Fatty acids, C8-12, isopentyl esters

Endpoint summary

Additional information

Genetic dose toxicity

Justification for read-across

There are no data regarding genetic toxicity available for Fatty acids, C8-12, isopentyl ester. In order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4, read-across from appropriate substances is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.

According to 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) “to avoid the need to test every substance for every endpoint”.

Fatty acids, C8-12, isopentyl ester is a multi-constituent substance specified by C8, C10 and C12 linear saturated fatty acids esterified with isopentanol resulting in monoesters which meets the definition of an UVCB substance. Thus, the test substance represents fatty acid esters which undergo to a high extent hydrolysis by ubiquitous expressed gastrointestinal enzymes into the free fatty acid components and the respective alcohol (Lehninger, 1970; Mattson and Volpenhein, 1972). Considering the common metabolism, the read-across approach is based on the presence of common functional groups, common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals, common functional groups, structural similarities and similar physico-chemical, toxicological and toxicokinetic behaviour. For further details on the read-across approach, please refer to the analogue justification in section 13 of the technical dossier.

As no data on genetic toxicity are available for Fatty acids, C8-12, isopentyl ester, read-across to reliable data on the analogue substances Isopropyl Myristate (CAS 110-27-0), Fatty acids, C8-16, 2-Ethylhexyl esters (CAS 135800-37-2), Hexyl Laurate (CAS 34316-64-8), Isopropyl Laurate (CAS 10233-13-3), Fatty acids, C16-18 and C18-unsatd., branched and linear, Butyl Esters (CAS 163961-32-8) and 2-Ethylhexyl Oleate (CAS 26399-02-0) was conducted for genetic toxicity. The substances were chosen based on their structural similarities regarding the branched alcohol component (Isopropyl Myristate (CAS 110-27-0), Isopropyl Laurate (CAS 10233-13-3), 2-Ethylhexyl Oleate and Fatty acids, C8-16, 2-Ethylhexyl esters (CAS 135800-37-2)) and / or the linear fatty acid component (Isopropyl Laurate (C12; CAS 10233-13-3, Isopropyl Myristate (C14; CAS 110-27-0), Fatty acids, C8-16, 2-Ethylhexyl esters (CAS 135800-37-2) and Hexyl Laurate (CAS 34316-64-8)). Furthermore, data of the structural analogue substances Fatty acids, C16-18 and C18-unsatd., branched and linear, Bu esters (CAS 163961-32-8) and Ethyl Oleate (CAS 111-62-6) were considered to allow a weight-of-evidence approach covering short and long chain fatty acids and saturated/unsaturated fatty acids.

Genetic toxicity in bacteria (Ames)

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. Appropriate solvent and positive controls were included into the test and gave the expected results. Based on these results, the test item was considered to be not mutagenic to bacteria under the conditions of the test. 

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 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 0, 8, 40, 200, 1000 and 5000 µg/plate in ethanol with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). 4-Nitro-o-phenylendiamine, Sodium azide and 9-Aminoacridine and 2-Aminoanthracene were used as positive controls without and with S9 mix, respectively. Two independent experiments were performed with triplicates each. No toxicity or precipitation of the test substance was observed.

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, 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 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 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. Mitomycin C and Cyclophosphamide were used as positive control substances. Vehicle (solvent) controls induced aberration frequencies within the range expected for normal human lymphocytes. 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 at 150 µg/mL (exposure period 24 h, fixation time 24 h, -S9 mix) and at 125 µg/mL (exposure period 48 h, fixation time 48 h, -S9 mix). The test material was therefore considered to be non-clastogenic to human lymphocytes 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 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 0, 312.5, 468.75 and 625 µg/mL in arachis oil were used for 24 hours of exposure without metabolic activation. 0, 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 0, 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 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 at 468.75 µg/mL without metabolic activation. 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 in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

CAS 26399-02-0

An in vitro mammalian chromosome aberration test was performed with 2-Ethylhexyl Oleate (CAS 26399-02-0) in primary human lymphocytes according to OECD guideline 473 and 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 incubated with test substance concentrations of 3, 10 and 33 µg/mL in ethanol for 3 hours with and without metabolic activation. In the second experiment cells were incubated with 3, 10 and 33 µg/mL for 24 hours followed by 24 hours expression time and 48 hours following 48 hours expression time without S9. 33 µg/mL was chosen as maximum dose due to limited solubility of the test substance. 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. Vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. 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 in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in-vitro

CAS 10233-13-3

An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and 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 without S9-mix and with 12% S9-mix (v/v) and without S9-mix, respectively. The test substance was tested up to precipitation, the following concentrations were tested: 0.01, 0.03, 0.1, 0.3, 1, 3, 5 and 10μg/mL. Cyclophosphamide and Methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. Positive and negative controls were valid and in 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. 

CAS 26399-02-0

An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and under GLP was performed with 2-Ethylhexyl Oleate (CAS 26399-02-0) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). Two independent experiments (with 3 or 24 hours of exposure) were performed in the absence and presence of S9-mix with test substance concentrations up to 100 μg/mL dissolved in ethanol. Precipitation was seen at 100 µg/mL and higher. Cyclophosphamide and Methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. Positive and negative controls were valid and in range of historical control data. No significant increase in mutation frequency occurred in any of the test conditions, indicating that 2-Ethylhexyl Oleate is not mutagenic in the 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 (heterozygous at the thymidine kinase locus) according to OECD guideline 476 and under GLP (Flanders, 2007). The cells were treated with the test substance in duplicate, together with vehicle (acetone) and positive controls. 4 hour exposure duration was used both with and without activation in Experiment l. In Experiment 2, the exposure duration without activation was increased to 24 hours. A confirmatory third experiment was performed due to a statistically significant response being observed in the lower / mid-dose range in the presence of metabolic activation in Experiment 2 that had not been seen in Experiment 1. The concentration range of test material 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. The confirmatory experiment 3 was performed using the concentration 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 that were within the normal range for the L5178Y cell line at the TK locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance 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 5000μg/mL. Thus, the test material was considered to be non-mutagenic to L5l78Y cells under the conditions of the test.

Conclusion on genetic toxicity

The available data do not provide evidence that the source substances exhibit mutagenic or clastogenic properties in bacteria or mammalian cells. Therefore, based on common functional groups and structural similarities, no potential for genetic toxicity is expected for Fatty acids, C8-12, isopentyl ester.

 

References

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within the CSR.

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, C8-12, isopentyl ester, data will be generated from information on 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 target substance information and 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.