(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 1988 - March 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

EC Number:

406-850-2

EC Name:

(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Cas Number:

133855-98-8

Molecular formula:

C17 H13 Cl F N3 O

IUPAC Name:

1-{[(2R,3R)-3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO cell line, cell bank of CCR
- Suitability of cells: The CHO cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation
rate (doubling time 16 - 20 h in stock cultures) and a high plating efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: not specified
- Number of passages if applicable: not specified
- Methods for maintenance in cell culture: The cells were subcultured twice weekly. Seeding was done with about 1 x 10^6 cells per flask in 15 mL of DMEM/F12 (mixture 1:1) medium supplemented with 10 % fetal calf serum.
- Doubling time: 16 - 20 h
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified

MEDIA USED
- Type and composition of media: DMEM/F12 (mixture 1:1) medium supplemented with 10 % fetal calf serum
- CO2 concentration: 11 %
- Humidity level: humdified incubator (not further specified)
- Temperature: 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of Aroclor 1254-induced Wistar rats
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/mL in the cultures. The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- concentration or volume of S9 mix in the final culture medium: 20 µL/mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): the metabolic capability was checked by positive controls

Test concentrations with justification for top dose:

7-h interval: 10.0; 50.0; 100.0; 140.0 µg/ml
24-h interval: 1.0; 5.0; 10.0; 50.0; 100.0; 140.0 µg/ml
30-h interval: 10.0; 50.0; 100.0; 140.0 µg/ml

In a pre-experiment for toxicity the colony forming ability of the CHO cells was reduced to about 40 % after treatment with the highest concentration (140.0 µg/mL) in the absence and presence of S9 mix. Higher concentrations precipitated in the culture medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells.
- Percentage of solvent in the final culture medium: The final concentration of the solvents in the culture medium did not exceed 1 % v/v.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. 0.6 - 1.5 x 10^5 cells/chamber
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 5, 21 and 27 h after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures for a duration of 2 h (7 h interval) or 3 h (24 h and 30 h interval).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After treatment with the spindle inhibitor, the cells on the slides in the chambers were treated with hypotonic solution (0.4 % KCl) at 37°C for 20 min. Afterwards the cells were fixed with 3:1 absolute methanol:glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 100 well spread metaphases per slide (200 per test group) were scored.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations.
- Determination of polyploidy: not specified
- Determination of endoreplication: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

The test article was classified as mutagenic if it induced either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.

A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points was considered non-mutagenic in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform. The aberration rates of the test groups after treatment with the test article were in the range of the control values.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Possibility of evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: Higher concentrations than 140 μg/mL resulted in test substance precipitation.
- Definition of acceptable cells for analysis: Only metaphases with characteristic chromosome number of 20 +/- 1 were included in the analysis.

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment on toxicity (colony forming ability) in the absence and presence of S9 mix treatment the highest concentration (140.0 µg/mL) reduced distinctly the colony forming ability. Higher concentrations precipitated in the culture medium.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see tables 1 - 3
- Results from cytotoxicity measurements: The mitotic index was reduced by about 60 % after treatment with the highest concentration (140.0 µg/mL) at fixation intervals 7 and 24 h.
- Genotoxicity results
o Definition for chromosome aberrations, including gaps : not specified
o The following dose levels were evaluated: 7-h interval: 140.0 µg/mL; 24-h interval: 10.0, 50.0 and 140 µg/mL; 30-h interval: 140 µg/mL
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : not specified

HISTORICAL CONTROL DATA
- Positive historical control data: not specified
- Negative (solvent/vehicle) historical control data: not specified

Any other information on results incl. tables

Tab. 1: Mutagenicity data (summary of results), fixation interval: 7 h after start of treatment

article	number of cells analysed	conc. per mL	S9 mix	% abberant cells
				incl. gaps	excl. gaps	exchanges
Solvent control	200	0.0 µg	-	4.00	3.00	0.50
Test article	200	140.0 µg	-	4.00	2.50	0.00
Solvent control	200	0.0 µg	+	6.00	1.50	0.00
Test article	200	140.0 µg	+	8.50	1.50	0.00

Tab. 2: Mutagenicity data (summary of results), fixation interval: 24 h after start of treatment

article	number of cells analysed	conc. per mL	S9 mix	% abberant cells
				incl. gaps	excl. gaps	exchanges
Negative control	200	0.0 µg	-	4.00	2.50	1.00
Solvent control	200	0.0 µg	-	1.50	1.00	0.00
Positive control EMS	200	0.72 mg	-	10.50	9.00	6.50
Test article	200	10.0 µg	-	0.50	0.50	0.50
Test article	200	50.0 µg	-	2.00	1.50	0.00
Test article	200	140.0 µg	-	3.50	2.50	0.00
Negative control	200	0.0 µg	+	4.00	2.50	1.00
Solvent control	200	0.0 µg	+	5.50	3.50	0.50
Positive control CPA	200	4.2 µg	+	20.00	17.00	10.00
Test article	200	10.0 µg	+	4.00	2.50	0.50
Test article	200	50.0 µg	+	4.50	3.50	1.00
Test article	200	140.0 µg	+	5.50	3.00	1.00

Tab. 3: Mutagenicity data (summary of results), fixation interval: 30 h after start of treatment

article	number of cells analysed	conc. per mL	S9 mix	% abberant cells
				incl. gaps	excl. gaps	exchanges
Solvent control	200	0.0 µg	-	0.50	0.50	0.00
Test article	200	140.0 µg	-	2.00	1.50	0.00
Solvent control	200	0.0 µg	+	4.00	3.50	1.50
Test article	200	140.0 µg	+	2.00	1.00	0.50