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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings(Deutschland) GmbH, BU Additives, Ludwig-Hermann-Strasse 100, 86368 Gersthofen, Germany

- Batch No.of test material: DEF2076375
- Expiration date of the batch: 17-May-2018
- Purity as per Certificate of Analysis: 98.65%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to + 25 °C)
- Stability under test conditions: The stability of the test item in the vehicle: 0.1%(w/v) Sodium Carboxymethyl cellulose (medium viscosity) in Milli-Q water was carried out under the Study No.: G12134. The test item was found to be stable and resuspendable in the vehicle for 4 days at room temperature, at the fortification levels of 1 mg/mL and 125 mg/mL.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel Mandal, Medak District, Telangana

- Age at study initiation: 13 to 14 weeks
- Weight at study initiation:
Mean body weight(g) Body weight range(g)
G1 : 243.089 ± 18.37 210.12 to 268.16
G2 : 239.855 ± 19.42 204.58 to 268.84
G3 : 239.042 ± 20.79 206.35 to 267.97
G4 : 239.115 ± 21.14 208.16 to 268.57

- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube.
Following was the housing pattern during different periods of the experiment:
i. Pre mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.
NOTE: The male rats and the remaining extra females were euthanized after mating procedure

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories, P.O.Box 44220, Madison Wi 53744-4220, was provided ad libitum to the rats.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: 08 December 2016 to 12 December 2016

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 64 – 66 %
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 19 December 2016 To: 09 January 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity at the initiation of treatment and at termination of treatment period.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored in the experimental room depending upon the obtained stability results. For each set, two replicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, two replicate samples from middle layer were drawn.
Samples were analyzed for Nylostab S-EED P content using analytical method which was validated under Advinus Study No. G12134. The analysis was carried out within the established stability period.

Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the replicate values) of assay of top, middle and bottom layers (RSD) was equal to or less than 10.0 %.

The dose formulation suspensions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits


Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 9 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
15 days
Frequency of treatment:
Gestation day 5 to Gestation day 19
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3 (Mid dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
Day '0" Pregnant rats : 24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As per the Sponsor’s suggestion, based on the results of a 28 day repeated dose study, the following doses were selected for the main embryo-fetal developmental toxicity study with Nylostab S-EED P in Sprague Dawley rats by oral route:
• Vehicle control (G1): 0 mg/kg/day
• Low dose(G2) : 50 mg/kg/day
• Mid dose(G3) : 250 mg/kg/day
• High dose(G4) : 1000 mg/kg/day

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20


OTHER:
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: Yes: [half per litter]
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption 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 were analyzed using Analysis of Convariance (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.

The incidence of with and without resorptions in dams will be tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated as * throughout the report.
Indices:
MATERNAL DATA:
Maternal Parameters and Formula used

> Mean number of corpora lutea
Total no. of CL
= ----------------------------------------
Total no. of pregnant animals

> Mean number of implantations/group
Total no. of implantations
= ----------------------------------------
Total no. of pregnant animals

> Embryonic resorption index (%)
No. of early resorptions
= --------------------------------- x 100
No. of implantations

> Fetal resorption index (%)
No. of late resorptions
= -------------------------------- x 100
No. of implantations

> Pre-implantation loss per group (%)
No. of CL - No. of implantations
= ----------------------------------------------- x 100
No. of CL

> Post-implantation loss per group (%)
No. of (early + late) resorptions
= --------------------------------------------- x 100
Total no. of implantations

> Implantation index (%)
No. of implantations sites
= ------------------------------------- x 100
No. of corpora lutea

> Corrected body weight (Carcass weight)
= Terminal body weight (body weight on GD20) - unopened uterine weight.

> Corrected body weight gain
= Corrected body weight – body weight on GD5

LITTER DATA:
Litter Parameters and Formula used

> Mean litter size per group
Total No. of fetuses
= ---------------------------------
Total No. of pregnant animals

> Percentage of abnormal fetuses
No. of abnormal fetuses
= ---------------------------------- x 100
Total No. of fetuses

> Percentage of live fetuses (Live fetus index)
No. of live fetuses
= ----------------------------- x 100
Total No. of fetuses

> Percentage of dead fetuses (Dead fetus index)
No. of dead fetuses
= ----------------------------- x 100
Total No. of fetuses

> Sex ratio (F : M)
Number of females
= ---------------------------
Number of males
Historical control data:
The results of the study were discussed taking into consideration the historical data of this lab.

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 throughout the experimental period 50, 250 and 1000 mg/kg/day dose groups.
Mortality:
no mortality observed
Description (incidence):
There was no morbidity or mortality throughout the experimental period in the 50, 250 and 1000 mg/kg/day dose groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The maternal group mean body weights were unaffected by the administration of test item Nylostab S-EED P at the doses of 50,
250 and 1000 mg/kg/day and were statistically comparable to vehicle control group.

At 50 and 250 mg/kg/day, the maternal body weight gain was comparable to vehicle control group during different periods of gestation.

At 1000 mg/kg/day, there was a significant reduction in maternal body weight gain during GD 14 to 17 (-21 %) and for entire gestation period (-14 %) as compared to vehicle control group.

The corrected body weight and corrected body weight gain was not statistically different from vehicle control group at all the tested doses.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The maternal food consumption was comparable to vehicle control group at all the doses tested during different periods of gestation, except for a significant reduction in food consumption during GD 5-8 (-15 %) at
1000 mg/kg/day.

The reduction in maternal body weights along with concomitant reduction in food consumption at 1000 mg/kg/day was considered as a treatment-related effect indicating maternal toxicity.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological findings in any of the tested doses

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The maternal data parameters comprising of gravid uterine weights, mean numbers of corpora lutea, implantations, early and late resorptions, pre and post-implantation loss rates and dams with any resorptions were unaffected by treatment with Nylostab S-EED P at all the doses tested.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other:
Remarks:
due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed during external observations of fetuses of dams treated up to 1000 mg/kg/day.
External malformations:
no effects observed
Description (incidence and severity):
Fetal, external observations were comparable to vehicle control group at all the doses tested.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Fetal, skeletal observations were comparable to vehicle control group at all the doses tested. Skeletal examinations revealed no signs of teratogenicity in any of the tested doses.
Visceral malformations:
no effects observed
Description (incidence and severity):
Fetal, visceral observations were comparable to vehicle control group at all the doses tested. Visceral examinations revealed no signs of teratogenicity in any of the tested doses.
Details on embryotoxic / teratogenic effects:
Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fetal developmental toxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

No Observed Adverse Effect Level (NOAEL) for:

- Maternal toxicity is 250 mg/kg/day due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day, 

 - Fetal developmental toxicity is 1000 mg/kg/day as the litter data parameters were unaffected by treatment up to the high dose of 1000 mg/kg/day and

 - Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

Table 1.       Details of the Experimental Design, Treatment Schedule, Maternal and Litter Data

Parameters

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

Total No.of rats found sperm positive/group

 

24

24

24

24

Duration of treatment(days)

 

GestationDays5to19(15days)

 

No.of rats sacrificed at caesarean section

24

24

24

24

No.of non-pregnantrats at caesarean section

1

3

0

0

No.of pregnant rats at caesarean section

23

21

24

24

No.of litters examined

23

21

24

24

Total no.of fetuses

289

266

288

270

Numberoffetusesevaluated

 

a. External examination

289

266

288

270

b. Visceral examination

144

133

144

135

c. Skeletal examination

145

133

144

135

 

Summary of Maternal Body Weight and Weight Gain

Table 2.              Summary of Maternal Group Mean Body Weight (g)

Group Mean body weight (g) on GD (gestation day)

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of rats

23

21

24

24

0

Mean

242.91

241.44

239.04

239.11

 

SD

18.76

19.15

20.79

21.14

3

Mean

258.64

255.01

252.80

252.77

 

SD

18.66

20.25

21.92

21.69

5

Mean

266.80

263.22

261.42

259.39

 

SD

21.01

21.59

24.63

22.65

8

Mean

276.27

273.15

271.55

265.58

 

SD

25.43

23.85

27.68

23.66

11

Mean

290.78

289.01

283.72

278.11

 

SD

26.07

24.69

29.96

23.60

14

Mean

305.86

303.03

299.04

291.91

 

SD

27.65

27.36

33.63

25.03

17

Mean

336.73

334.74

329.20

316.40

 

SD

28.87

28.31

41.28

28.44

20

Mean

371.30

370.13

363.57

349.36

 

SD

34.26

33.11

50.17

34.91

 

Table 3.              Summary of Maternal Body Weight Gain (g)

Period of treatment (days of gestation)

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of rats

23

21

24

24

0-3

Mean

15.73

13.57

13.76

13.66

 

SD

4.99

4.30

4.15

4.53

3-5

Mean

8.15

8.21

8.62

6.62

 

SD

5.06

3.97

5.42

2.92

5-8

Mean

9.47

9.93

10.12

6.19

 

SD

6.24

5.14

5.46

4.44

8-11

Mean

14.51

15.86

12.17

12.54

 

SD

4.37

3.54

5.21

4.39

11-14

Mean

15.08

14.02

15.32

13.79

 

SD

4.41

5.08

5.87

4.70

14-17

Mean

30.87

31.71

30.17

24.50*

 

SD

6.11

3.49

10.12

6.79

17-20

Mean

34.57

35.39

34.37

32.95

 

SD

8.98

8.39

11.20

8.07

Values at each interval group Mean +/- SD; * significant different from G1

  

Table 3. contd.                Summary of Maternal Body Weight Gain (g)

Period of treatment (days of gestation

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of rats

23

21

24

24

Pre-treatment period (days 0-5)

Mean

23.88

21.78

22.38

20.28

 

SD

8.00

6.76

6.76

6.0

Treatment period (days 5-20)

Mean

104.50

106.91

102.15

89.97

 

SD

17.39

16.24

30.22

17.52

Entire gestation (days 0-20)

Mean

128.38

128.69

124.53

110.24*

 

SD

21.53

18.33

33.71

18.27

Values at each interval group Mean +/- SD; * significant different from G1

 

Table 4.             Summary of Corrected Body Weight and Body Weight Gain (g)

Period of treatment (days of gestation

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of rats

23

21

24

24

Body weight

on GD 5

Mean

266.80

263.22

261.42

259.39

 

SD

21.01

21.59

24.63

22.65

Terminal Body weight on GD 20

Mean

371.30

370.13

363.57

349.36

 

SD

34.26

33.11

50.17

34.91

Uterine weight

Mean

76.72

75.43

72.77

69.71

 

SD

17.59

14.92

26.33

18.13

Carcass weight (corrected body weight on GD 20)

Mean

294.58

294.70

290.80

279.64

 

SD

32.86

25.79

32.29

27.55

Corrected body weight gain

Mean

27.78

31.48

29.38

20.25

 

SD

14.60

15.16

12.71

11.20

GD: Gestation Day

Corrected body weight on gestation day 20 (Caracass weight) = Terminal Body weight on GD 20 – unopended uterine weight.

Corrected body weight gain = caracass weight – body weight on Gestation day 5

Table 5.             Summary of Maternal data

 

Parameters

GroupNo.

Dose(mg/kg/day)

G1

0

G2

50

G3

250

G4

1000

 

No.of Rats

23

21

24

24

 

Gravid uterine weight(g)

 

Mean

 

76.72

 

75.43

 

72.77

 

69.71

 

SD

17.59

14.92

26.33

18.13

 

NumberofCorpora lutea

 

Mean

 

15.65

 

15.76

 

15.96

 

14.88

 

SD

2.40

3.08

4.42

2.98

 

Numberof Implantations

 

Mean

 

13.48

 

13.19

 

12.75

 

11.83

 

SD

2.79

2.46

4.42

3.38

 

Early Resorptions

 

Mean

 

0.87

 

0.43

 

0.71

 

0.50

 

SD

1.32

0.60

1.12

0.83

 

Late Resorptions

 

Mean

 

0.04

 

0.10

 

0.04

 

0.08

 

SD

0.21

0.30

0.20

0.28

 

Pre-implantation Loss

 

Mean

 

2.17

 

2.57

 

3.21

 

3.04

 

SD

2.39

2.87

3.18

3.43

 

Post-implantation Loss

 

Mean

 

0.91

 

0.52

 

0.75

 

0.58

 

SD

1.35

0.60

1.29

0.83

 

DamswithanyResorption

 

Total

 

12

 

10

 

11

 

10

 

 

Table 5. contd. Summary of Maternal data (% per litter)

 

Parameters

Group No.

Dose(mg/kg/day)

G1

0

G2

50

G3

250

G4

1000

 

No.of Rats

23

21

24

24

 

Early Resorptions

 

Mean

 

7.18

 

3.54

 

8.11

 

3.98

 

SD

13.93

4.97

17.03

6.33

 

Late Resorptions

 

Mean

 

0.26

 

0.87

 

0.60

 

0.97

 

SD

1.23

2.77

2.92

3.39

 

Pre-implantation Loss

 

Mean

 

13.54

 

14.94

 

21.24

 

19.65

 

SD

15.78

15.08

23.41

22.69

 

Post-implantation Loss

 

Mean

 

7.43

 

4.41

 

8.71

 

4.95

 

SD

13.96

5.09

19.43

6.59

 

Implantation Index

 

Mean

 

86.46

 

85.06

 

78.76

 

80.35

 

SD

15.78

15.08

23.41

22.69

 

Table 6.             Summary of Litter data

 

 

Parameters

Group No.

Dose(mg/kg/day)

G1

0

G2

50

G3

250

G4

1000

 

No.of Rats

23

21

24

24

 

 

No.of litters

 

 

 

23

 

 

21

 

 

24

 

 

24

 

Total no.of fetuses

 

 

289

 

266

 

288

 

270

 

Mean litter size

 

 

12.6

 

12.7

 

12.0

 

11.3

 

Total live fetuses

 

 

 

 

 

a.  Number

 

289

266

288

270

 

b.  Weight(g)

 

Mean

 

4.02

 

4.02

 

4.03

 

4.10

 

SD

0.18

0.35

0.29

0.42

 

Live male fetuses

 

 

 

 

 

a.  Number

 

141

145

132

154

 

b.  Weight(g)

 

Mean

 

4.11

 

4.13

 

4.15

 

4.15

 

SD

0.19

0.40

0.30

0.41

 

Live female fetuses

 

 

 

 

 

a. Number

 

148

121

156

116

 

b.  Weight(g)

 

Mean

 

3.93

 

3.89

 

3.87

 

3.94

 

SD

0.20

0.35

0.24

0.42

 

Sex Ratio-Male:Female

 

 

1:1.05

 

1:0.83

 

1:1.18

 

1:0.75

 

Table 7.             Summary of Gross Pathological findings

Group No.

G1

G2

G3

G4

Parameters / Dose(mg/kg/day)

0

50

250

1000

1.     No. of rats subjected to caesarian section

24

24

24

24

2.     No.of rats pregnant at caesarian section

23

21

24

24

3.     No.of rats showing gross pathology

0

0

0

0

Table 8.              Summary of Fetal External Observations

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of litters examined

23

21

24

24

No. of fetuses examined

289

266

288

270

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

 

 

 

 

 

 

 

 

 

 

 

 

 

Normal Variant

None

 

 

 

 

 

 

 

 

 

 

 

 

 

Minor Anomalies

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Small fetus

0

0.00

0.00

1

0.38

1.30

0

0.00

0.00

0

0.00

0.00

 

 

 

 

 

 

 

 

 

 

 

 

 

Major Malformations

None

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 9.              Summary of Fetal Visceral Observations (incidence and percentage)

 

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of litters examined

23

21

24

24

No. of fetuses examined

144

133

144

135

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

 

 

 

 

 

 

 

 

 

 

 

 

 

Normal Variant

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Kidney renal pelvis dila. (Rt/Lt/B) (+)

0

0.00

0.00

2

1.50

3.63

0

0.00

0.00

3

2.22

8.81

 

 

 

 

 

 

 

 

 

 

 

 

 

Minor Anomalies

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Kidney renal pelvis dila. (Rt/Lt/B) (++)

3

2.08

6.55

0

0.00

0.00

3

2.08

7.14

0

0.00

0.00

 

 

 

 

 

 

 

 

 

 

 

 

 

Major Malformations

None

 

 

 

 

 

 

 

 

 

 

 

 

 

+: Slight

++: moderate

Table 10.           Summary of Fetal Skeletal Observations (incidence and percentage)

              

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of litters examined

23

21

24

24

No. of fetuses examined

145

133

144

135

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

 

 

 

 

 

 

 

 

 

 

 

 

 

Normal Variant

 

DSO:  Stern: # 5

3

2.07

2.11

1

0.75

0.68

1

0.69

0.69

2

1.48

1.22

           Stern: # 6

0

0.00

0.00

1

0.75

0.60

0

0.00

0.00

7

5.19

4.88

           Stern: # 5,6

1

0.69

0.62

2

1.50

1.90

0

0.00

0.00

1

0.74

0.60

           CV:centra: 1/7

0

0.00

0.00

0

0.00

0.00

1

0.69

0.60

0

0.00

0.00

           CV:centra: 2/7

2

1.38

1.35

2

1.50

1.98

0

0.00

0.00

1

0.74

1.04

           CV:centra: 3/7

4

2.76

2.69

1

0.75

1.19

2

1.39

1.19

0

0.00

0.00

           CV:centra: 4/7

6

4.14

4.18

1

0.75

0.68

4

2.78

2.62

4

2.96

3.72

          CV:centra: 5/7

31

21.38

20.05

19

14.29

14.82

22

15.28

14.93

18

13.33

13.52

           CV:centra: 6/7

64

44.14

46.05

47

35.34

34.99

64

44.44

43.32

52

38.52

36.73

           CV:centra: 7/7

38

26.21

25.68

63

47.37

46.34

51

35.42

33.17

60

44.44

40.82

 

 

 

 

 

 

 

 

 

 

 

 

 

DSO:  TV:centra:1/13

1

0.69

0.72

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

           CdV:centra:1/4

3

2.07

1.79

14

10.53

9.15

7

4.86

3.41

21

15.56

12.67

           CdV:centra:2/4

1

0.69

0.62

0

0.00

0.00

0

0.00

0.00

1

0.74

0.60

           CdV:centra:4/4

0

0.00

0.00

1

0.75

0.95

0

0.00

0.00

0

0.00

0.00

           CdV:arch:1/2

4

2.76

2.33

6

4.51

3.87

10

6.94

5.19

17

12.59

10.24

           CdV:arch:2/2

0

0.00

0.00

1

0.75

0.95

0

0.00

0.00

0

0.00

0.00

           F limb:Metacarp.:1/4

6

4.14

3.57

11

8.27

7.53

6

4.17

6.71

9

6.67

5.41

           F limb:Pr. Phal:1/2

0

0.00

0.00

3

2.26

2.07

10

6.94*

5.82

11

8.15*

6.56

           F limb:Pr. Phal:2/2

2

1.38

1.16

4

3.01

3.11

2

1.39

1.39

9

6.67

5.70

           F limb:Dt. Phal:1/4

1

0.69

0.62

1

0.75

0.68

0

0.00

0.00

2

1.48

1.12

 

 

 

 

 

 

 

 

 

 

 

 

 

*: Significantly different from G1

Table 10. contd.              Summary of Fetal Skeletal Observations (incidence and percentage)

 

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of litters examined

23

21

24

24

No. of fetuses examined

145

133

144

135

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

 

 

 

 

 

 

 

 

 

 

 

 

 

Normal Variant

 

DSO: F limb:Dt. Phal:4/4

0

0.00

0.00

1

0.75

0.95

0

0.00

0.00

0

0.00

0.00

          H limb:Dt. Phal:1/5

1

0.69

0.54

0

0.00

0.00

2

1.39

1.12

3

2.22

1.79

          H limb:Dt. Phal:5/5

1

0.69

0.62

2

1.50

1.63

 

0.00

0.00

4

2.96

2.31

INO/PO:Stern:# 2,5

3

2.07

1.71

11

8.27

7.24

13

9.03

7.86

10

7.41

6.17

                Stern:# 1

0

0.00

0.00

0

0.00

0.00

1

0.69

0.60

0

0.00

0.00

                Stern:# 2

2

1.38

2.90

7

5.26

4.90

4

2.78

2.48

8

5.93

5.74

                Stern:# 4

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1

0.74

0.52

                Stern:# 5

24

16.55

14.61

18

13.53

13.02

27

18.75

16.40

30

22.22

19.11

                Stern:# 6

14

9.66

8.35

23

17.29

15.70

15

10.42

8.22

13

9.63

8.13

                CV:centra:1/7

24

16.55

15.10

12

9.02

8.76

19

13.19

12.22

18

13.33

12.54

INO/PO:CV:centra:2/7

1

0.69

0.62

2

1.50

2.38

2

1.39

1.43

2

1.48

1.64

                CV:centra:3/7

1

0.69

0.72

1

0.75

0.79

0

0.00

0.00

0

0.00

0.00

                TV:centra:1/13

0

0.00

0.00

1

0.75

0.68

1

0.69

0.42

0

0.00

0.00

Minor Anomalies

 

 

 

 

 

 

 

 

 

 

 

 

HYPOPLASTIC:    Stern:# 2

0

0.00

0.00

1

0.75

0.79

1

0.69

0.60

0

0.00

0.00

                               Stern:# 5

8

5.52

4.92

4

3.01

2.68

3

2.08

1.71

4

2.96

2.54

DB:  TV:centra:1/13

34

23.45

28.30

33

24.81

23.43

36

25.00

25.92

29

21.48

19.70

         TV:centra:2/13

17

11.72

10.94

11

8.27

7.97

19

13.19

15.47

14

10.37

9.49

         TV:centra:3/13

6

4.14

3.65

3

2.26

2.31

5

3.47

2.64

4

2.96

2.86

         TV:centra:4/13

1

0.69

0.62

2

1.50

1.36

3

2.08

1.63

4

2.96

2.71

 

 

 

 

 

 

 

 

 

 

 

 

 

 Table 10. contd.              Summary of Fetal Skeletal Observations (incidence and percentage)

 

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

50

250

1000

No. of litters examined

23

21

24

24

No. of fetuses examined

145

133

144

135

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

 

 

 

 

 

 

 

 

 

 

 

 

 

Minor Anomalies

 

DB: TV:centra:5/13

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1

0.74

0.52

        LV:centra:1/6

7

4.83

4.48

8

6.02

5.83

3

2.08

1.53

6

4.44

4.24

        LV:centra:2/6

1

0.69

0.62

0

0.00

0.00

0

0.00

0.00

1

0.74

0.83

        LV:centra:4/6

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1

0.74

0.52

SPLIT:TV:centra:1/13

2

1.38

1.24

4

3.01

2.95

1

0.69

0.60

1

0.74

1.04

ASY DB:     TV:centra:1/13

0

0.00

0.00

1

0.75

0.79

0

0.00

0.00

4

2.96

2.82

                   TV:centra:2/13

0

0.00

0.00

1

0.75

0.68

0

0.00

0.00

0

0.00

0.00

ASY OSSI:   stern:#4,5

1

0.69

0.62

0

0.00

0.00

0

0.00

0.00

1

0.74

0.69

                     stern:# 3,4

0

0.00

0.00

0

0.00

0.00

1

0.69

0.60

1

0.74

0.52

                     stern:# 3-5

1

0.69

2.17

2

1.50

1.59

1

0.69

0.83

1

0.74

0.69

ASY SPLIT:TV:centra:1/13

1

0.69

0.62

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

EXTRA: LV:centra & arch #7

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1

0.74

0.69

RUDIMENTARY: RIB(Rt/Lt/B):# 14

46

31.72

31.60

43

32.33

32.30

21

14.58*

12.99

50

37.04

37.05

ACCESSORY: RIB(Rt/Lt/B):# 14

2

1.38

1.45

1

0.75

0.60

1

0.69

0.60

1

0.74

0.60

WAVY:RIB(Rt/Lt/B)(+/++):2/13

1

0.69

0.62

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

 

 

 

 

 

 

 

 

 

 

 

 

 

Major Malformations

None

 

 

 

 

 

 

 

 

 

 

 

 

 

*: Significantly different from G1

+: Slight

++: moderate

Applicant's summary and conclusion

Conclusions:
Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

- Maternal toxicity is 250 mg/kg/day due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day,

- Fetal developmental toxicity is 1000 mg/kg/day as the litter data parameters were unaffected by treatment up to the high dose of 1000 mg/kg/day and
- Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

in Sprague Dawley rats when Nylostab S-EED P was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.
Executive summary:

The objective of this study was to evaluate theembryo-fetal developmentaltoxicity of test itemNylostabS-EED Pwhen administered to pregnant Sprague Dawley rats by oral route during gestation days (GD) 5 to GD19.The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item.

 

In this study, each group (G1, G2, G3 and G4) consisted of 24 presumed pregnant Sprague Dawley rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal plug was observed or vaginal smear was found sperm positive.

 

The test item,NylostabS-EED Pwas suspended in vehicle [0.1% (w/v) Sodium Carboxymethyl cellulose (medumviscosity) in Milli-Q water] and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 50, 250 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. The rats in the vehicle control (G1) group received the vehicle alone. A constant dose volume of 10 mL/kg body weight was administered to all groups.

 

The dose formulation suspensions were analyzed for active ingredient concentrations at the initiation and termination of treatment. The results of analysis of formulations revealed that the analyzed concentrations were within the acceptable limits.

 

The mated females were observed twice daily for clinical signs, mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. About 200 g (food input) was provided on Day ‘0’. The food left over was recorded and replenished to about 200 g on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation. The intermittent body weight gain and food intake was calculated and presented for rats found pregnant at caesarean section.

 

Caesarean section was performed for all the rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the dams was removed (by laparotomy) and the contents were examined.The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and deadfetuses. The number of corpora lutea was counted on each ovary. All thefetuseswere sexed, weighed and examined for external malformations.Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

 

Results of the study are presented below:

·        There were no mortalities and clinical signs at all the doses tested.

·        There was significant reduction in maternal body weights and food consumption at 1000 mg/kg/day as compared to vehicle control group indicating maternal toxicity.

·        The other maternal and litter data parameters were comparable to vehicle control group up to the high dose of 1000 mg/kg/day.

·        Fetal, external, visceral and skeletal observations were comparable to vehicle control group at all the doses tested. Visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested doses.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

·           Maternal toxicity is 250 mg/kg/day due to treatment-related significant reduction in maternal body weight gain and food consumption at 1000 mg/kg/day, 

·           Fetaldevelopmental toxicity is 1000 mg/kg/day as the litter data parameters were unaffected by treatment up to the high dose of
1000 mg/kg/day and

·           Teratogenicity is 1000 mg/kg/day as there were no signs of teratogenicity in any of the tested dose levels up to the high dose of 1000 mg/kg/day

 

in Sprague Dawley rats whenNylostabS-EED Pwas administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.