Registration Dossier

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 2016-08-04 to 2017-02-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

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

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
females and males
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Telangana, India. CPCSEA Registration No. 1117/PO/RcBiBt/S/07/CPCSEA.
- Age at study initiation: 15-16 weeks
- Weight at study initiation:
* Males : 289 g - 362 g
* Females : 201g - 239 g
- Fasting period before study: no
- Housing:
CAGES: Polycarbonate cages (size 31 cm (L) X 21 cm (W) X 20 cm (H)), cleaned and disinfected before the animals were brought in and cleaned at regular interval during the study.
* Acclimatation period: 1-3 rats /sex/cage
* During mating: 2 females and 1 male / cage
* Pregnant females, post-mating: 1 female/cage.
BEDDING MATERIAL: sterilized corn cob produced from pure corn, dried and free from dust, batch SPAR-33/2016, from Sparconn Life Sciences, Bangalore. Renewed as often as necessary to keep the animals dry and clean

STUDY ROOM: before initiation, the study room was cleaned and disinfected. During study: the floor of the study room and work tops were swept and mopped with disinfectant solution every day and when required.

- Diet (e.g. ad libitum): ad libitum: conventional laboratory pellet diet (batch 040716, 040816 and 040916) from Nutrivet Life Sciences, Pune.
- Water (e.g. ad libitum): ad libitum: aqua guard filtered drinking water in bottles. Samples of drinking water were subjected periodically to bacteriological tests and to chemical contaminant analysis (latests test results available)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22.4°C
- Relative Humidity (%): 45.4 to 66.5%
- Air changes (per hr): 12 time / hr, and filtered adequately
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 2016-08-05 to 2016-09-23

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily: the defined quantity of the test substance was transfered to the mortar and triturated using pestle. Vehicle is added in to mortar and mixed well.
The formulation was transfered to the measuring cylinder and make the volume up to desired quantity.
At the time of dosing, dose formulations were stirred continously on a magnetic stirrer for maitaining homogeneity of testing solutions.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was used based on the solubility testing
- Concentration in vehicle: up to 20 mg test item / bw
- Amount of vehicle (if gavage): dose volume not exceeding 0.4 ml/100g bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The testing solution samples at 0 mg/mL, 1.25 mg/mL, 2.5 mg/mL and 5 mg/L were analysed by HPLC. Two replicates of 2 ml samples from upper, middle and lower layer were sent to Analytical department for the determination of homogeneity and test substance content. The analysis was carried out 2 times during treatment at starting and end of the treatment period.

The analytical parameter has been validated with respect to the following parameters:
SPECIFICITY: evaluated by analysing the blank, standard, vehicle and sample.
LINEARITY: carried out by preparing and analysing standards solutions of 6 concentrations (covering the target analyte concentration). The coded calibration solutions were injected into the HPLC, a plot was drawn between the concentration and the peak area response. The correlation coefficient, slope and intercept of the linear regression were calculated.
ASSAY ACCURACY AND PRECISION: carried out by fortifying the standard in the vehicle at 2 levels (covering the target analyte concentration). Five preparations were carried out at each concentration level selected. One control at each concentration level was maintained. Mean, SD, % RSD were calculated.

The test item formulations were found to be homogeneous.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: until pregancy occurs or 2 weeks elapsed.
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: observation of sperm positive vaginal smear referred to as day 0 of gestation.
Duration of treatment / exposure:
From gestation day 5 to gestation day 19 (i.e. 15 days).
Frequency of treatment:
Once daily
Duration of test:
19 days from gestation day 0 (i.e. 20 days from gestation day 0).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
25 females per dose:
G1: 0 mg/kg bw/day
G2: 5 mg/kg bw/day
G3: 10 mg/kg bw/day
G4: 20 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range finding study
- Rationale for animal assignment: based on body weight, all animals were assigned in descending order. Highest body weight animals were selected for mating. Randomization was done based on day 0 body weight. After confirmation of mating by vaginal smear examination, animals were assigned in unbaised manner to control and treated groups. The inseminated by same male were evenly distributed across the group.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule : at arrival in the laboratory, after completion of the acclimatization period and before group allocation and D0, D3, D5, D8, D11, D14, D17 and D20 (day of scheduled killed) of pregnancy

FEED CONSUMPTION: Yes
- Time schedule : D0, D3, D5, D8, D11, D14, D17 and D20 (day of scheduled killed) of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20: Organs examined: gross observation of all maternal organes (for any structural abnormalities or pathological changes) with emphasis on uterus, number of live/viable foetuses, number of dead fetuses, number of corpora lutea, number of implantations sites, degree of resorption and weight of placenta recoded for the terminally sacrified females. The gravid uteri (including cervix) were weighed at necropsy

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 resorptions: Yes




Fetal examinations:
Each fetuses were marked, sexed, weighted and crown rump length was measured
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:
Raw data were processed using statistical software Sigma Plot 11.0: the mean and standard deviation were calculated using the excel and all data were summarized in tabular form.

Homogeneity checked for all continuous data (body weight, feed consumption, weight of uterus, relative weight of uterus, weight of ovaries, number of live fetuses, pre-implantation loss, number of implantation site, number of resorption, post-implantation loss, CL count, mean weight of fetus, mean weight of placenta, mean foetal CRL measurement) using Shapiro Wilk Test, followed by ANOVA and Kruskal Wallis one Way ANOVA
Data showing singificance in their variances were subjected to Holm-Sidak and Dunn's methods.
Indices:
Pre-implantation loss, post-implantation loss, mean numbers of resorption, embryonic deaths and total foetuses
Historical control data:
No

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed in any of the animals throughout the study period (see table 1).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were oberved among any of the groups of animals throughout the study period (see Table 2)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the body weight of dams of treated groups when compared to control group during study period.
There was no statistically significant difference in the percent body weight change of dams of treated groups when compared to control group during study period.
(see Tables 3 and 4).
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Even if not a feeding study, food consumption was examined: there was no statistically significant diffrence in feed consumption of dams of trested groups when compared to control group during the study period.
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal gross examination of all dams in control and all treated groupds did not reveal any abnormality of pathological significance (see Table 5).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
weight of uterus, weight of ovaries

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in pre- and post-implantation loss in any of the treated groups as compared to control group (see Table 7).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
no total litter loss by resorption was observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in number resorptions in any of the treated groups as compared to control group (see Table 7).
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in number of lives fetuses in any of the treated groups as compared to control group (see Table 7).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Intrauterine growth was unaffected by test item administration in G2, G3 and G4 compared to control group. All females were determined to be gravid, with the exception of 6 females in G1, 2 females in G2 and 4 females in G4.
(see Table 6).
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate of female in the group of control, and treated animals in G2, G3 and G4 was 76%, 92%, 100% and 84% respectively.
Other effects:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in absolute and relative weights of uterus, weight of ovaries, corpus luteum, number of implemantation site in any of the treated groups as compared to control group (see Table 7).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no test item related changes at the highest tested dose

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in mean weight of fetus of both sexes of any of the treated group fetuses as compared to control group (see Table 8).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in number of lives fetuses in any of the treated groups as compared to control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Male/female sex ratio of fetuses in the group of control, and treated animals at in G2, G3 and G4 was 101/100, 135/139, 141/157 and 107/125 respectively (see Table 12).
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The external gross examination of fetuses showed small fetuses:
- G1 : 2 fetuses
- G2 : 5 fetuses
- G3: 1 fetus
- G4: 2 fetuses

(see Table 9)

These observations were not dose dependent and inconsistent hence, not considered as treatment related, but considered as spontaneous in origin.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related fetal skeletal malformations noted at any of the dose levels tested.
(see Table 11)
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examination did not show any malformation in fetuses of treatment groups and control group (see Table 10).
Other effects:
no effects observed
Description (incidence and severity):
Head razor examination did not show any abnormality in treated group and control group (see Table 10)

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no test item induced adverse effects on structural development or growth in fetuses under the experimental conditions

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of General Clinical Signs Observation

Sex: Female

Group

Dose (mg/kg b. wt.)

No. of Animals

Animal Number

Gestation Day

Clinical Signs

G1

0

25

01-25

0-20

Normal

G2

5

25

26-50

0-20

Normal

G3

10

25

51-75

0-20

Normal

G4

20

25

76-100

0-20

Normal

 Keys:No. = Number, mg/kg b. wt. = milligram/ kilogram body weight

Table 2: Summary of Mortality and Morbidity

Sex: Female

Group

Dose

(mg/kg b. wt.)

No. of Animals

Gestation Day

Observation

Morning 

Evening

G1

0

25

0-20

No mortality/ morbidity

No mortality/ morbidity

G2

5

25

0-20

No mortality/ morbidity

No mortality/ morbidity

G3

10

25

0-20

No mortality/ morbidity

No mortality/ morbidity

G4

20

25

0-20

No mortality/ morbidity

No mortality/ morbidity

 Keys:No. = Number, mg/kg b. wt. = milligram/ kilogram body weight

Table 3: Summary of Body Weight (gram)

Sex: Female

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0

235.53

10.48

233.87

9.69

233.76

11.41

233.81

12.03

3

243.21

11.83

241.22

9.15

240.60

12.14

240.00

12.05

5

245.89

12.04

245.87

9.45

245.96

12.27

244.76

11.43

8

250.89

12.56

250.65

10.42

251.36

12.90

248.38

11.25

11

261.11

12.58

261.61

11.21

262.44

12.83

257.52

11.68

14

273.68

13.60

273.57

12.04

274.24

14.41

268.52

12.27

17

296.26

17.43

297.22

14.84

298.04

15.46

290.52

14.00

20

329.74

23.17

330.83

18.44

331.44

18.08

321.05

16.37

 Keys:N= Number of Animals, SD= Standard Deviation, mg/kg b. wt. = milligram/ kilogram body weight

Table 4: Summary of Body Weight Change (%)

Sex: Female

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0-3

3.27

2.19

3.16

1.09

2.93

1.22

2.66

1.49

0-5

4.41

2.33

5.16

1.65

5.22

1.33

4.72

1.81

0-8

6.52

2.38

7.19

1.99

7.53

1.63

6.28

2.07

0-11

10.88

2.91

11.89

3.15

12.29

2.04

10.20

2.50

0-14

16.21

3.11

17.00

2.98

17.32

2.59

14.92

3.26

0-17

25.79

5.12

27.07

3.09

27.53

3.55

24.34

3.98

0-20

40.02

8.12

41.41

4.11

41.85

5.53

37.42

5.29

Table 5: Summary of Individual Gross observation

Sex: Female

Group

Dose (mg/kg b. wt.)

No. of Animals

Animal Number

Macroscopic Observation

External

Internal

G1

0

25

01-25

NAD

NAD

G2

5

25

26-50

NAD

NAD

G3

10

25

51-75

NAD

NAD

G4

20

25

76-100

NAD

NAD

Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight

Table 6: Summary of Number of gravide uterus and pregnancy rate

Group (N)

G1 (25)

G2 (25)

G3 (25)

G4 (25)

Dose (mg/kg b. wt.)

0

5

10

20

No. of Gravid Uterus

19

23

25

21

Pregnancy Rate (%)

76

92

100

84

Keys:N= Number of animals,mg/kg b. wt. = milligram/ kilogram body weight, % = Percent

Table 7: Summary of Maternal Evaluation Data

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Parameters

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Weight of Dam (g)

329.737

23.1729

330.826

18.4407

331.44

18.081

321.048

16.3661

Weight of Uterus (g)

55.77

13.24

62.66

8.79

64.12

9.39

58.19

9.83

Relative Uterus Weight (%)

16.76

3.28

18.90

2.11

19.09

2.60

18.1017

2.8028

Weight of Ovaries (g)

0.16

0.04

0.16

0.02

0.15

0.02

0.14

0.03

CL Count

12.16

1.74

12.91

1.41

12.80

1.47

13.14

1.59

Number of Live Fetus

10.47

2.63

11.91

1.56

11.88

1.67

11.05

2.09

Number of Dead Fetus

0.05

0.23

0.00

0.00

0.04

0.20

0.00

0.00

Number of Implantation Site

11.79

2.02

12.61

1.75

12.72

1.40

12.00

2.24

Number of Resorption

1.21

1.32

0.65

0.78

0.76

0.97

0.95

0.97

Pre-Implantation Loss (%)

3.17

7.43

2.53

6.88

0.19

9.73

8.32

14.69

Post-Implantation Loss (%)

12.05

13.15

5.21

6.17

6.69

7.45

7.53

7.97

Keys:N = Number of Dams, n = Number of Dams including Dams with Complete Resorption, g = Gram, CL = Corpus Luteum, SD = Standard Deviation,% = Percent,mg/ kg b. wt. = milligram/ kilogram body weight

Table 8: Summary of Fetal weight (g), Placenta weight (g) and CRL Measurment (cm)

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Sex

Parameters

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Males

Fetus Weight (g)

3.4058

0.3107

3.4789

0.2757

3.5694

0.2939

3.5248

0.2209

Placenta Weight (g)

0.4700

0.0536

0.4494

0.0456

0.4412

0.0385

0.4359

0.0356

CRL (cm)

3.4288

0.1919

3.4309

0.1224

3.4588

0.1384

3.4644

0.1780

Females

Fetus Weight (g)

3.2420

0.2542

3.3270

0.2318

3.4079

0.2326

3.3480

0.2943

Placenta Weight (g)

0.4688

0.0948

0.4405

0.0464

0.4331

0.0460

0.4271

0.0493

CRL (cm)

3.3418

0.1759

3.3712

0.0975

3.4447

0.1538

3.3383

0.1725

Male and Female

Fetus Weight (g)

3.3323

0.2504

3.4010

0.2269

3.4747

0.2487

3.4331

0.2525

Placenta Weight (g)

0.4734

0.0802

0.4437

0.0439

0.4371

0.0390

0.4329

0.0409

CRL (cm)

3.3829

0.1762

3.4049

0.1032

3.4518

0.1443

3.3951

0.1690

Keys:N= Number of animals, SD= Standard Deviation, g = Gram, cm = Centimeter, CRL = Crown to Rump Length,mg/kg b. wt. = milligram/ kilogram body weight.

 Note:Fused placenta was observed in fetus number 09 and 10 of animal number 08. Hence placenta weight was not considered in mean weight of Placenta in male and female separately but considered in combined male and female.

Table 9: Summary of External, Visceral and Head Razor Evaluation Data

Group

Fetus

Litters

G1

G2

G3

G4

G1

G2

G3

G4

Dose (mg/kg b. wt.)

0

5

10

20

0

5

10

20

Number Examined Externally

201

274

298

232

19

23

25

21

Dead Fetus

1

0

1

0

1

0

1

0

Small Fetus

2

5

1

2

1

3

1

2

NAD

198

269

296

230

19

23

25

21

Number Examined Viscerally

104

142

156

123

19

23

25

21

NAD

104

142

156

123

19

23

25

21

Number Examined by

Head Razor

104

142

156

123

19

23

25

21

NAD

104

142

156

123

19

23

25

21

Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight

Table 10: Summary of Percent (%) External, Visceral and Head Razor Evaluation Data

Group

% Fetus

% Litters

G1

G2

G3

G4

G1

G2

G3

G4

Dose (mg/kg b. wt.)

0

5

10

20

0

5

10

20

Number Examined Externally

201

274

298

232

19

23

25

21

% Dead Fetus

0.50

0.00

0.34

0.00

5.26

0.00

4.00

0.00

% Small Fetus

1.00

1.82

0.34

0.86

5.26

13.04

4.00

9.52

% NAD

100

100

100

100

100

100

100

100

Number Examined Viscerally

105

142

156

123

19

23

25

21

% NAD

100

100

100

100

100

100

100

100

Number Examined by

Head Razor

105

142

156

123

19

23

25

21

% NAD

100

100

100

100

100

100

100

100

Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight,% = Percent

Table 11: Summary of Number and Percent (%) Skeletal Evaluation Data

Skeletal Malformations / Variations

Group: No. of Fetus (% foetus)

Group: No. of Litters (% litters)

G1

(96)

G2

(132)

G3

(137)

G4

(109)

G1

(19)

G2

(23)

G3

(25)

G4

(21)

Name of Bone

Malformation/ Variation

NAD

45

(46.88%)

61

(46.21%)

67

(48.91%)

73

(66.97%)

1

(5.26%)

2

(8.70%)

3

(12.00%)

7

(33.33%)

Hyoid

Unossified / Incomplete Ossification

5

(5.21%)

3

(2.27%)

3

(2.19%)

4

(3.67%)

4

(21.05%)

1

(4.35%)

2

(8.00%)

4

(19.05%)

Frontal/ Parietal/ Interparietal/ Occipital

Unossified/Incomplete Ossification

8

(8.33%)

15

(11.36%)

13

(9.49%)

3

(2.75%)

4

(21.05%)

5

(21.74%)

7

(28.00%)

3

(14.29%)

Zygomatic

Incomplete Ossification

1

(1.04%)

5

(3.79%)

7

(5.11%)

6

(5.50%)

1

(5.26%)

1

(4.35%)

4

(16.00%)

3

(14.29%)

Fused

0

(0.00%)

2

(1.52%)

2

(1.46%)

0

(0.00%)

0

(0.00%)

2

(8.70%)

1

(4.00%)

0

(0.00%)

Rib

Supernumerary

19

(19.79%)

20

(15.15%)

28

(20.44%)

17

(15.60%)

9

(47.37%)

10

(43.48%)

12

(48.00%)

6

(28.57%)

Wavy

0

(0.00%)

2

(1.52%)

4

(2.92%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Incomplete Ossification

0

(0.00%)

1

(0.76%)

2

(1.46%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Sternebra

Unossified/Incomplete Ossification

32

(33.33%)

41

(31.06%)

42

(30.66%)

21

(19.27%)

15

(78.95%)

18

(78.26%)

18

(72.00%)

10

(47.62

Sternoschisis

4

(4.71%)

2

(1.52%)

3

(2.19%)

3

(2.75%)

3

(15.79%)

2

(8.70%)

3

(12.00%)

2

(9.52%)

Bipartite Ossification

0

(0.00%)

2

(1.52%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

2

(8.70%)

1

(4.00%)

0

(0.00%)

Misaligned

0

(0.00%)

0

(0.00%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.00%)

0

(0.00%)

Thoracic centrum

Bipartite Ossification

0

(0.00%)

3

(2.27%)

2

(3.65%)

0

(0.00%)

0

(0.00%)

3

(13.04%)

2

(8.00%)

0

(0.00%)

Dumbell Ossification

0

(0.00%)

3

(2.27%)

5

(3.65%)

0

(0.00%)

0

(0.00%)

3

(13.04%)

3

(12.00%)

0

(0.00%)

Lumbar Vertebra

Unossified / Incomplete Ossification

0

(0.00%)

1

(0.76%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

0

(0.00%)

0

(0.00%)

Thoracic Vertebra

Incomplete Ossification

0

(0.00%)

1

(0.76%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

0

(0.00%)

0

(0.00%)

Ischium

Unossified / Incomplete Ossification

0

(0.00%)

2

(1.52%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Pubis

Unossified/Incomplete Ossification

0

(0.00%)

0

(0.00%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.00%)

0

(0.00%)

Squamosal

Incomplete Ossification

0

(0.00%)

3

(2.27%)

4

(2.92%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Metacarpals and Metatarsals

Incomplete Ossification

0

(0.00%)

(0.00%)

1

(0.76%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

 Keys:NAD = No Abnormality Detected,No. = Number, % = Percent.

Table 12: Summary of Fetal Sex Ratio

Group (N)

G1 (201)

G2 (274)

G3 (298)

G4 (232)

Dose

(mg/kg b. wt.)

0

5

10

20

Sex Ratio

(Male/Female)

101/100

135/139

141/157

107/125

Key:N= Number of Fetus,mg/kg b. wt. = milligram/ kilogram body weight

Applicant's summary and conclusion

Conclusions:
Based on the findings of prenatal developmental oral toxicity study of the test item in Wistar rat at dose level 0, 5, 10, 20 mg/kgbw, no test item related changes were observed in any of the doses tested hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/kg bw in Wistar rat. Since, there was no test item induced adverse effects on structural development or growth in the fetuses, the developmental No Observed Adverse Effect Level (NOAEL) of the test item in fetuses is considered to be 20 mg/kg bw under the experimental conditions.
Executive summary:

The objective of this study was to provide evaluations of prenatal developmental toxicity of the test item in Wistar rats.

The results of the study provides general information concerning the effects of prenatal exposure on the pregnant animal and fetus, also include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus and determination of the maternal and developmental No Observed Adverse Effect Level (NOAEL).

The animals were randomly allocated to the four groups (25 Females/group). The doses selected for groups were; 0, 5, 10 and 20 mg/kg body weight.

Control group animals received vehicle (Corn oil) aloneand treatment groups were administered with test item daily by oral route, throughout the study period (Gestation day5-19).

Test item formulation was found to be homogeneous. The mean active ingredient content of AY36 at 1.25, 2.5 and 5 mg/ml concentrationwas 1.197,2.377, 4.701 mg/ml and 1.186, 2.356, 4.730 mg/mlat first week (initialsample)and last week (finalsample)of treatment, respectively.The mean active ingredient content of BV3 at 1.25, 2.5 and 5 mg/ml concentrationwas1.205, 2.391, 4.759 mg/ml and 1.194, 2.393, 4.747mg/mlat first week (initialsample)and last week (finalsample)of treatment, respectively.

No mortality and morbidity were observed among any of the groups of animals throughout the study period.

No clinical signs were observed in any of the animals throughout the study period.

There was no statistically significant difference in the body weight of dams of treated groups when compared to control group G1 during study period.

There was no statistically significant difference in the percent body weight change of dams of treated groups when compared to control group G1 during study period.

There was no statistically significant difference in feed consumption of dams of treated groups when compared to control group during study period.

External and internal gross examination of all dams in control and all treated groups did not reveal any abnormality of pathological significance.All females were determined to be gravid, with the exception of 6, 2, 4 females in G1 (0 mg/kg body weight), G2 (5 mg/kg body weight) and G4 (20 mg/kg body weight), respectively.

There was no statistically significant difference in absolute and relative weights of uterus, weight of ovaries, number of live fetuses, pre-implantation loss, number of implantation site, number of resorptions, post-implantation loss, CL count in any of the treated groups as compared to control group. Intrauterine growth and survival of fetuses was unaffected by test item administration at 5 (G2), 10 (G3) and 20 (G4) mg/kg body weight. Pregnancy rate of female in the group of G1, G2, G3 and G4 was 76%, 92%, 100% and 84%, respectively.

There was no statistically significant difference in mean weight of fetus, mean weight of placenta and mean foetal CRL measurement in male, female and both sexes of any of the treated group fetuses as compared to control group.

The external gross examination of fetuses showed small fetuses 2 (G1), 5 (G2), 1 (G3), 2 (G4) and dead fetuses 1(G1), 1(G3). These Observations were not dose dependent and inconsistent hence, not considered as treatment related, but considered as spontaneous in origin. Male/ Female sex ratio of fetuses in the group G1, G2, G3 and G4 was 101/100, 135/139, 141/157 and 107/125, respectively.                      

Visceral examination did not show any malformation in the fetuses of treatment groups and control group.

Head razor examination did not show any abnormality in control and treatment groups.

There were no test item related fetal skeletal malformation noted at any of the dose levels tested. Skeletal malformations like Hyoid: Unossified/ Incomplete Ossification were noted for 5 (4), 3 (1), 3 (2), 4 (4) fetus (litter) in G1, G2, G3 and G4, respectively. Frontal/ Parietal/ Interparietal/Occipital: Unossified/Incomplete Ossification were noted for 8 (4), 15 (5), 13 (7), 3 (3) fetus (litter) in G1, G2, G3 and G4, respectively. Zygomatic: incomplete ossification were noted for 1 (1), 5 (1), 7 (4), 6 (3) fetus (litter) in G1, G2, G3 and G4, respectively and Zygomatic: fused were noted for 2 (2) and 2 (1) fetus (litter) in G2 and G3, respectively. Supernumerary rib were noted for 19 (9), 20 (10), 28 (12), 17 (6) fetus (litter) in G1, G2, G3 and G4, respectively. Wavy ribs were noted for 2 (1), 4 (1), fetus (litter) in G2 and G3 respectively. Incomplete ossification of ribs were noted for 1 (1), 2 (1), fetus (litter) in G2 and G3 respectively.

Sternebra: unossified/ Incomplete ossification were noted for 32 (15), 41 (18), 42 (18), 21 (10) fetus (litter) in G1, G2, G3 and G4, respectively. Sternebra: sternoschisis were noted for 4 (3), 2 (2), 3 (3), 3 (2) fetus (litter) in G1, G2, G3 and G4, respectively. Sternebra: bipartite ossification were noted for 2 (2), 1 (1) fetus (litter) in G2 and G3, respectively. Sternebra: misaligned was noted for 1 (1) fetus (litter) in G3. Thoracic centrum: Bipartite ossification were notedfor 3 (3), 2 (2), fetus (litter) in G2 and G3, respectively.

Thoracic centrum: dumbell ossification were noted for 3 (3), 5 (3) fetus (litter) in G2 and G3, respectively. Lumbar vertebra: Unossified/ Incomplete ossification was noted for 1 (1) fetus (litter) in G2. Thoracic vertebra: Incomplete ossification was noted for 1 (1) fetus (litter) in G2. Ischium: Unossified/ Incomplete ossification were noted for 2 (1), 1 (1) fetus (litter) in G2 and G3, respectively. Pubis: Unossified/ Incomplete ossification was noted for 1 (1) fetus (litter) in G3. Squamosal: Incomplete ossification were noted for 3 (1), 4 (1) fetus (litter) in G2 and G3, respectively. Metacarpals and Metatarsals: Incomplete ossification were noted for 1 (1), 1 (1) fetus (litter) in G2 and G3, respectively.

There were no test item related fetal skeletal developmental variations noted at any of the dose levels tested. These findings occured infrequently and were noted similarly in the concurrent control group, and /or were noted in a manner thatwas not considered to be treatment related. As these observations were not dose dependent therefore, these skeletal developmental variations were not considered to be test item related.

 

Conclusion

Based on the findings ofprenatal developmentaloral toxicity study of the item in Wistar rat at dose level 0, 5, 10 and 20 mg/kg body weight, no test item related changes were observed in any of the doses tested hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/ kg body weight in Wistar Rat. Since, there was no test item induced adverse effects onstructural development or growth in the foetuses, the developmental No Observed Adverse Effect Level (NOAEL) of test item in fetuses is considered to be 20 mg/ kg body weight under the experimental conditions.