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Ethyl ester - Mutagenicity in bacteria

 

An Ames test was performed with 9,12-Octadecadienoic acid (Z,Z)-, ethyl ester (CAS# 544 -35 -4) according to OECD guideline 471 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 rat liver S9-mix. The test substance slightly precipitates at concentrations ≥ 1000 µg/plate but induces no cytotoxic or genotoxic effects at any concentration neither in the presence nor in the abscence 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 strains.

 

Isopropyl ester - Mutagenicity in bacteria

 

The mutagenic potential of isopropyl myristate (CAS# 110-27-0) was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Gloxhuber, 1991) equivalent to OECD Guideline 471. Test substance concentrations of 0, 4, 20, 100, 500 and 2500 µg/plate in acetone were tested with and without the addition of a rat liver homogenate metabolising system (S9-mix).

No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. Thus, isopropyl myristate did not induce gene mutations in five tested Salmonella strains under the given test conditions.

 

Butyl ester - Mutagenicity in bacteria

 

Fatty acids, C16-18 and C18 unsatd. branched and linear, butyl esters (CAS# 163961-32-8) was tested for mutagenicity in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 using a plate incorporation according to OECD Guideline 471 (Bowles, 2002). 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. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation.

 

Ethylhexyl ester - Mutagenicity in bacteria

 

A Bacterial Reverse Mutation Assay was performed with Fatty acids, C8-16, 2-ethylhexyl esters (CAS# 135800-37-2) according to OECD Guideline 471 (Banduhn, 1990). Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with the test substance diluted in Tween 80/water (bidest.) using the plate incorporation method. Two independent experiments were performed with identical dose levels of 0, 8, 40, 200, 1000 and 5000 µg/plate. Both experiments were performed in triplicates with and without the addition of a rat liver homogenate metabolising system (S9-mix).

Cytotoxic effects were not observed. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation. Based on the study results, Fatty acids, C8-16, 2-ethylhexyl esters did not induce gene mutations in five tested Salmonella strains.

 

Fatty acids, C16-18, 2-ethylhexyl esters (CAS# 91031-48-0) was tested for mutagenicity in S. tyhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 according to OECD Guideline 471 (Banduhn, 1988). Test substance concentrations of 0, 8, 40, 200, 1000 and 5000 µg/plate in Tween 80/bidest. water were tested in triplicates in two independent experiments using the plate incorporation method with and without the addition of a rat liver homogenate metabolising system (S9-mix).

No cytotoxicity was observed. No increase in the frequency of revertant colonies compared to concurrent negative controls were observed in all tested strains, neither in the presence nor in the absence of metabolic activation.

 

Ethyl ester - In vitro cytogenicity in mammalian cells

 

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 (Verbaan, 2010) in two independent experiments. 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 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.

 

Isopropyl ester - In vitro cytogenicity in mammalian cells

 

An in vitro Mammalian Cell Gene Mutation Test according to OECD Guideline 476 was performed with Isopropyl Laurate (CAS# 10233 -13 -3) in mouse lymphoma L5178Y cells (Verspeek-Rip, 2010). The cells were treated for 3 hours without S9-mix, 3h with 8% (v/v) S9-mix, 24h without S9-mix and 3h with 12% (v/v) S9-mix with 0.01, 0.03, 0.1, 0.3, 1, 3, 5 and 10μg/mL of the test substance. 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.

 

Butyl ester - In vitro cytogenicity in mammalian cells

 

An in vitro mammalian chromosome aberration test was performed with Fatty acids, C16-18 and C18-unsatd., branched and linear, Bu esters (CAS# 163961-32-8) in primary human lymphocytes according to OECD Guideline 473 (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 followed by 20 hours expression time with metabolic activation. In the second experiment 0, 625, 1250 and 2500 µg/mL were used for 4 hours exposure followed by 20 hours expression time with and without S9. 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 metabolising 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 metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

Ethylhexyl ester - In vitro cytogenicity in mammalian cells

 

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 (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 test substance concentrations of 3, 10 and 33 µg/mL in ethanol were used for 3 hours of exposure with and without metabolic activation. In the second experiment 3, 10 and 33 µg/mL were used for 24 hours exposure followed by 24 hours expression time and 48 hours exposure following 48 hours expression time without S9. 33 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 metabolising 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 metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

Ethyl ester - In vitro mutagenicity in mammalian cells

 

The mutagenic activity of ethyl linoleate (CAS# 544-35-4) was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma 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 300μg/mL dissolved in dimethyl sulfoxide. At this dose level cytotoxicity occurred in the presence and absence of metabolic activation. 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.

 

Isopropyl ester - In vitro mutagenicity in mammalian cells

 

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 (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 test substance concentrations of 10, 33 and 100 µg/mL in ethanol were used for 3 hours of exposure with and without metabolic activation. In the second experiment 66, 150 and 250 µg/mL were used for 24 hours exposure followed by 24 hours expression time and 3, 125 and 150 µg/mL for 48 hours exposure following 48 hours expression time without S9. 250 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 metabolising 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 metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

Butyl ester - In vitro mutagenicity in mammalian cells

 

An in vitro Mammalian Cell Gene Mutation Test was performed with Fatty acids, C16-18 and C18-unsatd., branched and linear, Bu esters (CAS# 163961-32-8) in mouse lymphoma L5178Y cells (heterozygous at the thymidine kinase locus) according to OECD Guideline 476 (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.

 

Ethylhexyl ester - In vitro mutagenicity in mammalian cells

 

The mutagenic activity of 2 -ethylhexyl oleate (CAS# 26399 -02 -0) was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma 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. At this dose level 2-ethylhexyl oleate precipitated in the culture medium. 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.

 


Short description of key information:
Studies on the mutagenicity in bacteria were available for the following category members (CAS#): 110-27-0, 544-35-4, 135800-37-2, 91031-48-0, 163961-32-8
In none of these studies mutagenicity in bacteria could be observed.
Studies on the in vitro cytogenicity in mammalian cells were available for the following category members (CAS#): 10233-13-3, 544-35-4, 26399-02-0 and 163961-32-8.
In none of these studies clastogenic effects in mammalian cells could be observed.
Studies on the in vitro gene mutation in mammalian cells were available for the following category members (CAS#): 10233-13-3, 544-35-4, 26399-02-0 and 163961-32-8.
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 DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria for genetic toxicity, no classification is required.