1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Sep - 30 Oct 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

adopted in 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

other: in vivo micronucleus test in mice

Test material

Test material information

Constituent 1

Reference substance name:

1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-1,2,3,4-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

EC Number:

810-161-6

Cas Number:

1229654-66-3

Molecular formula:

C22 H16 Cl F3 N10 O2

IUPAC Name:

1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-1,2,3,4-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 35.0 ± 1.7 g (males), 28.5 ± 1.2 g (females)
- Assigned to test groups randomly: yes
- Housing: individual in Makrolon Type II (pre-test) / III (main study) cages with wire mesh top, granulated soft wood bedding
- Diet: certified standard diet, pelleted, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 42.8 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Corn oil was chosen due to its relative non-toxicity for the animals and as a smooth homogeneous suspension was attainable, which was not the case for several aqueous solvents/vehicles tested.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was suspended in corn oil. Grinding of the test item in a mortar was used to formulate the test item.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

24 and 48 h after last treatment

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

2 (pre-experiment on toxicity)
6 (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally once
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

Cell type: bone marrow erythroblasts

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Since no signs of toxicity were observed in a pre-experiment on toxicity a limit test was performed in two sexes using the maximum dose of 2000 mg/kg b.w.

TREATMENT AND SAMPLING TIMES: The animals of all dose groups, except the positive control, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.

DETAILS OF SLIDE PREPARATION: Following sacrifice the femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal bovine serum using a syringe. The cell suspension was centrifuged and a small drop of the re-suspended cell pellet was spread on a slide. The smear was airdried, stained with Giemsa and cover slips were mounted. One slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using light microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group above the laboratory’s historical solvent control data range. The primary point of consideration is the biological relevance of the results and statistical analysis was used as an aid in evaluating the results, if necessary. A test item that fails to produce a biological relevant increase in the number of micronucleated
polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the nonparametric Mann-Whitney test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: The animals treated with the test item did not express any clinical symptoms.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and gender after administration of the test item.
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of polychromatic erythrocytes of the vehicle control.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male animals

			Mean PCEs / 2000 erythrocytes at sampling time		Total micronuclei per 1000 PCEs at sampling time (range)		PCEs with micronuclei (%)
Test group	Number of animals	Dose [mg/kg bw]	24 h	48 h	24 h	48 h	24 h	48 h
Vehicle control (corn oil)	6	0	1160	n.d.	1 - 6	n.d.	0.150	n.d.
Positive control (cyclophosphamide)	6	40	1163	n.d.	12 – 60*	n.d.	1.900*	n.d.
Test substance	6	2000	1173	1178	1 - 4	1 - 4	0.117	0.117

n.d. = not determined; *statistically significant (p<0.001); PCE = polychromatic erythrocytes

Table 2: Results of the in vivo micronucleus assay in female animals

			Mean PCEs / 2000 erythrocytes at sampling time		Total micronuclei per 1000 PCEs at sampling time (range)		PCEs with micronuclei (%)
Test group	Number of animals	Dose [mg/kg bw]	24 h	48 h	24 h	48 h	24 h	48 h
Vehicle control (corn oil)	6	0	1149	n.d.	0 - 5	n.d.	0.083	n.d.
Positive control (cyclophosphamide)	6	40	1163	n.d.	33 – 89*	n.d.	2.625*	n.d.
Test substance	6	2000	1271	1212	0 - 3	0 - 4	0.108	0.083

n.d. = not determined; *statistically significant (p<0.001); PCE = polychromatic erythrocytes