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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
Study period:
Aug 1999 - Feb 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no data reported on dam thyroid weight and histopathological assessment, dam thyroid hormones and fetal anogenital distance

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)
Version / remarks:
Draft 1998 (for updating guideline of 1981)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
current version adopted in 2018
Deviations:
yes
Remarks:
no investigation of dam thyroid weight and histopathological assessment, dam thyroid hormones, fetal anogenital distance
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: M.A.F.F., Japan, No. 4200
Version / remarks:
1985
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

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 analytical verification of doses or concentrations:
Aqueous methylcellulose samples were diluted with acetonitrile. The quantification was performed by HPLC using UV detection at 277 nm and external standardization. Test substance formulations in aqueous methylcellulose were demonstrated to be stable over a 21-day period at 5°C ± 3°C. Concentrations checks were in a range of [90-106%] of the nominal values.
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
Dose / conc.:
0.25 mg/kg bw/day (actual dose received)
Dose / conc.:
0.5 mg/kg bw/day (actual dose received)
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Dose / conc.:
4 mg/kg bw/day (actual dose received)
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 (1999) 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

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All clinical signs were those commonly observed, recorded once only or distributed between different groups and were not considered to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Tables for maternal body weights and weight gains can be found in attached documents.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption showed similar significant effect when expressed in g/day or g/kg/day. At 4.0 mg/kg bw/day, mean food consumption was significantly reduced during each interval from GD 6 to GD 22 and significantly increased for GD 26 to 29. At 2.0 mg/kg bw/day, food consumption was significantly reduced for GD 10 to 14 and for GDI 4 to 18, At 0.25 and 0.5 mg/kg bw/day, mean food consumption was unaffected by treatment.
Tables for food consumption can be found in attached documents.
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
Description (incidence and severity):
Gravid uterine weight did not show significant differences between groups (please refer to the table in the attached document).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 4 mg/kg bw/day, accentuated lobular pattern of the liver was observed in females that aborted (10/11 females with abortions).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
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 indicating maternal toxicity as cause of the observed abortions. No abortion was observed in any other dose group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 4.0 mg/kg bw/day, the mean post-implantation loss percentage was increased (not statistically significant) when compared to the control group value. It was linked to an increased number of late resorptions (statistically significant for females with live foetuses) and an increased percentage of dead foetuses (no statistically significant).
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
At 4.0 mg/kg bw/day, two females were observed with resorptions only.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 4.0 mg/kg bw/day, an increased number of late resorptions was observed (statistically significant for females with live foetuses).
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
At 4.0 mg/kg bw/day, an increased percentage of dead foetuses was observed (not statistically significant).
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
0.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
development
Effect level:
0.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
development
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
dead fetuses
early or late resorptions
number of abortions
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
abortions starting at 2 mg/kg bw/day; increased post-implantation loss and late resorptions at 4 mg/kg bw/day. However, effects were regarded as secondary to the reduced food consumption of dams.

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
At 4.0 mg/kg bw/day, the number of live foetuses was slightly but not statistically significantly reduced when compared to control group value.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The number of runt foetuses (body weight < 28.0 g) was not affected by treatment. No foetal external findings were considered to be related to treatment as all findings concerned one foetus only per group.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
No malformations showed any dose-related increase. No marked ossification delay was noted but a few findings were slightly increased at 2.0 and 4.0 mg/kg bw/day (incomplete ossification of pubis, metacarpal and/or middle phalanges). All the other variations and anomalies were considered to be randomly distributed between all groups.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
The examination of heads of half of the number of foetuses did not reveal any treatment-related findings.
At visceral examination, a few malformations were observed but none of them was considered to be related to treatment except the two diaphragmatic hernia recorded at 4 mg/kg bw/day. All variations and anomalies were considered to be randomly distributed between treated and control groups without any treatment-related increase.
Other effects:
not examined

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall findings (reduction in number of fetuses (non-significant), diaphragmatic hernia (low incidence (2 fetuses in 2 litter)) and incomplete ossification (slight severity)
Dose descriptor:
NOEL
Effect level:
0.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: overall findings (reduction in number of fetuses (non-significant), diaphragmatic hernia (low incidence with 2/80 fetuses in 2 litter) and incomplete ossification (slight severity)

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
4 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
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

Table 3: Fetal observations (excerpt)

Group

1

5

6

2

3

4

1

5

6

2

3

4

Visceral anomalies

 

Number of fetuses examined

Number of litters examined

 

178

169

191

171

135

80

23

23

26

24

20

13

 

Number of heads examined

Number of heads examined

 

83

80

90

78

63

38

22

23

26

23

20

12

 

Number of fetuses affected (mean % of fetuses affected)

Number of fetuses affected (mean % of fetuses affected)

Diaphragmatic hernia

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

2 (1.6)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

2 (15.4)

Skeletal anomalies

 

Number of fetuses examined

Number of litters examined

 

178

169

191

171

136

80

23

23

26

24

20

13

 

Number of heads examined

Number of heads examined

 

95

89

101

93

73

42

23

23

26

24

20

13

 

Number of fetuses affected (mean % of fetuses affected)

Number of fetuses affected (mean % of fetuses affected)

Pubis (unilateral/bilateral): incomplete ossification

4 (3.1)

3 (1.2)

4 (2.5)

2 (1.0)

14 (8.2)

11 (11.0)

3 (13.0)

2 (8.7)

4 (15.4)

1 (4.2)

6 (30.0)

4 (30.8)

1stmetacarpal: incomplete ossification or unossified. Forepaws: 4thor/and 5thmiddle phalanges: unossified.

18 (9.8)

19 (11.4)

20 (12.1)

17 (10.6)

39 (27.6)

31 (35.4)

6 (26.1)

12 (52.2)

13 (50.0)

11 (45.8)

13 (650.)

8 (61.5)

Groups: 1 and 5 (control), 6 (0.25 mg/kg bw/day), 2 (0.5 mg/kg bw/day), 3 (2.0 mg/kg bw/day), 4 (4.0 mg/kg bw/day)

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 observed in dams. 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. Moreover, increased post-implantation loss and fetal death was observed at 4 mg/kg bw/day without reaching statistical significance. All other litter parameters did not show any significant treatment-related effects. Fetal 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.
Based on the results of the conducted study, a NOAEL of 0.5 mg/kg bw/day was derived for maternal systemic and maternal developmental toxicity. In regard to fetal toxicity, a NOAEL of 2 mg/kg bw/day was established taking into account the overall effects on fetuses (reduced number of live fetuses (non-significant), diaphragmatic hernia (low incidence with 2/80 fetuses) and incomplete ossification (slight severity)). Due to clear maternal systemic toxicity at this dose levels, the effects observed on maternal developmental and fetal parameters are considered as secondary, non-specific consequence of maternal toxicity.