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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1996
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines (1990)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
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.