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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:
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: Comparable to guideline study with acceptable restrictions. No discussion of results and no historical control data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no discussion of results and no historical control data.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no discussion of results and no historical control data.
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany
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): trade name
- Chemical identity: diester of 1,3-butandiol with C8/C10-fatty acids
- Physical state: clear slightly yellowish liquid
- Analytical purity: 97.9 area-%
- Purity test date: 1997-04-21
- Lot/batch No.: 704025
- Stability: > 1 year
- Storage: dark, without air humidity, under exhaust hood, at room temperature

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
MEM medium supplemented with 2 mM L-Glutamine and
- 4% (v/v) fetal calf serum (MEM4) or
- 0% (v/v) fetal calf serum (MEM0)
- 100 IU/mL penicillin/streptomycin
During exposure to the test substancewith S9 mix, MEM0 medium was used and replaced by MEM4 after 3 h after test substance administration.
Additional strain / cell type characteristics:
other: modal chromosome number of 22 and a cell cycle length of approx. 16.5 h
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:
18h treatment: 10, 40 and 80 µg/mL (without metabolic activation)
18h treatment: 10, 60, 80 and 100 µg/mL (with metabolic activation)
28h treatment: 80 µg/mL (without metabolic activation)
28h treatment: 100 µg/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM4 medium(resp. MEM0 medium in the test with S9 mix) containing 1% (v/v) ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 3 and 4 µg/mL in MEM0 medium, +S9; mitomycin C, 0.03 and 0.04 µg/mL in MEM4 medium, -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 and 28 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h treatment: 18 h; 28 h treatment: 28 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 replications each in independent experiments

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells per slide

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreduplicated cells: yes
Evaluation criteria:
The test chemical is to be considered clastogenic in this assay if:
- it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
- the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system
- positive results can be verified in an independent experiment.
Statistics:
The number of aberrant cells in each replica was used to establish acceptable homogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
systematic influence of the test compound which led to a reduction in the mitotic index.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was soluble in ethanol, in MEM4 medium containing 1% ethanol the solubility limit of the test substance was determined to be 100 µg/mL (homogeneous emulsion).

- Other confounding effects: In the first test without metabolic activation event the negative controls exhibited only a mitotic index of 2.0%. Therefore, test 1 without metabolic activation was completely repeated with the same doses and named test #1a.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary of data obtained in experiment #1a.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in % mean

with gaps

without gaps

Exposure period 18h, without S9 mix

Control, Ethanol

1.0% (v/v)

10.7

2.0

0.0

MMC

0.03

2.1

24.6

18.5*

MMC

0.04

2.9

40.0

31.9*

Test substance

10

8.0

3.5

1.5

40

6.7

3.0

0.5

80

5.3

5.0

0.0

Exposure period 18h, with S9 mix

Control, Ethanol

1.0% (v/v)

7.0

6.5

4.0

CP

3.0

2.7

45.0

34.0*

Test substance

10

6.7

3.5

1.5

60

6.1

4.5

1.0

100

5.7

4.0

2.0

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

*: significant, no statistical evaluation

Table 2. Summary of data obtained in experiment #2.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in % mean

with gaps

without gaps

Exposure period 18h, without S9 mix

Control, Ethanol

1.0% (v/v)

7.9

6.0

3.5

MMC

0.03

3.4

23.5

14.0*

Test substance

10

7.5

4.0

0.5

40

8.0

9.5

3.0

80

5.8

5.5

0.5

Exposure period 18h, with S9 mix

Control, Ethanol

1.0% (v/v)

6.3

8.0

2.0

CP

3.0

1.6

42.0

33.3*

Test substance

10

6.5

5.5

0.0

60

5.7

4.5

1.5

100

6.7

6.5

1.5

Exposure period 28h, without S9 mix

Control, Ethanol

1.0% (v/v)

7.9

3.5

0.0

MMC

0.03

4.4

43.5

32.5*

Test substance

80

5.8

4.5

0.5

Exposure period 28h, with S9 mix

Control, Ethanol

1.0% (v/v)

9.2

6.5

1.5

CP

3.0

6.0

36.5

27.5*

Test substance

100

8.4

3.5

1.0

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

 *: significant, no statistical evaluation

Applicant's summary and conclusion