Fatty acids, C16-18 and C18-unsatd., branched and linear, reaction products with diethanolamine

Endpoint summary

Additional information

Ames Test

Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Isostearamide DEA, using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. 

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the Salmonella tester strains (except TA98 dosed in the presence of S9-mix), initially from 500 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9‑mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. A test item film (creamy in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. 

Conclusion.The test item,IsostearamideDEA, was considered to be non-mutagenic under the conditions of this test.

 

Chromosome Aberration Test

Introduction. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design was also compatible with the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

 Methods. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of test item (µg/ml)
4(20)-hour without S9	25, 50, 100, 200, 400, 800
4(20)-hour with S9 (2%)	25, 50, 100, 200, 400, 800
24-hour without S9	12.5, 25, 50, 100, 200, 400
4(20)-hour with S9 (1%)	25, 50, 100, 200, 400, 800

 Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments using a dose range that included a dose level that was just outside the optimal 50% mitotic inhibition.

Conclusion. The test item, IsostearamideDEA, was considered to be non-clastogenic to human lymphocytesin vitro.

In vitro Gene Mutation

The potential mutagenicity of the test material on the thymidine kinase (TK +/-) locus of the L5178Y mouse lymphoma cell line was investigated in accordance with the standardised guidelines OECD 476. The test was assigned a reliability score of 1 in accordance with the criteria of Klimisch (1997).

Two independent experiments were performed; the maximum dose levels were limited by test material-induced toxicity. In Experiment 1, cells were treated with the test material at five dose levels (30 to 70 µg/mL in the absence of metabolic activation, 10 to 40 µg/mL in the presence of metabolic activation), in duplicate, together with solvent and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (1 % S9).

In Experiment 2, the cells were treated with the test material at five dose levels using a 24 hour exposure group in the absence of metabolic activation. The dose range was 20 to 50 µg/mL.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The vehicle and positive controls were acceptable.

Under the conditions of this study, the test material is considered to be non-mutagenic in the L5178Y mouse lymphoma assay.

Final Conclusion:

Isostearamide DEA does not show evidence of genetic toxicity in vitro in three separate in vitro assays.

Justification for selection of genetic toxicity endpoint
The mouse lymphoma assay was chosen as the key study. Additional supporting studies include results from one in vitro Ames study in bacterial cells and one in vitro chromosome aberration study in mammalian cells. All studies were negative for genetic toxicity.

Short description of key information:
One in vitro study in bacterial cells and 2 in vitro studies in mammalian cells are available. All studies were negative with respect to genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification:

All in vitro tests are negative