Fatty acids, C18 unsaturated, C12-14 (even numbered) alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tetradecyl myristate, a constituent of the registration substance, and 2-octyldodecyl isooctadecanoate (CAS No 93803-87-3), a structural analogue, showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The studies were performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvr A. Test concentrations up to the precipitation limit concentration of 1000 µg/plate were tested in the experiment. The test compounds proved to be not mutagenic to the bacterial strains.

2-Ethylhexyl octadec-9-enoate (CAS No. 26399-02-0) andOleyl oleate (CAS No. 3687-45-4), structural analogues, also yielded negative results inin vitrogene mutation studies (MLA and HPRT) in mammalian cells in concentration up to 100 µg/mL with and without metabolic activation.

2-Ethylhexyl octadec-9-enoate (CAS No. 26399-02-0), 2-octyldodecyl isooctadecanoate (CAS No 93803-87-3) and Oleyl oleate (CAS No. 3687-45-4), structural analogues, were assessed for their potential to induce chromosome aberrations in human lymphocytes andChinese hamster lung fibroblasts (V79) in vitro. The test items did not induce chromosome aberrations. Therefore, it is concluded that the registration substance is non-mutagenic in these in vitro chromosome aberration tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1981

Deviations:

yes

Remarks:

limited information on test material and methods; five S. typhimurium strains tested, but no TA 102 or E. coli strain included

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0.1, 1, 10, 100, 500 and 1000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (2 μg/plate, -S9, TA 100, TA 1535), nitrofluorene (2 µg/plate, -S9, TA 98, TA 1537, TA 1538), 2 -aminoanthracene (1 μg/plate, -S9, TA 98, TA 100, TA 1535, TA 1537, TA 1538; 1 μg/plate, +S9, TA 98, TA 100, TA 1535, TA 1537, TA 1538)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: 3 replicates in 1 experiment for TA 98, TA 100, TA 1535 and TA 1537. 3 replicates in 2 independent experiments for TA 1538.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10 µg/plate and above without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: in a range-finding study, a range of concentrations 0.1-1000 µg/plate was tested with TA 98 and TA 100, with and without metabolic activation. The solubility limit was 1000 µg/plate. More than 50% cytotoxicity was observed from 10 µg/plate in TA 100 and at 1000 µg/plate in TA 98; in both strain without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: more than 50% cytotoxicity was observed in the range-finding study with TA 98 from 10 µg/plate and with TA 100 from 1000 µg/plate; without metabolic activation for both strains (see Table 1).

Table 1: Cytotoxicity measured in two strains

 

	Survival/plate*
	TA 98		TA 100
Test substance (µg/plate)	-S9	+S9	-S9	+S9
Control	1.0	1.0	1.0	1.0
0.1	0.6	1.0	0.8	1.0
1	0.6	0.9	0.5	0.9
10	0.4	0.8	0	0.9
100	0.4	0.9	0	0.8
500	0.4	1.0	0	0.8
1000**	0.2	0.9	0	0.9

* survival ratio treated : control

** solubility limit

Table 2: Test results

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	TA 1538*	TA 98	TA 1537
-	Culture medium	162	7	10	25	5
–	Ethanol	153	7	10	25	4
-	0.05	-	-	-	33	5
-	0.1	191	6	12	26	4
-	0.5	173	7	10	29	4
–	1	199	6	11	30	3
–	5	173	6	13	26	4
–	10	171	4	12	-	-
-	40	-	-	-	-	-
-	160	-	-	-	-	-
–	640	-	-	-	-	-
–	1000	-	-	-	-	-
Positive controls, –S9	Name	SA	SA	NF	NF	NF
	Concentrations (μg/plate)	1.0	1.0	2.0	2.0	2.0
	Mean No. of colonies/plate (average of 3)	779	277	1840	1602	559
Positive controls, –S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1.0	1.0	1.0	1.0	1.0
	Mean No. of colonies/plate (average of 3)	215	7	13	40	8
+	Culture medium	148	8	11	34	6
+	Ethanol	150	6	14	33	6
+	0.05	-	-	-	-	-
+	0.1	-	-	-	-	-
+	0.5	-	-	-	-	-
+	1	-	-	-	35	5
+	5	-	-	-	-	-
+	10	175	6	13	37	7
+	40	176	6	15	35	4
+	160	163	5	17	38	3
+	640	149	6	15	40	6
+	1000	161	6	14	-	-
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1.0	1.0	1.0	1.0	1.0
	Mean No. of colonies/plate (average of 3)	1905	145	1478	1903	238

* 2 experiments, 6 plates in total

NF = nitrofluorene

SA = sodium azide

2AA = 2-Aminoanthracene

Conclusions:

Based on the number of revertant colonies it can be stated that the test item is not mutagenic in this bacterial test system either with or without metabolic activation.

Executive summary:

Tetradecyl myristate, a constituent of the registration substance, was tested for mutagenicity with five strains of S. thyphimurium, both in the presence and absence of S9 mix. Six different doses from 0.1 to 1000 µg/plate were used in the preincubation assay. The solubility limit was 1000 µg/plate. More than 50% cytotoxicity was observed from 10 µg/plate in TA 100 and at 1000 µg/plate in TA 98; in both strain without metabolic activation.

Based on the number of revertant colonies it can be stated that the test item is not mutagenic in this bacterial test system either with or without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

09 Apr - 10 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to cread across justification justification setcion 13.2

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to cread across justification justification setcion 13.2

4. DATA MATRIX
Please refer to cread across justification justification setcion 13.2

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

Remarks:

Migrated from field 'Test system'.

 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:

The test substance is considered to be non-mutagenic in this chromosome aberration assay.

Executive summary:

The study was performed to investigate the potential of 2 -ethylhexyl octadec-9-enoate, a structural analogue, to induce chromosome aberrations in cultured human lymphocytes. For each experiment two cell cultures were used.

The test article was dissolved in ethanol and tested at the following concentrations:

3, 10 and 33 µg/mL.

At a concentration of 33 µg/mL 2-ethylhexyl oleate precipitated in the culture medium. Both in the absence and presence of S9-mix 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 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. The substance is therefore considered to be non-mutagenic in this chromosome aberration assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

09 Apr - 27 Jul 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to cread across justification justification setcion 13.2

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to cread across justification justification setcion 13.2

4. DATA MATRIX
Please refer to cread across justification justification setcion 13.2

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.

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:

Based on the results obtained, the test item is considered as non-mutagenic both in the presence and absence of metabolic activation under the conditions applied.

Executive summary:

The test substance, 2-ethylhexyl octadec-9-enoate (CAS No. 26399 -02 -0), a structural analogue, was evaluated for gene mutation in mouse lymphoma L5178Y cells according to OECD guideline 476. The substance 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.

2 -Ethylhexyl octadec-9-enoate precipitated in the exposure medium at concentrations of 100 µg/mL and above. There was no concentration related or statistically significant increase in the mutant frequencies at any of the test item concentrations when compared with vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans. 

Additional information

There is no evidence for species specific effects of the substance. Therefore the results of the in vitro data are regarded as relevant for humans.

Justification for classification or non-classification

Fatty acids, C18 unsaturated, C12-14 (even numbered) alkyl esters does not have to be not classified for mutagenicity since constituents of the substance and structural analogues did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation, in in vitro gene mutation assays or in in vitro chromosome aberration studies.