Fatty acids, C16-18, 2-ethylhexyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance 2-ethylhexyl oleate (CAS No. 26399-02-0). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl oleate

EC Number:

247-655-0

EC Name:

2-ethylhexyl oleate

Cas Number:

26399-02-0

Molecular formula:

C26H50O2

IUPAC Name:

2-ethylhexyl octadec-9-enoate

Details on test material:

- Name of test material :2-Ethyl oleate
- Molecular formula: C26H50O2
- Molecular weight :394
- Substance type: clear light yellow liquid
- Physical state: Liquid
- Analytical purity: > 60% C18 unsaturated, < 5% 2 Ethylhexyl alcohol, <5 % Oleic Acid
- Lot/batch No.:9002
- Expiration date of the lot/batch: 03 March 2010 (allocated by NOTOX, 1 year after receipt of the test substance).
- Stability under test conditions: Stable
- Storage condition of test material:At room temperature in the dark
- Other: Acidity value - 0.05 mg KOH/g, Viscosity at 40°C -8.3 cSt, Flash point (COC- 220°C.

Method

Target gene:

Thymidine Kinase locus (TK gene)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were prepared from adult male Wistar rats which were treated with phenobarbital (89 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/ml - with/without S9 mix (Experiment 1 & 2).

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Migrated to IUCLID6: Used at a concentration of 7.5 µg/ml

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: Used at concentration of 15 and 5 µg/ml for a 3 and 24 hours treatment period.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
For 3 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum.
For 24hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum.
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum =20%, R20) and 5 µg/ml trifluorothymidine (TFT). Non selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of erum =20%, R20) .

DURATION
- Exposure duration: In experiment 1 - 3 hours. Experiment 2 - 24 hours (- S9 mix) and 3 hours (+S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2-yl]-2,5- diphenyltetrazolium bromide (MTT).

NUMBER OF REPLICATIONS:1

NUMBER OF CELLS EVALUATED: Whole wells counted

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth;

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency will be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evalulation factor (GEF) has been defined by the IWTG as the mean of the neqative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered postive (mutagenic) in the mutation assay if it induces a MF of more then MF(control) + 126 in a dose-dependent manner. An observed increase should be biologically relevent and will be compared with the historical control data range. The test substance will be considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
Thes test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controsl) + 126.
b) The resullts are confirmed in an indepedently reapeated test.

Results and discussion

Test results

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 finsing test was 333 µg/ml.
- Precipitation:2-Ethylhexyl oleate precipitated in the exposure medium at concentration of 100µg/ml and abouve.

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.
sku

Any other information on results incl. tables

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
MMS	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.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative