Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

Endpoint summary

Additional information

Gene mutation in bacteria

The potential of the substance to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium according to the OECD guideline 471.The study was conducted in compliance with Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system.

Both experiments were performed according to the direct plate incorporation. After 48 -72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Since the test item was found poorly soluble and non-cytotoxic in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines. The selected treatment-levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains used in both mutagenicity experiments.

A moderate to strong precipitate was observed in the petri plates at dose-levels equal or higher than 125 µg/plate. No noteworthy toxicity was noted at any dose-levels towards the five strains used, either with or without S9 mix.

In the two independent assays, no significant increase in the mean number of revertants was noted in the bacterial strains tested in the presence of the test substance neither with nor without metabolic activation.

Under these experimental conditions,the substance did not show any mutagenic activity in the bacterial reverse mutation test (Sire, 2012a).

Gene mutation in mammalian cells

An in vitro mouse lymphoma assay was conducted to evaluate forward mutation induction potential of the test substance at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The study was conducted according to the OECD Guideline 476 and in compliance with the principles of Good Laboratory Practices.

 After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system .

Cultures of 20 mL at 5 x 10⁵cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

 Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. The test item was dissolved in dimethylsulfoxide.

 Since the test item was cytotoxic but also poorly soluble in the culture medium, the highest dose‑level for the main test was selected on the basis of the level of precipitate.

The dose-levels were 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL for both mutagenicity experiments without S9 mix. A precipitate was observed in the culture medium at dose-levels ≥ 50 µg/mL at the end of the 3‑hour treatment, as well as, at the end of the 24-hour treatment. Following the 3-hour treatment, a slight toxicity was induced at 100 µg/mL, as shown by a 27% decrease in the Adjusted Relative Total Growth .Following the 24-hour treatment, a slight to severe toxicity was induced from 3.13 µg/mL, as shown by a 28 to 82% decrease in the Adjusted Relative Total Growth.

No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control, following either the 3- or the 24-hour treatment. Consequently, no statistical analysis was undertaken for the evaluation of the dose-response relationship and these results were not considered to meet the criteria of a positive response.

 The dose-levels were the same for both mutagenicity experiments with S9 mix. A precipitate was observed in the culture medium at dose-levels ≥ 50 µg/mL but no toxicity was noted as shown by the absence of any noteworthy decrease in the Adjusted Relative Total Growth.

No noteworthy increase in the mutation frequency was observed in comparison to the vehicle control, in either experiment. Consequently, no statistical analysis was undertaken for the evaluation of the dose-response relationship and these results were not considered to meet the criteria of a positive response.

 The mean Cloning Efficiencies (CE₂), suspension growths and mutation frequencies of the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered to be valid

The test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or in the absence of metabolizing system (Sire, 2012b).

Chromosomal aberrations in mammalian cells

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473 and EU Method B.10 in compliance with Good Laboratory Practices.

Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: 

-        Test 1 (3-h treatment with and without S9 mix) at dose levels of 0, 62.5, 125 and 250 µg/mL,

-        Test 2 ( 3-h treatment with S9 mix and 20 -hr treatment without S9 mix) at dose levels of: 0, 62.5, 125 and 250 µg/mL and (44 -h treatment with and without S9 mix) at 0 and 250 µg/mL .

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay (Sire, 2013).

Justification for selection of genetic toxicity endpoint
No study was selected, since results from all the available guideline-compliant in vitro tests were negative.

Short description of key information:
The available data from three in vitro assays suggests that the substance does not have a genotoxic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicity according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.