5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethylsulfinyl)-1H-pyrazole-3-carbonitrile

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:

08 Oct - 10 Dec 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1996

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: UKEMS Guidelines (1990)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

446-630-3

EC Name:

-

Cas Number:

181587-01-9

Molecular formula:

C13H9Cl2F3N4OS

IUPAC Name:

5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethanesulfinyl)-1H-pyrazole-3-carbonitrile

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL gentamycin. Phytohaemagglutinin (PHA) was included at a concentration of approximately 10 µg/mL of culture to stimulate the lymphocytes to divide.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

KCl-buffered post-mitochondrial fraction (S9-mix) supplemented with co-factors (glucose-6-phosphate, NADP), prepared from male Sprague Dawley rats treated with Aroclor 1254 (MolToxTM S-9, Molecular Toxicology Incorporated, Boone, USA)

Test concentrations with justification for top dose:

Experiment I – normal sampling time:
Cytotoxicity testing: 19.01, 25.34, 33.79, 45.05, 60.07, 80.09, 106.8, 142.4, 189.8, 253.1, 337.5, 450, 600 and 800 µg/mL
Chromosome aberration analysis: 253.1*, 450, 600 and 800 µg/mL (*this concentration was additionally tested in the absence of S9 mix)

Experiment II – normal sampling time:
Cytotoxicity testing: 106.8, 142.4, 189.8, 253.1, 337.5, 450, 600 and 800 µg/mL
Chromosome aberration analysis: 253.1*, 450, 600 and 800 µg/mL (*this concentration was additionally tested in the absence of S9 mix)

Experiment II – delayed sampling time:
Cytotoxicity testing: 106.8*, 142.4*, 189.8*, 253.1, 337.5, 450, 600 and 800 µg/mL (*this concentrations were additionally tested in the absence of S9 mix)
Chromosome aberration analysis: 800 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.1 mL per culture)
- Justification for choice of solvent/vehicle: Solubility data indicated that the test article was soluble (with warming to 37 °C) in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h (+S9), 20 and 44 h (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells):
3 h treatment (+S9): 20 and 44 h;
20 h treatment (-S9): 20 h;
44 h treatment (-S9): 44 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 1 µg/mL medium
STAIN (for cytogenetic assays): 4% (v/v) filtered Giemsa stain in pH 6.8 buffer

NUMBER OF REPLICATIONS: duplicate cultures for the treatment groups and quadruplicate culture for the solvent, two independent experiments

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The test article was to be considered as positive if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeded the normal range, and
3) the results were confirmed in the second experiment.
A positive result only at the delayed harvest in Experiment 2 was to be taken as evidence of clastogenicity, provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as "equivocal".

Statistics:

Heterogeneity between replicate cultures was determined using the binomial dispersion test. The proportion of cells with structural aberrations (excluding gaps) for each test treatment condition was compared with the proportion in concurrent negative controls using Fisher's exact test. Statistical significance was indicated at p ≤ 0.05.

Results and discussion

Test results

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: up to 337.5 µg/mL (-S9, 20 h) and ≥ 450 µg/mL (+S9, 3 h); Experiment II: ≥ 253 µg/mL (-S9, 20 h), up to 337.5 µg/mL (-S9, 44 h), 337.5 - 600 µg/mL (+S9, 3 h + 17 h recovery); 600 µg/mL (+S9, 3 h + 41 h recovery)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment 1, precipitation of the test substance was observed at ≥ 253.1 µg/mL (with or without S9). In experiment 2, precipitation of the test substance was observed at ≥ 253.1 µg/mL (with S9) and at ≥ 337.5 µg/mL (without S9).
- Biological significance of the effects: a small, but statistically significant (p ≤ 0.05) increase in cells with aberrations at the highest concentration (800 µg/mL) following treatment in the absence of S9 for 20 h in Experiment II was considered to be of no biological significance because:
(A) no statistically significant increase in numbers of cells with aberrations were observed under the same conditions in Experiment I
(B) no dose-response relationship was observed and
(C) the numbers of aberrant cells observed fell within the historical negative control range.

RANGE-FINDING/SCREENING STUDIES: A maximum concentration of 800 µg/mL was chosen as an appropriate top dose for the assay as being close to, but in excess of, the solubility limit (approximately 518 µg/mL) in culture medium. Further treatment concentrations for chromosome analysis were selected by evaluating the effect of the test substance on the mitotic index. The top dose for chromosome analysis from the 20 + 0 h (-S9) and 3 + 17 h (+S9) treatments in Experiments 1 and 2 was to be one at which a 50-80% reduction in mitotic index occurred.

COMPARISON WITH HISTORICAL CONTROL DATA: The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within normal ranges of the historical control data and thus within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The absence of a clear dose-relationship for mitotic inhibition was a reflection of an accumulation of cells in mitosis at > 337.5 µg/mL (-S9) in Experiment I.

Remarks on result:

other: no clear dose-response relationship for cytotoxic effects; highest concentrations did not yield 50% reduction in mitotic index; precipitation was observed at higher doses; no data on the number of accumulated cells in metaphase were given

Any other information on results incl. tables

Table 1. Test results of experiment I

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 20 h, fixation time 20 h, without S9 mix
DMSO	1.0% (v/v)	100	0.5	0
NQO	2.5	n.d.	8.5	7
Test substance	253.1	39	1.5	1
	450	68	2	1
	600	60	1.5	1.5
	800	58	3	1.5
Exposure period 3 h, fixation time 20 h, with S9 mix
DMSO	1.0% (v/v)	100	0.5	0
CP	12.5	n.d.	26	24
Test substance	450	62	1.5	0.5
	600	78	1	0.5
	800	69	1	0.5

n.d.: not determined;

NQO: 4-Nitroquinoline-1-oxide; CP: Cyclophosphamide (positive controls)

Table 2. Test results of experiment II

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 3 h, fixation time 20 h, with S9 mix
DMSO	1.0% (v/v)	100	1	0
CP	12.5	n.d.	18.5	13
Test substance	450	62	1.5	0.5
	600	85	1.5	0.5
	800	100	1	0.5
Exposure period 20 h, fixation time 20 h, without S9 mix
DMSO	1.0% (v/v)	100	0.5	0
NQO	1.25	n.d.	12.5	9.5
Test substance	253.1	34	1.1	0.6
	450	67	1.5	0
	600	69	2	0.5
	800	84	3.5	2.5
Exposure period 3 h, fixation time 44 h, with S9 mix
DMSO	1.0% (v/v)	100	1.5	1
Test substance	800	100	1.5	1.5
Exposure period 44 h, fixation time 44 h, without S9 mix
DMSO	1.0% (v/v)	100	0.5	0.5
Test substance	800	100	2	1.5

n.d.: not determined

NQO: 4-Nitroquinoline-1-oxide; CP: Cyclophosphamide (positive controls)

Applicant's summary and conclusion

Conclusions:

Under the conditions chosen, the test substance was concluded not to be clastogenic in peripheral human lymphocytes.