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

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 18 May 2010, in-life phase 24 May 2010 to 04 February 2011, experimental completion date 27 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 NohSan No. 8147

Deviations:

no

Principles of method if other than guideline:

Not applicable

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:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: HanRcc: WIST(SPF)
- Age at study initiation: (P) 7 wks; (F1) approx 4 wks
- Weight at study initiation: (P) Males: 205-257 g; Females: 136-176 g
- Fasting period before study: None
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Granulated 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: 24 May 2010 To: 04 February 2011

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Granulated standard Kliba-Nafag 3433 rat/mouse maintenance diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Every 3 weeks
- Mixing appropriate amounts with (Type of food): Granulated standard Kliba-Nafag 3433 rat/mouse maintenance diet
- Storage temperature of food: 17-23°C

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear, referred to as day 0 post coitum
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate samples for analyses of test item concentration and homogeneity in the feed were drawn at the start of the main pre-pairing periods and thereafter at approximately two-month intervals. The concentrations for all batches of diet preparations analysed were within 90.2-104.4% of the nominal concentration. The diets were homogenous, values did not deviate more than 7.8% from the corresponding mean. SYN545192 was found to be stable in diet when kept 28 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

SYN545192 was administered continuously throughout the study at dietary levels of 0 ppm (control group), 25, 100, 250 (females only) or 600 ppm (males only).

Frequency of treatment:

P generation received SYN545192 over a 70-day pre-pairing period and during the pairing and after pairing periods in males and during the pairing, gestation and lactation periods in females for breeding of the F1 litters. F1 generation received SYN545192 following weaning of the F1 litters on day 21 post partum. Treatment was considered to have commenced when the selected F1 animals were about four weeks of age but the animals were maintained on their respective diets from weaning. The test item was administered during growth of the F1 generation to adulthood (at least a 70-day pre-pairing period) and also during the pairing, gestation and lactation periods for breeding of the F2 litters.

Details on study schedule:

- F1 parental animals not mated until 90 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: approx 17 weeks (P), approximately 13 weeks (F1)

Doses / concentrations

Remarks:

Doses / Concentrations:
0 ppm (control group), 25, 100, 250 (females only) or 600 ppm (males only)
Basis:
nominal in diet

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on results of preliminary single generation study in Han Wistar rats, which used dietary levels of 0, 75, 200 and 400 ppm for the females and 0, 75, 400 and 600 ppm for the males with the same batch no. of SYN545192. 400 ppm in females was demonstrated to be too high for use in the two generation study due to significantly reduced body weight in dams and offspring. There was no significant effect on body weight at 200 ppm in females, although food consumption was occasionally reduced. All dietary concentrations were well tolerated in males.
- Rationale for animal assignment (if not random): Animals were assigned to the study by computer-generated random algorithm.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality and morbidity)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week

BODY WEIGHT: Yes
- Time schedule for examinations: First day of dosing and thereafter at weekly intervals, with the exception of the pairing period. After mating, females were weighed on days 0, 7, 14 and 21 post coitum. Dams which littered were weighed on days 1, 4, 7, 14 and 21 post partum and on the day the animals were sacrificed

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YES
- Time schedule: 3 periods per week at intervals of 2 or 3 days during the pre-pairing, gestation and lactation periods up to day 21 post partum.
- Compound intake calculated from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

For P and F1 generation, 21 days prior to pairing and throughout pairing until the smear was sperm-positive or a copulation plug was observed. A vaginal smear was taken immediately before termination of each female

Sperm parameters (parental animals):

Parameters examined:
- Motility (all adult males): 100 sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.
- Morphology (groups 1 and 4 only): 500 sperm/sample evaluated microscopically and classified into the following categories: normal complete sperm, normal head only (tail detached), complete sperm misshapen hook, complete sperm abnormally curved hook, complete sperm reversed head or abnormal head only (tail detached).
- Sperm, spermatid count (groups 1 and 4 only): Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- The following parameters were examined in F1 and F2 offspring: Number and sex of pups, viability/mortality (daily), sex ratio (days 0/1, 4 and 21 post partum), clinical signs (daily), body weights (days 0/1, 4, 7, 14 and 21 post partum).
- Sexual maturation (For the F1 selected for breeding of F2 generation, the age and body weight at which vaginal opening or preputial separation occurred were recorded).

Postmortem examinations (parental animals):

SACRIFICE
- All P and F1 adult animals selected for breeding were sacrificed when they were no longer necessary for the assessment of reproductive effects. Females for which no pregnancy was detected were sacrificed after a decision had been made that they were no longer required for a possible second mating. Females which lost their litters were sacrificed with the other dams after weaning.

GROSS NECROPSY
- All animals were sacrificed by an injection of sodium pentobarbital and examined macroscopically for any structural abnormalities or pathological changes. Special attention was directed at the organs of the reproductive system.

ORGAN WEIGHTS
- The following organ weights were recorded for all P and F1 parental males and females on day 21 post partum, or shortly thereafter: Brain (including entire brainstem), testes, kidney, seminal vesicles with coagulating glands and their fluids (as one unit), pituitary, epididymides (total weight as cauda separately), adrenal glands, prostate, liver, ovaries, spleen, uterus (including cervix, excluding oviducts).
- Paired organs were weighed separately.

HISTOPATHOLOGY
- The following tissues from all P and F1 parental males and females were collected at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution: Gross lesions, pituitary, adrenal glands, liver, prostate, seminal vesicle with coagulating gland, right testis and epididymis (in Bouin's fixative), ovaries, uterus and cervix, vagina, oviducts.
- The tissues from the control and high dose animals were examined histopathologically together with any gross lesions (from all groups) and tissues from animals which died spontaneously or had to be terminated in extremis. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
- In addition to qualitative examination of ovarian histopathology, a quantitative evaluation of primordial follicles, growing follicles and antral follicles from 10 sections per ovary, and corpora lutea from 1 section per ovary in 10 females of the F1 generations in groups 1 and 4 were included.
- If test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.
- Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning.
- Any pups found dead were examined macroscopically. F1 pups not selected for the F1 generation pairing and the remaining F2 pups were sacrificed after weaning. Dead pups, except those excessively cannibalized, were examined macroscopically. All dead pups were fixed in neutral phosphate buffered 4% formaldehyde solution. Brain, thymus, spleen and liver weights were recorded from one randomly selected male and female pup from each F1 and F2 litter (on day 21 post partum precisely). Any abnormal tissues, organs weighed at necropsy and thyroids were fixed in neutral phosphate buffered 4% formaldehyde solution. Abnormal tissues were also preserved from the remaining male and female pups per litter, which were given a macroscopic examination.

Statistics:

The following statistical methods were used to analyze food consumption, body weight and reproduction 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. 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. Organ weights were analyzed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. Ratios of organ weight/body weight and organ weight/brain weight are presented for information, but were not analyzed statistically.

Reproductive indices:

mean precoital interval, % females mating, female fertility index, conception rate, gestation index, mean number of corpora lutea, mean duration of gestation, post-implantation loss

Offspring viability indices:

birth index, viability index, weaning index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

no test-related mortalities or clinical signs

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

BODY WEIGHT (PARENTAL ANIMALS): At 600 ppm, body weight gain in the P generation was lower than control during the pre-pairing period and body weight was lower during the pre-pairing and after pairing periods. In the F1 generation, mean body weight and body weight gain were statistically significantly lower than control during the pre-pairing and after pairing periods.

At 250 ppm, body weight gain in the P and F1 generations was statistically significantly lower than control during the pre-pairing and gestation periods and recovered in the lactation period. Body weight was statistically significantly lower than control during the pre-pairing period and remained lower throughout the gestation and lactation periods.

At 100 and 25 ppm, any changes in mean body weight and body weight gain in males and females of either the P or F1 generation were considered not to be adversely affected by treatment with the test item.

FOOD CONSUMPTION AND UTILISATION (PARENTAL ANIMALS): At 600 ppm group, mean food consumption in the P generation was periodically statistically significantly lower than control during the pre-pairing and after pairing periods. In the F1 generation, mean food consumption was slightly lower. Mean food utilization was statistically significantly lower than control over 2 weeks of the pre-pairing period in the P generation and during 1 week in the F1 generation.

At 250 ppm dose group, mean food consumption in the P and F1 generations was periodically statistically significantly lower than control during the pre-pairing period. It was statistically significantly lower during the whole of the gestation period and over part of the lactation period. Mean food utilization in the P generation was statistically significantly lower than control over 1 week during the pre-pairing period and in the F1 generation over 2 weeks.

At 25 and 100 ppm groups, mean food consumption for P and F1 generation males and females was not affected by treatment with the test item. Food utilization in the P generation was occasionally statistically significantly reduced over the pre-pairing period and in the F1 generation, food utilization was statistically significantly decreased for 1 week.

ORGAN WEIGHTS (PARENTAL ANIMALS): At 600 ppm, the weight of the liver adjusted for body weight was statistically significantly increased in the P and F1 generations. The weight of the liver adjusted for body weight was statistically significantly higher than control in the F1 females at 250 ppm. All other values for organ weight adjusted for body weight and organ weight to body weight ratio were either within the range of the historical control data or did not correlate with any histopathological findings and were therefore considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS): Both in P and F1 generations, microscopic examination revealed centrilobular hepatocellular hypertrophy in males at 600 ppm. Hepatocellular glycogen deposits were decreased in females at 100 and 250 ppm.

OTHER FINDINGS (PARENTAL ANIMALS): At 250 ppm, an increased incidence of lactational dioestrus was associated with decreased food consumption and decreased mean body weight in the affected females and pups, which lead to a prolonged nursing period and a delay in the affected dams returning to estrous cyclicity. Therefore, the increased incidence of lactational diestrus at the top dose level is considered an indirect consequence of high-dose effects on pup and maternal body weight, and not a direct effect on the reproductive system. At 100 ppm the incidence was within normal range for animals of this strain at this laboratory.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: equivalent to 6.9 mg/kg/day during pre-pairing

2

Dose descriptor:

NOEL

Remarks:

reproduction

Effect level:

600 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: equivalent to 40.5 mg/kg/day for P generation males during pre-pairing

3

Dose descriptor:

NOEL

Remarks:

reproduction

Effect level:

250 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: SYS545192 had no effect on any parameter of reproduction across two generations at dose levels up to 250 ppm (females) and 600 ppm (males).

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

BODY WEIGHT (OFFSPRING): The body weight of the P generation pups in the 250 ppm dose group was statistically significantly lower than control during the lactation period. In the F1 generation, body weight was statistically significantly lower.

SEXUAL MATURATION (OFFSPRING): The time until preputial separation was statistically significantly longer in the males in the 600 ppm dose group, however, the body weight at the time of sexual maturation was similar to the control value and other treated group values. This delay in sexual maturation in selected F1 males at 250 ppm is considered to reflect the lower body weight of these animals rather than a direct effect of SYN545192. The time until vaginal patency in the F1 female pups was not affected by treatment with the test item. The anogenital distance was not affected by treatment with the test item.

ORGAN WEIGHTS (OFFSPRING): In the 250 ppm F1 males, the weight of the spleen adjusted for the body weight was statistically significantly reduced. In addition, the weight of the liver adjusted for body weight was statistically significantly increased. In the F2 male pups, the organ/body weight ratio of the brain was statistically significantly increased. In the 250 ppm females in both generations, the weight of the liver adjusted for body weight was statistically significantly increased.

Effect levels (F1)

Dose descriptor:

NOEL

Remarks:

general toxicity

Generation:

F1

Effect level:

100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: equivalent to 7.8 mg/kg/day during pre-pairing

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

 Table 2: Intergroup comparison of mean cumulative body weight gain (g) (selected timepoints – P generation)

Day	Dietary Concentration of SYN545192 (ppm)
	Males				Females
	0	25	100	600	0	25	100	250
8 pre-pairing	40	40	40	36	20	20	17	17
22	101	99	99	91*	43	46	42	38
29	121	117	120	113	54	56	53	47*
70	201	190	197	185	95	91	85	75**
8 post-pairing	9	8	6*	8	-	-	-	-
45	44	37	42	43	-	-	-	-
7 gestation	-	-	-	-	22	22	22	20
14	-	-	-	-	49	47	48	39**
21	-	-	-	-	117	116	113	93**
4 lactation	-	-	-	-	9	11	16*	15
7	-	-	-	-	17	20	24	25
14	-	-	-	-	16	24	34**	29**
21					13	14	24**	22

 

Table 3: Intergroup comparison of mean cumulative body weight gain (g) (selected timepoints – F1 generation)

Day	Dietary Concentration of SYN545192 (ppm)
	Males				Females
	0	25	100	600	0	25	100	250
8 pre-pairing	47	47	46	43*	21	21	21	21
15	94	92	90	85*	40	40	40	37
22	130	129	129	122	57	56	56	51*
70	262	251	259	244	119	112	114	101**
8 post-pairing	10	9	9	6*	-	-	-	-
29	37	34	34	29**	-	-	-	-
40	46	52	52	43
7 gestation	-	-	-	-	23	21	19	18**
14	-	-	-	-	48	48	48	39**
21	-	-	-	-	117	119	121	92**
14 lactation	-	-	-	-	30	31	34	43**
21					19	18	20	41**

 

Table 4: Intergroup comparison of mean food consumption (g/animal/day) (selected timepoints – P generation)

Day	Dietary Concentration of SYN545192 (ppm)
	Males				Females
	0	25	100	600	0	25	100	250
1-3 pre-pairing	20.9	20.7	20.9	18.0**	14.9	15.4	14.8	12.8**
24-26	23.3	22.6	22.7	22.4	16.7	17.0	18.2	16.7
31-33	23.5	23.1	23.3	22.8	18.7	18.1	17.7	16.7**
52-54	23.4	22.7	23.3	22.7	18.4	18.3	17.3	15.8**
68-70	23.7	22.5	23.0	22.1*	16.6	16.9	17.1	15.7
1-3 post-pairing	22.9	22.5	21.8	21.4*	-	-	-	-
40-43	20.3	19.5	20.3	20.2	-	-	-	-
0-2 gestation	-	-	-	-	17.7	17.6	17.3	15.6**
9-11	-	-	-	-	22.6	22.0	21.9	18.7**
18-21	-	-	-	-	22.9	23.1	22.0	18.1**
1-3 lactation	-	-	-	-	25.0	26.2	27.2	24.8
7-9	-	-	-	-	47.0	47.0	47.3	41.3**
14-16	-	-	-	-	58.4	56.1	58.0	50.4**
19-21					62.6	64.7	64.1	56.8

 

Table 5: Intergroup comparison of mean food consumption (g/animal/day) (selected timepoints – F1 generation)

Day	Dietary Concentration of SYN545192 (ppm)
	Males				Females
	0	25	100	600	0	25	100	250
3-5 pre-pairing	21.0	21.1	20.9	19.5*	15.8	16.0	15.5	15.2
24-26	24.1	24.3	24.2	23.1	17.5	17.8	17.6	16.0*
47-50	24.1	22.6*	23.1	22.0**	17.7	17.1	17.2	15.8**
68-70	24.7	24.0	24.2	23.6	17.7	18.0	17.4	16.0**
10-12 post pairing	23.4	23.5	22.0	21.3**	-	-	-	-
36-38	22.0	21.5	22.3	20.6	-	-	-	-
0-2 gestation	-	-	-	-	17.6	17.9	18.3	15.7*
9-11	-	-	-	-	22.8	22.5	23.2	20.0**
18-21	-	-	-	-	24.2	24.7	24.8	17.7**
1-3 lactation	-	-	-	-	25.9	28.0	26.3	17.8**
9-11	-	-	-	-	54.5	54.3	54.0	48.0**
14-16	-	-	-	-	58.1	58.5	57.6	50.6**
20-21					62.8	66.4	61.3	55.8*

   * Statistically significant difference from control group mean, p<0.05 (Dunnett test)

** Statistically significant difference from control group mean, p<0.01 (Dunnett test)

Tables 6 -17 included in Overall remarks field

Applicant's summary and conclusion

Conclusions:

SYN545192 had no effect on any parameter of reproduction across two generations at dose levels up to 600 ppm.

Executive summary:

In a two-generation reproduction study, four groups of 25 male and 25 female, young adult HanRcc:WIST(SPF) rats (P generation) received SYN545192 (purity 97%), in the diet for 10 weeks and were then paired (one male with one female) for mating.

The F1generation animals were selected from the weaned F1 litters. The F1 parents were maintained on test diets for at least 90 days and were then paired for mating. The F2 offspring were sacrificed at weaning. Each group consisted of 25 male and 25 female rats. SYN545192 was administered orally, by ingestion, continuously throughout the study at dietary levels of 0 ppm (control group), 25, 100, 250 (females only) or 600 ppm (males only).

All dams and remaining pups were sacrificed on day 21 post partum and males were sacrificed when they were no longer needed for reproduction.

SYN545192 had no effect on any parameter of reproduction across two generations at dose levels up to 600 ppm.

Based on the results of this study, the parental NOAEL (No Observed Adverse Effect Level) was considered to be 100 ppm (equivalent to 6.9 mg/kg/day for P generation males during pre-pairing) and the offspring NOEL (No Observed Effect Level) for general toxic effects was 100 ppm (equivalent to 7.8 mg/kg/day for F1 generation males during pre-pairing). The NOEL for reproduction was 600 ppm (equivalent to 40.5 mg/kg/day for P generation males during pre-pairing).