Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2004 to 12 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Not indicated by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5% (v/v)
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing
- Suitability of cells: recommended in guideline
- Cell cycle length, doubling time or proliferation index: 12 hours
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: not specified
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I, 4 hours exposure without metabolic activation: 0.025, 0.050, 0.100, 0.150, 0.200, 0.300 µL/mL
Experiment II, 18 hours exposure without metabolic activation: 0.006, 0.013, 0.025, 0.050, 0.100, 0.200 µL/mL
Experiment II, 24 hours exposure without metabolic activation: 0.013, 0.025, 0.050, 0.100 µL/mL
Experiment I, 4 hours exposure with metabolic activation: 0.003, 0.006, 0.013, 0.025, 0.050, 0.100 µL/mL
Experiment II, 4 hours exposure with metabolic activation: 0.013, 0.025, 0.050, 0.100, 0.200 and 0.400 µL/mL
Repetition of Experiment II, 4 hours exposure with metabolic activation, due to missing test item precipitation: 0.040, 0.080, 0.160, 0.310, 0.630, 1.250, 2.500, 5.000 µL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: 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
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 40000 cells/slide

METABOLIC ACTIVATION: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl, 33 mM KCI, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

DURATION
- Preincubation period: 48 hours
- Exposure duration: Experiment I, 4 hours with and without metabolic activation; Experiment II, 18 and 28 hours without metabolic activation; Experiment II, 4 hours with metabolic activation
- Expression time (cells in growth medium): Experiment I, 14 hours with and without metabolic activation; Experiment II, 14 or 28 hours with metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment I 18 hours with and without metabolic activation, Experiment II, 18 or 28 hours with and without metabolic activation.

SPINDLE INHIBITOR (cytogenetic assays): colcemid for 2.5 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Per experiment two slides per group were prepared.

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:
The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. 5 µL/mL of CBI were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 0.04 and 5 µL/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item after 4 hours treatment was observed at 0.16 µL/mL and above in the absence of S9 mix and at 0.04µL/mL in the presence of S9 mix.

Since no clear toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. Therefore, 0.3µL/mL was chosen as top treatment concentration in the absence of S9 mix and 0.1 µL/mL in the presence of S9 mix.

Dose selection of experiment Il was also based on toxicity data and the occurrence of precipitation. In the range finding experiment clearly reduced cell numbers were observed after 24 hours exposure with 0.04 to 0.63 µL/mL. Therefore, 0.2 µL/mL and 0.1 µL/mL were chosen as top treatment concentrations for continuous treatment (18 hrs and 28 hours, respectively) in the absence of S9 mix. In the presence of S9 mix 0.4 µL/mL were chosen as top treatment concentration with respect to the results obtained in experiment l. Due to missing test item precipitation, this experimental part was repeated with a top test item concentration of 5 µL/mL.

Evaluation criteria:
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 the number of structural chromosome aberrations is observed.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (9) (p < 0.05).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: tested up to cytotoxic and/or precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation:

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment, precipitation of the test item in culture medium was observed after 4 hours treatment with 0.16 µL/mL and above in the absence of S9 mix and with 0.04 µL/mL and above in the presence of S9 mix. In addition, after 24 hours continuous treatment precipitation occurred with 0.04 µL/mL and above in the absence of S9 mix. No relevant influence of the test item on the pH value was observed (solvent control: pH 7.3 versus pH 7.3 at 5 µL/mL). Due to technical problems at measuring the osmolarity of the highest concentration with the oily phase on the top the osmolarity was measured at 2.5 µL/mL (371 mOsm versus 425 mOsm in the solvent control). No relevant influence on the osmolarity was observed.


HISTORICAL CONTROL DATA
- Positive historical control data: within the ranges for positive control data
- Negative (solvent/vehicle) historical control data: within the ranges for negative control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the cytogenetic experiments, toxic effects indicated by reduced cell numbers of below 50 % of control were observed in experiment Il after 18 hours continuous treatment with O. 100 µL/mL in the absence of S9 mix. In all experimental parts of this study with 4 hours treatment with and without S9 mix no clear toxic effects indicated by reduced mitotic indices or cell numbers were observed up to the highest tested test item concentration.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.8 - 2.3 %) as compared to the rates of the solvent controls (1.1 - 1.9 %).

Any other information on results incl. tables

See attachment for results table.

Applicant's summary and conclusion

Conclusions:
The test substance has been tested for ability to cause chromosome aberrations in Chinese hamster ovary cells according to OECD TG 473 and under GLP (RCC, 2004). No increase in the number of cells with aberrations was observed either with or without metabolic activation in Chinese hamster V79 cells. Appropriate solvent, positive and negative controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of this study.