2-ethylhexyl benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun - 7 Jul 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl benzoate

EC Number:

226-641-8

EC Name:

2-ethylhexyl benzoate

Cas Number:

5444-75-7

Molecular formula:

C15H22O2

IUPAC Name:

2-ethylhexyl benzoate

Details on test material:

- Name of test material (as cited in study report): 2-Ethylhexyl benzoate
- Physical state: clear colourless liquid
- Analytical purity: 99.734%
- Impurities (identity and concentrations): 2-Ethyl-4-methyl-1-pentyl benzoate 0.209%, unknown octylbenzoate 0.023%, Dioctylphthalate 0.004%, unknown impurities 0.03%
- Lot/batch No.: 9915-140-2
- Expiration date of the lot/batch: 2000-12-14
- Storage condition of test material: room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
RPMI 1640 supplemented with
- 10% fetal calf serum (FCS)
- penicillin / streptomycin (20 IU/mL / 20 µg/mL)
- 2 mM glutamine

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:

First experiment
3 h treatment: 18.3, 36.6, 73.1, 146.3, 292.5, 585, 1170 and 2340 µg/mL without metabolic activation (for mitotic index data)
3 h treatment: 292.5, 585 and 1170 µg/mL with and without metabolic activation

Second experiment
21 h treatment: 146.3, 585, 1170 and 2340 µg/mL without metabolic activation
3 h treatment: 585, 1170 and 2340 µg/mL with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle and exposure concentrations: the test substance was found to form a dosable suspension in DMSO at 100 mg/mL. On dosing at 1% (v/v) into aqueous tissue culture medium, giving a final concentration of 1000 µ/mL, a precipitate was observed. A precipitate was also observed at 125 µg/mL, but was soluble after 3 h incubation. Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al., 1987). Therefore, concentrations greater than 5000 µg/mL or 10 mM are not used in this test system.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

cyclophosphamide, 25 µg/mL in water (3 h exp), +S9; mitomycin C, 0.8 µg/mL in water (3 and 21 h exp), -S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 21 h; 21 h treatment: 21 h
SPINDLE INHIBITOR (cytogenetic assays): colcemid (Sigma) 0.1 µg/mL medium
STAIN (for cytogenetic assays): Giemsa 10% (v/v) in buffered water (pH 7.2)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells (except for positive control treated cultures; only dose level causing a decrease in mitotic index of at least 50% of the solvent control or if there was no decrease, the maximum concentration was used as the highest dose level for the metaphase analysis)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
It induced a dose-related statistically significant increase (p < 0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) at one or more test concentrations.
The increases exceed the negative control range of the laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

Fisher´s test, p < 0.01, p < 0.001

Results and discussion

Test results

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

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:

COMPARISON WITH HISTORICAL CONTROL DATA:
In the second experiment (-S9), the test material caused a small statistically significant increase in the proportion of cells with chromosomal aberrations at 1170 µg/mL in comparison to the solvent control. This increase, to 3.5% (p < 0.01), lied just outside the upper 99% confidence limit of the historical control range (2.5%). However, it lied just within the upper limit (3.68%) when not applying the 99% level. To further investigate this response, a higher dose level (2340 µg/mL) was subsequently analysed. As no response was seen at this higher dose level, the increase was not considered indicative of a clastogenic response.

Any other information on results incl. tables

Table 1. Results of Experiment 1 and 2.

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Experiment 1 Exposure period 3 h, fixation time 21 h, without S9 mix
DMSO		100	0	0
MMC	0.8	-	20***	20***
Test substance	292.5	86	0	0
	585	53	2	2
	1170	48	0.5	0.5
Exposure period 3 h, fixation time 21 h, with S9 mix
DMSO		100	0,5	0,5
CP	25	-	34***	34***
Test substance	585	91	1	1
	1170	66	1	1
	2340	49	1	1
Experiment 2 Exposure period 21 h, fixation time 21 h, without S9 mix
DMSO		100	0.5	0
MMC	0.8	-	30***	30***
Test substance	146.3	90	3	1
	585	51	4	2
	1170	51	5**	3.5**
	2340	56	4	2
Exposure period 3 h, fixation time 21 h, with S9 mix
DMSO		100	0,5	0.5
CP	25	-	1	0.5
Test substance	585	93	1	0.5
	1170	89	1,5	1
	2340	62	27***	25***

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

**: p < 0.01, ***: p < 0.001

Table 2. Mitotic index data of Experiment 1 and 2.

Concentration of test substance (µg/mL)
	DMSO	18.3	36.6	73.1	146.3	292.5	585	1170	2340
Exposure period 3 h, fixation time 21 h, without S9 mix
Relative mitotic index (%)	100	107	90	96^a	78^a	86^a	53^a	48^b	43^b
Exposure period 3 h, fixation time 21 h, with S9 mix
Relative mitotic index (%)	100	89	82	79^a	90^a	87^a	91^a	66^b	49^b
Exposure period 21 h, fixation time 21 h, without S9 mix
Relative mitotic index (%)	100	n.d.	n.d.	86	90^a	48^a	51^a	51^b	56^b
Exposure period 3 h, fixation time 21 h, with S9 mix
Relative mitotic index (%)	100	n.d.	n.d.	n.d.	n.d.	84^a	93^b	89^b	62^b

a: Precipitate apparent on dosing, not apparent at end of treatment (3 h)

b: Precipitate apparent on dosing, still apparent at end of treatment (3 h)

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative