Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

The Short Chain Alcohol Esters (SCAE C2-C8) category covers esters from a fatty acid (C8-C29) and a C2-C8 alcohol (ethanol, isopropanol, butanol, isobutanol, pentanol, iso-pentanol, hexanol, 2-ethylhexanol or octanol). This category includes both well-defined mono-constituent substances as well as related UVCB substances with varying fatty acid chain lengths.

Fatty acid esters are generally produced by chemical reaction of an alcohol (e.g. isopropanol) with an organic acid (e.g. stearic acid) in the presence of an acid catalyst (Radzi et al., 2005). The esterification reaction is started by a transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The carboxylic acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to a carbonyl carbon of acid. An intermediate product is formed. This intermediate product loses a water molecule and proton to give an ester (Liu et al, 2006; Lilja et al., 2005; Gubicza et al., 2000; Zhao, 2000). Monoesters are the final product of esterification.  

The rationale for grouping the substances in the SCAE C2-C8 category is based on similarities in physicochemical, ecotoxicological and toxicological properties.

 

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, 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).

 

In this particular case, the similarity of the SCAE C2-C8 category members is justified, in accordance with the specifications listed in Regulation (EC) No. 1907/2006 Annex XI, 1.5

Grouping of substances and read across, based on representative molecular structure, physico-chemical properties, tox-, ecotoxicological profiles, supported by a robust set of experimental data and QSAR calculations. There is no convincing evidence that any one of these chemicals might lie out of the overall profile of this category, respectively.

 

Grouping of substances into this category is based on:

 Similar/overlapping structural features or functional groups: All category members are esters of primary alcohols (C2-C8) and fatty acids (C8-C29), with 13 to 32 carbons in total.

 Common precursors and the likelihood of common breakdown products via biological processes: All category members are subject to enzymatic hydrolysis by pancreatic lipases (Mattson and Volpenhein, 1972; and references therein). The resulting free fatty acids and alcohols are absorbed from the intestine into the blood stream. Fatty acids are either metabolised via the beta-oxidation pathway in order to generate energy for the cell or reconstituted into glyceride esters and stored in the fat depots in the body. The alcohols are metabolised primarily in the liver through a series of oxidative steps, finally yielding carbon dioxide (Berg et al., 2002).

 Similar physico-chemical properties: The log Kow and log Koc values of all category members are high (log Kow > 4, log Koc > 3), increasing with the size of the molecule. The substances are poorly soluble in water and have low vapour pressure. 

 Common properties for environmental fate & eco-toxicological: Based on experimental data , all substances are readily biodegradable and do not show toxic effects up to the limit of water solubility.

 Common levels and mode of human health related effects:All available experimental data indicate that the members of the SCAE C2-C18 category are not acutely toxic, are not irritating to the skin or to the eyes and do not have sensitizing properties. Repeated dose toxicity was shown to be low for all substances. None of the substances showed mutagenic effects, and toxicity to reproduction was low throughout the category.

 

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 considered as a category 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 substances listed below are allocated to the category of SCAE C2-C8.

 

 

Table 1: Members of the SCAE C2-C8 Category

EC No.

CAS No.

Chemical name

Alcohol Carbon No.

Fatty acid Carbon No.

Total Carbon

MW

208-868-4

544-35-4

ethyl linoleate or ethyl octadeca-9,12-dienoate

2

18

20

308.50

203-889-5

111-62-6

Ethyl oleate

2

18

20

310.52

293-054-1

91051-05-7

Fatty acids, essential, ethyl esters

2

14 - 22

16 - 24

252.39-368.64

233-560-1

10233-13-3

Isopropyl laurate

3

12

15

242.41

203-751-4

110-27-0

Isopropyl Myristate

3

14

17

270.46

205-571-1

142-91-6

Isopropyl palmitate

3

16

19

298.51

269-023-3

68171-33-5

Isopropyl isostearate

3

18

21

326.56

203-935-4

112-11-8

Isopropyl oleate

3

18

21

324.55

292-962-5

91031-58-2

Fatty acids, C16-18, isopropyl esters

3

16 - 18

19 - 21

312.54-326.57

264-119-1

63393-93-1

Fatty acids, lanolin, isopropyl esters

3

10 - 29

13 - 32

214.34-480.85

204-666-5

123-95-5

butyl stearate

4

22

22

340.59

267-028-5

67762-63-4

Fatty acids, tall-oil, butyl esters

4

18

22

423.72

287-039-9

85408-76-0

Fatty acids, C16-18, Bu esters

4

16 - 18

20 - 22

312.53-340.58

284-863-0

84988-74-9

Fatty acids, C16-18 and C18-unsatd., Bu esters

4

16 - 18

20 - 22

312.53- 340.58

 

163961-32-8

Fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters

4

16 - 18

20 - 22

312.54- 340.58

211-466-1

646-13-9

Isobutyl stearate

4

18

22

340.59

288-668-1

85865-69-6

Fatty acids, C16-18, iso-Bu esters

4

16 - 18

20 - 22

312.54- 340.60

84988-79-4

84988-79-4

Fatty acids, C16-18 and C18-unsatd., iso-Bu esters

4

16 - 18

20 - 22

312.54- 340.60

228-626-1

6309-51-9

Isopentyl laurate

5

12

17

270.46

694-886-1

1365095-43-7

Fatty acids, C8-10, 3-methylbutyl esters

5

8 - 10

13 - 15

214.344- 242.40

251-932-1

34316-64-8

Dodecanoic acid, hexyl ester

6

12

18

284.49

218-980-5

2306-88-9

octyl octanoate

8

8

16

256.42

 

84713-06-4

Dodecanoic acid, isooctyl ester

8

12

20

312.53

243-697-9

20292-08-4

2-Ethylhexyl laurate

8

12

20

312.53

692-946-1

649747-80-8

Fatty acids, C8-10, 2-ethylhexyl esters

8

8 - 10

16 - 18

256.42-284.48

603-931-6

135800-37-2

Fatty acids, C8-16, 2-ethylhexyl esters

8

12 - 14

20 - 22

256.42-368.65

249-862-1

29806-73-3

2-ethylhexyl palmitate

8

16

24

368.64

 

22047-49-0

2-Ethyl hexyl Stearate

8

18

26

396.69

295-366-3

92044-87-6

Fatty acids, coco, 2-ethylhexyl esters

8

12 - 18

20 - 26

312.53-340.60

292-951-5

91031-48-0

Fatty acids, C16-18, 2-ethylhexyl esters

8

16 - 18

24 - 26

368.65-396.70

285-207-6

85049-37-2

Fatty acids, C16-18 and C18-unsatd., 2-ethylhexyl esters

8

16 - 18

24 - 26

368.65-396.70

247-655-0

26399-02-0

2-Ethylhexyl oleate

8

18

26

394.67

 

In order to avoid the need to test every substance for every endpoint, the category concept is applied for the assessment of environmental fate and environmental and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, 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 the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

Table 2: Data matrix 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

544-35-4 (b)

negative

negative

negative

--

10233-13-3 (b)

RA: 110-27-0

negative

negative

--

110-27-0 (b)

negative

RA: 10233-13-3

RA: 10233-13-3

--

85865-69-6 (a)

negative

RA: 163961-32-8

RA: 163961-32-8

--

163961-32-8 (b)

negative

negative

negative

--

34316-64-8 (a)

 

negative

RA: 10233-13-3

RA: 26399-02-0

RA: 10233-13-3

RA: 26399-02-0

--

2306-88-9 (a)

negative

RA: 10233-13-3

RA: 26399-02-0

RA: 10233-13-3

RA: 26399-02-0

--

20292-08-4 (a)

 

negative

RA: 10233-13-3

RA: 26399-02-0

RA: 10233-13-3

RA: 26399-02-0

RA: 135800-37-2

135800-37-2 (b)

negative

--

--

negative

91031-48-0 (b)

negative

RA: 26399-02-0

RA: 26399-02-0

RA: 135800-37-2

85049-37-2 (b)

negative

RA: 26399-02-0

RA: 26399-02-0

RA: 135800-37-2

26399-02-0 (b)

 

--

negative

negative

--

(a) Category members subject to registration are indicated in bold font. Only for these substances a full set of experimental results and/or read-across is given.

(b) Substances not subject to registration are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

Gentox - Isopropyl esters 

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 110-27-0

AMES, OECD 471, negative

The mutagenic potential of Isopropyl Myristate (CAS# 110-27-0) was tested in a Salmonella typhimurium reverse mutation assay equivalent to OECD Guideline 471 (Gloxhuber, 1991). 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, 4, 20, 100, 500 and 2500 µg/plate in acetone with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9 mix). 4-Nitro-o-phenylendiamine and Sodium Azide and 2-Aminoanthracene were used as positive controls without and with S9 mix, respectively.

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, Isopropyl Myristate did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

 

 

Gentox – Butyl esters

CAS 85865-69-6

AMES, OECD 471, negative

The mutagenic potential of Fatty acids, C16-18, Isobutyl Esters (CAS# 85865-69-6) 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, C16-18, Isobutyl Esters did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

 

 

Gentox - Hexyl esters

CAS 34316-64-8

AMES, OECD 471, negative

The mutagenic potential of Fatty acids, C16-18, 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, 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.

 

CAS 20292-08-4

AMES, OECD 471, negative

2-Ethylhexyl Laurate (CAS# 20292-08-4) was tested in a bacterial reverse mutation assay, according to OECD Guideline 471 and GLP at concentrations of 0, 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation (Henkel, 1983). 2-Ethylhexyl Laurate did not induce point mutations by base-pair changes or frame-shifts in the bacterial reverse mutation assay.

 

Micronucleus Test in vivo, OECD 474, negative

Male and female mice were treated with 0, 1.25, 2.5, 5.0 mL/kg 2-Ethylhexyl Laurate (CAS# 20292-08-4) by gavage (Henkel). The study was conducted under GLP. The mouse micronucleus test did not reveal increases in the frequency of micronucleated polychromatic erythrocytes in treated animals and therefore no indications of induced chromosome damage were found.

 

Gentox - 2-Ethylhexyl esters

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.

 

 

Gentox - Isobutyl ester

CAS 2306-88-9

AMES, OECD 471, negative

The mutagenic potential of Octyl Octanoate (CAS# 20292-88-9) was tested in a Salmonella typhimurium reverse mutation assay equivalent to OECD Guideline 471 and under GLP (Wollny, 2000). The following strains were used: TA 1535, TA 1537, TA 100, TA 98 and TA 102. Tester strains were incubated up to the limit concentration with the test material: 33, 100, 333, 1000, 2500 and 5000 µg/plate in DMSO with and without the addition of a metabolic activation system (phenobarbital- and ß-naphthoflavone-induced rat liver S9 mix). Experiment I was carried out as a plate incorporation assay, whereas II and III were done as pre-incubation assays. 4-Nitro-o-phenylendiamine, Sodium Azide and Methylmethanesulfonate and 2-Aminoanthracene were used as positive controls without and with S9 mix, respectively. Three 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. Due to slightly induced numbers of revertant colonies for strain TA1535 without metabolic activation in the second experiment a third experiment was carried out for strain TA1535 under the same conditions and concentrations of 333, 1000, 2500 and 5000 µg/plate. No relevant increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the second experiment was judged as biologically irrelevant.

No increase in the frequency of revertant colonies compared to concurrent negative controls was observed in all other strains tested, neither in the presence nor in the absence of metabolic activation. Thus, Octyl Octanoate did not induce point mutations by base-pair changes or frame-shifts in the genome of the strains used.

 

 

Gentox – Read Across Substances

CAS 544-35-4

AMES, OECD 471, negative

A bacterial reverse mutation assay was performed with 9,12-Octadecadienoic acid (Z,Z)-,Ethyl Ester (CAS# 544-35-4) according to OECD guideline 471 and GLP with the S. typhimurium strains TA98, TA100, TA1535 and TA1537 and the E. coli strain WPA2uvr A (Verbaan, 2010). The bacterial tester strains were treated with 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate of the test substance in absence and presence of metabolic activation by phenobarbital- and ß-naphthoflavone-induced rat liver S9-mix. Two independent experiments were performed with triplicates each. Sodium Azide, 9-Aminoacridine, 2-Nitrofluorene, Methylmethanesulfonate and 4-Nitroquinoline-N-oxide and 2-Aminoanthracene were used as positive controls without and with metabolic activation, respectively. 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. The test substance slightly precipitated at concentrations ≥ 1000 µg/plate but induced no cytotoxic or genotoxic effects at any concentration neither in the presence nor in the absence of metabolic activation. Based on the study results,

9,12-Octadecadienoic acid (Z,Z)-,Ethyl Ester did not induce gene mutations in four tested Salmonella and one tested E. coli strain.

 

In vitro mammalian chromosome aberration test, OECD 473, negative

The ability of Ethyl Linoleate (CAS# 544 -35 -4) to induce chromosome aberrations in cultured peripheral human lymphocytes was tested according to OECD guideline 473 and under GLP (Verbaan, 2010). Test substance concentrations of up to 800 µg/mL dissolved in DMSO were tested in the presence and absence of metabolic activation. At concentrations of 333 µg/mL and higher precipitation of test substance occurred. The first experiment was incubated for 3 hours and 24 hours fixation with and without metabolic activation, whereas the second experiment was incubated for 3 hours together with 48 hours fixation (with S9 mix) and 24 and 48 hours incubation without S9 mix followed by 24 and 48 hours fixation, respectively. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, Mitomycin C and Cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Ethyl Linoleate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed.

 

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 Ethyl Linoleate (CAS# 544-35-4-13-3) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). The cells were treated for 3 and 24 hours with 5% (v/v) and without S9-mix and with 10% (v/v) and without S9-mix, respectively. The test substance was tested up to 300μg/mL dissolved in DMSO. Precipitation was seen at 100µg/mL and higher. At this concentration cytotoxicity occurred in the presence and absence of metabolic activation. 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 Ethyl Linoleate is not mutagenic in the mammalian cells in vitro.

 

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.

 

 

 

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 5000μg/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 1250μg/mL.

A precipitate of test material was observed at and above 78.13μg/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 5000μg/mL. Thus, the test material was considered to be non-mutagenic to L5l78Y 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 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 properties

In summary, all available datainvestigating the genetictoxicity indicate that members of the category Fatty acid C2-8 esters have no genotoxic potential and classificationaccording to EU classification criteria for genetic toxicity is not required.

 

 

References:

*Berg, J.M., Tymoczko, J.L. and Stryer, L., 2002, Biochemistry, 5thedition, W.H. Freeman and Company

*Gubicza, L., Kabiri-Badr, A., Keoves, E., Belafi-Bako, K. (2000): Large-scale enzymatic production of natural flavour esters in organic solvent with continuous water removal. Journal of Biotechnology 84(2): 193-196

*Lilja, J. et al. (2005). Esterification of propanoic acid with ethanol, 1-propanol and butanol over a heterogeneous fiber catalyst. Chemical Engineering Journal, 115(1-2): 1-12.

*Liu, Y. et al. (2006). A comparison of the esterification of acetic acid with methanol using heterogeneous versus homogeneous acid catalysis. Journal of Catalysis 242: 278-286.

*Mattson, F.H. and Volpenheim, R.A. (1972): Relative rates of hydrolysis by rat pancreatic lipase of esters of C2-C18 fatty acids with C1-C18 primary n-alcohols,Journal of Lipid Research, 10, 1969

*Radzi, S.M. et al.(2005). High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica. Electronic Journal of Biotechnology 8: 292-298.

*Tocher, D.R. (2003):Metabolism and function of lipids and fatty acids in teleost fish,Reviews of Fisheries Science, 11 (2), 197

*Zhao, Z. (2000). Synthesis of butyl propionate using novel aluminophosphate molecular sieve as catalyst. Journal of Molecular Catalysis 154(1-2): 131-135.


Short description of key information:
In none of these studies mutagenicity in bacteria could be observed.
In none of these studies clastogenic effects in mammalian cells could be observed.
In none of these studies mutagenicity in mammalian cells could be observed.

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 group concept is applied to the members of the SCAE C2-C8 category, data will be generated from representative reference substance(s) within the category to avoid unnecessary animal testing. Additionally, once the group concept is applied, substances will be classified and labeled on this basis.

The available data on genetic toxicity is conclusive but not sufficient for classification.