Bis(2-ethylhexyl) succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A well reported non-GLP study conducted to sound scientific principles with the read across substance, bis(2-ethylhexyl) adipate.

Data source

Reference

Reference Type:

publication

Title:

Responses of the L8178Y tk+/tk- Mouse Lymphoma Cells Forward Mutation Assay: III. 72 Coded Chemicals

Author:

McGregor D B, Brown A, Cattanach P, Edwards I, McBride D, Riach C, Casary W J

Year:

1988

Bibliographic source:

Environmental and Molecular Mutagenesis 12:85-154

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(study lacked a longer exposure time)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) adipate

EC Number:

203-090-1

EC Name:

Bis(2-ethylhexyl) adipate

Cas Number:

103-23-1

Molecular formula:

C22H42O4

IUPAC Name:

bis(2-ethylhexyl) adipate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): di(2-ethylhexyl)adipate

Method

Target gene:

Thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The tissue culture medium was maintained in Fischer’s medium. Fischer’s medium was supplemented with 2 mM L-glutamine, 110 µg/mL sodium pyruvate, 0.05 % pluronic F68 antibiotics, and 10 % heat-inactivated donor horse serum (v/v).
- Properly maintained: Yes. L5178Y TK+/- cells were stored in a liquid nitrogen freezer. Following removal from liquid nitrogen, the cultures were kept at 37 °C on gyratory tables
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 312.5, 625, 1250, 2500, 5000 µg/mL - Trial 1 (without metabolic activation)
0, 1800, 2600, 3400, 4200, 5000 µg/mL - Trial 2 (without metabolic activation)
0, 1000, 2000, 3000, 4000, 5000 µg/mL - Trials 1 and 2 (with metabiolic activation)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHODOLOGY
Each exposed culture consisted of 6 x 10^6 cells in a final volume of 10 mL Fischer's medium in a 30 mL plastic tube. The tube was incubated for 4 hours on a horizontal axis roller drum rotating at 10 rpm. At the end of the incubation time, the cells were sedimented by centrifugation at 500 g.av. for 10 minutes, washed and resuspended in 20 mL Fischer's medium. the cell suspensions (3 x 10^5 cells/mL) were incubated for a 2 day expression period, the cell population density being adjusted back to 20 mL of 3 x 10^5 cells/mL after 24 hours. After 48 hours, the cell population densities were estimated and culture volumes containing 3 x 10^6 cells adjusted to 15 mL with Fischer's medium, giving a cell population density of 2 x 10^5 cells/mL.

CLONING EFICIENCY
A 0.1 mL sample of the cell suspension was withdrawn and diluted 1:100. Three 0.1 mL samples (200 cells) of the diluted cultures were transferred to 30 mL tubes, mixed with 25 mL Fischer's medium containing 20 % heat-inactivated horse serum containing 0.35 % Noble agar and poured into 90 mm Petri plates.

MUTANT SELECTION
Three aliquots (each containing 10^6 cells) of the remaining culture were distributed to 30 mL tubes, mixed with 20 mL cloning medium to give final concentrations of 0.35 % Noble agar and 3µg trifluorothymidine/mL, then poured into 90 mm Petri plates.

INCUBATION
The agar was gelled at 4 °C for 5 - 10 minutes, then the plates were incubated for 11 - 14 days in 5 % CO2: 95 % air at 37 °C.

NUMBER OF REPLICATIONS
Vehicle controls were performed in quadruplicate, positive controls in duplicate and test concentrations in duplicate

COLONY COUNTING
Colonies were counted using at Artek 880 Automates Colony Counter.

CALCULATIONS
Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG) which was determined as follows:

RTG = (total suspension growth x cloning efficiency) in dosed culture / (total suspension growth x cloning efficiency) in control culture

Mutant fraction (MF) was calculated as follows:

MF = 200 x (mutant clones per plate / total clones per plate)

= mutants / 10^6 clonable cells

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
A positive response is obtained when there is a statistically significant dose-related increase at one of the three highest acceptable doses.

CRITERIA FOR A NEGATIVE RESPONSE
A negative response is obtained when there is no reproducible statistically significant dose-related increase in mutant frequency.

Statistics:

The statistical analysis consisted of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. The significance level was set at 5 %.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Precipitation of the test material was observed at 1000 µg/mL, but testing was continued up to 5000 µg/mL. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occurred in a second experiment. The Lowest Observed Effect Dose in this experiment was 2000 µg/mL, higher than the precipitation concentration. Significant toxicity was observed in all experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results (without metabolic activation)

Without S9 Trial 1						Without S9 Trial 2
Conc (µg/mL)	CE	RTG	MC	MF	Mean MF	Conc (µg/mL)	CE	RTG	MC	MF	Mean MF
0	78	96	60	26	24	0	92	100	75	27	22
	84	85	49	19			63	97	31	16
	91	105	65	24			103	104	61	20
	85	114	67	26			84	98	63	25
312.5	75	74	64	28	26	1800	88	26	41	16	18
	80	80	57	24			74	25	47	21
625	88	75	35	13	20	2600	73	24	51	23	18
	80	70	64	27			88	23	36	14
1250	69	25	58	28	23	3400	97	20	48	17	16
	75	38	39	17			93	15	44	16
2500	79	13	50	21	27	4200	78	15	35	15	20
	70	16	68	32			69	13	50	24
5000	87r	13	48	18		5000	74	12	37	17	17
	LETHAL						72	13	36	17
15 (MMS)	32	25	112	119	118*	15 (MMS)	44	29	110	83	83*
	44	24	156	118			39	34	97	84

Table 2: Results (with metabolic activation)

With S9 Trial 1						With S9 Trial 2
Conc (µg/mL)	CE	RTG	MC	MF	Mean MF	Conc (µg/mL)	CE	RTG	MC	MF	Mean MF
0	80	98	72	30	33	0	102	116	82	27	37
	76	109	55	24			109	101	114	35
	70	98	88	42			100	101	111	37
	73	94	78	36			68	82	102	50
1000	65	39	47	24	32	1000	93	70	116	42	40
	75	38	90	40			100	67	115	38
2000	50	27	53	36	33	2000	111	28	294	88	73*
	48	22	43	30			101	32	176	58
3000	59	18	55	31	34	3000	93	23	226	81	85*
	59	19	65	37			91	21	242	89
4000	56	16	61	37	39	4000	78	20	187	80	80*
	53	22	66	41			100	26	238	79
5000	40	16	37	31	34	5000	94	28	240	85	81*
	48	13	53	37			77	27	177	77
2.5 (MCA)	29	16	187	214	222*	2.5 (MCA)	55	11	603	364	363*
	32	21	223	231			55	11	592	361

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the experiment it was concluded that the test material was not mutagenic in this system.

Executive summary:

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in a study which was conducted to a methodology which was similar to that outlines in the standardised guideline OECD 476. L51788Y TK^+/-mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxillary metabolic system (S9 mix) in two independent experiments. Mutant colonies were scored for all cultures in each experiment. The test material was tested up to a maximum concentration of 5000 µg/mL in the presence and absence of metabolic activation. This concentration is approximately equivalent to the limit concentration for this assay.

Under the conditions of the study precipitation of the test material was observed at 1000 µg/mL, but testing was continued up to 5000 µg/mL. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occurred in a second experiment. The Lowest Observed Effect Dose in this experiment was 2000 µg/mL, higher than the precipitation concentration. Significant toxicity was observed in all experiments. It was therefore concluded that the test material was not mutagenic in this system.