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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; supplied in 4 cohorts
- Age at study initiation: 13-15 weeks
- Weight at study initiation: 2133-3302 g on gestation day (GD) 1.
- Housing: singly in type 12.2395, C stainless steel wire mesh cages
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water of tap water quality from water bottles, ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

aqueous 1% CMC solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at maximum intervals of 7 days, which took into account the period of established stability.
To prepare the dose formulations, the specific amount of test substance was weighed, topped up with 1% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and mixed intensely with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The HPLC analyses were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in 1% CMC for a period of at least 7 days at room temperature were carried out before the study was initiated.
Samples of the test substance preparations were sent to the analytical laboratory twice
during the study period (at the beginning and towards the end) for verification of the
concentrations.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
The day of pairing was designated as gestation day (GD) 0. Presumed pregnant animals were supplied one day after mating; this day was referred to as GD 1 and the following day as GD 2.

Duration of treatment / exposure:

GD6 - GD28

Frequency of treatment:

daily

Duration of test:

Sacrifice at GD29

Doses / concentrations

Remarks:

Doses / Concentrations:
40, 100, 250 mg/ kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

31 does per treatment (control: 32)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of doses for the present examination was based on the results of 2 preceding range-finding maternal toxicity studies. Taking into account the results of these range-finding studies, and trying to avoid excessive maternal toxicity the following doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits:
40 mg/kg body weight/day: as low-dose level (expected NOAEL)
100 mg/kg body weight/day: as mid-dose level with possible effects on mean body weight gain
250 mg/kg body weight/day: as high-dose level with effects on mean food consumption and/or body weight without being excessive
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

- Rationale for species selection:The Crl:KBL(NZW) rabbit was selected to provide data in a nonrodent species. The New Zealand White strain was selected because background data are available from the literature and previous studies.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 1-29). Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 1-29).

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 1, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated. The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
The food consumption was determined daily on GD 2–29.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order.

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
- Number of dead fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test
(one-sided) for the hypothesis of equal proportions.
Proportions of fetuses with malformations, variations and/or unclassified observations in each
litter: Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians.
All other parameters: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means.

Indices:

The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals)/ (number of fertilized animals) x 100
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula:
(number of corpora lutea – number of implantations)/ (number of f corpora lutea) x 100
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula:
(number of implantations – number of live fetuses)/ (number of implantations) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
CLINICAL EXAMINATIONS & MORTALITY
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice on GD 29 were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.
In this study, the following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d):
• 9 females – not pregnant
• 2 females – died/sacrificed after gavage error
Test group 1 (40 mg/kg bw/d):
• 4 females – not pregnant
• 1 female – sacrificed after abortion
• 1 female – died after gavage error
Test group 2 (100 mg/kg bw/d):
• 3 femalea – not pregnant
• 1 female – died intercurrently
• 1 female – sacrificed moribund
Test group 3 (250 mg/kg bw/d):
• 5 females – not pregnant
• 1 female – died after gavage error

BODY WEIGHT
The mean body weights and the average body weight gain of the low-, mid- and high-dose rabbits (40; 100 and 250 mg/kg bw/d) were comparable to the concurrent control values. The observable, insignificant differences between the test substance-treated groups and the controls were biologically not relevant.
Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all groups.

FOOD CONSUMPTION
The mean food consumption was comparable between all test groups during the entire study. Statistically significant differences to the control group were detected between GD 5-6 (pretreatment) for the low- and mid-dose groups (40 and 100 mg/kg bw/d) and between GD 7-8 for the low-dose group. These findings were assessed as spontaneous in nature because a dose-response relationship was missing.

POST-MORTEM EXAMINATIONS
Uterus weight
The mean gravid uterus weights of test groups 1, 2, and 3 (40; 100 or 250 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.

Necropsy findings
At necropsy, spontaneous findings occurred in single females of every test group. No test substance-related findings were observed after sacrifice.

Reproduction data of does
The conception rate reached 72% in the control group, 84% in test group 3 (250 mg/kg bw/d), 87% in test group 1 (40 mg/kg bw/d) and 90% in test group 2 (100 mg/kg bw/d).
Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 21-26 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice.
There were no test substance-related and/or biologically relevant differences between the control and all dosed groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age.
One control doe had no live fetuses at all but only one early resorptions in the uterus.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Sex distribution of fetuses
The sex distribution of the fetuses in test groups 1-3 (40; 100 and 250 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.

Weight of placentae
The mean placental weights in test groups 1, 2 and 3 (40; 100 and 250 mg/kg bw/d) were comparable to the controls.

Weight of fetuses
The mean fetal weights of all treated groups were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between the test substance-treated groups and the controls.

Fetal external malformations
External malformations were recorded for single fetuses of test groups 1; 2 and 3 (40; 100 or 250 mg/kg bw/d. Some of the malformations are present in the historical control data. The total incidences of external malformations were comparable to the historical control data. None of the individual malformations showed a dose-response relationship and no malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.

Fetal external variations
One external variation (paw hyperflexion) occurred in one fetuse each of the low and the high dose group. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data.
Thus an association of this finding to the treatment is not assumed.

Fetal external unclassified observations
One unclassified external observation, polyhydramnios, was recorded for one litter of test group 1 (40 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance-induced effect is not assumed.

Fetal soft tissue malformations
The examination of the soft tissues revealed several malformations in fetuses of all test groups including control (0; 40; 100; and 250 mg/kg bw/d). No statistically significant differences between the test groups and the control were observed. Neither a dose response-relationship nor a malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.

Fetal soft tissue variations
A number of soft tissue variations, such as dilated cerebral ventricle, malpositioned carotid branch, short innominate, narrowed carotid or pulmonary trunk, absent lung lobe (lobus inferior medialis) and dilated aortic arch, was detected in each test group including the controls. Total incidences were without a relation to dosing, neither statistically significant differences between the test groups nor differences to the historical control data were noted.

Fetal soft tissue unclassified observations
Unclassified soft tissue observations, such as fluid filled abdomen, infarct of liver and blood coagulum around urinary bladder, were recorded for single fetuses of all test groups 0, 1, 2 and 3 (0; 40; 100 and 250 mg/kg bw/d). A relation to dosing is not present if normal biological variation is taken into account. Therefore, a test substance induced effect is not assumed.

Fetal skeletal malformations
Malformations of the fetal skeletons were noted in fetuses of all test groups including control (0; 40, 100 and 250 mg/kg bw/d). Neither statistically significant differences between treated groups and the control were calculated nor a dose-response relationship was observed. Most of the individual malformations are present in the historical control data. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.

Fetal skeletal variations
For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were without a relation to dosing.
On a fetus per litter basis one specific skeletal variation, supernumerary 13th rib with present cartilage, was statistically significant higher than the concurrent control in the high-dose group. This statistical difference was not present for the litter incidence
which is lower in the treated groups than in the control. None of the incidences (fetal, litter, affected fetuses/litter) showed a dose response relationship. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain, and are present in the historical control data. Thus an association of these findings to the treatment is not assumed.

Fetal skeletal unclassified cartilage observations
Additionally, isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing, were comparable to historical control data and, therefore, were regarded to be spontaneous in nature.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: toxicity on fetuses: no adverse effects observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified