Fatty acids, C12-18 (even-numbered), 3-methylbutyl esters

Endpoint summary

Additional information

Justification for read-across

Data on the genetic toxicity of Fatty acids, C12-18, even numbered, 3-methylbutyl esters (CAS 1314963-50-2) are not available. The assessment was therefore based on studies conducted with analogue (source) substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 110-27-0

Isopropyl myristate (CAS 110-27-0) was investigated for mutagenicity to bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Oleon, 2014). The Salmonella typhimurium strains TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvrA were exposed to concentrations ranging from 52 - 5000 µg/plate in ethanol with and without the addition of metabolic activation system (S9-mix at concentrations of 5% and 10% v/v ) in three independent assays. The experiments were conducted according to the plate incorporation methodology. Cytotoxicity was observed in tester strain TA 100 in the presence of S9-mix, where a moderate to extreme reduction of the revertant colonies was observed at test substance concentrations of 512 µg/plate and above. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the other tester strains in the absence and presence of S9-mix. In tester strain TA 98 a 3.3-fold increases in the number of revertant colonies compared to the solvent control in the absence of S9-mix was observed. Since this increase was observed at a precipitating dose level and was not reproducible in two other experiments, this increase was not considered to be toxicologically relevant. Solvent and positive controls were included into the test and were shown to be valid. Based on these results, the test substance was considered to be not mutagenic to bacteria under the conditions of the test. 

CAS 163961-32-8

The mutagenic potential of Fatty acids, C16-18 and C18 unsaturated, branched and linear, butyl esters (CAS 163961-32-8) was tested in a Salmonella typhimurium reverse mutation assay equivalent to OECD guideline 471 and in compliance with GLP (Bowles, 2002). The following strains were used: TA 1535, TA 1537, TA 100, TA 98 and TA 102. Test substance concentrations of 50 - 5000 µg/plate in acetone were tested in two separate experiments with and without S9 mix. A globular, oily, opaque precipitate was observed at 5000 µg/plate, but this did not inhibit the scoring of revertant colonies. Cytotoxicity was not observed. The positive controls were shown to be valid. No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all strains tested, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C16-18 and C18 unsaturated, branched and linear, butyl esters did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

CAS 34316-64-8

The mutagenic potential of Hexyl laurate (CAS 34316-64-8) was tested in a Salmonella typhimurium reverse mutation assay equivalent to OECD guideline 471 and in compliance with GLP (Schröder, 1996). The following strains were used: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. Tester strains were incubated with test material concentrations of 8 - 5000 µg/plate in ethanol with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). Two independent experiments were performed with triplicates each. No toxicity or precipitation of the test substance was observed.

The 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 to concurrent negative controls was observed in all strains tested, neither in the presence nor in the absence of metabolic activation. Thus, Fatty acids, C16-18, hexyl laurate did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 163961-32-8

An in vitro mammalian chromosome aberration test was performed with Fatty acids, C16-18 and C18-unsaturated, branched and linear, butyl esters (CAS 163961-32-8) in primary human lymphocytes according to OECD guideline 473 and in compliance with GLP (Durward, 2004). For each experiment, whole blood was drawn from the peripheral circulation of a volunteer who had previously been screened for suitability. The cell cycle length of 17 h was determined by BrdU incorporation. Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix).

In the first experiment test substance concentrations of 312.5, 468.75 and 625 µg/mL in arachis oil were used for 24 hours of exposure without metabolic activation. 625, 1250 and 2500 µg/mL were used for 4 hours of exposure with metabolic activation followed by 20 hours expression time. In the second experiment 625, 1250 and 2500 µg/mL were used for 4 hours exposure with and without S9 followed by 20 hours expression time. 2500 µg/mL was chosen as the maximum dose due to limited solubility. Mitomycin C and cyclophosphamide were used as positive control substances. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material demonstrated only modest cytotoxicity. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolizing system. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

CAS 10233-13-3

An in vitro mammalian chromosome aberration test was performed with Isopropyl laurate (CAS 10233-13-3) in primary human lymphocytes according to OECD guideline 473 and in compliance with GLP (Buskens, 2010). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix).

In the first experiment cells were exposed for 3 hours to test substance concentrations of 10, 33 and 100 µg/mL in ethanol with and without metabolic activation. In the second experiment cells were exposed for 24 hours to 66, 150 and 250 µg/mL followed by 24 hours expression time. Additionally, 3, 125 and 150 µg/mL were the concentrations used for 48 hours exposure followed by 48 hours expression time. Both incubations were done without metabolic activation.

250 µg/mL was chosen as maximum dose due to limited solubility. The vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. The positive control materials induced statistically significant increases in aberration frequencies indicating the satisfactory performance of the test and of the activity of the metabolizing system. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material demonstrated only modest cytotoxicity. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 163961-32-8

An in vitro Mammalian cell gene mutation test was performed with Fatty acids, C16-18 and C18-unsaturated, branched and linear, butyl esters (CAS 163961-32-8) in mouse lymphoma L5178Y cells according to OECD guideline 476 and in compliane with GLP (Flanders, 2007). The cells were treated with the test substance in duplicate, together with vehicle (acetone) and positive controls. 4-hour exposures were used both with and without activation in Experiment l. In Experiment 2, the exposure time without metabolic activation was increased to 24 hours. A confirmatory third experiment was performed because a statistically significant response was observed in the lower / mid-dose range in the presence of metabolic activation in Experiment 2 that was not seen in Experiment 1. The concentration range in the first and second experiment was 156.25 to 5000 μg/mL following the results of a preliminary toxicity test without evidence of toxicity. Experiment 3 was performed using the dose range of 39.06 to 1250 μg/mL. A precipitate of test material was observed at and above 78.13 μg/mL. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5l78Y cell line at the TK locus. The positive control materials induced marked increases in the mutant frequency indicating the validity of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant increases in the mutant frequency at any dose level in any of the exposure groups, which included dose levels up to and including the maximum recommended dose level of 5000 μg/mL. Thus, the test material was considered to be non-mutagenic to L5l78Y cells under the conditions of the test.

 

CAS 10233-13-3

An in vitro Mammalian cell gene mutation assay according to OECD guideline 476 and in compliance with GLP was performed with Isopropyl laurate (CAS 10233-13-3) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). The cells were treated for 3 and 24 hours with 8% (v/v) and 12 (v/v) S9 -mix and without S9 -mix, respectively. The test substance was tested up to the precipitation, the following concentrations were tested: 0.01, 0.03, 0.1, 0.3, 1, 3, 5 and 10μg/mL. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. The 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 isopropyl laurate either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the isopropyl laurate treated cultures were comparable to the numbers of small and large colonies of the solvent controls. It was concluded that isopropyl laurate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

Overall conclusion for genetic toxicity

There are no available studies on the genetic toxicity of the target substance Fatty acids, C12-18, even numbered, 3-methylbutyl esters (CAS 1314963-50-2). Therefore analogue read-across from source substances was applied from in vitro studies on cytogenicity and gene mutation in bacteria and mammalian cells, using 4 source substances. The results of the available in vitro studies were negative. Based on the available data and following the analogue approach, Fatty acids, C12-18, even numbered, 3-methylbutyl esters is considered to be not mutagenic nor 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): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, TA 1538, TA 102 and E.coli WP2 uvr A.
Chromosome aberration (OECD 473): negative in human lymphocyte cells with and without metabolic activation.
Gene mutation in mammalian cells (OECD 476): negative in L5178Y mouse lymphoma 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 Fatty acids, C12-18, even numbered, 3-methylbutyl esters (CAS 1314963-50-2), 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 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.