2-ethylhexyl nonanoate

Endpoint summary

Additional information

There are no data available for the genetic toxicity of 2 -ethylhexyl nonanoate (CAS 59587 -44 -9). In order to fulfil the standard information requirements set out in Annex 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 for genetic toxicity

CAS #	Genetic toxicity (mutagenicity) in bacteria in-vitro	Genetic toxicity (cytogenicity) in mammalian cells in-vitro	Genetic toxicity (mutagenicity) in mammalian cells in-vitro	Genetic toxicity in vivo
59587-44-9 Target substance	RA: 135800-37-2 RA: 163961-32-8	RA: 10233-13-3 RA: 26399-02-0	RA: 10233-13-3 RA: 26399-02-0	RA: 135800-37-2
10233-13-3	--	negative	negative	--
163961-32-8	negative	--	--	--
135800-37-2	negative	--	--	negative
26399-02-0	--	negative	negative	--

The above mentioned substance is considered to be similar on the basis of structural similarity resulting in similar properties and/or activities. The available endpoint information is used to predict the same endpoints for 2-ethylhexyl nonanoate (CAS 59587-44-9).

A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Discussion:

CAS 135800-37-2

AMES, OECD 471, negative

The mutagenic potential of Fatty acids, C8-16, 2-Ethylhexyl Esters (CAS# 135800-37-2) was tested in a Salmonella typhimurium reverse mutation assay equivalent to OECD Guideline 471 and under GLP (Banduhn, 1990). 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, 100 and 5000 µg/plate in Tween 80/bidest water 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 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, Fatty acids, C8-16, 2-Ethylhexyl Esters did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

Micronucleus Test in vivo, OECD 474, negative

The ability to induce chromosome aberrations in vivo in mouse bone marrow cells was tested according to OECD guideline 474 and GLP (Paika, 1991). Male and female mice were treated with 0, 1075, 2150 and 4300 mg/kg bwFatty acids, C8-16, 2-Ethylhexyl Esters (CAS# 135800-37-2)by single intraperitoneal injection and sacrificed for examination at 24 h intervals for up to 72 hours. Cyclophosphamide was used as a positive control. The mouse micronucleus test did not reveal increases in the frequency of micronucleated polychromatic erythrocytes inFatty acids, C8-16, 2-Ethylhexyl Esterstreated animals and therefore no indications of induced chromosome damage were found.

CAS 163961-32-8

AMES, OECD 471, negative

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 under GLP (Bowles, 2002). The following strains were used: TA 1535, TA 1537, TA 100, TA 98 and TA 102. Test substance concentrations of 50, 150, 500, 1500 and 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.2-Aminoanthracene, Benzo(a)pyrene and 1,8 Dihydroxyanthraquinone were used as positive controls with S9 mix, whereas N-ethyl-N'-nitro-N-nitrosoguanidine, Mitomycin C, 4-Nitroquinoline-1-oxide and 9-Aminoacridine were tested as positive controls without S9 mix. 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 10233-13-3

In vitro mammalian chromosome aberration test, OECD 473, negative

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. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

In vitro mammalian cell gene mutation test, OECD 476, negative

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% (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

In vitro mammalian chromosome aberration test, OECD 473, negative

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.

 

In vitro mammalian cell gene mutation test, OECD 476, negative

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 100μg/mL dissolved in ethanol. Precipitation was seen at 100µg/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.

Conclusion for genetic toxicity

Two studies assessing the potential genetic toxicity using analogue based read-across from structure related substances Fatty acids, C8-16, 2-Ethylhexyl Esters (CAS 135800-37-2) and Fatty acids, C16-18 and C18 unsaturated, branched and linear, Butyl Esters (CAS 163961-32-8) in bacteria (Ames test) were available. In vitro chromosomal aberration tests were performed with 2 -Ethylhexyl oleate (CAS 26399-02-0) and Isopropyl Laurate (CAS 10233-13-3). Two in vitro mammalian cell gene mutation assays were also available with 2-ethylhexyl oleate (CAS 26399-02-0) and Isopropyl Laurate (CAS 10233-13-3). The in vivo mammalian erythrocyte micronucleus test in bone marrow was performed with Fatty acids, C8-16, 2-ethylhexyl esters (CAS 135800-37-2). The results of all the tests were negative. The available data on genetic toxicity, both in vitro and in vivo, indicates that the structural related substances do not have genetic toxicity potential.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across based on an analogue approach. No study was selected since all available in vitro and in vivo 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 substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Negative results in several Salmonella typhimurium strains , with and without metabolic activation (OECD 471, GLP, analogue approach).
Negative results in mammalian chromosomal aberration test (OECD 473, GLP, analogue approach).
Negative results in mammalian cell gene mutation tests (OECD 476, GLP, analogue approach).
Negative results in in vivo micronucleus test (OECD 474, GLP, analogue approach).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on read-across from structurally similar substances, 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.