Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

Endpoint summary

Additional information

Gene mutations

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix at 10 % (v/v)) and without metabolic activation at concentrations of 25, 75, 200, 600, 1800 and 5000 µg/plate (restesting of strain TA1537 with metabolic activation at various S9 mix concentrations (i.e. 5, 10 or 15 % (v/v)) was done at 25, 75, 200, 600, 1800, 2500, 3333, and 5000 µg/plate using) in the plate incorporation assay. Toxicity was observed beginning at 1800 µg per plate up to the highest dose tested. No precipitate was observed.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

The results of this study was confirmed in another bacterial reverse mutation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 with (induced rat liver S9 mix at 16 % (v/v)) and without metabolic activation (MA). Without MA dose levels tested were 0.3, 1, 4, 20, 100 and 300 µg/plate, with MA tests were performed at concentrations of 1, 4, 20, 100, 300 and 1000 µg/plate in the plate incorporation assay.

Assessment of the potential of the test item to induce gene mutations in mammalian cells in vitro was conducted using Chinese hamster ovary cells looking for forward mutations at the HGPRT locus. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation.

The test article was tested with the following concentrations:

without S9-mix: 10, 50, 100, 175, and 250 µg/ml (5 hours treatment with test material)

with S9-mix: 1, 10, 100, 500, and 1000 µg/ml (5 hours treatment with test material)

According to the preliminary experiment for toxicity the concentration ranges were selected. Starting from 100 µg/ml relative initial survival was slightly decreasing with increasing concentrations tested, reaching cytotoxicity effect level (i.e. only 10 -20% survival of control) at concentrations of 333 µg/ml in the test without metabolic activation and at 1000 µg/ml in these tests with various concentrations of the S-9 mix. Nevertheless up to the highest concentration tested in the mutagenicity assay no distinct decrease of the cloning efficiency was observed. Up to the highest investigated dose no increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, the test item does not induce gene mutations in the HGPRT-test with Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described. Therefore the test item is considered to be non-mutagenic in this HGPRT assay.

The findings in mammalian cells were supported by the results of an in vitro rat hepatocyte DNA repair assay (in vitro UDS assay). DNA damaging effects indicate for mutagenic events which may occur thereafter. The test item was therefore tested for the ability to induce unscheduled DNA synthesis. Results of both the preliminary and the replicate assay demonstrated that the test item is not a genotoxic agent under the conditions tested in this mammalian cell assay.

Chromosomal aberrations assay

To investigate cytogenetic potential of the submission substance an in vivo rat bone marrow assay was performed comparable to OECD TG 475.

The submission substance dosed at 770 mg/kg did not produce any evidence of chromosome damage as measured by increases in chromosome aberrations, altered mitotic index (as measure for cytotoxic effects), or chromosome number as compared to concurrent controls in this assay. The positive control substance, cyclophosphamide, produced significant increase in the percent of cells displaying chromosome aberrations, average number of aberrations and decreased the mitotic index and chromosome number, confirming senstitivity of the test system applied. Under the conditions of this assay,the submisson substance is not cytogenetically active.

Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro and in vivo studies were negative.

Short description of key information:
The genotoxic potential of the submission substance has been assessed in five GLP studies (in vitro and in vivo),
- including a bacterial reverse mutation assay (Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 uvr A pKM 101; according to OECD test guideline 471),
- a second bacterial reverse mutation assay (S. tphimurium strains TA 1535, TA 1537, TA 98 and TA 100; similar to OECD test guideline 471),
- a mammalian gene mutation assay (CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay; similar to OECD test guideline 476),
- an assay to evaluate the potential to induce unscheduled DNA synthesis (primary rat hepatocyte cultures; similar to OECD test guideline 482) and
- a mammalian chromosome aberration test (in vivo rat bone marrow; equivalent to OECD test guideline 475).
In all studies negative results were reported in the presence and absence of metabolic activation (if applicable).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In a reliable set of various bacterial mutation assay and gene mutation test in mammalian cells in vitro and in vivo the test item is considered to be non-mutagenic.

Based on the available data no classification according to Regulation (EC) No. 1272/2008 and Council Directive 67/548/EEC on mutagenicity is warranted.