Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

There are no data available for the genetic toxicity of Fatty acids, soya, 2 -ethylhexyl esters (CAS# 93572 -14 -6). 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,

the substance listed below are selected as reference substances for hazard assessment.

Overview for genetix 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

93572-14-6 Target substance

RA: 91031-48-0

RA: 163961-32-8

RA: 163961-32-8

RA: 26399-02-0

RA: 163961-32-8

RA: 26399-02-0

RA: 135800-37-2

91031-48-0

negative

--

--

--

163961-32-8

negative

negative

negative

--

135800-37-2

--

--

--

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 Fatty acids, soya, 2-ethylhexyl esters (CAS 93572-14-6).

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

Discussion:

CAS 91031-48-0

AMES, OECD 471, negative

The mutagenic potential of Fatty acids, C16-18, 2-Ethylhexyl Esters (CAS# 91031-48-0) was tested in a Salmonella typhimurium reverse mutation assay equivalent to OECD Guideline 471 and GLP (Banduhn, 1988). 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 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, 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, C16-18, 2-Ethylhexyl Esters did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

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.

 

In vitro mammalian chromosome aberration test, OECD 473, negative

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. 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.

 

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

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 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 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 5000 μg/mL. Thus, the test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

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 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 135800-37-2

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 bw Fatty 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.

Conclusion for genetic toxicity

Two studies assessing the potential genetic toxicity using analogue based read-across from structural related substances Fatty acids, C16-18, 2-Ethylhexyl Esters (CAS 91031-48-0) 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 Fatty acids, C16-18 and C18 unsaturated, branched and linear, Butyl Esters (CAS 163961-32-8). Two in vitro mammalian cell gene mutation assay were also available with 2-ethylhexyl oleate (CAS 26399-02-0) and Fatty acids, C16-18 and C18 unsaturated, branched and linear, Butyl Esters (CAS 163961-32-8). 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. On the basis of the read across from structurally related substances, 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).

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.