Fatty acids, C14-18 and C18-unsatd., branched and linear, 2-ethylhexyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available datas for in vitro bacteria reverse mutation, mammalian cytogenicity and mutagenicity tests of the source substances of the category, no genotoxicity was observed for both the tested substances. Hence, the registered substance 2-ethylhexyl isostearate could be considered as not mutagenic for bacteria strain and mammalian cells and not cytogenic using this category approach and according to the CLP criteria.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Key result

Species / strain:

other: TA 1535, 1537, 102, 100, 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Results from key studies on source chemicals of the category for in vitro mutagenicity tests.

ID#	CAS	In vitro gene mutation study in bacteria (Ames test)
Fatty acids, C8-16, 2- ethylhexyl esters	135800-37-2	Experimental result: Negative
Fatty acids, coco, 2-ethylhexyl esters	92044-87-6	No data
2-Ethylhexyl palmitate	29806-73-3	No data
Fatty acids, C16-18 and C18-unsatd., 2- ethylhexyl esters	85049-37-2	No data
Fatty acids, C16-18, 2-ethylhexyl esters	91031-48-0	Experimental result: Negative
2-Ethylhexyl oleate	26399-02-0	No data
2-Ethyl hexyl stearate	22047-49-0	No data

 

Similar toxicokinetic behavior and toxicity profile

None of the tested source substances of the category were positive to Ames test (OECD TG 471). They were not mutagenic for bacteria strains.

Based on the structural similarities between source and target chemicals and their common toxicokinetic behavior, the target substance was considered as not mutagenic in in vitro system.

 

Conclusions:

Based on the available datas for in vitro bacteria reverse mutation of the members of the category substances, nogenotoxicity was observed for both the tested substances. Hence, the registered substance 2-ethylhexyl isostearate could be considered as not mutagenic for bacteria strain using this category approach and according to the CLP criteria.

Executive summary:

According to the Regulation (EC) NO. 1907/2006, Annex XI, 1.5, A Read-Across Category for Long Chain Fatty Acid was performed in order to provide informations on the Isostearate Ethyl Hexyl.

This category was based on common and shared physico-chemical and structural properties as:

-       common functional group,

-       common precursors and the likehood of common impurities as well as common breakdown products via biological processes, which are chemically structurally similar, and

-       constant pattern in the changing of the potency of the properties across the category.

The fatty acids linked with 2-ethylhexyl esters have a common metabolic fate in organisms as glycolytic and fatty acid pathways after first hydrolysis step which led in breakdown products. The common toxicokinetic properties and behavior are expected due to the constant pattern (ethylhexyl esters and the fatty acid chain). The toxicological profiles between the members of the category are expected to be the same.

Several studies were performed on substances Fatty acids, C16-18, 2-ethylhexyl esters, 2-ethylhexyl oleate and Fatty acids, C8-16, 2-ethylhexyl ester in order to investigate the mutagenicity potential properties in bacteria. None of these experimental studies showed positive effect. Hence, the members of the category can be considered to have no mutagenic effects on bacteria strain.

 

Based on the structural similarities betweensource and target chemicals and their common toxicokinetic behavior, the target substance was considered as not mutagenic ini n vitro system using bacterias strain.

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Key result

Species / strain:

primary culture, other: Primary human lypmphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Results from key studies on source chemicals of the category for in vitro mutagenicity tests.

ID#	CAS	In vitro cytogenicity study in mammalian cells
Fatty acids, C8-16, 2- ethylhexyl esters	135800-37-2	No data
Fatty acids, coco, 2-ethylhexyl esters	92044-87-6	No data
2-Ethylhexyl palmitate	29806-73-3	No data
Fatty acids, C16-18 and C18-unsatd., 2- ethylhexyl esters	85049-37-2	No data
Fatty acids, C16-18, 2-ethylhexyl esters	91031-48-0	No data
2-Ethylhexyl oleate	26399-02-0	Experimental result: Negative
2-Ethyl hexyl stearate	22047-49-0	No data

 

Similar toxicokinetic behavior and toxicity profile

 

The tested source substance was not considered as cytogenic for mammalian cells.

Based on the structural similarities between source and target chemicals and their common toxicokinetic behavior, the target substance was considered as not clastogenic in in vitro system.

 

Conclusions:

Based on the available data for in vitro cytogenicity test on mammalian cells on the member of the category, no genotoxicity was observed for both the tested substances. Hence, the registered substance 2-ethylhexyl isostearate could be considered as not cytogenic for mammalian cells using this category approach and according to the CLP criteria.

Executive summary:

According to the Regulation (EC) NO. 1907/2006, Annex XI, 1.5, A Read-Across Category for Long Chain Fatty Acid was performed in order to provide informations on the Isostearate 2-Ethyl Hexyl.

This category was based on common and shared physico-chemical and structural properties as:

-       common functional group,

-       common precursors and the likehood of common impurities as well as common breakdown products via biological processes, which are chemically structurally similar, and

-       constant pattern in the changing of the potency of the properties across the category.

The fatty acids linked with 2-ethylhexyl esters have a common metabolic fate in organisms as glycolytic and fatty acid pathways after first hydrolysis step which led in breakdown products. The common toxicokinetic properties and behavior are expected due to the constant pattern (ethylhexyl esters and the fatty acid chain). The toxicological profiles between the members of the category are expected to be the same.

One key study was performed on source chemical of the category, the 2 -ethylhexyl oleate in order to investigate the clastogenicity potential properties in mammalian cells. No positive effect was observed. Hence, the members of the category can be considered to have no clastogenic effects in mammalian cells.

 

Based on the structural similarities between source and target chemicals and their common toxicokinetic behavior, the target substance was considered as not clastogenic in this in vitro mammalian cytogenicity test

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Results from key studies on source chemicals of the category for in vitro mutagenicity tests.

ID#	CAS	In vitro mutation study in mammalian cells
Fatty acids, C8-16, 2- ethylhexyl esters	135800-37-2	No data
Fatty acids, coco, 2-ethylhexyl esters	92044-87-6	No data
2-Ethylhexyl palmitate	29806-73-3	No data
Fatty acids, C16-18 and C18-unsatd., 2- ethylhexyl esters	85049-37-2	No data
Fatty acids, C16-18, 2-ethylhexyl esters	91031-48-0	No data
2-Ethylhexyl oleate	26399-02-0	Experimental result: Negative
2-Ethyl hexyl stearate	22047-49-0	No data

 

Similar toxicokinetic behavior and toxicity profile

The tested source substance was not considered as mutagenic for mammalian cells.

Based on the structural similarities between source and target chemicals and their common toxicokinetic behavior, the target substance was considered as not mutagenic in in vitro mutation test on mammalian cells.

 

Conclusions:

Based on the available datas for in vitro mutation test on mammalian cells, no genotoxicity was observed for both the tested substances. Hence, the registered substance 2-ethylhexyl isostearate could be considered as not mutagenic for L517Y cells using this category approach and according to the CLP criteria.

Executive summary:

According to the Regulation (EC) NO. 1907/2006, Annex XI, 1.5, A Read-Across Category for Long Chain Fatty Acid was performed in order to provide informations on the Isostearate 2-Ethyl Hexyl.

This category was based on common and shared physico-chemical and structural properties as:

-       common functional group,

-       common precursors and the likehood of common impurities as well as common breakdown products via biological processes, which are chemically structurally similar, and

-       constant pattern in the changing of the potency of the properties across the category.

The fatty acids linked with 2-ethylhexyl esters have a common metabolic fate in organisms as glycolytic and fatty acid pathways after first hydrolysis step which led in breakdown products. The common toxicokinetic properties and behavior are expected due to the constant pattern (ethylhexyl esters and the fatty acid chain). The toxicological profiles between the members of the category are expected to be the same.

One key study was performed on source chemical of the category, the 2 -ethylhexyl oleate in order to investigate the mutagenicity and genotoxicity potential properties in mammalian cells Mammalian cell gene mutation). No positive effect was observed. Hence, the members of the category can be considered to have no mutagenic effects in mammalian cells.

 

Based on the structural similarities between source and target chemicals and their common toxicokinetic behavior, the target substance was considered as not mutagenic in this in vitro mammalian mutagenicity test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the available key study on a source chemical from the category, the target substance was considered as not mutagenic in in vivo test sytem.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read across category

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Results from key studies on source chemicals of the category for in vivo mutagenicity tests.

ID#	CAS	In vivo mutagenicity test
Fatty acids, C8-16, 2- ethylhexyl esters	135800-37-2	Experimental result: Negative
Fatty acids, coco, 2-ethylhexyl esters	92044-87-6	No data
2-Ethylhexyl palmitate	29806-73-3	No data
Fatty acids, C16-18 and C18-unsatd., 2- ethylhexyl esters	85049-37-2	No data
Fatty acids, C16-18, 2-ethylhexyl esters	91031-48-0	No data
2-Ethylhexyl oleate	26399-02-0	No data
2-Ethyl hexyl stearate	22047-49-0	No data

 

Similar toxicokinetic behavior and toxicity profile

 

All category members are subject to enzymatic hydrolysis by pancreatic lipases resulting in free acids and alcohol. Based on current literature, when absorbed from intestines and carried through blood stream, fatty acids are oxidized by beta-oxydation pathway in order to provide energy for cell and stored as glycerides esters in fat deposit. The alcohols are primarily metabolized in the liver.

Hence, it can be stated that the members of the category have the same toxicity due to the same metabolic pathways when absorbed in the organisms.

Based on the available data for in vivo micronucleus test, no genotoxicity was observed on the tested source substance including in the category. Hence, according to the physico-chemical similarities between the substances of the category, the registered substance 2-ethylhexyl isostearate could be considered as not genotoxic in in vivo micronucleus test.

Conclusions:

 Based on the available data for in vivo micronucleus test, no genotoxicity was observed. Hence, the registered substance 2-ethylhexyl isostearate could be considered as not genotoxic in in vivo micronucleus test

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification and rationale of the category approach for the isostearate ethyl hexyl :

This category group covers 2-ethyl hexyl esters linked with fatty acid chains (C8 to C18) unsatured and satured. This category includes monoconstituent substances and UVCB substances varying fatty acid chain length. This category was made in order to provide sufficient information for physicochemical, ecotoxicological and toxicological caracterisation of the Fatty acids, C14-18 and C18-unsatd., branched and linear, 2-ethylhexyl esters (CAS No 85186-76-1)

The category group includes:

- 2-Ethyl hexyl stearate (CAS No 22047-49-0)

- Fatty acids, C8-16, 2-ethylhexyl esters (CAS No 135800-37-2)

- Fatty acids, C16-18 and C18-unsatd., 2-ethylhexyl esters (CAS No 85049-37-2)

- Fatty acids, coco, 2-ethylhexyl esters (CAS No 92044-87-6)

- Fatty acids, C16-18, 2-ethylhexyl esters (CAS No 91031-48-0)

- Fatty acids, C8-16, 2-ethylhexyl esters (CAS No 135800-37-2)

- 2-Ethylhexyl oleate (CAS No 26399-02-0)

Target substance for Category Approach : Fatty acids, C14-18 and C18-unsatd., branched and linear, 2-ethylhexyl esters (CAS No 85186-76-1)

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, environmental fate and ecotoxicity, 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)”. Therefore, the available experimental data were collected and evaluated according to Annex XI requirements.

Summary of the available studies

Fatty acids, C8 -C16, 2 -ethylhexyl esters CAS 135800 -37 -2

The mutagenic potential of Fatty acids, C8-16, 2-Ethylhexyl Esters 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. 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. 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.

A in vivo micronucleus test was performed. 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 of Fatty acids, C8-16, 2-Ethylhexyl Esters 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 in Fatty acids, C8-16, 2-Ethylhexyl Esters treated animals and therefore no indications of induced chromosome damage were found.

Fatty acids, C16-18, 2-Ethylhexyl Esters CAS 91031-48-0

The mutagenic potential of Fatty acids, C16-18, 2-Ethylhexyl Esters 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 . 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.

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.

2-Ethylhexyl oleate CAS 26399-02-0

An in vitro mammalian chromosome aberration test was performed with 2 -Ethylhexyl oleate 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. 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.

An in vitro Mammalian Cell Gene Mutation Assay according to OECD Guideline 476 and under GLP was performed with 2-Ethylhexyl Oleate 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.