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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Aug 2013 - 18 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2004

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan, notification 12 Nousan N°8147 guideline

Version / remarks:

adopted in 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

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:

rat

Strain:

other: Crl:CD (SD) Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: 239 - 328 g (at mating)
- Housing: individual in suspended polycarbonate wire mesh cages
- Diet: A04C-10 pelleted rodent diet (Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water from the municipal water supply, ad libitum
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Two dosing solutions were prepared during the study by dissolving appropriate amounts of the test material in an aqueous solution of methylcellulose 400 at 0.5% yielding final concentrations of 6.25, 25 and 100 g/L. A complementary formulation at the highest dose level for the last two days of the study was prepared for the treatment of one animal.

VEHICLE
- Concentration in vehicle: 6.25, 25 and 100 g/L
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The first formulation (F1) prepared for the study was evaluated for homogeneity and concentration using HPLC-UV analysis. Triplicate samples were collected of the lowest and the highest dietary concentrations from the top, the middle and the bottom of the formulation. In addition triplicate samples of the intermediate dose level (F1) and all dose levels of the second formulation (F2) were collected from the surface, middle and the bottom of the formulation for concentration analysis. One of the three collected samples was analyzed and the others were stored for possible verification analysis. Data indicate homogenous mixture of the test material in the vehicle. The measured values for the individual homogeneity samples ranged between 97 and 101% of nominal. Additionally, samples were at the targeted concentration (98 – 100% of nominal). The stability of test item in 0.5% aqueous methylcellulose was demonstrated in a previous study at concentrations of 0.3 and 250 g/L for up to 28 days under similar conditions of usage and storage to those of the current study (2012).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear or sperm plug in situ referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 - 20 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

day 21 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

23 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of doses has been selected in agreement with the Sponsor Representative and based on results obtained in a range-finding study where pregnant rats received the test substance at 0, 100, 400 or 800 mg/kg/day from GD 6 through 20 (2010). In this study, reduced mean maternal body weight gain between GD 6 and 8 when compared to controls was observed at 800 mg/kg bw/day. Mean maternal corrected body weight change (body weight gain between GD 6 and 21 independent of the gravid uterus weight recorded at cesarean section) was decreased by 60% in comparison to controls (not statistically significant). Decreased mean maternal corrected body weight change was observed at the mid and low dose (not statistically significant). Therefore, 0, 62.5, 250 and 1000 mg/kg/day were selected as the dose levels for this study. A high dose of 1000 mg/kg/day is a limit dose, which based on the range-finding study was not expected to exceed a maximal tolerated dose (MTD) in the dams. A mid dose of 250 mg/kg/day provided a 4 fold factor between the dose levels. A low dose of 62.5 mg/kg/day was expected to be a No Observed (Adverse) Effect Level for both maternal and fetal toxicity, based on the range-finding data.
- Other: If possible, those females having been paired with the same male were not allocated to the same group. Body weight means were checked after the mating period to ensure similar means among all groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once daily at weekends and public holiday)
- Cage side observations included: mortality, moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, day 0 - 21 of gestation

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 8, 10, 12, 14, 16, 18 and 21 of gestation

FOOD CONSUMPTION: Yes
Full feeder weights were measured on GD: 1, 6, 8, 10, 12, 14, 16 and 18. Empty feeder weights were measured on GD: 6, 8, 10, 12, 14, 16, 18 and 21. From these records the mean daily consumption was calculated.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: visceral organs (macroscopic examination), liver (weighed), reproductive tract (weighed and dissected; gravid uterine weight), uterine horn(s) without visible implantations (immersed in a 10% solution of ammonium sulfide to visualize any sites which were not apparent)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of live and dead fetuses, sex of live fetuses, individual weights of live fetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

For most maternal and litter based endpoints Bartlett test was performed to compare the homogeneity of group variances. If the Bartlett test was not significant (p>0.05), means were compared using the analysis of variance (ANOVA). If the ANOVA was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the ANOVA was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunnett test (2-sided). If the Bartlett test was significant (p≤0.05), group means were compared using the non- parametric Kruskal-Wallis test. If the Kruskal-Wallis test was not significant (p>0.05), the group means were considered to be homogeneous and no further analysis was performed. If the Kruskal-Wallis test was significant (p≤0.05), means of the exposed groups were compared to the mean of the control group using the Dunn test (2-sided). If the Bartlett test was significant for average food consumption data were transformed using the log transformation the Bartlett test was applied to the transformed data. If the Bartlett test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded as mentioned above (the ANOVA test was also performed with transformed data). For fetal body weight the Levene test was used followed by the ANOVA test in case of non-significance. In case of significance (p≤0.05) data were transformed using the log transformation before the Levene test was applied to the transformed data. If the Levene test was significant (p≤0.05) even after log transformation, statistical analysis was proceeded wth the ANOVA test or the Kruskal-Wallis test as mentioned above (the ANOVA test was also performed with transformed data). If one or more group variance(s) equaled 0, means were compared using non-parametric procedures.

Indices:

- Corrected body weight change (CBWC): CBWC = (BW on day 21 of gestation – BW on day 0 of gestation) – (gravid uterine weight)
- Carcass weight: BW on day 21 of gestation – uterus weight
- Total resorptions = early + late resorptions
- Pre-implantation loss %: (number of corpora lutea – number of implantations / number of corpora lutea) x 100
- Post-implantation loss %: (number of implantations – number of live fetuses / number of implantations) x 100
- Percentage of dead fetuse/ fetal death: number of dead fetuses / total number of fetuses x 100
- Percentage of male fetuses: number of live male fetuses / total number of live male and female fetuses x 100
- Mean fetal body weight: sum of individual weights of live fetuses / number of weighed live fetuses
- Mean fetal body weight per sex (example male fetuses): sum of individual weights of live male fetuses / number of weighed live male fetuses
- Fetal death per litter (%): number of litters with dead fetuses / total number of litters x 100
- For external, visceral and skeletal fetal findings, the percentage of fetuses affected per group for a given parameter: sum of live fetuses affected / number of live fetuses examined x 100
- For external, visceral and skeletal fetal findings, the percentage of litters affected per group for a given parameter: sum of litters with live fetuses affected / number of litter with live fetuses examined x 100

Historical control data:

Historical control data from studies conducted in-house were referred to in order to allow comparison with concurrent controls. The data were generated from sixteen rat studies between 2003 and 2012 and are presented in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

For details on results including tables see the attached document.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

For details on results including tables see the attached document.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: decreased mean fetal body weight (combined and per sex) compared to control (4%, non-adverse)
(see table 3)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SKELETAL FETAL VARIATIONS
1000 mg/kg bw/day: increased incidence of of 2 spontaneous variations; “5th and 6th sternebrae: incomplete ossification” at the fetal level and “5th and/or 6th sternebrae: unossified” at the fetal and litter levels (non-adverse)

Details on embryotoxic / teratogenic effects:

For details on results including tables see the attached document.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

2

Key result

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skeletal fetal variations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

For details on results including tables see the attached document.