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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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

96a

78a

86a

53a

48b

43b

Exposure period 3 h, fixation time 21 h, with S9 mix

Relative mitotic index (%)

100

89

82

79a

90a

87a

91a

66b

49b

Exposure period 21 h, fixation time 21 h, without S9 mix

Relative mitotic index (%)

100

n.d.

n.d.

86

90a

48a

51a

51b

56b

Exposure period 3 h, fixation time 21 h, with S9 mix

Relative mitotic index (%)

100

n.d.

n.d.

n.d.

n.d.

84a

93b

89b

62b

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