Ethylene distearate

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

Study period:

17 Jun 2010 - 17 Aug 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see "Remarks"

Remarks:

GLP-Guideline study, tested with the source substance 91031-31-1. According 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18, esters with ethylene glycol

EC Number:

292-932-1

EC Name:

Fatty acids, C16-18, esters with ethylene glycol

Cas Number:

91031-31-1

Molecular formula:

C18H36O3, C20H40O3, C34H66O2, C36H70O4, C38H74O2

IUPAC Name:

Fatty acids, C16-18, esters with ethylene glycol

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamin amd 10% (v/v) heat-inactivated horse serum (24 h exposure); for 3 h exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium and 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphtoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1:
With and without S9-mix: 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL (3 h)

Experiment 2:
In the absence of S9-mix: 3.0, 10.0, 33.0, 100.0, 125.0, 140.0 and 175.0 µg/mL for 24h
In the presence of 12% (v/v) S9-mix: 0, 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL for 3h

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide, 7.5 µg/mL, with S9; methylmethanesulfonate, 15 µg/mL and 5 µg/mL without S9, for 3 h and 24 h treatment, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 48 h

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine
- Selection time (if incubation with a selection agent): 11-12 days

NUMBER OF REPLICATIONS: 2 replications each in 5 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth, relative survival, suspension growth, relative suspension growth, growth rate

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50E-06 and ≤ 170E-06.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500E-06, and fro CP not below 700E-06.

The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequeny (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: The test substance precipitated in the exposure medium at concentrations of 100 µg/mL and above. The test 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 ending test was 333 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 66% at the test substance concentration of 333 µg/mL compared to the relative suspension growth of the solvent control. In the presence of S9-mix, no toxicity in the relative 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.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls.

Any other information on results incl. tables

Table 1: Cytotoxic and mutagenic responses

Treatment	Concentration [µg/mL]	Cloning efficiency [%]	Relative total growth [%]	Mutation frequency x 10^-6
				total	small colonies	large colonies
3 h treatment without S9-mix
Solvent control (mean)	--	86	100	61	42	19
Test substance	0.1	101	114	56	29	25
	0.3	101	108	52	28	23
	1.0	118	140	66	48	16
	3.0	91	109	63	46	15
	10.0	95	108	66	32	33
	33.0	97	104	76	46	28
	100.0*	91	97	81	32	46
	333.0*	102	77	64	48	14
MMS	15	60	45	685	521	118
3 h treatment with 8% (v/v) S9-mix
Solvent control (mean)	--	91	100	67	36	29
Test substance	0.1	108	111	74	47	25
	0.3	89	94	71	45	24
	1.0	108	113	64	42	20
	3.0	98	107	78	53	23
	10.0	95	100	87	54	29
	33.0	108	96	55	32	22
	100.0*	98	89	83	60	21
	333.0*	94	92	63	23	39
CP	7.5	60	32	1074	829	144
24 h treatment without S9-mix
Solvent control (mean)	--	115	100	51	26	23
Test substance	3	135	92	58	42	14
	10	137	109	51	39	11
	33	133	85	71	32	35
	100*	110	30	100	35	60
	125*	118	31	70	31	36
	140*	118	20	103	51	47
	175*	102	9	134	53	72
MMS	5	101	73	865	463	233
3 h treatment with 12% (v/v) S9-mix
Solvent control (mean)	--	109	100	72	47	23
Test substance	0.1	110	100	73	49	21
	0.3	123	117	72	47	23
	1.0	104	105	82	52	27
	3.0	97	104	94	62	29
	10.0	115	126	87	57	26
	33.0	107	108	85	59	23
	100.0*	131	123	80	51	26
	333.0*	97	83	92	64	24
CP	7.5	70	59	979	621	221

*precipitation of test substance in the exposure medium

MMS = methylmethanesulfonate

CP = cyclophosphamide

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative