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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
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:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
The acceptability criteria of the positive controls were augmented according to the latest IWGT recommendations. This deviation had no detrimental impact on the outcome of the study.
Principles of method if other than guideline:
The acceptability criteria of the positive controls were augmented according to the latest IWGT recommendations.
This deviation had no detrimental impact on the outcome of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
-Name of test material (as cited in study report): C-SAT 060033
- Physical state: Colorless to slight yellow, liquid
- Analytical purity: ca 80%
- Impurities (identity and concentrations): see confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: Not provided
- Lot/batch No.:CR51230002
- Expiration date of the lot/batch: 3-5-2007
- Stability under test conditions: Not provided
- Storage condition of test material: at room temperature


Method

Target gene:
mouse lymphoma thymidine kinase (TK)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: HAT & RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: thymidine Kinase TK+/-
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Experiment I
without S-9: 18.8; 37.5; 75.0; 112.5; and 150.0 µg/mL
with S-9: 18.8; 37.5; 75.0; 150.0; 225.0 µg/mL

Experiment IA
without S-9: 125.0; 150.0; 175.0; and 200.0 µg/mL
with S-9: 200.0; 225.0; 250.0; 275.0; and 300.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: highly soluble
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:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: without S-9
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:
Migrated to IUCLID6: with S-9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension in medium

DURATION
- Preincubation period: 3 days (1 day in RPMI-HAT medium and 2 days recovery in RPMI 1640 medium)
- Exposure duration: Experiment I and Experiment IA with and without S-9 =4 hours
- Expression time (cells in growth medium):48 hours
- Selection time (if incubation with a selection agent):10-15 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth(RTG);Relative suspension growth (RSG)
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10+6 cells above the corresponding solvent control.

A relevant increase of the mutation frequency should be dose-dependent.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration.

Results of test groups are rejected if the relative total growth, and the cloning efficiency 1 is less than
10 % of the vehicle control.
Statistics:
A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance should be considered together.

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:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion it can be stated that under experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of the test item, Butanedioic acid, sulfo-, 1,4 -bis(1,3 -dimethylbutyl) ester, sodium salt (ca. 80% act. ingr.) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range.

The highest applied concentration in the pre-test on toxicity (5000µg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was limited by toxicity of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Under experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.