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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:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
-Name of test material (as cited in study report): C-STAT 030041
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: Not provided
- Physical state: colorless, liquid
- Analytical purity: 80% active ingredient
- Impurities (identity and concentrations): Not provided
- Composition of test material, percentage of components: See confidential details
- Isomers composition: Not provided
- Purity test date: Not provided
- Lot/batch No.:EI90-00981
- Expiration date of the lot/batch: 06-11-2003
- Stability under test conditions: Not provided
- Storage condition of test material: at room temperature
- Other:

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 (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Experiment 1 (-S-9): 50, 100, 200, 400, 600 and 800 µg/mL
Experiment 1 (+S-9): 100, 200, 400, 600, 800 and 1000 µg/mL
Experiment 2 (–S-9): 50, 100, 200, 300, 400 and 600 µg/mL
Experiment 2 later sampling time (–S-9): 200, 300, 400 and 600 µg/mL
Experiment 2 (+S-9): 50, 100, 200, 400, 600 and 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its
relative nontoxicity to cell cultures
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in the -S-9 group
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the +S-9 group
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0-4.0%
aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0-4.0% aberrant cells, exclusive
gaps).
and
- either a concentration-related or a significant increase of number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0-8.5% polyploid cells).

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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality:no effect

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes


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

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells ( Chinese hamster cell line) in vitro.
Therefore, Butanedioic acid, sulfo, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (80% active ingredient) is considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.
Executive summary:

The test item Butanedioic acid, sulfo, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (80% active ingredient), dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments up to 5000 µg/mL(approx. 10 mM of the active ingredient). In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations and 500 cells were scored for polyploidy. Toxic effects indicated by reduced cell numbers and/or mitotic indices of about and below 50% of control were observed in all experimental parts. However, in experiment I in the absence and the presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The observed statistical significance's and dose-dependency are regarded as being biologically irrelevant. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastagenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.