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Toxicological information

Developmental toxicity / teratogenicity

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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:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: M.A.F.F., Japan, No. 4200
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
446-630-3
EC Name:
-
Cas Number:
181587-01-9
Molecular formula:
C13H9Cl2F3N4OS
IUPAC Name:
5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-(ethanesulfinyl)-1H-pyrazole-3-carbonitrile
Details on test material:
- Name of test material (as cited in study report): RPA 107382
- Analytical purity: 927 g/kg (92.7%)
- Batch No.: CDR-9706

Test animals

Species:
rabbit
Strain:
other: New Zealand White Crl: Kbl/BR (SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Saint Aubin les Elbeuf, France
- Weight at study initiation: 2.48 - 3.69 kg
- Housing: individually, in plastic cages on a perforated cage floor
- Diet: Certified Rabbit Pellet diet UAR 110 C-10, 250 ± 5 g
- Water: filtered water, ad libitum
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 29 Aug 1999
To: 09 Dec 1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqueous solution of 0.5% methylcellulose 400 (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: An appropriate amount of the test substance was suspended (w/v) in the vehicle and stored at approximately 5 °C (± 3 °C). Homogeneity of the suspensions was checked for all concentrations. Stability of the test substance in suspension was determined before the begin of the study at concentrations of 62.5, 75 and 15000 mg/L and was found acceptable within a period of 21 days.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: artificial insemination
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day of insemination referred to as day 0 of pregnancy.
Duration of treatment / exposure:
Day 6 - 28 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
29 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.50, 2 and 4 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.25 mg/kg bw/day
Basis:
actual ingested
This dose group and a concurrent control group were added to the initial study due to the abortion rate in the highest dose group in order to provide a study with three treatment groups and at least 20 pregnant females at scheduled sacrifice.
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses based on results obtained in a previous range-finding study (company study no. M-192052-01-2; report no. SA 99078) in rabbits.
- Rationale for animal assignment (if not random): The rabbits were allocated to the different groups using a body weight dependent procedure to ensure that the females inseminated with the same male were distributed among the different groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon); on weekends and public holidays once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days (GD) 0, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: full feeder weights were measured on GD 1 and daily thereafter through GD 28; empty feeder weights were measured on GD 2 and every day through GD 29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 29
- Organs examined: macroscopic examination of the visceral organs, recording of the number of ribs
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
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
- individual body weights of live foetuses
Statistics:
For maternal and litter parameters, Dunnett's test was used to compare exposed groups to controls when the overall effect was statistically significant (p<0.05 via the ANOVA F-test). The Generalized Estimating Equations (GEE) linear regression model was used for analyzing fetal body weight and fetal death status, and the GEE logistic model was used for analysing fetal sex. Although fetal death status is a binary outcome (dead vs. live foetus), this parameter was still analysed via a linear regression model because the distribution of the variable was too sparse for computations required for logistic regression. All GEE regression models assumed exchangeable intralitter correlations and used a robust variance estimator for regression parameters. Individual t-tests for pairwise comparisons to control were evaluated when the overall treatment effect was statistically significant (p<0.05 via a Wald chi-square test).
Indices:
Percentage of pre-implantation loss: [(no. of corpora lutea - no. of implantations) / no. of corpora lutea] * 100

Percentage of post-implantation loss: [(no. of implantations - no. of live foetuses) / no. of implantations] * 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: 4 mg/kg bw/day: body weight loss, significant reduced food consumption and high abortion rate; 2 mg/kg bw/day: significant reduced food consumption

Details on maternal toxic effects:
Mortality:
During the study period, no treatment-related deaths were observed. In the control group, 3/60 females were found dead on GD 10, 12 and 21. The necropsy revealed a gavage error in two cases and in the other case no abnormal findings were observed. In the 0.25 mg/kg bw/day group, 2/30 females died on GD 8 and 10 due to a gavage error.

Abortion:
In the 4 mg/kg bw/day group, 11/28 pregnant females aborted between GD 21 and 27. Nearly all of this females (10/11) had pale and/or accentuated lobular pattern of the liver. In the 2 mg/kg bw/day group, 2/24 pregnant females aborted on GD 25 and 28.
All females aborted after reduced food consumption and body weight loss. No abortion was observed in any other dose group.

Pregnancy rate:
The pregnancy rate was between 80 and 97% across all groups. At least 20 females per group treated with up to 2 mg/kg bw/day had live foetuses at cesarean section (see table 1). In the 4 mg/kg bw/day group, 13 females had live foetuses.

Clinical signs:
All clinical signs were those commonly observed, recorded once only or distributed between different groups and were not considered to be treatment-related.

Body weights:
At 4 mg/kg bw/day, the mean body weight changes for all intervals during the dosing period were significantly lower than the corresponding control values. From GD 6 through 18, a body weight loss of -0.27 kg was observed compared to a gain of 0.21 kg in the control group. At 2 mg/kg bw/day, the body weight changes were reduced for many intervals, showing statistical differences for GD 6-8, 6-14, 6-18, 6-22 and 6-26. At 0.25 and 0.5 mg/kg bw/day, the body weight changes were considered to be unaffected by treatment when compared to the controls. The corrected body weight change did not show any statistical differences between treated and control groups.

Food consumption:
At 4 mg/kg bw/day, mean food consumption was significantly reduced during each interval from GD 6 to 22 and significantly increased for GD 26 to 29. At 2 mg/kg bw/day, food consumption was significantly reduced for GD 10 to 14 and for GD 14 to 18. At 0.25 and 0.5 mg/kg bw/day, mean food consumption was unaffected by treatment.

Necropsy:
No treatment-related adverse effects were reported.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
0.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
0.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: 4 mg/kg bw/day: visceral malformations, incomplete ossification of bones; 2 mg/kg bw/day: incomplete ossification of bones

Details on embryotoxic / teratogenic effects:
Litter data:
In the 4 mg/kg bw/day group, the percentage of mean post-implantation loss was increased (not statistically significant) in comparison to the control value. This effect was linked to a statistically significant increased number of late resorptions for females with live foetuses and an increased percentage of dead foetuses (not statistically significant). The number of live foetuses in the high dose group was slightly reduced when compared to control group. The litter parameter of all other groups were not affected by treatment. The mean foetal body weight did not show any significant differences between treated and control groups (see Table 2).

External observations:
In the control group and the 4 mg/kg bw/day group 1/347 and 1/80 foetuses had omphalocele. At 2 mg/kg bw/day, in 1/136 foetus a short snout in combination with a protruding tongue was observed. One other foetus of the same group showed a folded dorsal skin. The abnormal external findings are not considered to be treatment-related. The number of runt foetuses with a body weight less than 28 g was not affected by treatment (10/347 foetuses in the control group and 4/191, 1/171, 7/136 and 4/80 foetuses at 0.25, 0.5, 2 and 4 mg/kg bw/day, respectively).

Visceral observations:
In the 4 mg/kg bw/day group, 2/80 foetuses had diaphragmatic hernia, which was considered to be treatment-related. All other abnormal findings were considered to be randomly distributed between treated and control groups without any treatment-related increase.

Head examinations:
The examination of the heads of one half of the foetuses did not reveal any treatment-related findings.

Skeletal observations:
There was no dose-related increase in malformations observed and no marked ossification delay was noted. In the 2 and 4 mg/kg bw/day groups incomplete ossification of pubis in 14/136 and 11/80 foetuses and incomplete ossification of metacarpal and/or middle phalanges in 39/136 and 31/80 foetuses, respectively, was observed. In the control group 7/347 and 37/347 foetuses showed incomplete ossification of pubis or of metacarpal and/or middle phalanges, respectively.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Animal status and pregnancy rate

 

 

Control

dose groups [mg/kg bw/day]

 

0.25

0.5

2

4

No. of inseminated females

60

30

30

30

30

No. of death females

3

2

0

0

0

No. of abortions

0

0

0

2

11

No. of pregnant females

53

29

26

24

28

 - with live foetuses

46

26

24

30

13

 - with resorptions only

0

0

0

0

2

 - with implantations only

4

1

2

2

2

No. of non-pregnant females

7

1

4

6

2

Pregnancy rate [%]

88

97

87

80

93

 

 

Table 2: Litter response and foetal parameters

 

 

Control

dose groups [mg/kg bw/day]

 

0.25

0.5

2

4

No. of all pregnant dams observed

50

27

26

22

17

No. of Corpora Lutea per dam

10.0

10.3

9.3

9.5

9.4

0.3

0.4

0.5

0.6

0.7

No. of Implantation Sites per litter

8.0

7.6

7.4

7.0

6.3

0.4

0.7

0.5

0.7

0.9

Pre-implantation loss per litter [%]

22.2

26.2

19.8

27.8

35.1

3.2

5.7

4.2

4.9

7.5

No. of early resorptions per litter

0.340

0.519

0.423

0.182

0.294

0.133

0.188

0.168

0.084

0.143

No. of late resorptions per litter

0.020

0

0.017

0

0.176

0.020

0

0.053

0

0.128

Post-implantation loss per litter [%]

17.1

9.8

17.9

16.9

38.7

3.9

4.2

5.8

6.3

10.0

No. of all pregnant dams with live foetuses

46

26

24

20

13

No. of Corpora Lutea per dam

10.4

10.3

9.8

10.1

10.5

0.2

0.5

0.3

0.5

0.6

No. of Implantation Sites per litter

8.3

7.9

7.9

7.5

7.6

0.4

0.7

0.4

0.6

0.9

Pre-implantation loss per litter [%]

21.0

23.7

18.5

26.9

28.6

3.3

5.3

3.9

5.3

7.3

No. of early resorptions per litter

0.370

0.538

0.458

0.200

0.231

0.144

0.194

0.180

0.092

0.166

No. of late resorptions per litter

0.022

0

0.083

0

0.231

0.022

0

0.058

0

0.166

Post-implantation loss per litter [%]

9.9

6.3

11.0

8.6

19.8

1.9

2.4

3.7

3.0

6.8

No. of live foetuses per litter

7.5

7.3

7.1

6.8

6.2

0.4

0.6

0.5

0.6

0.9

No. of dead foetuses per litter

0.3

0

0.1

0.4

0.8

0.1

0

0.1

0.2

0.4

No. of foetuses examined

359

191

174

143

90

Dead foetuses [%]

3.3

0

1.7

4.9

11.1

0.9

0

0.9

2.7

5.3

Male foetuses [%]

45.8

46.1

46.2

52.9

51.3

2.8

2.8

4.7

4.1

4.3

Fetal body weight [g]

39.7

39.8

40.5

37.4

38.3

0.5

0.7

0.6

1.0

1.4

Applicant's summary and conclusion

Conclusions:
No deaths were considered to be treatment-related. Abortions occurred at 2 and 4 mg/kg bw/day with a high incidence at 4 mg/kg bw/day. All the abortions were between GD 21 and 28 and in all cases after a reduction of food intake for several days and a body weight loss. Additionally, nearly all of the high dose group females that aborted had pale and/or accentuated lobular pattern of the liver.
Body weight changes and food consumption at 2 and 4 mg/kg bw/day were significantly reduced for many intervals during the dosing period, when compared to control values. The mean number of late resorptions was slightly increased at 4 mg/kg bw/day. All other litter parameters did not show any significant treatment-related effect. Foetal body weights were considered unaffected by treatment. No malformations at external, visceral and skeletal examination were considered to reflect any abnormal development linked to treatment except two foetuses at 4 mg/kg bw/day with a diaphragmatic hernia. Incomplete ossification was noted for a very few bones; it was limited to 2 and 4 mg/kg bw/day and did not reflect any general ossification delay.
The No Observed Effect Levels (NOEL) for maternal and developmental toxicity are 0.5 mg/kg bw/day under the conditions of this study.
In conclusion, the test substance had no effect on intrauterine development at doses up to 0.5 mg/kg bw/day.

CLP: not classified
DSD: not classified