Reaction products from the esterification of neopentylglycol with fatty acids, C16-18 (even numbered) and C18-unsatd. and fatty acids, C18-unsaturated, dimers

Endpoint summary

Additional information

Justification for read-across

There is no data available addressing the genetic toxicity of Reaction products from the esterification of neopentylglycol with fatty acids, C16-18 (even numbered) and C18-unsatd. and fatty acids, C18-unsaturated, dimers. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for the read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a comparable pattern as a result of structural similarity, the substances 2,2-dimethyl-1,4-propanediyl dioleate (CAS 42222-50-4), Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane (CAS 403507-18-6), Heptanoic acid, ester with 2,2-dimethyl-1,3-propanediol (CAS 68855-18-5) and Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5)are selected as source substances. 

In vitro mutagenicity in bacteria

CAS 147256-33-5

A bacterial gene mutation assay (Ames test) with Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5) was performed according to OECD guideline 471 and under GLP conditions (Sokolowski, 2012). The study included two independent experiments both in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, the direct plate incorporation procedure was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and the Escherichia coli strain WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, the same concentrations and exposure duration were applied for treatment of bacteria, but an additional pre-incubation period of 60 min was included in the test. In both experiments, no cytotoxicity was evident up to the limit concentration of 5000 µg/plate in all bacterial strains. In the first experiment, precipitation was observed at concentrations ≥ 333 µg/plate with metabolic activation, and at ≥ 1000 µg/plate without metabolic activation. In the second experiment precipitation was evident at ≥ 1000 µg/plate in the presence and absence of metabolic activation. The mean number of revertant colonies was not increased in the bacteria at any concentrations tested. The positive and negative controls included were shown to be valid in each experiment. Under the conditions of this assay, Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane did not induce mutagenicity in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli WP2 uvrA in the absence and presence of metabolic activation.

CAS 403507-18-6

The mutagenic potential of Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane (CAS 403507-18-6) was assessed in a reverse mutation assay comparable to OECD guideline 471 and under GLP conditions (Bowles, 2002). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were used. Tester strains were incubated with test substance dissolved in acetone at concentrations of 50, 150, 500, 1500 and 5000 µg/plate with and without the addition of a metabolic activation system (phenobarbitale and beta-naphthoflavone induced rat liver S9 mix). Vehicle and appropriate positive controls were included. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared with vehicle controls was observed in all strains treated with the test substance, neither in the presence nor in the absence of metabolic activation. No cytotoxicity was observed, but the test substance was tested up to precipitating concentrations (5000 µg/plate with and without metabolic activation) . Thus, Fatty acids, C16-18 and C18-unsatd., branched and linear ester with trimethylolpropane did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains tested.

In vitro cytogenicity in mammalian cells

CAS 147256-33-5

In an in vitro chromosome aberration test performed according to OECD guideline 473 and under GLP conditions, cultured peripheral human lymphocytes were treated with Fatty acids, C18-unsatd., dimers, mixed esters with oleic acid and trimethylolpropane (CAS 147256-33-5) at concentrations ranging from 0.01 to 5 µL/mL in the presence and absence of metabolic activation (S9 mix) in two independent experiments (Bohnenberger, 2012). These concentrations were selected based on a preliminary cytotoxicity test, in which no toxicity was observed up to the maximum concentration. In the first experiment, cells were exposed to the test substance for 4 h with and without S9 mix, while exposure periods of 22 h (without S9 mix) and 4 h (with S9 mix) were chosen in the second experiment. All fixation times were 22 h. No cytotoxicity was observed up to the maximum concentration of 5 µL/mL with and without S9 mix. Based on these results, concentrations of 0.01, 2.86 and 5 µL/mL were selected for chromosome analysis in the absence and presence of metabolic activation in the first and second experiment. No increase in the number of cells with chromosomal aberrations was observed compared to controls in any of the experiments performed. At the end of treatment, phase separation was observed in the first experiment in the absence and presence of S9 mix and in the second experiment in the absence of S9 mix at 0.02 μL/mL and above. In the presence of S9 mix, phase separation was observed at 0.03 μL/mL and above in the second experiment. It is assumed that this did not affect the reliability of the results. The positive controls included in both experiments showed the expected results and thus verified the sensitivity of the assay. Based on the negative results obtained in this chromosome aberration assay, it was concluded that the test substance did not show clastogenic activity in cultured peripheral human lymphocytes in the presence or absence of metabolic activation.

CAS 68855-18-5

A chromosome aberration test was conducted with Heptanoic acid, ester with 2,2-dimethyl-1,3-propanediol (CAS 68855-18-5) according to OECD guideline 473 and under GLP conditions in human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatments conditions were used for the study: in Experiment 1, 4 h in the presence of an induced rat liver homogenate metabolising system (S9 mix), at a 2% final concentration with cell harvest after a 20-h expression period and a 4 h exposure in the absence of metabolic activation (S9 mix) with a 20-h expression period. In Experiment 2, the 4 h exposure with addition of S9 mix was repeated (using a 1% final S9 mix concentration), while in the absence of metabolic activation the exposure time was 24 h. The dose levels used in the main experiments, selected using data from the preliminary toxicity test, were 12.5, 25, 50, 100, 200, 400 µg/mL. 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 the satisfactory performance of the test and the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in the absence and the presence of metabolic activation, in two separate experiments. In conclusion, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

In vitro gene mutation in mammalian cells

CAS 42222-50-4

An in vitro mammalian cell gene mutation assay completed according to OECD guideline 476 and und GLP conditions was performed with 2,2-dimethyl-1,3-propanediyldioleate (CAS 42222-50-4) in mouse lymphoma L5178Y cells (Flügge, 2012). The test substance was tested at 312.5, 625, 1250, 2500 and 5000 μg/mL in the absence and presence of S9 mix. The cells were treated for 3 h with and without S9 mix in the first experiment, for 24 h without S9 mix in the second experiment and again for 3 h with S9 mix in the third experiment. All cultures were incubated for 7 days after exposure. No toxicity was observed. Positive and negative controls were valid and within the range of historical control data. No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9 mix. Therefore, it was concluded that 2,2-dimethyl-1,3-propanediyldioleate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

Conclusion for genetic toxicity in vitro

There are no studies available on the in vitro and in vivo genetic toxicity of the target substance Reaction products from the esterification of neopentylglycol with fatty acids, C16-18 (even numbered) and C18-unsatd. and fatty acids, C18-unsaturated, dimers. Therefore analogue read-across from source substances was applied from in vitro studies on bacterial and mammalian cells, using four source substances. The results of the available in vitro studies on source substances did not provide evidence indicative for either mutagenic and/or clastogenic potential. Based on the available data, and following the analogue approach Reaction products from the esterification of neopentylglycol with fatty acids, C16-18 (even numbered) and C18-unsatd. and fatty acids, C18-unsaturated, dimers is not expected to be mutagenic and clastogenic in vitro.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues. All available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Genetic toxicity in vitro:
Ames test (OECD 471, WoE): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102 and in E. coli WP2 uvrA
Chromosome aberration (OECD 473, WoE): negative in cultured peripheral human lymphocytes with and without metabolic activation
Gene mutation in mammalian cells (OECD 476): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Reaction products from the esterification of neopentylglycol with fatty acids, C16-18 (even numbered) and C18-unsatd. and fatty acids, C18-unsaturated, dimers, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.