3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Reg.No. 5069089
- Analytical purity: 99.2 weight% (following the Certificate of Analysis)
- Lot/batch No.: L80-68
- Physical state: Solid/pink
- Storage condition of test material: Refrigerator
- pH value: 2.5 (undiluted test substance, moistened with water)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Mean weight at study initiation: 28.1 g
- Housing: Makrolon cages, type M I; single housing
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: Drinking water from bottles was available ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was suspended in corn oil. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and than shaken thoroughly. All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the
test substance and the vehicle. The animals were sacrificed 24 hours (all test substance
concentrations, vehicle, both positive controls) and 48 hours (highest test substance
concentration, vehicle) after the treatment, respectively.

Frequency of treatment:

once

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 males per treatment

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP): 20 mg/kg bw
Vincristine sulfate (VCR): 0.15 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow cells from the animals femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, at 2000 mg/kg bw recommended as the highest dose according to the OECD Guideline all animals (male and female) survived showing weak clinical signs. The clinical sign only observed was piloerection.

DETAILS OF SLIDE PREPARATION:
- The slides were stained with eosin and methylene blue (modified May-Gruenwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance ( 9), i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Summary of results
Test	group	Sacrifice	Animal	Micronuclei in PCE		Number
		interval	No.	total a	large b	of NCE c
		[hrs]		[‰]	[‰]
Vehicle	control	24	5	1.2	0	3663
corn	oil
Test	substance	24	5	1.0	0	5607
500	mg/kg bw.
Test	substance	24	5	0.9	0.1	5328
1000	mg/kg bw.
Test	substance	24	5	0.8	0.1	4328
2	0
Positive	control	24	5	14.4**	0	4090
cyclophosphamide
20	mg/kg bw.
vincristine	sulfate	24	5	60.5**	19.2**	5077
0.15	mg/kg bw.
Vehicle	control	48	5	0.8	0	3670
corn	oil
Test	substance	48	5	1.3	0.1	5550
2000	mg/kg bw.