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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:
from 17 December 2009 to 29 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bioagri Laboratorios - DF / BRAZIL
- Age at study initiation: 11 weeks old at mating
- Weight at study initiation:
- Housing:
Each animal was housed individually, except during acclimation and cohabitation. During the acclimation period, male rats were housed up to 4 animals/cage and females up to 2 animals/cage. After acclimation one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually.
The rats were housed in polypropylene cages (41x34x19cm) with wire mesh tops and bedding material (wood shavings).
- Diet: Nuvilab CR-1 for rodents (ad libitum)
- Water: filtered drinking water (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.0-22.2°C
- Humidity: 31.9-66.9%
- Air changes: 10-20 times per hour
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: from 1 June 2010 to 7 July 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionised
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test suspensions were prepared under inert atmosphere. Before preparation, the vehicle (deionised water) was degassed by sonication for 15 minutes, then saturated with inert gas, and kept under inert atmosphere for 15 minutes. The prepared doses were stored for no longer than 2 hours at room temperature before administration to the animals. Test suspensions were stirred continuously during the administration period to maintain homogeneity.

VEHICLE
- Concentration in vehicle: 1 to 10 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test item in samples of each control and test suspensions prepared for use was determined after each new preparation. The acceptance criteria used for the analyses was the nominal value ± 10% for the actual concentration. All values were within the acceptable range.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: from about 4:30pm to about 8:00am the following day
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from Gestation Day (GD) 6 to GD19
Frequency of treatment:
once daily
Duration of test:
from mating to GD20
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30, 100 mg/kg bw/day
Basis:
other: nominal dose
No. of animals per sex per dose:
25 mated female rats per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels were selected based upon the results of a preliminary prenatal and development toxicity study by oral route in rats where:
- the highest dose tested (200 mg/kg/day) induced: maternal toxicity characterized by body weight loss from GD6 to GD9 associated with lower food consumption, slightly lower body weight gain from GD12 to GD20 and lower corrected body weight gain and fetal toxicity characterized by lower male and female fetal body weight;
- the intermediate dose tested (100 mg/kg/day) induced maternal toxicity characterized by lower body weight gain associated with lower food consumption from GD6 to GD9, slightly lower body weight gain from GD12 to GD20 and lower corrected body weight gain and fetal toxicity characterized by lower male and female fetal body weight;
- the lowest dose tested (50 mg/kg/day) induced maternal toxicity characterized by lower body weight gain from GD6 to GD9, slightly lower body weight gain from GD12 to GD20 and lower corrected body weight gain and fetal toxicity characterized by lower male and female fetal body weight.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily on weekends
- Cage side observations checked: changes in skin and fur, eyes and mucous membranes, effects on respiratory, circulatory, autonomic and central nervous systems, motor activity and behavioral patterns

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation

FOOD CONSUMPTION: Yes
- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: no data
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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Quantitative variables such as fetal and maternal body weights, maternal body weight gain, food consumption, number of implantations, fetuses and corpora lutea were analyzed by One-Way Analysis of Variance (ANOVA), followed by Dunnett’s test if significance was detected. For qualitative or non-parametric data such as fetal variations and malformations, comparison was carried out using the Chi-Square Test. When necessary, additional statistical analysis was carried out using suitable methods (Wilcoxon followed by Kruskall – Wallis (Mann Whithbey U test). The level of significance was set at 5% and the statistical program used was SAS Software.
Indices:
% pre-implantation loss = ( number of corpora lutea – number of implantations ) x 100 / number of corpora lutea
% post-implantation loss = ( number of implantations – number of live fetuses ) x 100 / number of implantations

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Only pregnant dams were used for calculations of maternal body weights, food consumption, and gestation parameters.
No mortality and no clinical signs were observed whatever the group.
Statistically significant lower mean body weights were observed at 10 mg/kg/day on GD15 and GD20 and at 30 mg/kg/day on GD18 and GD20. These differences were very slight (<10%) and not dose-related.
Body weight gains were lower from GD6 to GD9 when compared to the control group whatever the dose with a dose-response (statistically significant at 30 and 100 mg/kg/day). From GD9, body weight gains were slightly lower when compared to the control group whatever the dose (statistically significant at 30 mg/kg/day from GD18 to GD20). Global body weight gains (from GD6 to GD20) were slightly lower whatever the dose (statistically significant at 30 and 100 mg/kg/day) without dose relationship. These effects were dose-related just after the initiation of treatment (from GD6 to GD9) and were slight and not dose-related from GD9. Therefore, they were considered related to the treatment with the test item and inducing a slight maternal toxicity.
Statistically significant lower food consumption was observed from GD6 to GD15 at 100 mg/kg/day, from GD6 to GD20 at 30 mg/kg/day and from GD6 to GD20 at 10 mg/kg/day (< 10%). These effects were dose-related just after the initiation of treatment (from GD6 to GD9) and decrease and were not dose-related from GD9. Global food consumption (from GD6 to GD20) was slightly statistically lower at each dose without dose relationship. Therefore, they were considered related to the treatment with the test item and inducing a slight maternal toxicity.
Statistically slightly lower (< 10%) carcass weight was observed at 10 and 100 mg/kg/day without dose relationship. This effect is considered not related to treatment with the test item.
Corrected maternal body weight gain was statistically lower in treated groups when compared to the controls. This effect is dose-related and is considered related to treatment with the test item.
No changes in gestation parameters were observed between the treated groups and the control.
No macroscopic lesion was observed in dams.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No statistically significant finding was found on mean placental or fetal body weight. However, slightly lower mean fetal body weight was observed at 100 mg/kg/day with higher standard deviation. This difference may indicate a slight effect related to treatment with the test item and maternal toxicity.
The external examination of the fetuses revealed no variations or malformations in control or treated groups.
Soft tissue malformations were observed in the fetuses in either the control or treated groups. Soft tissue variations were limited to kidney (unilateral renal pelvis dilatation) and observed in all groups including control group. This is a very common finding in this kind of study; therefore considered not related to treatment with the test item.
No skeletal malformations were observed.
Statistically significant higher fetal incidences of short supernumerary ribs were observed at 30 and 100 mg/kg/day. This observation is not dose-related and is a common finding in this kind of study. Thus, it is considered not related to the treatment with the test item.
Higher fetal and litter incidences of sternebrae not or incompletely ossified were observed at 30 and 100 mg/kg/day. This effect is dose-related, associated with maternal toxicity and concerned mainly 5th and 6th sternebrae which is a common finding in this kind of study. Therefore, it could be considered related to the treatment with the test item but not adverse.
Other skeletal retardations observed were isolated and not dose-related, thus, they were considered not related to the treatment with the test item.
The observation of fetal ossification level revealed no differences between fetuses from control and treated dams.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: summary of gestation parameters

 

Dose mg/kg/day

0 (control)

10

30

100

Number of females pregnant

21

19

21

20

With premature birth

0

0

0

0

With viable fetuses

20

18

20

20

With all resorption

1

1

1

0

Pregnant at necropsy

21

19

21

20

mean±SD / total

No. of corpora lutea

12.52 ± 2.06

263

11.63 ± 2.97

221

11.90 ± 3.79

250

12.85 ± 1.93

257

No. Of implantation

10.05 ± 3.80

211

8.53 ± 5.02

162

8.81 ± 4.68

185

9.30 ± 3.21

186

Fetuses

 

 

 

 

Dead

0.00 ± 0.00

0

0.00 ± 0.00

0

0.00 ± 0.00

0

0.00 ± 0.00

0

Live

9.52 ± 3.79

200

7.89 ± 4.83

150

7.86 ± 4.43

165

8.40 ± 3.33

168

Males

4.24 ± 2.59

89

3.74 ± 2.54

71

4.14 ± 2.35

87

4.25 ± 2.00

85

Females

5.29 ± 2.94

111

4.16 ± 3.04

79

3.71 ± 2.83

78

4.15 ± 2.18

83

Resorption

 

 

 

 

Total

0.52 ± 0.60

11

0.63 ± 1.01

12

0.95 ± 1.43

20

0.90 ± 1.33

18

Early

0.48 ± 0.60

10

0.53 ± 1.02

10

0.90 ± 1.34

19

0.80 ± 1.32

16

Late

0.05 ± 0.22

1

0.11 ± 0.32

2

0.05 ± 0.22

1

0.10 ± 0.31

2

Mean ± SD

% preimplantation loss

21.43 ± 24.67

32.01 ± 31.41

24.84 ± 29.65

27.57 ± 23.02

% postimplantation loss

9.62 ± 21.75

12.16 ± 24.95

15.30 ± 25.05

9.55 ± 15.67

No statistical differences between the test groups and the control (p>0.05).

 

Table 2: Mean placental and fetal body weights (grams)

 

Parameters

Dose mg/kg/day

0 (control)

10

30

100

No. of litters evaluated

20

18

20

20

mean±SD (N)

Placental weight

Male

Female

0.507± 0.046

0.524 ± 0.059

0.493 ± 0.046

0.590± 0.126

0.597 ± 0.129

0.559 ± 0.121

0.593± 0.190

0.600 ± 0.187

0.524 ± 0.110

0.538± 0.107

0.545 ± 0.095

0.540 ± 0.135

Fetal weight

Male

Female

3.779± 0.513

3.916 ± 0.546

3.670 ± 0.526

3.799± 0.543

3.918 ± 0.588

3.692 ± 0.588

3.667± 0.532

3.716 ± 0.546

3.604 ± 0.561

3.641± 0.739

3.768 ± 0.793

3.513 ± 0.752

No statistical differences between the test groups and the control (p>0.05).

 

Table 3: Summary of soft tissue variations

 

Dose mg/kg/day

0 (control)

10

30

100

 

Fetuses evaluated

95

71

78

79

Litters evaluated

20

17

19

20

Total variations

Fetal incidence (%)

Litter incidence (%)

12 (12.6)

10 (50.0)

2 (2.8)*

2 (11.8)*

10 (12.8)

8 (42.1)

11 (13.9)

5 (25.0)

Kidney (bilateral)

Renal pelvic dilatation

Fetal incidence (%)

Litter incidence (%)

 

1 (1.1)

1 (5.0)

 

0 (0.0)

0 (0.0)

 

2 (2.6)

2 (10.5)

 

3 (3.8)

3 (15.0)

Kidney (unilateral)

Renal pelvic dilatation

Fetal incidence (%)

Litter incidence (%)

 

11 (11.6)

9 (45.0)

 

2 (2.8)*

2 (11.8)*

 

8 (10.3)

8 (42.1)

 

8 (10.1)

3     (15.0)*

* bold entriessignificantly different from the control (p<0.05).

 

Table 4: Summary of fetal skeletal variations

 

Dose mg/kg/day

0 (control)

10

30

100

 

Fetuses evaluated

105

79

87

89

Litters evaluated

20

18

20

20

Total variations

Fetal incidence (%)

Litter incidence (%)

11 (10.5)

8 (40.0)

14 (17.7)

9 (50.0)

30 (34.5)*

12 (60.0)

27 (30.3)*

11 (55.0)

Rib

Short supernumerary

Fetal incidence (%)

Litter incidence (%)

 

11 (10.5)

8 (40.0)

 

14 (17.7)

9 (50.0)

 

30 (34.5)*

12 (60.0)

 

27 (30.3)*

11 (55.0)

* bold entriessignificantly different from the control (p<0.05).

 

Table 5: Summary of significant fetal skeletal retardation

 

Dose mg/kg/day

0 (control)

10

30

100

 

Fetuses evaluated

105

79

87

89

Litters evaluated

20

18

20

20

Total retardations

Fetal incidence (%)

Litter incidence (%)

86 (81.9)

16 (80.0)

67 (84.8)

15 (83.3)

81 (93.1)*

19 (95.0)

78 (87.6)

20 (100.0)*

Exoccipital

Incomplete ossification

Fetal incidence (%)

Litter incidence (%)

 

19 (18.1)

3 (15.0)

 

17 (21.5)

3 (16.7)

 

7 (8.0)*

4 (20.0)

 

8 (9.0)

3 (15.0)

Interparietal

Incomplete ossification

Fetal incidence (%)

Litter incidence (%)

 

74 (70.5)

15 (75.0)

 

56 (70.9)

13 (72.2)

 

72 (82.8)*

17 (85.0)

 

66 (74.2)

16 (80.0)

Mandible

Incomplete ossification

Fetal incidence (%)

Litter incidence (%)

 

15 (14.3)

4 (20.0)

 

21 (26.6)*

4 (22.2)

 

12 (13.8)

3 (15.0)

 

1 (1.1)*

1 (5.0)

Maxilla

Incomplete ossification

Fetal incidence (%)

Litter incidence (%)

 

15 (14.3)

4 (20.0)

 

22 (27.8)*

6 (33.3)

 

11 (12.6)

4 (20.0)

 

9 (10.1)

3 (15.0)

Squamosal

Incomplete ossification

Fetal incidence (%)

Litter incidence (%)

 

8 (7.6)

2 (10.0)

 

7 (8.9)

2 (11.1)

 

0 (0.0)*

0 (0.0)

 

10 (11.2)

3 (15.0)

Sternebrae

Not ossified

Fetal incidence (%)

Litter incidence (%)

 

18 (17.1)

8 (40.0)

 

21 (26.6)

8 (44.4)

 

28 (32.2)*

10 (50.0)

 

30 (33.7)*

12 (60.0)

* bold entriessignificantly different from the control (p<0.05).

 

Applicant's summary and conclusion

Conclusions:
On the basis of the effects observed in this study, the LOEL for maternal toxicity was determined at 10 mg/kg/day (effects on maternal body weight) and the NOAEL for fetal and developmental toxicity at 30 mg/kg/day (growth retardation at 100 mg/kg bw/day).
Executive summary:

In a developmental toxicity study according to OECD testing guideline 414 (GLP study, scored as validity 1 according to Klimisch criteria), the test item was administered to 25 female Wistar rats per dose by gavage at dose levels of 0, 10, 30 and 100 mg/kg bw/day from days 6 through 19 of gestation.

 The administration of the test item produced no mortality or clinical signs. No toxicologically significant effects were observed on gestation parameters.

When compared to controls, a lower mean maternal body weight was observed at 100 mg/kg/day, associated with a lower mean corrected maternal body weight gain and a lower mean food consumption. A slightly lower mean fetal body was also observed and these changes may indicate slight fetal retardation growth at this dose associated with frank maternal toxicity. Higher fetal and litter incidences of sternebrae not or incompletely ossified were also observed.

At 30 mg/kg/day, lower maternal body weight gain was observed during the treatment period, associated with lower corrected maternal body weight gain and lower maternal food consumption. Higher fetal and litter incidences of sternebrae not or incompletely ossified were observed but considered non adverse.

At 10 mg/kg/day, slightly lower mean maternal body weight gain was observed during the treatment period associated with lower corrected maternal body weight gain.

On the basis of the effects observed in this study, the LOEL for maternal toxicity was determined at 10 mg/kg/day (effects on maternal body weight) and the NOAEL for fetal and developmental toxicity at 30 mg/kg/day (growth retardation at 100 mg/kg bw/day).