Fatty acids, C16-18, 2-butyloctyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-14 to 2015-02-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction, derived from male Wistar rats, phenobarbital (80 mg/kg bw) / ß-naphthoflavone (100 mg/kg bw) induced for three consecutive days by oral route

Test concentrations with justification for top dose:

Pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate
Experiment I: 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate
Experiment II: 0.0158, 0.050, 0.158, 0.5, 1.58, 5 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

A. dest., treated in the same way as all dose groups

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, treated in the same way as all dose groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: S. typhimurium strains TA 100, TA 1535: sodium azide (10µg/plate); S. typhimurium strains TA 98, TA 1537: 4-nitro-o-phenylene-diamine (10 µg/plate for TA 98, 40 µg/plate for TA 1537); E.coli WP2 uvrA: methylmethanesulfonate (1 µg/plate)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

A. dest., treated in the same way as all dose groups

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, treated in the same way as all dose groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic actiavation, S thyphimurium TA 98, TA 1537

Untreated negative controls:

yes

Remarks:

A. dest., treated in the same way as all dose groups

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, treated in the same way as all dose groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: E. coli WP 2 uvrA

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene (2.5 µg/plate for all S. typhimurium strains, 10 µg/plate for E. coli WP2 uvrA)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in the pre-experiment and in experiment I, preincubation in experiment II

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control

OTHER:
The properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al..
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH, Germany). Tester strains with a low spontaneous mutation frequency (TA 1535 and TA 1537) were counted manually.

Evaluation criteria:

Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice
as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible
biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system.

Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data ranges
-corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.
- at least five different concentrations of each tester strain are analysable.

Statistics:

A statistical evaluation of the results was not regarded as necessary as the biological relevance of the results is the criterion for the interpretation of results (according to guideline).

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment I toxic effects of the test item were observed in tester strain TA 100 at concentrations of 2.5 μg/plate and higher (with metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determind with tester stains TA 98 and TA 100. Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The control plates with and without S9 mix were within the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item were noted in any of the five tester strains used up to the highest
dose group evaluated with and without metabolic activation in experiment I and II, with one exception: In experiment I toxic effects of the test item were observed in tester strain TA 100 at concentrations of 2.5 μg/plate and higher (with metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Fatty acids, C16 -18, 2 -butyloctyl esters was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. A correction factor of 1.022 was applied to consider purity of the test item.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: Experiment I: 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate Experiment II: 0.0158, 0.050, 0.158, 0.50, 1.58 and 5.0 μL/plate Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II, with one exception: In experiment I toxic effects of the test item were observed in tester strain TA 100 at concentrations of 2.5 μg/plate and higher (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Fatty acids, C16-18, 2-butyloctyl esters at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Fatty acids, C16-18, 2-butyloctyl esters did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Fatty acids, C16-18, 2-butyloctyl esters is considered to be nonmutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

09 Apr - 27 Jul 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the structural similarities and consistent trend in toxicokinetic and (eco)toxicological behaviour, the selected source substances are considered suitable and systemic human health effects and ecotoxicological effects can be directly read-across in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.

Reason / purpose for cross-reference:

read-across source

Vehicle / solvent:

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

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 2-Ethylhexyl oleate was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 µg/mL.
- Precipitation: 2-Ethylhexyl oleate precipitated in the exposure medium at concentrations of 100 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: In the dose finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24-hour treatment period and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours treatment and 24 and 48 hours subculture, no toxicity in the suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix.
After 24 hours of treatment and 24-hour subculture, in the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentration of 333 µg/mL compared to the solvent control.


COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the
minimum and maximum value of the historical control data range. See Table 1 to 4.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Table 1. Experiment 1: Cytotoxic and mutagenic response of 2-Ethylhexyl oleate in the mouse lymphoma L5178Y test system.

Dose (µg/mL)	RSG (%)	CE^day2(%)	RS^day2 (%)	RTG (%)	Mutation Frequency x 10-6
					Total	Small	Large
	Without metabolic activation (3-hour treatment)
SC1	100	89	100	100	100	69	28
SC2	100	108	100	100	99	67	28
0.03	106	105	107	113	87	63	20
0.1	102	101	102	104	76	50	23
0.3	88	86	88	77	109	71	34
1	107	99	101	108	99	73	22
3	106	97	98	104	93	64	25
10	103	105	107	110	88	62	22
33	82	111	113	92	133	86	38
100^(1)	82	120	121	100	93	67	22
MMS	66	56	57	37	1463	939	292
	With 8% (v/v) metabolic activation (3-hour treatment)
SC1	100	65	100	100	68	41	26
SC2	100	67	100	100	58	32	25
0.03	96	66	100	96	73	45	26
0.1	102	63	95	97	71	39	30
0.3	93	67	102	94	71	46	24
1	107	66	100	107	71	40	29
3	108	58	88	95	74	53	20
10	107	53	80	85	74	50	22
33	95	62	94	89	74	43	29
100^(1)	100	54	81	81	68	44	23
CP	57	44	66	38	752	574	137

Note: all calculations were made without rounding off.

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = Ethanol; MMS Methylmethanesulfonate; CP = Cyclophosphamide.

(1) = 2-Ethylhexyl oleate precipitated in the exposure medium.

Table 2. Experiment 2: Cytotoxic and mutagenic response of 2-Ethyl oleate in the mouse lymphoma L5178Y test system.

Dose (µg/mL)	RSG (%)	CE^day2(%)	RS^day2 (%)	RTG (%)	Mutation Frequency x 10-6
					Total	Small	Large
	Without metabolic activation (24-hour treatment)
SC1	100	85	100	100	59	35	22
SC2	100	93	100	100	63	35	26
0.03	119	93	104	124	67	36	29
0.1	134	91	103	138	56	34	21
0.3	121	115	129	156	40	20	20
1	124	97	109	135	47	35	12
3	105	89	100	105	57	38	18
10	124	94	106	131	52	27	24
33	119	99	112	133	58	31	25
100^(1)	129	97	109	140	44	28	15
MMS	106	81	92	97	503	305	152
	With 12% (v/v) metabolic activation (3-hour treatment)
SC1	100	76	100	100	102	51	47
SC2	100	101	100	100	76	38	35
0.03	96	81	92	88	82	44	36
0.1	100	80	91	91	82	44	35
0.3	103	95	108	111	97	52	41
1	106	101	114	121	70	41	27
3	102	83	94	95	85	40	42
10	96	88	99	95	76	46	28
33	99	81	92	91	89	55	31
100^(1)	98	86	98	96	73	37	34
MMS	62	66	75	46	1337	776	310

Table 3. Historical control data of the spontaneous mutation frequencies of the solvent controls of the mouse lymphoma assay.

	-S9-mix		+S9-mix
	3-hour treatment	24-hour treatment
Range	[51-120] x 10^-6	[50-127] x10-^6	[50-170] x 10^-6
Mean	77 x 10^-6	80 x 10^-6	92 x 10^-6
SD	18 x 10^-6	19 x10^-6	33 x10^-6
n	88	82	141

SD = Standard deviation

n = Number of observation

The above mentioned historical control data range of the solvent controls were obtained by collecting all data of the solvent control values (media, DMSO, and ethanol) over the period of June 2006 to June 2009.

Table 4. Historical control data of the spontaneous mutation frequencies of the positive controls for the mouse lymphoma assay.

	-S9-mix		+S9-mix
	3-hour treatment	24-hour treatment
Range	[518-2052] x 10^-6	[578-1533] x10-^6	[724-3715] x 10^-6
Mean	1004 x 10^-6	1063 x 10^-6	1597 x 10^-6
SD	356 x 10^-6	232 x10^-6	712 x10^-6
n	45	34	81

 

The above mentioned historical control data range of the positive controls were obtained by collecting all data over the period of June 2006 to June 2009.

Conclusions:

Interpretation of results (migrated information):
negative

The source substance 2-ethylhexyl oleate was in the mouse lymphoma test according to OECD 476 negative.

Executive summary:

The source substance 2-ethylhexyl oleate was in the mouse lymphoma test according to OECD 476 negative.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Due to the structural similarities and consistent trend in toxicokinetic and (eco)toxicological behaviour, the selected source substances are considered suitable and systemic human health effects and ecotoxicological effects can be directly read-across in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 33 µg/mL 2-ethylhexyl oleate precipitated in the culture medium
- Other confounding effects: 2-ethylhexyl oleate showed no significant effects on the number of polyploid cells

RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity, inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the duplicate cultures of the 3h exposure time.

Remarks on result:

other: strain/cell type: cultured human peripheral lymphocytes

Table 1: Cytotoxic and Genotoxic observations

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 3 h, fixation time 24 h, without S9 mix
Ethanol	1.0% (v/v)	100	2	2
MMC	0.5	67	31	30
Test substance	3	99	1	1
	10	98	2	2
	33	92	1	1
Exposure period 3 h, fixation time 24 h, with S9 mix
Ethanol	1.0% (v/v)	100	1	1
CP	0.5	51	38	38
Test substance	3	101	1	1
	10	108	0	0
	33	103	3	3
Exposure period 24 h, fixation time 24 h, without S9 mix
Ethanol	1.0% (v/v)	100	1	1
MMC	0.2	54	34	33
Test substance	3	99	1	1
	10	103	3	3
	33	70	4	3
Exposure period 48 h, fixation time 48 h, without S9 mix
Ethanol	1.0% (v/v)	100	2	0
MMC	0.1	120	51	49
Test substance	3	108	1	0
	10	100	0	0
	33	99	2	2
Exposure period 3 h, fixation time 48 h, with S9 mix
Ethanol	1.0% (v/v)	100	0	0
CP	10.0	--	44	44
Test substance	3	100	2	2
	10	94	2	2
	33	97	1	0

MMC: Mitomycin          CP: Cyclophosphamide

                    

Conclusions:

Interpretation of results (migrated information):
negative

Both in the absence and presence of S9-mix the similar substance 2-ethylhexyl oleate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of 2-ethylhexyl oleate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the similar substance 2-ethylhexyl oleate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report according to OECD 473.

Executive summary:

Both in the absence and presence of S9-mix the similar substance 2-ethylhexyl oleate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of 2-ethylhexyl oleate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that ethylhexyl oleate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The target substance was considered to be non-mutagenic in a bacterial reverse mutation assay (Ames test) performed according to OECD Guideline 471, with and without metabolic activation.

Both in the absence and presence of S9-mix the source substance 2-ethylhexyl oleate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of 2-ethylhexyl oleate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed in the absence and presence of S9-mix. Therefore it can be concluded that the source substance 2-ethylhexyl oleate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report performed according to OECD Guideline 473.

The source substance 2-ethylhexyl oleate was in the mouse lymphoma test according to OECD Guideline 476 negative.

In conclusion, the results of the available in vitro studies on target and source substances were negative. Based on the available data on target and source substances, and following the analogue approach, Fatty acids, C16-18, 2-butyloctyl esters is not expected to be mutagenic and clastogenic.

Justification for selection of genetic toxicity endpoint
In all available genetic toxicity tests the test substance and the source substances with and without metabolic activation were not mutagenic.

Short description of key information:
In all available genetic toxicity tests the test substance and the source substances with and without metabolic activation were not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance and the similar substances were not mutagenic in any of the available in vitro genetic toxicity tests. Therefore a classification according to Directive 67/548/EEC and Regulation (EC) No 1272/2008 is not required.