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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 July 1997 to 19 August 1997
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
When comparing to the OECD TG 473 (1997 version), the following deviations were reported: - karyotype stability and absence of mycoplasme contamination were not reported; - cytotoxicity measurements are missing; - test-item confounding factors (e.g. pH, osmolality) are not mentioned; - historical control data are not available; - the exposure condition without S9Mix was 24 and 48 hrs instead of 3-6 and 24 hrs.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Exposure time without metabolic activation was 24 and 48 hrs, instead of 3-6 and 24 hrs. Reporting is not sufficient to assess the deviations (cf. rationale for reliability for further details).
Principles of method if other than guideline:
Chromosome aberration test with CHL/IU cells
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Blue powder
- Batch number: 25 C
Specific details on test material used for the study:
- Storage condition of test material: ambiant temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable at ambient temperature
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not reported
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not reported
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reported

- Treatment of test material prior to testing (e.g. warming, grinding): ultrasonication was conducted in the preparation of the test substance formulation
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: 50 mg/mL (highest concentration of test substance formulation)
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): suspension prepared using ultrasonication

FORM AS APPLIED IN THE TEST (if different from that of starting material): not applicable

- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: none


Species / strain
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and source of cells: CHL/IU obtained from Dainipon Pharmaceutical Co., Ltd.
- Suitability of cells: standard cell line as per OECD TG 473
- Normal cell cycle time (negative control): not reported
- Absence of Mycoplasma contamination: not reported
- Number of passages if applicable: 16
- Methods for maintenance in cell culture: plastic plate, 37°C, 5% CO2
- Cell cycle length, doubling time or proliferation index : doubling time ca. 15 hours
- Modal number of chromosomes: 25
- Periodically checked for karyotype stability: not reported
- Periodically ‘cleansed’ of spontaneous mutants: not reported

- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's MEM, 10%. 37°C, 5% CO2.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Kikkoman Corporation (Lot No. RAA-363)
- method of preparation of S9 mix: Amount in 1 mL of S9 mix
S9 0,3 mL;
MgCl2 5 µmol;
KCl 33 µmol;
D-glucose 6-phosphate 5 µmol;
NADP 4 µmol;
Buffer (HEPES) 4 µmol;
Distilled water Added up to 1 mL.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% S9 / 1.32 mg/mL S9 protein.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported
Test concentrations with justification for top dose:
concentration [mg/ml] : 0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, 2.500, 5.000.
Based on the results of the preliminary test (no cytotoxicity).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% CMC-Na solutions

- Justification for choice of solvent/vehicle: Vehicle of the test substance was selected based on the result of a solubility test. In the solubility test, physiological saline and dimethyl sulfoxide were used as vehicles. As a result, the test substance was not dissolved in physiological saline at 50 mg/mL or in dimethyl sulfacide at 500 mg/mL. Te test substance suspended with ultrasonication in 1% CMC-Na solutions at 50 mg/mL. Base don these results, 1% CMC-Na solutions with which the test substance suspense in better condition could be prepared, was selected as the vehicle of the test substance.

- Justification for percentage of solvent in the final culture medium: 1%, based on preliminary solubility experiment. In line with OECD TG 473.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
- Number of cultures per concentration (single, duplicate, triplicate) duplicate
- Number of independent experiments: 3 (24-hrs -S9 / 48-hrs -S9 / 6-hrs + S9)

- Cell density at seeding (if applicable): 2x10E03 cells/well
- Test substance added in medium

- Preincubation period, if applicable: n/a
- Exposure duration/duration of treatment: 24-hrs -S9 / 48-hrs -S9 / 6-hrs + S9
- Harvest time after the end of treatment (sampling/recovery times): 24-hrs -S9 / 48-hrs -S9 / 24-hrs + S9

- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. Colcemid at 0.2 µg/mL, for 2 hours exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): not reported
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): duplicate cultures. 200 cells per experiments.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The cells were fixed (10% formaline for 15 minutes), stained (0.1% crystal violet for 15 minutes), and extracted (acetic acid : 50% ethanol = 1:99) and the optical absorption (620 nm) was measured with a microplate reader.
- Determination of polyploidy: not reported
- Determination of endoreplication: not reported

- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: cell growth index
- Any supplementary information relevant to cytotoxicity: none
Rationale for test conditions:
Selection of concentration was based on the result of the preliminary test. Because cytotoxicity was not recognised in the treatment with and without metabolic activation, 5 mg/mL was set as the highest concentration in each treatment condition. Five concentrations, by 4 step dilution with a common ratio of 2 from the highest concentration, were set.
Evaluation criteria:
Chromosomal aberration was classified as "gap", "break", "exchange", and "others" (fragmentation etc.) with chromatid type and chromosome type. A "gap" was defined as a clear achromatic region on an axis of a chromatid and the width of the achromatic

region was equal to or larger than the width of the chromatid.

In the chromosomal aberration test, judgment criteria described below were set for total incidence of aberrant cells. Data was shown in two ways; including and excluding "gap". The final judgment was conducted with data including "gap".

Negative (-): less than 5%
Inconclusive (±): 5% or more and less than 10%
Positive (+): 10% or more

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
cytotoxicity was not recognized in the treatment with and without metabolic activation, 5 mg/mL was set as the highest concentration in each treatment condition
Vehicle controls validity:
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not soluble
- Precipitation and time of the determination: not reported
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: not reported

- Concurrent vehicle negative and positive control data: Number of chromosomal aberrant cells increased in the positive control group (MMC treatment group without S9 and B[a]P treatment with S9 group) and, therefore, these results showed that the test was conducted appropriately.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index. Not available
- Genotoxicity results (for both cell lines and lymphocytes): Number of chromosomal aberrant cells (structural aberrant cells and polyploid cells) did not increase in the treatment without or with metabolic activation. (cf. table of results attached to this ESR)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: not reported

Applicant's summary and conclusion

The test substance did not have the ability to induce chromosomal aberration under the test conditions.
Executive summary:

A Chromosome aberration test with CHL/IU cells was conducted with the test substance suspended in 1% CMC-Na at concentrations up to 5 mg/mL .

The number of chromosomal aberrant cells (structural aberrant cells and polyploid cells) did not increase in the treatment without or with metabolic activation. Number of chromosomal aberrant cells increased in the positive control group (MMC treatment group without S9Mix and B[a]P treatment group with S9Mix) and, therefore, these results showed that the test was conducted appropriately.

Based on the above results, it was judged that the test substance did not have the ability to induce chromosomal aberration.