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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD guideline 487 under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test )
Deviations:
yes
Remarks:
minor changes to culture conditions with no impact on study outcome
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C-SAT 100018
- Analytical purity: 100%
- Lot/batch No.: CF00460029
- Expiration date of the lot/batch: February 26, 2011
- Storage condition of test material: room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
without S9 mix: 4.5, 7.8, 13.6, 23.9, 41.8, 73.1, 127.9, 223.9, 391.8, 685.7, 1200, 2100 µg/ml
with S9 mix: 13.6, 23.9, 41.8, 73.1, 127.9, 223.9, 391.8, 685.7, 1200, 2100 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF (tertahydrofuran)
- Justification for choice of solvent/vehicle: solubility properties and its relative non-toxicity to the cell cultures
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: Demecolcin and Mitomycin C; with S9: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index

Evaluation criteria:
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data and
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Chi-square test

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In a test according to OECD Guideline 487 under GLP, the test substance was negative (with and without metabolic activation) as it did not induce micronuclei in human lymphocytes in vitro.
Executive summary:

The test substance, dissolved in THF, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix acc. to OECD guideline 487 under GLP.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The chromosomes were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 2100.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the current OECD Draft Guideline 487 for the in vitro mammalian cell micronucleus test.

In Experiment I, visible precipitation of the test item in the culture medium was observed at 13.6 µg/mL and above in the absence of S9 mix and at 73.1 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 23.9 µg/mL and above and in the presence of S9 mix at 73.1 µg/mL and above at the end of treatment. No relevant influence in the osmolarity or pH value was observed.

In this study, at both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by a clearly CBPI expressed as cytostasis could be observed.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.2 -1.0 % micronucleated cells) were close to the range of the solvent control values (0.55 - 0.9 % micronucleated cells) and within the range of the laboratory´s historical control data. In Experiment II in the absence of S9 mix, one single value slightly exceeded this range (0.45 % micronucleated cells), but since no statistical significance is observed this finding is considered as being biologically irrelevant.

In both experiments, either Demecolcin (100 ng/mL), MMC (1.0 µg/mL) or CPA (12.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in human lymphocytesin vitro,when tested up to precipitating concentrations.