Isopentyl oleate

Endpoint summary

Additional information

Justification for grouping of substances and read-across

There are only limited data available on genetic toxicity of isopentyl oleate (CAS 627-89-4). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted. 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 grouping of substances and 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 regular pattern as a result of structural similarity.

Overview of genetic toxicity

CAS	Chemical name	Molecular weight [g/mol]	Gene mutation in vitro (Ames)	Chromosome aberration in vitro	Gene mutation in vitro (MLA)
627-89-4 (a)	Isopentyl oleate	326.56 - 354.61	WoE: RA: CAS 163961-32-8 RA: CAS 85865-69-6	WoE: RA: CAS 163961-32-8 RA: CAS 26399-02-0	WoE: RA: CAS 163961-32-8 RA: CAS 26399-02-0
163961-32-8 (b)	Fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters	312.53-340.58	Experimental result: negative	Experimental result: negative	Experimental result: negative
26399-02-0 (b)	2-ethylhexyl oleate	394.67	--	Experimental result: negative	Experimental result: negative
85865-69-6 (b)	Fatty acids, C16-18, isobutyl esters	312.53 – 340.58	Experimental result: negative	--	--

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substances are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for isopentyl oleate (CAS 627-89-4). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

No data on genetic toxicity is available with isopentyl oleate (CAS 627-89-4). Therefore, read across from the structurally analogue substances fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS 163961-32-8), 2-ethylhexyl oleate (CAS 26399-02-0) and fatty acids, C16-18, isobutyl esters (CAS 85865-69-6) was applied.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 163961-32-8

A reliable bacterial gene mutation study (Ames test) performed according to OECD TG 471 and in compliance with GLP with fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS 163961-32-8) is available (Bowles, 2002). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I+II) procedure in the absence and presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 50 to 5000 µg/plate (experiment I and II). No cytotoxicity was observed in a preliminary toxicity test performed with tester strain TA 100 after treatment with the test item up to 5000 µg/plate in the absence and presence of metabolic activation. Precipitation was recorded at a concentration of 5000 µg/plate. Appropriate solvent (acetone) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of the test.

CAS 85865-69-6

A reliable bacterial gene mutation study (Ames test) performed equivalent or similar to OECD TG 471 and in compliance with GLP with fatty acids, C16-18, isobutyl esters (CAS 85865-69-6) is available (Banduhn, 1990). The strains Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were tested according to the plate incorporation (experiment I+II) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 8 to 5000 µg/plate. No cytotoxicity was recorded in any tester strain treated with the test material up to and including the highest dose of 5000 µg/plate. Appropriate solvent (Tween 80/bidest. water) and positive controls were included and gave the expected results. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Therefore, the test material was considered to be non-mutagenic under the conditions of the test. 

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 163961-32-8

A reliable in vitro chromosome aberration test in primary human lymphocytes was performed with fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS 163961-32-8) according to OECD TG 473 and in compliance with GLP (Durward, 2004). Human peripheral lymphocytes were cultured and treated with the test material or vehicle (arachis oil) in the absence or presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix) in duplicates at concentrations of 625, 1250 and 2500 µg/mL for 4 h (with and without S9-mix) and 312.5, 468.75 and 625 µg/mL for 24 h (without S9-mix). Fixation and staining of the cells were performed 24 hours after start of exposure with the test material. Cytotoxicity was assessed by determination of the mitotic index. Appropriate solvent and positive controls were included in the test and gave the expected results. Neither precipitation nor cytotoxicity was recorded at any concentration during the course of the study. The test substance did not cause a statistically significant, dose-related increase in chromosome aberrations under the tested conditions. Based on the results of the study the test material is considered not to be clastogenic to primary human lymphocytes in this chromosome aberration test in vitro.

CAS 26399-02-0

A reliable in vitro chromosome aberration test in primary human lymphocytes was performed with 2-ethylhexyl oleate (CAS 26399-02-0) according to OECD TG 473 and in compliance with GLP (Buskens, 2010). Human peripheral lymphocytes were cultured and treated with the test material or vehicle (1% (v/v) ethanol) in the absence or presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix) in duplicates at concentrations of 3, 10 and 33 µg/mL µg/ml for 3 h (with 1.8% (v/v) and without S9-mix), 24 h and 48 h (without S9-mix). In experiment I fixation and staining of the cells were performed 24 hours after start of exposure (3 h) with the test material. In the second experiment, fixation and staining was performed 24 (without metabolic activation) and 48 hours (with and without metabolic activation) after start of exposure with the test material. Cytotoxicity was assessed by determination of the mitotic index but could not be detected at any test concentration either in the presence or in the absence of metabolic activation. Precipitation of the test material was limited to the highest concentration of 33 µg/mL tested. Appropriate solvent and positive controls were included in the test and gave the expected results. The test substance did not cause a statistically significant, dose-related increase in chromosome aberrations under the tested conditions. In addition, no polyploidy did occur in a significant manner neither in the treatment groups nor in the negative control group. Based on the results of the study the test material is considered not to be clastogenic to primary human lymphocytes in this chromosome aberration test 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 (heterozygous at the thymidine kinase locus) according to OECD TG 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 exposures were used both with and without activation in Experiment l. In Experiment 2, the exposure time 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 dose 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 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 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 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 5000 μg/mL. Thus, the test material was considered to be non-mutagenic to L5l78Y cells under the conditions of the test.

CAS 26399-02-0

A reliable in vitro gene mutation test in Mouse lymphoma L5178Y cells was performed with 2-ethylhexyl oleate (CAS 26399-02-0) according to OECD TG 476 and in compliance with GLP (Verspeek-Rip, 2010). Mouse lymphoma cells were treated with the test material or vehicle (ethanol) in two independent experiments in the absence or presence of a metabolic activation system (phenobarbitone/β-naphthoflavone-induced rat liver S9-mix) at concentrations from 0.03 to 100 µg/mL for 3 hours (without and with 8% (v/v) S9-mix, experiment I; with 12% (v/v) S9-mix, experiment II) and 24 h (without S9-mix, experiment II). After exposure with the test material mouse lymphoma cells were cultured in microtiter plates containing 5 µg/mL trifluorothymidine selective medium for additional 11 - 12 days. Fixation of the cells was performed 11 to 12 days after start of exposure with the test material. Appropriate solvent and positive controls were included in the test and gave the expected result. No cytotoxicity was observed in a preliminary toxicity test with L5178Y mouse lymphoma cells treated with concentrations from 3 to 333 µg/mL for 3 h (with and without metabolic activation) and 24 h (without metabolic activation). Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the mutagenicity assays considering the highest dose level was the 100 µg/mL due to solubility in the culture medium. No evidence of marked toxicity following exposure to the test item was recorded in the presence or absence of metabolic activation in both experiments. Precipitation was recorded at a test material concentration of 100 µg/mL. The test substance did not cause a statistically significant, dose-related increase in mutant frequency either in the absence or in the presence of metabolic activation. Therefore, the test substance is considered not to be mutagenic in the mouse lymphoma L5178Y test system under the conditions of the test.

Taken together, the available data on genetic toxicity from structural analogue substances do not indicate any mutagenic and clastogenic potential. Therefore, according to EU classification criteria, the target substance isopentyl oleate (CAS 627-89-4) is not to be classified.

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.

Short description of key information:
Genetic toxicity in vitro:
Bacterial reverse mutation assay (WoE, Ames test / OECD 471): negative (RA CAS 163961-32-8 and RA CAS 85865-69-6)
In vitro chromosome aberration test in human lymphocytes (WoE, CA / OECD 473): negative (RA CAS 163961-32-8 and RA CAS 26399-02-0)
In vitro gene mutation assay in mouse lymphoma cells (WoE, MLA / OECD 476): negative (RA CAS 163961-32-8 and RA CAS 26399-02-0)

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 isopentyl oleate (CAS 627-89-4), 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.