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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Oct 2003 - 06 Jul 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 2000
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted in 1998
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone
EC Number:
609-256-3
Cas Number:
365400-11-9
Molecular formula:
C14H13F3N2O4S
IUPAC Name:
(5-hydroxy-1,3-dimethyl-1H-pyrazol-4-yl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Prof. G. Speit, University of Ulm, Germany
- Cell cycle length, doubling time or proliferation index: population doubling time of 10 to 14 h
- Methods for maintenance in cell culture: cultures were maintained in plastic tissue culture vessels at 37°C in a humidified atmosphere containing approximately 5% CO2. Exponential growth of cell cultures was maintained by subculturing at least twice a week. For cell detachment in order to subculture, a solution consisting of 0.1% trypsin and 0.04% EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid) in phosphate buffered saline (PBS) has been employed.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: hypoxanthine-free Eagle's Minimal Essential Medium with non-essential amino acids, 2 mM L-glutamine, MEM-vitamins, NaHCO3, 100 units/mL penicillin, 100 µg/mL streptomycin and 10% heat-inactivated fetal calf serum. During treatment with the test substance the serum content was reduced to 2%. For selection of mutants, a hypoxanthine-free culture medium containing 10 µg/mL of 6-thioguanine was used.
- 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:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 and 2
With and without S9 mix: 30, 60, 120, 240, 480 and 960 µg/mL
The selection of the concentrations was based on the results of the preliminary cytotoxicty test. Although no cytotoxic effects were observed in the pretest up to 2400 µg/mL, the highest concentration in the main study was set to 960 µg/mL due to relevant alterations of pH at 312.5 µg/mL and above.
Vehicle / solvent:
- Vehicle/solvent used: DMSO (final concentration in the medium: 1% (v/v))
- Justification for choice of solvent/vehicle: The test substance is not sufficiently soluble in deionized water. In DMSO the test substance was soluble up to 2400 µg/mL.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: dimethylbenzanthracene
Remarks:
- S9: ethylmethanesulphonate (900 µg/mL) + S9: dimethylbenzanthracene (20 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 6 to 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 14 days

SELECTION AGENT: 10 µg/mL 6-thioguanine (6-TG)

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments (-S9) or in three independent experiments (+S9); one dish lost in the 2nd experiment due to contamination

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
A trial will be considered positive if a concentration-related and an in parallel cultures reproducible increase in mutant frequencies is observed. The increase should be at least two to three times that of the highest negative or vehicle control value observed in the respective trial. A positive result will only be considered relevant, if no significant change in osmolality compared to the vehicle control can be observed.
A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.
Statistics:
Mutant frequencies are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both wit Poisson derived weights. The weighted analysis of variance in all acceptable groups is followed by pairwise comparisons to the vehicle control on a nominal significance level of a = 0.05 using the Dunnett test. The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there is a significant concentration related increase of the mutant frequency (a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Changes in pH were observed at 312.5 µg/mL and above.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 2400 µg/mL.
- Precipitation: Precipitation of the test substance in the cell culture medium was not observed.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test (relative cloning efficiency) was conducted with and without metabolic activation using concentrations of the test substance ranging from 18.8 to 2400 µg/mL. No cytotoxicity effect was observed.

HISTORICAL CONTROL DATA
All results were within the range of historical control data.

Applicant's summary and conclusion