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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:
weight of evidence
Study period:
04 Sep 2018 to 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 98%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0000145096
- Expiration date of the lot/batch: 03-01-2020
- Purity test date: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC)
- Solubility and stability of the test substance in the solvent/vehicle: Based on results of validation study, the test material formulations prepared at 1.0 and 250 mg/mL in the vehicle (Corn oil) are stable and resuspendable in the vehicle for 3 days when stored at room temperature at both the concentrations.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd., No.93, Solur, Thally Road, Anekal, Bengaluru-562106, India
- Age at study initiation: 7 months young adult, nulliparous and non-pregnant females at the initiation of the study and adult males (for mating purpose only).
- Weight at study initiation: G1: 2.975 ± 0.20, G2: 2.938 ± 0.21, G3: 2.984 ± 0.20, G4: 2.995 ± 0.20
- Housing: The rabbits were housed individually, except during cohabitation, when the females rabbits were cohabited with the males in 1:1 ratio in rabbit cages (approximate size: Length 65 cm x BWidth 65 cm x Height 45 cm) with shallow cage body and facilities for providing pelleted food (Stainless steel feed hopper) and drinking water in bottle fitted with sipper tube. The waste collection tray was changed daily (except on Sundays and public holidays). - Diet: Rabbit feed manufactured by Special Diets Services, Essex, England (Batch No.3439) was provided ad libitum in stainless steel feed hoppers to rabbits.
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier was provided ad libitum in polycarbonate bottles with stainless steel sipper tubes to rabbits.
- Acclimation period: After clinical examination for good health and suitability for the study, the rabbits were acclimatized for five days before initiation of mating. During the acclimatization period, all rabbits were observed at least once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 64-65, The relative humidity in the experimental rooms was calculated daily from dry and wet bulb temperature recordings.
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material dose formulations were prepared prior to treatment and used within the stability period. Required quantities of the test item were weighed separately for each dose group and transferred into separate mortars. The test item was triturated gently using a pestle to obtain a fine powder. A small portion of the vehicle (Corn oil) was added and the contents was triturated well to obtain a suspension. This suspension was transferred into a pre-marked beaker (Previously calibrated to the desired volume). The mortar and pestle was rinsed with the vehicle and the contents were transferred to the same pre-marked beaker. The suspension was made up to the intended volume with the vehicle. The homogeneity of the the test material dosing suspension during oral gavage was maintained by constant stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as vehicle for preparing the test material formulations in this study, as the same vehicle was used in pilot rabbit study without any effects.
- Concentration in vehicle: 0 mg/mL, 2.5 mg/mL, 7.5 mg/mL, 25 mg/mL
- Amount of vehicle: 2 mL/kg bw
-The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation. The actual volume administered was calculated based on the most recent body weight on first day of treatment (Gestation Day 6) and was adjusted according to the most recent body weights recorded till Gestation Day 28. Dose formulations were continuously stirred during dose administrations. The animals in the vehicle control group were handled and administered vehicle in an identical manner to the treatment groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and concentration analysis of test material, the prepared formulations were sampled at the initiation of treatment (GD 6) and at termination of treatment period (GD 28). Samples were drawn at each dose level (two samples from the top, middle and bottom layers) in duplicate sets. Similarly, samples only from middle layer was collected from vehicle control. The analyses was done using a validated analytical method. One set of samples was analyzed for concentration and other set of samples was kept as a backup at room temperature for reanalysis. These samples were discarded as the results of first set of samples were within the acceptable limits. Formulations were considered acceptable as the mean result for all layers was within ± 15% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: During the mating period, females were cohabited randomly with males in a 1:1 ratio
- Proof of pregnancy: After confirmation of mating by visual examination, the day was referred to as day 0 of pregnancy



Duration of treatment / exposure:
The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation.
Frequency of treatment:
Once per day
Duration of test:
All animals surviving until scheduled euthanasia were euthanised on day 29 post-coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
Total of 92 female rabbits were mated and allocated to a control group and 3 treatment groups (23 female rabbits/ group). Male rabbits were used only for mating.
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of Dose Levels and Dose Justification: A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with test material dosed at 5, 15 and 50 mg/kg bw/day along with the concurrent vehicle control group. The rabbits were treated once daily with test item formulations by oral gavage at a dose volume of 2 mL/kg body weight from GD 6 to 28 and observed for clinical signs and mortality. The test material at 5 mg/kg bw/day did not affect body weight, food consumption, and maternal and litter data parameters. At 15 mg/kg bw/day, body weight and food consumption were not affected, but there was reduction in mean fetal weights observed. Treatment at 50 mg/kg bw/day resulted in reduction in food consumption and there was reduction in uterine weight observed further causing reduction in the mean litter weights. These findings were considered test material treatment related. Gross evaluation of the placenta revealed no findings. No abnormality was noticed on external observations of fetuses at any of the doses tested. Based on the results of the dose range finding study and in consultation 0, 5, 15 and 50 mg/kg/day dose levels were selected for the definitive study.

Examinations

Maternal examinations:
CLINICAL SIGNS AND MORTALITY:
- Clinical Signs and Mortality: All rabbits were observed for morbidity and mortality twice daily, i.e. once in the morning and once in the afternoon. Rabbits were observed for clinical signs during the treatment period of the study: pre dose and post dose (within 1- 2 hours of administration). On 20 September 2018, post dose observation was delayed by approximately 4.00 hrs. Aborted rabbits were necropsied, and gross observations observed were recorded.

BODY WEIGHT:
- All the females included in the study (G1 to G4) were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29. The intermittent body weight gain was calculated for GD 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 - 24, 24 - 27 and 27 - 29. Further the body weight gain for pre-treatment period (GD 0 to 6), treatment period (GD 6 to 28), and for entire gestation period (GD 0 to 29) was derived and statistically analyzed only for rabbits pregnant at caesarean section. The corrected body weight (carcass weight) was obtained by subtracting the unopened uterine weight from terminal body weight (body weight on GD 29). The corrected body weight gain was calculated by subtracting the body weight on GD 6 from corrected body weight.

FOOD CONSUMPTION:
- About 600 g of food (food input) was provided on GD 0. The left over food was recorded and replenished to about 600 g on GD 3, 6, 9, 12, 15, 18, 21, 24 and 27. Prior to terminal sacrifice, left over food was recorded on GD 29 of presumed gestation. There was no spillage of food noticed during the entire study period. Intermittent food consumption i.e GD 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 -24, 24 - 27 and 27 - 29 was calculated. Further, food intake for pre-treatment (GD 0 to 6), treatment (GD 6 to 29), and for entire gestation period (GD 0 to 29) was derived and statistically analyzed only for rabbits pregnant at caesarean section.

NECROPSY:
- Prior to caesarean section, random numbers were generated for coding to avoid bias during caesarean section and subsequent fetal evaluations. The animal code was written on the ear and the permanent accession number was striked out. On GD 29, all the rabbits were sacrificed by intravenous injection of sodium thiopentone. Gross pathological changes of all external and visceral organs of dams, including uterine contents were recorded.
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
- Other: gross evaluation of placenta
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
Statistics:
The data on maternal body weight and food consumption, interval body weight changes, gravid uterine weight, body weight change corrected to gravid uterine weight were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney/Wilcoxon test pair wise comparisons of the treated groups with the control group was performed, when the group differences were significant. The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.
Indices:
All the indices can be found in the field 'Any other information on materials and methods, incl. tables'.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed at any of the doses tested during the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no change in mean maternal (corrected) body weights and (corrected) body weight gains during the different periods of gestation (GD 0-6; GD 6-29; GD 0-29) of the rabbits treated at 5 and 15 mg/kg/day compared to the vehicle control group. At 50 mg/kg/day there was statistically significant decrease in body weight gain during GD 6-29 (116%) and GD 0 – 29 (79%). In addition, there was a trend towards a decrease in mean maternal (corrected) body weight and corrected body weight gain, which was not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 5 and 15 mg/kg/day. Though not significant, there was a trend for a decrease in food consumption during different periods of gestation with stastistical significance during GD 21-24 at 50 mg/kg/day. The reduction in body weight and food consumption at 50 mg/kg/day is considered a treatment-related effect.
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:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was a decrease in uterine weight (25%).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological findings in any of the treated groups except for a single occurrence of pale liver at 15 and 50 mg/kg/day.
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, non-treatment-related
Description (incidence and severity):
There were incidental occurrences of abortion in each of the low mid and high dose groups. One animal per group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was an increase in post implantation loss.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Two pregnant dams at the high dose of 50 mg/kg/day had complete resorptions.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was an increase in dams with resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were found in any of the dose groups
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The number of rabbits sacrificed at term were 23, 22, 22 and 22, respectively, of which 2, 2, 2 and 3 rabbits were non pregnant. The number of pregnant rabbits in the vehicle control, low, mid and high dose groups were 21, 20, 20 and 19, respectively.
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
15 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:
maternal developmental toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
pre and post implantation loss
total litter losses by resorption
necropsy findings

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day, there was a significant reduction in mean fetal weights (-8.3%) at both the sexes (males and females) as compared to the vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The total number of fetuses were 135, 122, 118 and 94 at 0, 5, 15 and 50 mg/kg/day, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Information can be found in Table 1 in the field 'Any other information on results, incl. tables'.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related major abnormality was observed during external observations of the fetuses at any of the doses. Anomaly of fore limb flexed at wrist (moderate) was observed in one fetus of a litter with 5 fetuses at 50 mg/kg bw/day and in one fetus of a litter with 8 fetuses in the vehicle control group. These minor changes were considered incidental and not adverse as these observations commonly occur in animals of this test model. The incidence is attributed to random occurrence and not treatment related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related skeletal malformations observed in fetuses of dams treated up to 50 mg/kg/day. Variants and anomalies observed across vehicle and treated groups were comparable and are consistent with concurrent and historical data. The variants and anomalies were consistent with concurrent and historical data.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related major visceral malformations observed in fetuses of dams treated up to 50 mg/kg/day. Anomalies such as gall bladder hypoplastic in vehicle and test material treated groups, and bilobed gall bladder in the vehicle control group were observed. These findings were not considered adverse as these observations commonly occur in animals of this test model and the incidence of occurrence were consistent with concurrent or historical controls. At 50 mg/kg bw/day, visceral evaluation showed incidences of unilateral absence of kidney and ureter in one dam and unilateral dilated ureter with hydroneprosis in another dam. These findings (2/94 fetuses and 2/17 litters) were considered developmental anomalies.
Other effects:
no effects observed
Description (incidence and severity):
Gross evaluation of the placenta revealed no findings. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental Toxicity
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

For homogeneity and concentration analysis of test material it was found that the relative standard deviation of assay of top, middle and bottom layer (total 6 samples) was less than or equal to 10%.

Table 1. Summary of Litter data

Sex: female

G1

0

mg/kg/day

G2

5

mg/kg/day

G3

15

mg/kg/day

G4

50

mg/kg/day

Pregnant

N

21

20

21

19

Total number of fetuses

Mean 

N

Sum

6.4

21

135.0

6.0

20

119.0

5.9

20

118.0

5.5

17

94.0

% Male Fetuses

 

60.0

45.4

48.3

43.6

Dead

Sum

0

0

0

0

Live Male

Sum

81

55

57

41

Mean fetal [c] weight (M) (g)

Mean

SD

N

39.61

5.20

21

39.82

4.77

20

38.02

5.59

20

35.94

7.38

16

Live Female

Sum

54

67

61

53

Mean fetal [c1] weight (F) (g)

Mean

SD

N

40.22

6.24

21

39.83

4.78

20

37.13

4.22

20

36.03

7.26

17

Live fetuses

Sum

135

122

118

94

Mean fetal [c2] weight (both) (g)

Mean

SD

N

39.81

5.32

21

39.79

4.47

20

37.50

4.38

20

36.51*

7.21

17

[c] – Anova/Anova&Dunnett

{Covariate(s): Number of Live Male Fetuses}

[c1] - Anova/Anova&Dunnett

{Covariate(s): Number of Live Female Fetuses}

[c2] - Anova/Anova&Dunnett

{Covariate(s): Number of both Fetuses}; *=p<0.05

Table 2.            Summarized Details of Necropsy 

Parameters 

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

5

15

50

Total No. of rabbits / group

23

23

23

23

Duration of treatment 

GD 6 to 28 (total 23 days)

Caesarean section

(day of presumed gestation)

29

Number of rabbits sacrificed at caesarean section

23

22

22

22

Number of rabbits aborted

0

1

1

1

Number of rabbits non-pregnant at caesarean section

2

2

2

3

Number of rabbits pregnant at caesarean section

Number of rabbits with complete resorption

21

0

20

0

20

0

19

2

Number of litters examined

21

20

20

17

Total number of fetuses

135

122

118

94

 

 

 

 

 

Number of fetuses evaluated

 

 

 

 

External, visceral and skeletal

135

122

118

94

Number of fetus for decapitation

61

54

55

44

Number of intact fetuses

74

68

63

50

 

  Table 3. Summary of Maternal Body Weight of Pregnant Rabbits

Sex: Female

 

Day(s)

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Body Weight (kg)

0 [a]

Mean

SD

N

2.9731

0.2061

21

3.0035

0.2150

20

2.9701

0.2116

20

2.9880

0.1671

19

3 [a]

Mean

SD

N

3.0049

0.2171

21

3.0608

0.2312

20

2.9842

0.2261

20

3.0354

0.2011

19

6 [a]

Mean

SD

N

3.0279

0.2159

21

3.0947

0.2496

20

3.0148

0.2489

20

3.0541

0.1908

19

9[a]

Mean

SD

N

2.9963

0.2104

21

3.0166

0.2102

20

2.9551

0.2423

20

2.9587

0.1932

19

12 [a]

Mean

SD

N

3.0370

0.2183

21

3.0246

0.2512

20

2.9803

0.2381

20

2.9600

0.2331

19

15 [a]

Mean

SD

N

3.0925

0.2588

21

3.0756

0.2707

20

2.9968

0.2844

20

2.9672

0.1995

19

18 [a]

Mean

SD

N

3.1110

0.2546

21

3.1034

0.2673

20

2.9996

0.2799

20

2.9928

0.2294

19

21 [a]

Mean

SD

N

3.1415

0.2842

21

3.1024

0.2706

20

3.0086

0.2724

20

2.9965

0.2373

19

24 [a]

Mean

SD

N

3.1610

0.2899

21

3.1264

0.2659

20

3.0384

0.2709

20

3.0274

0.2524

19

27 [a]

Mean

SD

N

3.1636

0.3142

21

3.1256

0.2557

20

3.0443

0.2493

20

3.0205

0.2254

19

29 [a]

Mean

SD

N

3.1781

0.3041

21

3.1374

0.2504

20

3.0505

0.2179

20

3.0307

0.2454

19

 [a] – Anova & Dunnett(Log)

  

Table 4. Summary of Maternal Body Weight Gain of Pregnant Rabbits

Sex: Female

 

Day(s)

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Absolute Weight Gain (kg)

0 -> 3 [a]

Mean

SD

N

0.0318

0.0641

21

0.0573

0.0374

20

0.0140

0.0830

20

0.0475

0.0734

19

 

3 -> 6 [a1]

Mean

SD

N

0.0230

0.0527

21

0.0340

0.0395

20

0.0307

0.0494

20

0.0186

0.0431

19

 

6 -> 9 [a1]

Mean

SD

N

-0.0316

0.0778

21

-0.0781

0.0876

20

-0.0598

0.0743

20

-0.0953

0.0759

19

 

9 -> 12 [a1]

Mean

SD

N

0.0407

0.0855

21

0.0080

0.0967

20

0.0252

0.0856

20

0.0012

0.0840

19

 

12 -> 15 [a]

Mean

SD

N

0.0554

0.0740

21

0.0510

0.0720

20

0.0165

0.0925

20

0.0072

0.1001

19

 

15 -> 18 [a1]

Mean

SD

N

0.0185

0.0664

21

0.0279

0.0513

20

0.0028

0.0775

20

0.0256

0.0610

19

 

18 -> 21 [a]

Mean

SD

N

0.0306

0.0493

21

-0.0010

0.0447

20

0.0090

0.0757

20

0.0036

0.0461

19

 

21 -> 24 [a1]

Mean

SD

N

0.0194

0.0457

21

0.0240

0.0361

20

0.0298

0.0584

20

0.0309

0.0598

19

 

24 -> 27 [a1]

Mean

SD

N

0.0026

0.0701

21

-0.0007

0.0483

20

0.0059

0.0570

20

-0.0069

0.0486

19

 

27 -> 29 [a1]

Mean

SD

N

0.0145

0.0401

21

0.0118

0.0612

20

0.0062

0.0560

20

0.0102

0.0465

19

 

0 -> 6 [a]

Mean

SD

N

0.0548

0.0832

21

0.0912

0.0583

20

0.0447

0.0950

20

0.0661

0.0705

19

 

6 -> 29 [a1]

Mean

SD

N

0.1502

0.2006

21

0.0427

0.1361

20

0.0357

0.1844

20

-0.0234*

0.1614

19

 

0 -> 29 [a]

Mean

SD

N

0.2050

0.2326

21

0.1339

0.1331

20

0.0804

0.1645

20

0.0427*

0.1894

19

 

Corrected/adjusted Body weight (kg)

GD29 - gravid uterine. Wt [a2]

Mean

SD

N

2.8132

0.2704

21

2.7880

0.2443

20

2.7093

0.1993

20

2.7586

0.2638

19

 

Corrected/adjusted Body weight Gain

Corrected Bwt-GD6 [a1]

Mean

SD

N

-0.2147

0.1931

21

-0.3067

0.1271

20

-0.3055

0.1611

20

-0.2954

0.1820

19

 

 

[a] – Anova & Dunnett: *= p<0.05

[a1] – Anova & Dunnett(Rank): *= p<0.05

[a2] – Anova & Dunnett(Log)

  

Table 5. Summary of Maternal Data

Sex: Female

 

Day(s) Relative to mating

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Group size

GD 29

N

23

23

23

23

Pregnant

GD 29

N

21

20

20

19

Gravid Uterus (g)

GD 29 [a]

Mean

SD

N

364.9

79.9

21

349.4

62.5

20

341.3

64.4

20

286.1**

88.2

17

Number of CorporaLutea

GD 29 [k]

Mean

SD

N

8.2

1.4

21

8.1

1.4

20

8.4

2.0

20

8.2

1.9

17

Number of Implantation

GD 29 [k]

Mean

SD

N

7.0

1.7

21

6.6

1.2

20

6.6

1.8

20

6.4

2.0

17

With Early resorption

GD 29

Total

N

21

4

20

5

20

6

17

6

Number of Early Deaths

GD 29 [k]

Mean

SD

N

Sum

0.2

0.5

21

5.0

0.3

0.6

20

6.0

0.4

0.6

20

7.0

0.4

0.5

17

6.0

With Late Resorptions

GD 29

Total

N

21

4

20

4

20

6

17

7

Number of Late deaths

GD 29 [k]

Mean

SD

N

Sum

0.3

0.7

21

6.0

0.2

0.4

20

4.0

0.3

0.5

20

6.0

0.5

0.6

17

8.0

With Resorptions

GD 29

N

N-ve

N+ve

21

13

8

20

11

9

20

9

11

17

5

12

Total number of resorptions

GD 29 [f]

Mean

SD

N

Sum

Trend

0.5

0.8

21

11.0

   .

0.4

0.6

20

10.0

   .

0.7

0.7

20

13.0

0.1969.

0.8

0.6

17

14.0

0.0454*

Pre-imp Loss / Animal

GD 29 [k]

Mean

SD

N

1.24

0.94

21

1.50

1.05

20

1.85

1.25

20

1.82

1.63

17

[a] – Anova & Dunnett(Log)

[k] – Kruskal-Wallis & Wilcoxon 

[f] – Cochran Armitage, Chi-Squared & Chi squared - Pearson

 

 

Applicant's summary and conclusion

Conclusions:
In this study, which conducted based on OECD TG 414 and under GLP conditions, prenatal developmental toxicity of the test material was evaluated in New Zealand White rabbits following daily administration by oral gavage at 0, 5, 15 and 50 mg/kg/day during gestation days 6 to 28. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity. Based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) are as follows:
- NOAEL maternal general toxicity is 15 mg/kg/day
- NOAEL maternal developmental toxicity is 15 mg/kg/day
- NOAEL developmental toxicity is 15 mg/kg/day
Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test material when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits.This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. Mated female rabbits were assigned to four groups with 23 animals in each group. The day on which mating had occurred was considered as gestation Day 0 (GD 0) for each individual female rabbit. Test material in vehicle (Corn oil) was administered at 0, 5, 15 and 50 mg/kg/day. The control group received the vehicle only. Dose formulation analysison initiation and termination of treatment periodindicated all samples were within 15% of the nominal concentrations. All rabbits were observed for clinical signs, morbidity and mortality, body weight changes and food consumption.Caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses.The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. The main findings of the study are presented below:

There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested except for incidental abortions in each of the, low, mid and high dose groups. Mean body weight gain at 50 mg/kg/day was lower compared to the vehicle control animals. Food consumption at 50 mg/kg/day was reduced during treatment period (GD 6 to 28). At 50 mg/kg/day there was a reduction in uterine weight, increase in post implantation loss and two dams with complete resorptions. At 50 mg/kg/day a significant reduction in litter weight was observed. Visceral evaluation at 50 mg/kg/day showed incidences (2/94 fetus) of unilateral absence of kidney and ureter and ureter dilated with hydronephrosis.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) for maternal and fetal developmental toxicity is 15 mg/kg/day.