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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-11-11 to 1985-12-13
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C14 H6 Cl2 F4 N2 O2
Test material form:
solid: crystalline


Target gene:
The gene for HGPRT located on the X-chromosome of the V79 cells.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and source of cells: cellbank of test laboratory
- Suitability of cells: The V79 cell line has been used for many years in experiments with success.
- Absence of Mycoplasma contamination: not specified
- Methods for maintenance in cell culture: subcultured twice a week
- Cell cycle length, doubling time or proliferation index : doubling time 12-16 h
- Modal number of chromosomes: 22

- Type and composition of media, CO2 concentration, humidity level, temperature: MEM medium supplemented with 10% fetal calf serum (FCS). Cells have been cultured at 37°C, 5% CO2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: rat liver
- method of preparation of S9 mix: The S9 liver microsomal fraction is obtained from the liver of 8 - 12 weeks old male Wistar rats, strain CFHB (weight ca. 150-200 g) which had been given a single i. p. injection of 500 mg/kg bw Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of 5 animals are removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1:3 in KCl centrifuged cold at 9000 g for 10 minutes. The supernatant which contains microsomes is frozen in ampoules of 2 or 5 mL and stored in liquid nitrogen. For standardization a protein estimation is made.

- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/mL in the cultures. The concentration in the S9 preparation is between 30 and 45 mg/mL. The composition of the cofactor solution is concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2; 33 mM KCl; 5 mM glucose-6-phosphate; 5 mM NADP; 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix is stored in an ice bath.

- quality controls of S9: not specified
Test concentrations with justification for top dose:
5, 10, 25 and 50 µg/mL
The test substance could be solubilized up to 50 µg/mL, higher concentrations precipitated in the medium.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because it proved to be relatively nontoxic in the concentrations applied.
- Justification for percentage of solvent in the final culture medium: final concentration in the medium 1 % is non-toxic.
Controlsopen allclose all
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
with S9 mix, 15.4 µg/mL
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
without S9 mix, 1 mg/mL
Details on test system and experimental conditions:
- Number of independent experiments: 2

- Cell density at seeding: 1x 10E6 cells per flask in 30 mL medium for mutagenicity test
- Test substance added in medium

- Exposure duration/duration of treatment: 4 hours at 37°C

Treatment scheme:
Day 1: Subculturing of a log-phase culture
a) About 400 cells in 5 mL medium/25 cm2-plastic-flask for plating efficiency; in duplicate per experimental point
b) 1 x 10E6 cells in 30 mL medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point
Day 2: Treatment of a) and b)
Day 5: Subculturing of b) in 175 cm2-plastic flasks.
Day 8: Fixation and staining of colonies in a)-flasks = plating efficiency and dose relationship
Day 9: Subculturing of b) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 6 x 10E5 cells/flask): subculturing of b) in two 25 cm2-flasks for plating efficiency (about 500 cells/flask). For the selection of mutants the medium was supplemented with 11 µg/mL thioguanine.
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9.
Staining was done with 10% methylene blue in 0.01 n-KOH solution. The stained colonies with more than 50 cells were counted with a preparation.

- Method: plating efficiency
Evaluation criteria:
The test substance is classified as mutagenic if it induces with at least one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.

The test substance is classified as mutagenic if there is a reproducible concentration - related increase in the mutation frequency. Such evaluation may be considered independently of the enhancement factor for the induced mutants.

However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found (10-30 mutants per 10E6 cells) a seemingly concentration-related increase of the mutations within this range will be regarded as to be irrelevant.
Not necessary to perform as there is no reproducible increase of the mutation rates in the test groups treated with the test substance .

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
other: no cytotoxicity, but tested up to maximum solubilisable concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Precipitation and time of the determination: The test substance could be solubilized up to 50 µg/mL. Higher concentrations precipitated in the medium.

- The toxicity of the test substance was determined in a pre-experiment by establishing the concentration related plating efficiency. According to these data the concentration range of 5, 10, 25 and 50 µg/mL was chosen in the main test with and without S9 mix.

- Concurrent vehicle negative and positive control data: The sensitivity of the test system was demonstrated by the enhanced mutation rates found in the groups treated with the positive control substances compared to the negative control.

- Results from cytotoxicity measurements: No cytotoxicity was detected, plating efficiency was 84.3 - 106.7% in test substance-treated cultures across the two experiments performed.

- Genotoxicity results: With no concentration of the test substance a reproducible enhancement of the mutation rate over the range of the negative controls was found, neither without nor with metabolic activation by S9 mix.

HISTORICAL CONTROL DATA: "spontaneous mutation rate [...] normally found" was given as 10-30 mutants per 10E6 cells.

Applicant's summary and conclusion