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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:
1999-10-06 to 2000-01-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
February 1998, adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
EEC Directive 92/69, L 383 A, dated December 29, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver microsome preparations (S9 mix); ß-naphthoflavone/phenobarbital induced
Test concentrations with justification for top dose:
2860 µg/mL ( = 10 mM) were applied as top concentration for treatment of the cultures in the pre-test.
Test item concentrations between 22.3 and 2860 µg/mL (with and without S9-mix) were chosen for the evaluation of cytotoxicity.


Vehicle / solvent:
- Solvent used:
Ethanol (E. MERCK, D-64293 Darmstadt, Germany; purity 99.8 %). On the day of the experiment (immediately before treatment ), the test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5% (v/v).

- Justification for choice of solvent:
The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
Ethylmethane sulfonate (without metabolic activation), Cyclophosphamide (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation

Preparation of the Cultures:
16 h and 26 h, respectively after the start ofthe treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt). Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 1 0 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

DETERMINATION OF CYTOTOXICITY
The chromosomes were prepared 18 h and 28 h after start of treatment with the test item. In experiment I, the exposure period was 4 h with and without metabolic activation. In experiment li the exposure period was 4 h with S9 mix and 18 h and 28 h without S9 mix. The chromosomes were prepared 18 h (exp. I and II) and 28 h (exp. lI) after start of treatment with the test item.
In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

OTHER EXAMINATIONS:
- Analysis of Metaphase Cells: Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives.
Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as
well but not included in the calculation of the aberration rates. 100 weil spread metaphase plates per culture were scored for cytogenetic darnage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cellline polyploid means a near tetraploid karyotype).

NUMBER OF REPLICATIONS: triplicate

OTHER: Two independent tests were performed with and without metabolic activation.



Evaluation criteria:
Evaluation of the cultures was performed using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic darnage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of
polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cellline polyploid means a near tetraploid karyotype).

The chromosome aberration assay was considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historicallaboratory control data: 0.00 %- 4.00 %.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxic effects indicated by reduced cell numbers were observed after 4 h treatment with 44.7 µg/mL and above in the absence of S9 mix and with 2860 µg/mL in the presence of S9 mix. In addition, 24 h continuous treatment with 22.3 µg/ml and above in the absence of S9 mix induced a toxic effect.
Turbidity of the test item in culture medium was observed after treatment with 89.4 µg/mL and above in the absence of S9 mix and at 178.8 µg/mL and above in the presence of S9 mix in the pre-test.
No relevant influence of the test item on the pH value or osmolarity was observed (solvent contro: 404 mOsm, pH 7.2 versus 346 mOsm and pH 7.3 at 2860 µg/mL).
In experiment I, in the absence of S9 mix clearly reduced mitotic indices were observed at the 18 h interval after 4 h treatment with 400 µg/mL (33.0 % of control). In experiment II, in the absence of S9 mix the mitotic indices were reduced after treatment with 25 µg/mL (39.7 % of control) at the 18 h interval and 200 µg/mL (9.9 % of control) at the 28 h interval. In the presence of S9 mix only slightly reduced mitotic indices were observed after 4 h treatment with 2860 µg/mL (64.7 % of control).
In both experiments, EMS (600 and 1000 µg/mL, respectively) and CPA (0.71 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
It can be stated that in the study described and under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cellline ).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion