Teflubenzuron

Genetic toxicity: in vitro

Administrative data

Endpoint:

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

1983

Deviations:

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

Test material information

Constituent 1

Reference substance name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

EC Number:

617-441-5

Cas Number:

83121-18-0

Molecular formula:

C14 H6 Cl2 F4 N2 O2

IUPAC Name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

Test material form:

solid: crystalline

Method

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):

CELLS USED
- 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

MEDIA USED
- 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.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

with S9 mix, 15.4 µg/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix, 1 mg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- 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.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- 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.

Statistics:

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity, but tested up to maximum solubilisable concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test substance could be solubilized up to 50 µg/mL. Higher concentrations precipitated in the medium.

RANGE-FINDING/SCREENING STUDIES
- 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.

STUDY RESULTS
- 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.