxic effects were noted in the presence of metabolic activation.

The mutation frequency of the solvent controls (ethanol) was 9.02 and 10.83 x 10^-6clonable cells without metabolic activation. Under these conditions, the mutation frequency of the test cultures ranged from 5.71 to 12.76 x 10^-6clonable cells. In the presence of metabolic activation, the mutation frequency of the solvent controls (ethanol) was 11.09 and 7.18 x 10^-6clonable cells, and the mutation frequency of the test cultures ranged from 6.29 to 10.74 x 10^-6clonable cells. Thus, both in the absence and presence of metabolic activation, the mutation frequency of the cells treated with the test substance was within the normal range of the solvent controls. The positive controls, ethyl methanesulfonate (without S9 mix) and 9,10-dimethyl-1,2-benzanthracence (with S9 mix), included in the experiments showed the expected results (ca. 10- to 20-fold increase in mutation frequency relative to solvent control).

In conclusion, the test substance was negative in the HPRT-V79 mammalian cell mutagenicity test at up to 5,000 µg/mL both with and without metabolic activation (Study director 2003b).

 

The potential clastogenic activity of the test substance was assessed in a study carried out in accordance with OECD guideline 473 and GLP. The test substance dissolved in ethanol was assayed in this in vitro cytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation (Aroclor 1254-induced rat S9 fraction). Cell were exposed to the test substance at up to 5,000 (with metabolic activation) and 2,500 µg/mL medium (without metabolic activation) for 4 h, as well as at up to 1,000 µg/mL medium (without metabolic activation) for 24 h. The spindle inhibitor colcemid was added to the cell cultures at 10 µg/mL 22 h after start of exposure, and cells were harvested 2 h later. The study was conducted in duplicate.

Pronounced cytotoxicity was observed at the top concentrations mentioned above after the corresponding exposure times.

In the tests without metabolic activation, the mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the test substance ranged from 0.5 to 2.5% and no polyploidy was noted. The results were considered to be within the normal range of the solvent control, where a mean incidence of chromosomal aberrations (excluding gaps) of 0.0% (4-h exposure) and 1.0% (24-h exposure) were observed. A marginal, but not significant, increase in the incidence of aberrations to 3.7% was observed at the cytotoxic concentration of 1000 µg/mL after 24 h of exposure. This increase was considered to be related to the cytotoxic effect and not to the test substance.

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the test substance in the presence of metabolic activation ranged from 1.0 to 3.0%. No polyploidy was observed. The results were also considered to be within the normal range of the solvent control, where a mean incidence of chromosomal aberrations (excluding gaps) of 1.0-1.5% was noted.

According to the testing laboratory’s background data, the incidence of chromosomal aberrations (excluding gaps) of the references items with and without metabolic activation ranged from 0.0 to 5.0% for the 30 previous experiments. The positive control substances, mitomycin C (0.1 and 0.2 µg/mL without S9 mix) and cyclophosphamide (10 and 20 mg/mL with S9 mix), included in the experiments showed the expected results.

In conclusion, there were no indications for mutagenic properties with regard to chromosomal or chromatid damage after exposure of cultured human lymphocytes to the test substance up to cytotoxic concentrations in the presence and absence of metabolic activation (Study director 2003c).

 

Short description of key information:
In vitro genotoxicity:
Negative results in the Ames test (S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli) with and without metabolic activation.
Negative results in a gene mutation study in mammalian cells (HPRT mutation in V79 cells) with and without metabolic activation.
Negative results in a cytogenicity study in mammalian cells (chromosomal aberrations in cultured human lymphocytes) with and without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on the genetic toxicity of the test substance is conclusive but not sufficient for classification according to DSD (67/548/EEC) and GHS (CLP, 1272/2008/EC).