Calcium 1,4-bis(2-ethylhexyl) bis(2-sulphosuccinate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

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. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium dihexylsulfosuccinate

IUPAC Name:

sodium dihexylsulfosuccinate

Constituent 2

Reference substance name:

Sodium 1,4-bis(1,3-dimethylbutyl) sulphonatosuccinate

EC Number:

219-147-9

EC Name:

Sodium 1,4-bis(1,3-dimethylbutyl) sulphonatosuccinate

Cas Number:

2373-38-8

Molecular formula:

C16H30O7S.Na

IUPAC Name:

sodium 1,4-bis(1,3-dimethylbutoxy)-1,4-dioxobutane-2-sulfonate

Details on test material:

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%
- Lot/batch No.:CR51230002
- Expiration date of the lot/batch: 3-5-2007
- 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:
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes - spontaenous mutant are reduced by growing teh cells for one day in RPMI 1640-HAT medium, followed by recovery of 2 days in RPMI 1640 medium containing hypoxyanthine and thymiden only. After this period, cells were returned to normal RPMI 1640 medium.

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Experiment I
without S-9: (4.7; 9.4); 18.8; 37.5; 75.0; 112.5; and 150.0 µg/mL
with S-9: (9.4); 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, (225.0) 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:

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: without S-9

Controls 2

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with S-9

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 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

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The highest concentration used in the pre-test was chosen with regard to the purity (80 % active substance) and the molecular weight of the test item (400 g/mol). Test item concentrations between 39.1 and 5000 μg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Following 4 hour treatment distinct toxic effects leading to RSG (relative suspension growth) values below 50 % were observed at 156.3 μg/mL and above in the absence and at 312.5 μg/mL and above in the presence of metabolic activation. Following continuous treatment (24 hours) a reduced relative suspension growth was determined at 312.5 μg/mL and above. The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was noted at 2500 μg/mL and above at the end of the 4 h treatment. Following 24 hours treatment, precipitation was observed at the maximum concentration of 5000 μg/mL.

The dose range of the first main experiment was selected according to the data generated in the pre-experiment. However, the onset of toxicity shifted and the analysable toxic range was not covered in the first experiment. Therefore, this experiment was repeated at higher concentrations without metabolic activation and at more narrowly spaced concentrations in the presence of metabolic activation. To overcome problems with possible deviations in toxicity both main experiments were started with more than four concentrations.

Relevant toxic effects indicated by a relative cloning efficiency 1 (survival) and/or a relative total growth (RTG) of less than 50 % in both parallel cultures were observed at 150 μg/mL and above in experiment IA without metabolic activation. The recommended toxic range of approximately 10 – 20 % of survival or RTG was covered. In the presence of metabolic activation toxic effects as described above were noted at 300 μg/mL. Although the recommended 10 – 20 % range of toxicity was not covered severe toxic effects occurred. The cell growth compared to the corresponding solvent control was severely reduced 24 h after treatment (20.8 - 21.3%). The surviving cells recovered and grew at a quite normal rate resulting in higher values of survival and RTG. Still, a reduction of the cell population down to about 20 % of the initial value is indicating severe toxicity, any further reduction may lead to undesired selection processes resulting in irreproducible artefacts.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The threshold of 126 plus each solvent control count was not reached or exceeded at any test point even at toxicity levels below 10 % of survival or RTG.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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 abscence and presence of metabolic activation.

Executive summary:

C-SAT 06003 was studied in 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 4 h. 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 (5000 μg/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 observed up to the maximum concentration 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. In conclusion it can be stated that during the mutagenicity test described and under the 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. Therefore, C-SAT 060033 is considered to be non-mutagenic in this mouse lymphoma assay.