2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Remarks:

no blood sample to show bone marrow exposure occurred and no systemic effects observed, 2000 PCE/animal instead of 4000 & historical mnPCE<0.1% in males, humidity <40%

Adequacy of study:

key study

Study period:

From February 21 to July 3, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no blood sample to show bone marrow exposure occurred & no effects observed & no systemic effects observed, 2000 PCE/animal instead of 4000 & historical MN PCE<0.1% in males, humidity <40%

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:

1997 (old)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

EC Number:

815-961-9

Cas Number:

1374760-95-8

Molecular formula:

C17H17N3O2S

IUPAC Name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state : White powder
- Storage condition of test material: Dry place, protected form light, (15 to 30°C)

Specific details on test material used for the study:

The Sponsor indicated that protection from light was not required for the bulk test article and for the test article and vehicle control dosing formulations.

Test animals

Species:

mouse

Strain:

other: Crl:CD-1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 40 days old
- weight: males are expected to weigh 24-34 grams and the females 18-28 grams
- Housing: Upon arrival: 3 per cage by sex for at least 3 days. After : all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, ad libitum
- Acclimation period: 11-day and 18-day acclimation period for the range-finding and definitive phases, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.3 to 21.5 °C
- Humidity: 35.4% to 39.0%
- Air changes: Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From February 21 to April 17, 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% methylcellulose [400 cps] in deionized water.
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle: 50; 100,150 and 200 mg/ml.
- Amount of vehicle (if gavage or dermal): 10 mL/kg (gavage)
- Lot/batch no. (if required): Methylcellulose, 400 cps (lot no. 2BF0758, retest date: 17 May 2013, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of test article was combined with a small amount of vehicle in a mortar and ground until a uniform mixture was obtained. The mixture was transferred to a glass container, the appropriate amount of vehicle was added to the container, and the mixture was stirred until the test article was wetted. The remaining vehicle was added, the mixture was homogenized using a Silverson L4RT or L4RT-A homogenizer until a uniform mixture was obtained, and the test article formulations were then stirred overnight. The test article formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. Prior to dose administration, the test article formulations were visually inspected to ensure that the formulations were visibly homogenous and acceptable for dosing.
Dosing solutions were single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C).

Duration of treatment / exposure:

Three consecutive days (Positive control formulation administered to Group 5 animals once on study day 2 (third day of dosing)).

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Range-finding test: 3 males and 3 females / dose
Main test: 6 Males and 6 Females / dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide monohydrate
- Doses / concentrations: 60 mg/kg bw
- Route of administration: Oral

Examinations

Tissues and cell types examined:

The objective of this study was to assess the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCEs) in CD-1 mouse bone marrow.Bone marrow smears were prepared and the coded slides were counted for polychromatic, normochromatic, and micronucleated polychromatic erythrocytes following the final bone marrow sample collection on study day 3.

Details of tissue and slide preparation:

Bone marrow was aspirated or flushed 2 to 3 times from each femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). Animals not used for collection of bone marrow were euthanized by inhalation of carbon dioxide and discarded. The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 2 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time. The slides were stored and shipped at ambient temperature to WIL Research Skokie for analysis.

CRITERIA FOR DOSE SELECTION:
Dosage levels for the range-finding phase were selected based on the recommendation of OECD Guideline 474, mammalian erythrocyte micronucleus test (July 1997), to dose up to the limit dosage of 2000 mg/kg/day. Additionally, the Sponsor has data which demonstrate that administration of the test item at 1000 mg/kg/day for 15 days did not result in maternal toxicity or fetal abnormalities (Charlap, 2013, WIL-884019), and a 28-day range finding study conducted in rats using an analog at doses of 10, 30, and 100 mg/kg did not result in any toxicity (Diehl, Draft, Study No. 20026750). In addition, no effects on survival, body weights, food consumption, or clinical observations were noted following administration of 500 to 2000 mg/kg/day for 3 consecutive days during the range-finding phase. Therefore, dosage levels of 1000, 1500, and 2000 mg/kg/day were selected for the definitive phase based on the lack of effects in the range-finding phase and the recommendation of the OECD Guideline 474.

TREATMENT AND SAMPLING TIMES:
For the range-finding and definitive phases, as appropriate, the vehicle and test article formulations were administered orally by gavage via an appropriately sized flexible Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily for 3 consecutive days, through the day prior to the scheduled euthanasia. The positive control formulation for the definitive phase was administered in the same manner once on study day 2, the day prior to the scheduled euthanasia, to the positive control group animals. The dose volume for all groups was 10 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage. Adjusted doses became effective the day of collection of the body weights. The first day of dosing was study day 0.

DETAILS OF SLIDE PREPARATION:
Bone marrow was collected from the first 5 of 6 animals in each sex/group at the time of euthanasia (at least 18 to 24 hours following dose administration) from both femurs of animals euthanized by inhalation of carbon dioxide. Bone marrow was aspirated or flushed 2 to 3 times from each femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). Animals not used for collection of bone marrow were euthanized by inhalation of carbon dioxide and discarded. The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 2 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time. The slides were stored and shipped at ambient temperature to WIL Research Skokie for analysis.

METHOD OF ANALYSIS:
Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution (Hayashi et al., 1983) and placed in numerical order using the animal numbers. Two separate evaluations were made for each slide: 1) a total of 1000 erythrocytes (both polychromatic erythrocytes [PCEs] and normochromatic erythrocytes [NCEs]) per animal were counted and the PCE:total erythrocytes [TE] ratio was determined; and 2) the number of micronucleated PCEs from a total of 2000 PCEs was scored per animal.

OTHER:

Evaluation criteria:

See section “Any other information on materials and methods incl. tables”

Statistics:

For the range-finding phase, data were not analyzed statistically due to the absence of a concurrent control group. For the definitive phase, analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the vehicle control group by sex. Body weight, body weight change, and food consumption data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the vehicle control group. The positive control data were evaluated using the 2-sample t-test (Sokal and Rohlf, 1981) and compared to the vehicle control group.

For the definitive phase, the percentages of micronucleated cells in PCEs and in the ratio of PCEs to TEs for the test article-treated and vehicle control group (Group 1) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p≤0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the vehicle control group (Group 1). In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA. Statistical significance was assessed at a 95% confidence level (p≤0.05).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500, and 2000 mg/kg/day
- Solubility: The analyzed dosing formulations met the protocol-specified acceptance criteria for suspensions (80% to 120% of target concentration) and were homogeneous. In addition, dosing formulations at concentrations of 100 and 200 mg/mL were demonstrated to be stable for 3 days at room temperature.
- Clinical signs of toxicity in test animals: All animals survived to the scheduled euthanasia. There were no test article-related clinical observations or effects on body weights or food consumption.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes (MPCE) or micronucleated normochromatic erythrocytes (MNCE) in male or female rats. The coded positive control demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): Test item did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female rats.
- Appropriateness of dose levels and route: All animals survived to the scheduled euthanasia. There were no test article-related clinical observations. All clinical findings in the test article-treated groups were noted with similar incidence in the vehicle control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory mice of this age and strain.

Any other information on results incl. tables

Table 7.6.2/1: Summary of results and statistical analysis

Treatment	Dosage	%MN PCEs	MN PCEs/2000 PCEs	PCE:TE Ratio
Male data
Vehicle	0	0.08 (0.09)	1.6 (1.8)	0.67 (0.04)
Test item	1000 mg/kg/day	0.03 (0.03)	0.6 (0.55)	0.63 (0.08)
	1500 mg/kg/day	0.04 (0.02)	0.8 (0.45)	0.63 (0.06)
	2000 mg/kg/day	0.04 (0.04)	0.8 (0.84)	0.66 (0.06)
Cyclophosphamide^a	60 mg/kg	1.15 (0.44*)	23 (8.8)	0.64 (0.19)
Female data
Vehicle	0	0.03 (0.03)	0.6 (0.55)	0.68 (0.13)
Test item	1000 mg/kg/day	0.00 (0.00)	0 (0)	0.64 (0.08)
	1500 mg/kg/day	0.03 (0.03)	0.6 (0.55)	0.62 (0.07)
	2000 mg/kg/day	0.01 (0.02)	0.2 (0.45)	0.66 (0.12)
Cyclophosphamide^a	60 mg/kg	0.82 (0.32*)	16.4 (6.3)	0.66 (0.08)

 

Vehicle: Basal diet

PCE: Polychromatic erythrocytes

TE: Total erythrocytes

MN: Micronucleated

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

*p< 0.05 (significant)

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female Crl:CD-1(ICR) mice.

Executive summary:

In an in vivo micronucleus test conducted according to OECD 474 guideline and in compliance with GLP, induction micronuclei by test item in bone marrow cells of male and female Crl:CD-1(ICR) mice was assessed. Mice (6 / sexe / dose) were exposed to the test substance (suspended in vehicle (1% methylcellulose [400 cps] in deionized water) by oral administration (gavage) at the dose levels of 500, 1000, 1500, and 2000 mg/kg/day for 3 days. A concurrent vehicle control group (Group 1) received the vehicle on a comparable regimen. A positive control group received a single oral dose of 60 mg/kg cyclophosphamide monohydrate (CPS) on study day 2, the day prior to the scheduled euthanasia. A dose range-finding study was performed in 3 animals / sexe / dose in order to determine the appropriate concentrations. All animals were observed for mortality and moribundity, clinical examinations, detailed physical examinations and body weights and individual food weights were recorded. Bone marrow collection for micronucleus evaluation was performed for 5 animals/sex/group. Bone marrow smears were prepared and the coded slides were counted for polychromatic, normochromatic, and micronucleated polychromatic erythrocytes. At least one smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in mice at any dose level, compared to vehicle control values. The coded positive control slides demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

 

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female rats.