Fatty acids, C14-22, 2-ethylhexyl esters, epoxidized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genetic toxicity studies in bacteria (OECD 471) and mammalian cells (OECD 487) are available and report universally negative results.

In the presence of a negative OECD 471 (Annex VII) and OECD 487 (Annex VIII) an in vitro gene mutation study in mammalian cells will be comissioned.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 15, 1992 - June 25, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP compliant. According to guidelines. Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils and esters (linseed, soybean,9-octadecanoate propylene glycol ester and 2-ethylhexyl tallate ester ETP). The C14-C22, 2-ethylhexylesters are listed as similar products on the market to ETP based on fatty acids from other naturally occurring fatty acids This group of epoxies are identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil, ELO epoxidised Linseed oil and ETP epoxidised 2ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters, epoxidised is commonly utilised in the preparation of this dossier. Read-across bridges are used for members of the EOD group where appropriate, is justified based on similar toxicity profiles and structural and functional similarities.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk gene locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK +/- mouse lymphoma cells were obtained from the American Type Culture Collection. They were stored as frozen stocks in liquid nitrogen. Each batch of cells was purged of TK mutants, checked for spontaneous mutant frequency and that they were mycoplasma free. For each experiment the vials were thawed rapidly, the cells were diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mammalian liver post-mitochondrial fraction

Test concentrations with justification for top dose:

The concentrations were selected using a cytotoxicity range-finder. See Table 1 below.

Vehicle / solvent:

Acetone was used as a solvent. The test item was added and the culture was then vortexed for approx. 10 seconds to obtain a good emulsion. A top dose of 5000 ug/ml was achievable using an emulsion.

Negative solvent / vehicle controls:

yes

Remarks:

Comprised of solvent, acetone, diluted 100-fold in the treatment medium.

Positive controls:

yes

Remarks:

Please see Below

Positive control substance:

benzo(a)pyrene

Remarks:

The positive controls were also made to certain concentrations please see table 2 below.

Positive controls:

yes

Remarks:

Please see Below

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

The positive controls were also made to certain concentrations please see table 2 below.

Details on test system and experimental conditions:

METHOD OF APPLICATION:

Preparations of cofactor solutions with and without S-9

Quantity (ml)
With S-9 Without S-9
Glucose-6-phosphate (180 mg/ml) 1.0 -
NADP (25 mg/ml) 1.0 -
150 mM KCL 1.0 5.0
Rat Liver S-9 2.0 -

The above were used at the rate of 1.0 ml per 19 ml of cell culture containing the test chemical (to achieve the required final concentration in a total of 20 ml).


Three types of RPMI 1640 medium were prepared as follows:
Final Concentration in:
RPMI A RPMI 10 RPMI 20
Horse Serum (heat inactivated) 0 % v/v 10 % v/v 20 % v/v
Gentamycin 100 ug/ml 100 ug/ml 100 ug/ml
Fungizone 2.5 ug/ml 2.5 ug/ml 2.5 ug/ml
Pluronic 0.5 ug/ml 0.5 ug/ml -


DURATION
- Exposure duration: 9- 15 days
- Expression time (cells in growth medium): 2 days

NUMBER OF CELLS EVALUATED: 1 x E7 cells per flask

DETERMINATION OF CYTOTOXICITY
Following adjustment of the cultures to 2 x E5 cells/ml after treatment, samples from these were diluted to 8 cells/ml as seen in table 3. using a 8-channel pipette, 0.2 ml of concentration C of each culture was placed into each well of two 96-well microtitre plates (192 wells, at an average of 1.6 cells per wall). The plates were incubated at 37 C in a humidified incubator gassed with 5 % v/v CO2 in air until scorable (9-15 days). Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

At the end of the expression period the cell densities in the selected cultures were adjusted to 1 x E4/ml. TFT (300 ug/ml) was diluted 100-fold into these suspensions to give a final concentration of 3 ug/ml. Using a 8-channel pipette, 0.2 ml of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x E3 cells per well). Plates were incubated until scorable (13 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for doses of test chemical showing a significant increase in mutant frequency over the negative control.

Statistics:

All calculations were performed on a microcomputer.

Determination of survival or viability: Poison distribution, plating efficiency, % relative survival
Determination of mutant frequency: Mutant frequency, plating efficiency,
Statistical significance of mutant frequencies.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Please see Tables 5 & 6 below

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No marked toxicity was seen after the application of the highest dose. Please see Table 4

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Please see table 4 below

Table 4: raw plate counts and % relative survival for Epoxidised Soybean Oil in the cytotoxicity range-finder.

Treatment ug/mL)	In the absence of S-9		In the presence of S-9
	Survival (1) at Day 0*	% Relative survival	Survival (1) at day 0*	% relative survival
0	18	100.0	23	100.0
78.125	21	129.2	21	84.2
156.25	16	83.8	17	59.7
312.5	18	100.0	19	71.0
625	15	76.5	15	49.9
1250	15	76.5	15	49.9
2500	16	83.8	18	65.2
5000	19	109.0	23	100.0

(1) 1.6 cells/well plated

* 32 wells scored

Table 5: Summary of Results Experiment 1

Treatment (ug/ml)	Absence of S-9		Treatment (ug/mL)	Presence of S-9
	% RS	Mutant Frequency		% RS	Mutant Frequency
0	100.0	312.57	0	100.0	401.83
312.5	95.8	389.82 NS	312.5	97.9	484.86 NS
625	86.6	326.20 NS	625	87.9	563.19 NS
1250	79.9	439.82 NS	1250	86.0	518.57 NS
2500	131.9	520.41 *	2500	152.9	426.40 NS
5000	143.9	467.96 *	5000	178.7	433.74 NS
Linear Trend		**	Linear Trend		NS
NQO			BP
0.05	83.5	1676.58	2	55.2	1794.55
0.1	45.3	2078.73	3	39.4	2122.84

 

Table 6: Summary of Results Experiment 2

Treatment (ug/ml)	Absence of S-9		Treatment (ug/mL)	Presence of S-9
	% RS	Mutant Frequency		% RS	Mutant Frequency
0	100.0	410.98	0	100.0	304.98
312.5	112.9	334.82 NS	312.5	91.7	331.46 NS
625	87.7	466.23 NS	625	82.6	331.03 NS
1250	71.4	404.74 NS	1250	87.7	313.73 NS
2500	84.5	461.15 NS	2500	84.5	283.11 NS
5000 $$	100.7		5000	83.2	311.46 NS
Linear Trend		NS	Linear Trend		NS
NQO			BP
0.05	67.5	1136.27	2	59.5	883.78
0.1	37.7	1143.11	3	32.1	1443.04

 

NS Not significant

$$ Treatment excluded due to excessive heterogeneity

*, **, *** Significant at 5 %, 1% and 0.1 % level respectively

Conclusions:

Negative ESBO failed to demonstrate an ability to induce mutation at the tk locus of mouse lymphoma L5178Y cells.

It is concluded that, under the conditions employed in this study, ESBO failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, ESBO is not considered to be mutagenic.
According to Directive 67/548/EEC, no classification is warranted.
According to Regulation (EC) No. 1272/2008, no classification is warranted.

Executive summary:

Epoxidised Soybean Oil (ESBO) was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

Following a wide range of treatments in the range-finder experiment, separated by 2-fold intervals and ranging from 78.125 to 5000µg/ml, cells survived all doses of ESBO yielding 109.0 % relative survival in the absence and 100.0 % relative survival in the presence of S-9 at the top dose.

Accordingly, 5 doses were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top doses plated yielded 143.9 % and 178.7 % relative survival in the absence and presence of S-9. In the second experiment the same dose range was selected. The top dose plated in this experiment was again 5000µg/ml in the absence and presence of S-9, which yielded 100.7 % and 83.2 % relative survival respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In the absence of S-9, reproducible statistically significant and dose-related increases in mutant frequency were not observed in the 2 experiments over the dose range 312.5 to 2500µg/ml. At 5000µg/ml, a positive point was obtained in Experiment 1 and due to heterogeneity in the data this dose was excluded from analysis in Experiment 2. However, if each of the replicate cultures at 5000µg/ml in Experiment 2 are considered in turn, neither yields a statistically significant increase in mutant frequency. This, combined with the fact that there were no absolute increases in mutant numbers in Experiment 1 at 5000µg/ml and that carry over of the test compound was a problem at this dose, suggests that the increased mutant frequency seen in experiment 1 was not the result of chemically induced mutation.

In the presence of S-9, no statistically significant increases in mutant frequency were observed at any dose level tested in Experiment 1 or 2.

It is concluded that, under the conditions employed in this study, ESBO failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, ESBO is not considered to be mutagenic.

According to Directive 67/548/EEC, no classiification is warranted.

According to Regulation (EC) No. 1272/2008, no classiification is warranted.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 August 2021 to 10 September 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Sponsor
- Lot/batch number of test material: OC061290
- Purity: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Assumed stable for the duration of the test. Homogeneity not determined.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Miscible in acetone at 500 mg/mL, assumed stable for the duration of the test.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: The test item was formulated at 500 mg/mL and dosed at 0.5% to achieve a maximum concentration of 2500 μg/mL. However, due to excessive precipitate during the solubility test, the maximum concentration added to the cells in the preliminary toxicity test was 320 μg/mL. In the main test the highest tested concentration was 80 μg/mL as this was the lowest precipitating concentration observed in the blood-free cultures at the end of the exposure period for all three exposure groups. There was no significant change in pH or osmolality.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human peripheral blood from female donors, aged 28 & 31 years.
- Suitability of cells: Used in line with OECD 487 guidance and fully compatible with the test system.
- Normal cell cycle time (negative control): ca. 16 hours

For lymphocytes:
- Sex, age and number of blood donors: Two female donors, one for the preliminary toxicity test (aged 28) and one for the main experiment (aged 31).
- Whether whole blood or separated lymphocytes were used: Whole blood
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI medium (supplemented with L glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% foetal bovine serum (FBS)), heparinised whole blood, phytohaemagglutinin (PHA) and lithium heparin. Lymphocyte cultures were prepared in test flasks or exposure tubes after 44 to 48 hours incubation at ca. 37 ºC, 5% CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B (formulated in DMSO), added to all cultures, after washing at the end of the exposure period, at a final concentration of 6 μg/mL for a period of 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Purchased for a commercial supplier
- method of preparation of S9 mix: Contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium: Final concentration when dosed at 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes

Test concentrations with justification for top dose:

4-hour exposure (-S9): 0, 2.5, 5, 10, 20, 40 and 80 μg/mL
4-hour exposure (+S9): 0, 2.5, 5, 10, 20, 40 and 80 μg/mL
24-hour continuous exposure (-S9): 0, 2.5, 5, 10, 20, 40 and 80 μg/mL.

Based on results from the preliminary toxicity test, the maximum concentration selected for the main experiment was 80 μg/mL as this was the lowest precipitating concentration observed in the blood-free cultures at the end of the exposure period for all three exposure groups.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test item was miscible in acetone at 500 mg/mL.

- Justification for percentage of solvent in the final culture medium: 0.5% to prevent any potential solvent vehicle toxicity.

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

mitomycin C

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

cyclophosphamide

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: Demecolcine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: One main independent experiment (with a prior preliminary toxicity test).

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: Yes, dosed at 0.5% (v/v).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4-hour exposure (-S9), 4-hour exposure (+S9) & 24-hour continuous exposure (-S9).
- Harvest time after the end of treatment (sampling/recovery times): 24-hours after the end of treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure. Cytochalasin B (formulated in DMSO) at 6 μg/ml was added to all cultures, after washing, at the end of the exposure for a period of 24 hours.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Lymphocytes were re-suspended in fresh fixative, centrifuged, and re-suspended in a small amount of fixative. Drops were placed onto clean, wet microscope slides and left to air dry with gentle warming. Dry slides were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of 500 cells/culture were scored for the incidence of
mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a
percentage of the solvent controls. The micronucleus frequency in 1000 binucleated cells/per replicate culture (i.e. 2000 binucleated cells/test item concentration or positive control and 4000 binucleated cells/solvent control). In this study, an additional 1000 binucleate cells were scored for the 4-hour exposure group (+S9) from the main experiment to compensate for the heterogeneity. An additional 2000 binucleate cells were also scored from the B replicates of the solvent control in the 4-hour exposure group (-S9) as initial slides were unsuitable for scoring.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Micronuclei were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was ca. less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was ca. no greater than one quarter of the nuclear diameter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

Rationale for test conditions:

All three exposure groups were tested at six concentrations 0(solvent), 2.5, 5, 10, 20, 40 & 80 μg/mL based on data from the preliminary toxicity test where precipitate was observed at the end
of exposure at 80 μg/mL (in blood-free cultures) as required by OECD 487. No marked concentration-related toxicity was observed in any exposure group. Three test concentrations were evaluated for micronuclei in binucleate cells, these were: 0 (solvent), 20, 40 & 80 μg/mL.

Evaluation criteria:

A test item is considered to be clearly negative if:
1. None of the test item concentrations exhibits a statistically significant increase
compared with the concurrent negative control.
2. There is no concentration-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test item is then considered to be unable to induce chromosome breaks and/or gain or
loss in this test system.

A test item may be considered to be clearly positive if:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks
and/or gain or loss in this test system.

Where the response is neither clearly negative nor clearly positive, the data should be evaluated by expert judgement and/or further investigations to determine the biological relevance of a result.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent solvent control value using the Chi-squared Test on observed numbers of cells
with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a concentration-related increase in the frequency of binucleate cells with micronuclei.
The concentration-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with concentration values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Key result

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Remarks:

Acetone

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Mitomycin C: 0.2 μg/mL (4-hour -S9) Cyclophosphamide: 6 μg/mL (4-hour +S9) Demecolcine: 0.0375 μg/mL (24-hour -S9)

Table 1             Summary of Results 

Exposure Condition	Treatment/Concentration (μg/mL)	Mean Cytostasis  (%)	% Binucleated cells containing micronuclei (a)
			Mean	p-value (b)	Trend test p-value (d)
4-hour -S9	Solvent (Acetone) †	0	0.80	-	0.265
	20	4	0.30	-
	40	5	0.25	-
	80 P	1	0.25	-
	MMC 0.2	19	2.45	1.7814E-07***	-
4-hour +S9	Solvent (Acetone) §	0	0.30	-	0.157
	20 §	5	0.33	-
	40 §	5	0.50	0.0875
	80 P §	14	0.43	-
	CP 6	22	1.40	9.4978E-10***	-
24-hour -S9	Solvent  (Acetone)	0	0.45	-	0.502
	20	5	0.55	-
	40	9	0.35	-
	80 P	12	0.65	0.3084
	DC 0.0375	62	1.75	3.9130E-07***	-

a The percentage of micronucleated cells determined in a sample of 2000 / 4000 binucleate cells
b p-values are for comparison with the control using Chi-square test
d Trend test p-values using Linear regression model applied to control and test item concentrations
P Precipitate observed at end of exposure period in blood-free cultures
§ Additional 1000 binucleate cells scored
† 2000 binucleate cells scored from B replicates due to loss of A replicates
MMC Mitomycin C
CP Cyclophosphamide
DC Demecolcine
*** P<0.001

 

Cytostasis and Micronucleus Data – Main Experiment - 4-Hour Exposure without Metabolic Activation (S9)

Treatment/ Concentration (μg/mL)	Replicate	Mean Cytostasis (%)	Binucleated cells containing micronuclei
			%	Mean %	p-value (b)	Trend test p-value (d)
Solvent         (Acetone)	A1	0	-	0.80	-	0.265
	A2		-
	B1		0.95 §
	B2		0.65 §
20	A	4	0.10	0.30	-
	B		0.50
40	A	5	0.00	0.25	-
	B		0.50
80 P	A	1	0.10	0.25	-
	B		0.40
MMC 0.2	A	19	2.70	2.45	1.7814E-07***	-
	B		2.20

b p-values are for comparison with the control using Chi-square test
d Trend test p-values using Linear regression model applied to control and test item concentrations
§ 2000 binucleate cells scored due to loss of ‘A’ replicates
P Precipitate observed at end of exposure period in blood-free cultures
MMC Mitomycin C
*** P<0.00

 

Cytostasis and Micronucleus Data – Main Experiment - 4-Hour Exposure with Metabolic Activation (S9)

Treatment/ Concentration (μg/mL)	Replicate	Mean Cytostasis (%)	Binucleated cells containing micronuclei
			%	Mean %	p-value (b)	Trend test p-value (d)
Solvent         (Acetone) §	A1	0	0.35	0.30	-	0.157
	A2		0.25
	B1		0.35
	B2		0.25
20 §	A	5	0.30	0.33	-
	B		0.35
40 §	A	5	0.70	0.50	0.0875
	B		0.30
80 P §	A	14	0.50	0.43	-
	B		0.35
CP 6	A	22	1.40	1.40	9.4978E-10***	-

b p-values are for comparison with the control using Chi-square test
d Trend test p-values using Linear regression model applied to control and test item concentrations
§ Additional 1000 binucleate cells scored
P Precipitate observed at end of exposure period in blood-free cultures
CP Cyclophosphamide
*** P<0.001

 

Cytostasis and Micronucleus Data – Main Experiment - 24-Hour Exposure without Metabolic Activation (S9)

Treatment/ Concentration (μg/mL)	Replicate	Mean Cytostasis (%)	Binucleated cells containing micronuclei
			%	Mean %	p-value (b)	Trend test d p-value (d)
Solvent         (Acetone)	A1	0	0.50	0.45	-	0.502
	A2		0.40
	B1		0.60
	B2		0.30
20	A	5	0.60	0.55	-
	B		0.50
40	A	9	0.50	0.35	-
	B		0.20
80 P	A	12	0.40	0.65	0.3084
	B		0.90
DC 0.0375	A	62	1.40	1.75	3.9130E-07***	-

b p-values are for comparison with the control using Chi-square test
d Trend test p-values using Linear regression model applied to control and test item concentrations
P Precipitate observed at end of exposure period in blood-free cultures
DC Demecolcine
*** P<0.001

Conclusions:

Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

The potential of ‘Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised’ to induce clastogenic and/or aneugenic activity in human lymphocytes was evaluated in line with OECD 487. As the test item was freely miscible in acetone at 500 mg/ml, the highest selected dose level in the preliminary toxicity test was 2500 µg/mL when dosing at 0.5% (v/v) in the absence and presence of metabolic activation. On the basis of results from the preliminary toxicity test the highest dose for the main test was 80 µg/mL as this was the lowest dose with precipitate at the end of exposure.

The test item was evaluated in a single independent experiment, with and without a metabolic activation system and tested at a final concentration of 2% in culture media.

The concentrations selected for the main experiment were 0(solvent), 2.5, 5, 10, 20, 40 & 80 µg/mL) across all three treatment regimes (4-hr +/- S9) & 24-hr (-S9). At the end of the exposure period, all cultures were washed and incubated for 24 hours in the presence of Cytochalasin B (6 µg/mL). Ca. 500 cells/culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value (to calculate cytostasis) expressed as a percentage of the solvent controls. To evaluate micronucleus frequency, 1000 binucleated cells/replicate culture, i.e. 2000 binucleated cells/test item concentration or positive control and 4000 binucleated cells for the solvent control were evaluated. An additional 1000 binucleate cells were scored for the 4-hour (+S9) exposure group in the main experiment to compensate for heterogeneity.  An additional 2000 binucleate cells were also scored from the ‘B' replicates of the solvent control in the 4-hour (-S9) exposure group as ‘A’ cultures slides were unsuitable for scoring, 4000 binucleate cells were analysed for the solvent control in this exposure group. 

All solvent (acetone) controls had frequencies of cells with micronuclei within or close to the range expected for normal human lymphocytes and were considered acceptable. Positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses compatible with those in the laboratory historical positive control data range.  The sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei. All values were within the 95% control limits of the historical control data and no concentration dependent trend was observed for any treatment regime. 

Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised was therefore considered to be negative, i.e. non-clastogenic and
non-aneugenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 August 2021 to 6 September 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Sponsor
- Lot/batch number of test material: OC061290
- Purity: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Assumed stable for the duration of the study. Homogeneity not determined.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Miscible in acetone at 100 mg/mL, assumed stable for the duration of the study.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Vortex mixed in high purity acetone. All test item preparation and dosing was performed under yellow safety lighting.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: Using the plate incorporation method, precipitate was noted at 1500 and 5000 µg/plate in both the presence and absence of S9-mix (Experiment 1). Using the pre-incubation method, precipitate was noted at 5000 μg/plate only in both the presence and absence of S9-mix. Precipitate did not prevent the scoring of revertant colonies.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Bacteria were obtained from the British Industrial Biological Research Association on 17 August 1987, and Trinova Biochem GmbH on 27 June 2017.
- Suitability of cells: Bacterial strains selected in line with OECD guidance and fully compatible with the test system.
- Normal cell cycle time (negative control): Not stated.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Top agar was prepared using 0.6% Bacto agar (lot No. 0189156, exp. date 03/2025) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin (for S. typhimurium strains) or 1.0 mM tryptophan solution (for E.coli strain) added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot No's. 59071, exp. date 09/2021 and 59481 exp. date 10/2021). All plates were incubated at 37 ±3°C for between 48 and 72 hours.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Purchased from a commercial supplier.
- method of preparation of S9 mix: The metabolic activation system was lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix reconstituted with cold, sterile water to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix. Lot No's 4438 (Experiments 1 & 2) and 4352 (Experiment 2 rpt).
- concentration or volume of S9 mix and S9 in the final culture medium: 10% (v/v)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes

Test concentrations with justification for top dose:

The recommended maximum test concentration of 5000 µg/plate (for soluble non-cytotoxic substances) in accordance with OECD 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and DMSO at 50 mg/mL but was fully miscible in acetone at 100 mg/mL. Acetone was therefore selected as the solvent.

- Justification for percentage of solvent in the final culture medium: 100 μL (Experiment 1) and 50 μL (Experiment 2) as acetone is toxic to bacterial cells at 0.1 mL (100 μL).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two (plate-incorporation, Experiment 1 & pre-incubation, Experiment 2).

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not stated
- Test substance added in agar: Yes, using the plate-incorporation and pre-incubation methods.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Incubated at 37 ±3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
- Exposure duration/duration of treatment: All plates were incubated at 37 ±3°C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: A lack of any notable cytotoxicity was observed.

Rationale for test conditions:

Concentrations were tested up to the recommended maximum test concentration of 5000 µg/plate (for soluble non-cytotoxic substances) in accordance with OECD 471. In the plate incorporation assay concentrations of 1.5 to 5000 µg/plate were selected, in the pre-incubation assay concentrations of 15 to 5000 µg/plate were selected. Triplicate cultures were used throughout and incubated at standard incubation conditions. Precipitate was observed at 1500 and 5000 µg/plate +/- S9 (Plate incorporation) and 5000 µg/plate +/- S9 (Preincubation). Precipitate did not prevent the scoring of revertant colonies. A lack of any notable cytotoxicity was observed at the limit guideline concentration.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.
A test item is considered non-mutagenic (negative) if the above criteria are not met.

Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was not used in this study.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

-S9 only

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

-S9 only

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

-S9 only

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Remarks:

-S9 only

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

-S9 only

Positive controls validity:

valid

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From:17 August 2021					To: 20 August 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	89 117 96	(101) 14.6#	11 13 34	(19) 12.7	18 17 11		(15) 3.8	12 17 19	(16) 3.6	5 14 4	(8) 5.5
	1.5 µg	103 102 108	(104) 3.2	14 15 16	(15) 1.0	20 10 10		(13) 5.8	16 19 10	(15) 4.6	9 12 9	(10) 1.7
	5 µg	84 111 127	(107) 21.7	12 15 7	(11) 4.0	15 8 14		(12) 3.8	21 14 18	(18) 3.5	14 18 8	(13) 5.0
	15 µg	126 138 104	(123) 17.2	12 24 20	(19) 6.1	13 20 13		(15) 4.0	11 17 16	(15) 3.2	13 4 9	(9) 4.5
	50 µg	120 114 110	(115) 5.0	13 4 19	(12) 7.5	14 14 7		(12) 4.0	15 23 15	(18) 4.6	9 9 4	(7) 2.9
	150 µg	120 124 137	(127) 8.9	36 18 15	(23) 11.4	19 14 15		(16) 2.6	14 17 20	(17) 3.0	9 7 11	(9) 2.0
	500 µg	138 121 120	(126) 10.1	13 20 8	(14) 6.0	18 9 15		(14) 4.6	16 21 14	(17) 3.6	11 4 12	(9) 4.4
	1500 µg	106 P 116 P 117 P	(113) 6.1	10 P 20 P 25 P	(18) 7.6	14 P 14 P 10 P		(13) 2.3	17 P 19 P 20 P	(19) 1.5	12 P 9 P 6 P	(9) 3.0
	5000 µg	111 P 127 P 124 P	(121) 8.5	16 P 18 P 16 P	(17) 1.2	22 P 13 P 18 P		(18) 4.5	10 P 23 P 17 P	(17) 6.5	9 P 2 P 10 P	(7) 4.4
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	515 522 565	(534) 27.1	164 230 157	(184) 40.3	188 148 162		(166) 20.3	212 171 194	(192) 20.6	140 96 317	(184) 117.0

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO          4-Nitroquinoline-1-oxide
9AA             9-Aminoacridine
P                 Test item precipitate
#                 Standard deviation

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From:17 August 2021					To: 20 August 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	107 122 113	(114) 7.5#	10 11 19	(13) 4.9	15 29 22		(22) 7.0	20 16 27	(21) 5.6	10 10 9	(10) 0.6
	1.5 µg	115 97 118	(110) 11.4	9 12 13	(11) 2.1	33 21 28		(27) 6.0	25 17 21	(21) 4.0	4 7 6	(6) 1.5
	5 µg	130 125 118	(124) 6.0	8 7 15	(10) 4.4	23 15 17		(18) 4.2	19 34 17	(23) 9.3	8 10 9	(9) 1.0
	15 µg	117 132 138	(129) 10.8	13 9 11	(11) 2.0	13 16 18		(16) 2.5	21 32 20	(24) 6.7	19 5 6	(10) 7.8
	50 µg	146 131 126	(134) 10.4	10 8 11	(10) 1.5	15 14 8		(12) 3.8	25 20 20	(22) 2.9	7 16 12	(12) 4.5
	150 µg	135 142 118	(132) 12.3	5 10 5	(7) 2.9	21 11 15		(16) 5.0	25 16 22	(21) 4.6	15 9 9	(11) 3.5
	500 µg	133 108 118	(120) 12.6	11 18 18	(16) 4.0	15 26 19		(20) 5.6	21 18 24	(21) 3.0	9 20 14	(14) 5.5
	1500 µg	105 P 157 P 97 P	(120) 32.6	14 P 15 P 7 P	(12) 4.4	16 P 18 P 21 P		(18) 2.5	25 P 26 P 31 P	(27) 3.2	20 P 19 P 9 P	(16) 6.1
	5000 µg	144 P 146 P 124 P	(138) 12.2	10 P 9 P 17 P	(12) 4.4	13 P 20 P 23 P		(19) 5.1	23 P 21 P 36 P	(27) 8.1	17 P 9 P 8 P	(11) 4.9
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1643 1602 1036	(1427) 339.2	135 130 105	(123) 16.1	110 125 172		(136) 32.3	203 199 218	(207) 10.0	62 78 59	(66) 10.2

BP            Benzo(a)pyrene
2AA          2-Aminoanthracene
P              Test item precipitate
#              Standard deviation

Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 27 August 2021					From: 30 August 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	91 81 94	(89) 6.8#	17 13 17	(16) 2.3	22 13 19		(18) 4.6	29 21 16	(22) 6.6	7 7 8	(7) 0.6
	15 µg	79 98 83	(87) 10.0	7 8 15	(10) 4.4	12 22 19		(18) 5.1	25 15 14	(18) 6.1	5 9 5	(6) 2.3
	50 µg	84 87 77	(83) 5.1	22 14 7	(14) 7.5	11 20 19		(17) 4.9	26 16 16	(19) 5.8	4 3 7	(5) 2.1
	150 µg	97 73 101	(90) 15.1	11 16 14	(14) 2.5	13 10 20		(14) 5.1	23 20 16	(20) 3.5	8 11 11	(10) 1.7
	500 µg	104 88 79	(90) 12.7	11 11 13	(12) 1.2	18 25 23		(22) 3.6	20 25 17	(21) 4.0	9 8 9	(9) 0.6
	1500 µg	101 87 98	(95) 7.4	10 15 12	(12) 2.5	16 11 18		(15) 3.6	15 25 19	(20) 5.0	9 9 12	(10) 1.7
	5000 µg	81 P 93 P 95 P	(90) 7.6	11 P 14 P 16 P	(14) 2.5	15 P 17 P 20 P		(17) 2.5	15 P 10 P 13 P	(13) 2.5	10 P 12 P 6 P	(9) 3.1
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	544 540 497	(527) 26.1	376 427 319	(374) 54.0	526 503 464		(498) 31.3	166 189 196	(184) 15.7	565 335 286	(395) 149.0

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO          4-Nitroquinoline-1-oxide
9AA             9-Aminoacridine
P                 Test item precipitate
#                 Standard deviation

Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 27 August 2021 From: 02 September 2021 †					From: 30 August 2021 From: 05 September 2021 †
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98 †		TA1537
	Solvent Control (Acetone)	153 136 150	(146) 9.1#	11 7 8	(9) 2.1	15 23 26		(21) 5.7	17 22 23	(21) 3.2	9 7 4	(7) 2.5
	15 µg	124 128 147	(133) 12.3	12 8 6	(9) 3.1	20 31 19		(23) 6.7	21 22 24	(22) 1.5	13 12 15	(13) 1.5
	50 µg	106 110 99	(105) 5.6	17 9 13	(13) 4.0	18 22 24		(21) 3.1	19 21 20	(20) 1.0	11 14 10	(12) 2.1
	150 µg	95 107 87	(96) 10.1	17 9 13	(13) 4.0	18 15 19		(17) 2.1	35 23 21	(26) 7.6	5 8 8	(7) 1.7
	500 µg	103 78 104	(95) 14.7	13 6 19	(13) 6.5	22 19 26		(22) 3.5	24 17 15	(19) 4.7	13 10 8	(10) 2.5
	1500 µg	113 91 111	(105) 12.2	10 17 7	(11) 5.1	21 21 19		(20) 1.2	28 34 22	(28) 6.0	13 10 6	(10) 3.5
	5000 µg	100 P 112 P 114 P	(109) 7.6	12 P 12 P 10 P	(11) 1.2	17 P 20 P 27 P		(21) 5.1	25 P 25 P 28 P	(26) 1.7	10 P 19 P 9 P	(13) 5.5
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1268 1343 1305	(1305) 37.5	195 211 166	(191) 22.8	380 400 408		(396) 14.4	157 150 128	(145) 15.1	197 175 207	(193) 16.4

†              Experimental procedure repeated at a later date due to contamination in the original test
BP            Benzo(a)pyrene
2AA          2-Aminoanthracene
P              Test item precipitate
#              Standard deviation

Conclusions:

Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised was considered non-mutagenic.

Executive summary:

The potential of the test item ‘Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised’ to induce reverse mutation was evaluated in five bacterial strains in line with OECD 471 and other current international guidelines. The study was GLP compliant. As the test item was freely miscible in acetone at 100 mg/ml, the highest selected dose level was 5000 µg/plate, in line with regulatory guidelines.

The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix was prepared from lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (Mutazyme TM) to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix. The first experiment used the plate incorporation method at concentrations of 1.5 - 5000 µg/plate, the second experiment used the pre-incubation method at concentrations of 15 - 5000 µg/plate. Four strains of bacteria Salmonella typhimurium: TA1535, TA1537, TA98, TA100 and a single strain of E. coli (WP2 urvA) were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level) and all were incubated at standard incubation conditions for 48 to 72 hours after which revertant colonies were scored. The preincubation period in experiment two was for 20 minutes (with shaking) followed by standard incubation for 48-72 hours. Appropriate positive controls were also used. Plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity). 

The number of revertants for the vehicle and positive controls was within an acceptable range and therefore the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first experiment, precipitate was observed at 1500 & 5000 µg/plate, in the second experiment precipitate was observed at 5000 µg/plate only. In both experiments there was no notable reduction in the growth of the bacterial background lawn at any dose level (even at precipitating concentrations), either in the presence or absence of S9-mix.

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Fatty acids, C14-C22, 2-ethyl hexyl esters, epoxidised was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data are available and none are required based on the negative responses seen in studies of genetic toxicity in vitro.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data do not indicate any evidence of genetic toxicity. According to Regulation (EC) No. 1272/2008, no classification is warranted.