benzovindiflupyr (ISO); N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In life 05 April 2010 to 30 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant, non-guideline animal experimental study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

This study was to evaluate the effects on the pregnant rat and development of the embryo and foetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undeca-2,4,6-trien-3-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

EC Number:

691-719-4

Cas Number:

1072957-71-1

Molecular formula:

C18H15Cl2F2N3O

IUPAC Name:

N-[11-(dichloromethylidene)tricyclo[6.2.1.0²,⁷]undeca-2,4,6-trien-3-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide

Details on test material:

- Name of test material (as cited in study report): SYN545192
- Physical state: Beige powder
- Analytical purity: 97.0%
- Expiration date of the lot/batch: End February 2013
- Storage condition of test material: <30°C

Test animals

Species:

rat

Strain:

other: RccHan: WIST(SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: RccHan: WIST(SPF)
- Age at study initiation: 11 weeks
- Weight at study initiation: 185-229 g
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilised standard softwood bedding
- Diet: Pelleted standard Kliba-Nafag 3433 rat/mouse maintenance diet ad libitum
- Water: Community tap-water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Air changes: 10-15 air changes per hour
- Photoperiod: 12 hour fluorescent light/12 hour dark

IN-LIFE DATES: From: 05 April 2010 To: 30 April 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The dose formulations were made weekly in seven aliquots for each group using SYN545192 as supplied by the Sponsor. SYN545192 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v) and homogenised before the remaining vehicle was added.
- Separate formulations were made for each concentration.
- Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer for at least 30 min. before administration.
- Before the first treatment and during the last week of treatment, duplicate samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken for analysis of concentration and homogeneity. Duplicate samples of about 2 g of each concentration were taken from the middle only from the first suspension prepared to confirm stability (4 hrs and 7 days).
- The samples were analysed by HPLC coupled to an UV detector.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of SYN545192 in the dose formulations ranged from 94.0% to 101.70% of target.
Homogeneous distribution of SYN545192 in the dose preparations was demonstrated and individual results did not deviate more than 0.6% from the corresponding mean.
Stability of SYN545192 in dose formulations of 0.1 mg/mL and 20 mg/mL, for at least 8 days at 2-8°C, was validated in a preliminary analytical study with SYN545192 in 0.5% carboxymethylcellulose aqueous solution (C80036). In this study the test item was found to be stable in application of dose preparations when kept 4 hours and 7 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Details on mating procedure:

- Impregnation procedure: Mated rats were supplied by the breeder. After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually when the daily vaginal smear was sperm positive or a copulation plug was observed.
- Proof of pregnancy: The day of mating was designated day 0 post coitum.

Duration of treatment / exposure:

Days 6-20 post coitum

Frequency of treatment:

Once daily by gavage on days 6-20 post coitum

Duration of test:

To day 21 post coitum

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 20 and 30 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous non-GLP toxicity study in non-pregnant female Han Wistar rats at the same laboratory, using dose levels of 0, 10, 20, 40, 75 and 100 mg/kg/day.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for signs of viability and mortality twice daily.
- Cage side observations: Daily cage-side clinical observations were made once daily during acclimatisation and up to day of necropsy.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was recorded at 3 day intervals on days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post coitum
- Post mortem examination included gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed and the data recorded.
- The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
- If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Fetal examinations:

- External examinations: Yes:
- The foetuses were weighed individually and examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- At least 50% of the foetuses from each litter were fixed in Bouin's fixative and examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage.
- The skeletons were examined and all abnormal findings and variations were recorded.

Statistics:

The following statistical methods were used to analyse food consumption, body weight, body weight gain, reproduction and foetal examination data:
• Means and standard deviations of various data were calculated.
• All statistical tests were two-sided.
• Statistical significance between groups was evaluated by Analysis of Variance (ANOVA). In the case where variances were non-homogenous, appropriate transformations were applied (e.g. log, square root, or double arcsine) to stabilize the variances before the ANOVA. The Dunnett many-one t-test was then used to compare each group to control based on the error mean square in the ANOVA.
• Fisher’s exact-test was applied if the variables could be dichotomized without loss of information.
• For statistical tests on foetal data, comparisons were made between groups for number of foetuses affected and number of litters affected, for completeness. The litter was considered the proper unit of measurement for overall study evaluation.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Test item-related clinical signs were observed in seven animals in the 30 mg/kg/day dose group. Hunched posture, uncoordinated movements, circling movements, decreased activity and ruffled fur were seen. No clinical signs or observations were noted in any other dose group during the study.
Body weight and mean body weight gain were statistically significantly lower at 30 mg/kg/day during the treatment period. The corrected body weight gain was also statistically significantly reduced in this dose group. At 20 mg/kg/day, mean body weight gain was statistically significantly reduced on day 8 post coitum only.
Mean food consumption was statistically significantly lower at 30 mg/kg/day during the treatment period. At 20 mg/kg/day, food consumption was statistically significantly reduced for days 6-9 and 15-18 and was slightly lower during the whole treatment period when compared to the current control.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Intergroup comparison of bodyweight gain (g) – selected timepoints

	Dose level of SYN545192 (mg/kg bw/day)
day	0 (control)	10	20	30
1	-16	-17	-15	-17
7	2	4	0	-11**
14	32	31	28	15**
21	102	90	95	72**
** Statistically significant difference from control group mean at 1% (Dunnett test)

 

Table 2: Intergroup comparison of food consumption (g/animal/day) – selected timepoints

	Dose level of SYN545192 (mg/kg bw/day)
days	0 (control)	10	20	30
0-2	19.6	19.2	19.2	20.1
6-9	20.7	20.2	17.4**	12.1**
12-15	22.8	22.1	20.5	18.7**
15-18	25.0	22.0	21.3**	18.2**
18-21	24.0	28.5	22.2	17.9**
** Statistically significant difference from control group mean at 1% (Dunnett test)

 

Applicant's summary and conclusion

Conclusions:

The dose level of 30 mg/kg body weight/day produced obvious maternal toxicity whilst slight toxicity was seen at 20 mg/kg body weight/day. There were no external, visceral or skeletal abnormalities associated with treatment with the test item. Based on the results of this preliminary study, dose levels of 0, 7.5, 15 and 30 mg SYN545192/kg body weight/day were selected for use in a prenatal developmental toxicity study in the rat.

Executive summary:

In a dose range-finding prenatal developmental study, SYN545192 was administered to groups of ten mated female RccHan: WIST(SPF) rats at dose levels of 0, 10, 20 and 30 mg/kg bw/day), from day 6 to day 20 post coitum (the day prior to caesarean section). A standard dose volume of 10 mL/kg body weight was used. Control animals were dosed with the vehicle alone (0.5% CMC).

The dose level of 30 mg/kg body weight/day produced obvious maternal toxicity whilst slight toxicity was seen at 20 mg/kg body weight/day. There were no external, visceral or skeletal abnormalities associated with treatment with the test item.

Based on the results of this preliminary study, dose levels of 0, 7.5, 15 and 30 mg SYN545192/kg body weight/day were selected for use in a prenatal developmental toxicity study in the rat.