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:
2016-06-09 to 2017-08-20
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)
Version / remarks:
adopted on January 22, 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
white crystalline material
chemically pure
Specific details on test material used for the study:
Chemical name (IUPAC): [Bis[3,3-bis-[4`-hydroxy-3`-tert-butylphenyl) butanoicacid] glycolester]

CAS No.: 32509-66-3

Batch No.: DEF2109009

Manufactured date: 23.06.2014

Expiry date: 22.06.2020

Purity as per Certificate of Analysis : 97.4 area %

Physical appearance: White powder

Storage conditions: Ambient (+15 to +25ºC)

Test animals

Species:
rat
Strain:
Wistar
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
G1 : 212.38 ± 13.74 192.21 to 239.45 g
G2 : 212.41 ± 13.80 193.21 to 242.46 g
G3 : 212.42 ± 13.71 193.86 to 239.28 g
G4 : 212.38 ± 14.18 190.26 to 241.21 g

- Housing: 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
- Diet: Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Envigo (previously referred to as Harlan Laboratories), P.O.Box 44220, Madison Wi 53744-4220;ad libitum
- Water: yes 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; ad libitum
- Acclimation period: 5 days; Females used in this study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C,
- Humidity (%): 65 – 68 %
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2016-06-09 To: 2016-07-05 - 2016-07-08 (sacrifice)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item forms good suspension in sunflower oil as indicated by the study sponsor and the same vehicle was used in the previous studies carried out by study sponsor and hence the same vehicle is selected for the present study.

The dose formulations were prepared at an interval of 3 to 4 days and were used within the established stability period. The prepared dose formulation when not in use were stored in the experimental room.
The homogeneity of the Hostanox O 3 P formulation during treatment/sampling was maintained by constant stirring using a magnetic stirrer.
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 in duplicate sets at the initiation of treatment and at termination of treatment period.The prepared formulations were sampled in duplicate sets. One set was used for analysis and another was kept as back up set which was stored in the experimental room. For each set, duplicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, duplicate samples from middle layer were drawn. The test item Hostanox O 3 P in the dose formulations was determined using High Performance Liquid Chromatograph (HPLC) with UV Detector. Dose formulation samples were analyzed as per the Analytical method validated under Advinus Study No.G11434. The results of analysis of formulations were within the acceptable limits. The test item was found to be homogeneous in all the dose formulation samples which met the acceptance limits of variation (85 to 115%) from the claimed concentrations and % RSD was less than 10 %.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: If there was presence of sperm in a vaginal smear or if of vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as Day ‘0’ of gestation.
Duration of treatment / exposure:
gestation day 5 to gestation day 19
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1, vehicle control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2, low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3, mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4, high dose
No. of animals per sex per dose:
24 pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were considered to be best choice based on the outcome of available repeated dose studies.
Based on the body weight, day ‘0’ pregnant rats were allotted a serial number in the ascending order. These rats were then assigned to the groups starting from control to treatment groups and then in the reverse order of dose groups to control.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.
- Cage side observations checked in table.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each rat was observed twice daily for morbidity and mortality i.e., once in the morning and once in the afternoon.

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 AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined:
The gravid uterus along with the cervix including ovaries was excised, weighed and immediately examined.

Ovaries and uterine content:
The following maternal data were recorded.
a. Pregnancy status
b. Gravid uterine weight
c. No. of corpora lutea
d. No. of implantation sites
e. No. of early resorptions
f. No. of late resorptions
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, gravid uterine weight, corrected body weight on gestation day 20 (carcass weight), corrected body weight gain, maternal food consumption, number of corpora lutea, number of implantations, total number of fetuses, male and female fetus number and weight including combined sex was analyzed using ANOVA model, after testing for homogeneity of intra group variance using Levene’s test. When 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.

Incidences of pre-implantation loss, post implantation loss, number of early and late resorptions were analyzed using Kruskal Wallis test.

Overall percentage of minor external, visceral and skeletal malformations, Sex ratio, number of dams with any resorptions and number of dams with complete resorptions were analyzed using 2 X 2 Contingency Table.
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:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day, there was one incidence of poorly formed faeces containing mostly water in one dam (Rs4625) on GD 20.

At 1000 mg/kg/day, reddish discharge from vagina was observed in most of the rats between GD 13 to 20 which can be correlated to resorptions noted at caesarean section.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights were unaffected by treatment at 100 and 300 mg/kg bw/day.
At 1000 mg/kg/day, the mean body weight was significantly lower on GD 11 (-5%), 14 (-11%), 17 (-18%) and 20 (-26%) as compared to vehicle control group.

At 100 mg/kg/day, the maternal body weight gains were comparable to vehicle control group.
At 300 mg/kg/day, the maternal body weight gains were significantly lower during GD 14-17 (- 31%), during treatment period GD 5-20 (-22%) and entire gestation period GD 0-20 (-17%) as compared to vehicle control group. The body weight change corrected for gravid uterine weight was lower at 300 mg/kg/day (81%) compared to control.

At 1000 mg/kg/day, there was a significant reduction in maternal body weight gain during GD 5-8 (-66%), GD 8-11 (-71%), GD 11-14 (-157%), GD 14-17 (-87%) and GD 17-20 (-118%). The maternal body weight gains were also significantly lower during treatment period GD 5-20 (-102%) and entire gestation period GD 0-20 (-87%) including the corrected body weight gains (-172%) as compared to vehicle control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 100 and 300 mg/kg/day, the maternal food consumption was comparable to vehicle control group.

At 1000 mg/kg/day, there was a significant reduction in maternal food consumption during GD 5-8 (-25%), GD 8-11 (-41%), GD 11-14 (-48%), GD 14-17 (-31%) and GD 17-20 (-34%). The maternal food consumption was also significantly lower during treatment period GD 5-20 (-36%) and entire gestation period GD 0-20 (-26%) as compared to vehicle control group.
The significant reduction in food consumption at 1000 mg/kg/day was considered to be treatment-related and indicates maternal toxicity.
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:
effects observed, treatment-related
Description (incidence and severity):
There were no gross pathological findings in control and 100 mg/kg/day dose group.

At 300 mg/kg/day, one dam (Rs4630) showed bilaterally enlarged adrenals.

At 1000 mg/kg/day, there were incidences of thymus small (10/24)/not prominent (9/24) and bilaterally enlarged adrenals (12/24).

The gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg/day and small / not prominent thymus at 1000 mg/kg/day are indicative of maternal stress at these doses.

There were no abnormalities during gross evaluation of placenta.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
At 300 and 1000 mg/kg/day, there were treatment-related significant reduction in maternal body weight gains during the treatment period GD 5-20 (-22% and -102%, respectively) and entire gestation period GD 0-20 (-17% and -87%, respectively). The body weight change corrected for gravid uterine weight was lower at 300 mg/kg/day (81%) and was significantly lower at 1000 mg/kg/day (-172%) compared to controls, indicating maternal toxicity.

The significant reduction in food consumption at 1000 mg/kg/day was considered to be treatment-related and indicates maternal toxicity.

The gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg/day and small / not prominent thymus at 1000 mg/kg/day are indicative of maternal stress at these doses.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Mean numbers of pre-implantation losses in all treated groups were comparable to the vehicle control group.
There was significant increase in post-implantation loss at 300 and 1000 mg/kg/day.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
There were two dams out of 24 with total resorptions observed at the 300 mg/kg /day group.
There was significant increase in dams with complete resorptions at 1000 mg/kg/day (17/24).
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Maternal data parameters comprising of early resorptions in all treated groups were comparable to the vehicle control group.
There was significant increase in late resorptions at 300 and 1000 mg/kg/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Pregnancy duration is not relevant for the study design of the OECD 414 study.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregnancy duration is not relevant for the study design of the OECD 414 study.
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
At 300 and 1000 mg/kg/day, there were treatment-related significant reduction in maternal body weight gains during the treatment period GD 5-20 (-22% and -102%, respectively) and entire gestation period GD 0-20 (-17% and -87%, respectively). The body weight change corrected for gravid uterine weight was lower at 300 mg/kg/day (81%) and was significantly lower at 1000 mg/kg/day (-172%) compared to controls, indicating maternal toxicity.

The significant reduction in food consumption at 1000 mg/kg/day (-28%) compared to controls was considered to be treatment-related and indicates maternal toxicity.

The gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg/day and small / not prominent thymus at 1000 mg/kg/day are indicative of maternal stress at these doses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
pre and post implantation loss
total litter losses by resorption
early or late resorptions

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the litter data parameters such as the total number of fetuses and weight of male and female fetuses were significantly lower and were considered to be treatment-related developmental toxic effects. At 100 and 300 mg/kg/day the litter parameters such as total number of fetuses, number and weight of live fetuses and sex ratio were statistically comparable to the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Due to the study design of the OECD 414 body weight change can not be determined in fetals.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the litter data parameters such as the total number of fetuses were significantly lower. At 100 and 300 mg/kg/day the litter parameters such as total number of fetuses were statistically comparable to the vehicle control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio at at all dose groups was statistically comparable to the vehicle control group.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day group the litter size were reduced.
Changes in postnatal survival:
not examined
Description (incidence and severity):
Not relevant due to the study design of the OECD 414.
External malformations:
no effects observed
Description (incidence and severity):
There were no signs of external fetal malformations in any of the treatment groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no signs of teratogenicity / malformations in any of the treatment groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no incidences of major malformations in any of the treatment groups.

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Remarks on result:
other:
Remarks:
At 1000 mg/kg bw/d final body weight (including uterus) was 26% reduced compared to control group. Significant decreased maternal body weight gains during GD 5-20 at dose groups 300 and 1000 mg/kg/day (-22% and -102%, respectively) as well as weight change corrected for gravid uterine weight at 1000 mg/kg/day was significantly lower (-171%), indicating maternal toxicity. Significantly reduced food consumption at 1000 mg/kg/day group during GD 5 to GD 20 (-39%) and entire gestation period 0 to 20 (-28%) compared to controls indicating maternal toxicity. Due to before mentioned effects the following observations are considered of secondary nature: Decreased gravid uterine weights and significant increase in late resorptions and post implantation loss at 300 and 1000 mg/kg/bw is observed along with significant increase in dams with any resorption and complete resorptions.

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

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

100

300

1000

Total No. of rats found sperm positive / group

24

24

24

24

Duration of treatment (days)

Gestation Day 5 to 19 (15 days)

No. of rats sacrificed at caesarean section

24

24

24

24

No. of non-pregnant rats at caesarean section

1

0

0

0

No. of pregnant rats at caesarean section

23

24

24

24

No. of litters examined

23

24

22

7

Total no. of foetuses

252

267

203

24

 

 

 

 

 

Number of fetuses evaluated

 

 

 

 

a. External examination

252

267

203

24

b. Visceral examination

126

134

102

12

c. Skeletal examination

126

133

101

12

 

 

 

 

 

TABLE 2.           Summary of Clinical Signs, Physical examination and Mortality

Observations

 

 

 

 

Group No.

Dose (mg/kg/day)

Total No. of mated rats/group

G1

G2

G3

G4

0

100

300

1000

24

24

24

24

 

 

 

 

 

Mortality

None

 

 

 

 

 

Clinical signs 

 

 

 

 

Poorly formed faeces containing mostly water

0

0

1

0

Reddish discharge from vagina

0

0

0

16

 

 

 

 

 

 

 

TABLE 3.           Summary of Maternal Body Weight and Weight Gain

Table 3A: Summary of maternal group mean body weight (g)

Group No.

Dose

(mg/kg/day)

No. of

Dams

Group mean body weight (g) on day

 

0

3

5

8

11

14

17

20

G1

0

23

Mean

212.58

221.07

226.12

231.76

244.28

254.43

277.73

302.53

 

 

 

SD

14.02

15.38

15.59

15.90

16.87

18.50

20.66

22.59

 

 

 

 

 

 

 

 

 

 

 

 

G2

100

24

Mean

212.41

221.44

226.59

233.31

246.44

256.44

280.57

305.70

 

 

 

SD

13.80

15.46

14.88

15.39

15.44

15.82

16.68

22.76

 

 

 

 

 

 

 

 

 

 

 

 

G3

300

24

Mean

212.42

221.00

226.92

232.37

243.78

252.23

268.41

286.72

 

 

 

SD

13.71

15.01

16.51

18.10

17.48

19.25

22.60

33.15

 

 

 

 

 

 

 

 

 

 

 

 

G4

1000

24

Mean

212.38

221.43

225.97

227.89

231.52*

225.69*

228.81*

224.38*

 

 

 

SD

14.18

16.08

15.77

16.37

20.60

22.79

26.27

29.24

 

 

 

 

 

 

 

 

 

 

 

 

*: Significantly different from vehicle control group

TABLE 3. contd. Summary of Maternal Body Weight and Weight Gain

 

Table 3B: Summary of maternal body weight gain (g)

Period of treatment (days of gestation)

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of Dams

23

24

24

24

 

 

 

 

 

 

0-3

 

Mean

8.49

9.03

8.59

9.05

 

 

SD

3.02

3.07

2.84

3.33

 

 

 

 

 

 

 

3-5

 

Mean

5.05

5.15

5.91

4.54

 

 

SD

1.93

2.20

3.40

1.80

 

 

 

 

 

 

 

5-8

 

Mean

5.64

6.72

5.46

1.92*

 

 

SD

2.68

2.34

3.76

3.67

 

 

 

 

 

 

 

8-11

 

Mean

12.52

13.13

11.40

3.63*

 

 

SD

2.43

2.21

4.99

8.46

 

 

 

 

 

 

 

11-14

 

Mean

10.15

10.00

8.46

-5.83*

 

 

SD

4.27

2.85

4.99

9.31

 

 

 

 

 

 

 

14-17

 

Mean

23.30

24.13

16.18*

3.12*

 

 

SD

3.95

4.41

9.64

11.57

 

 

 

 

 

 

 

17-20

 

Mean

24.80

25.13

18.31

-4.43*

 

 

SD

4.00

10.04

14.47

11.81

 

 

 

 

 

 

 

*: Significantly different from vehicle control group

 

TABLE 3. contd. Summary of Maternal Body Weight and Weight Gain

 

Table 3C: Summary of maternal body weight gain (g)

Period of treatment (days of gestation)                     

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of Dams

23

24

24

24

 

 

 

 

 

 

Pre-treatment period (Days 0-5)

Mean

13.55

14.18

14.50

13.58

SD

3.98

3.45

4.76

3.88

 

 

 

 

 

 

Treatment Period (Days 5-20)

Mean

76.40

79.11

59.80*

-1.59*

SD

10.45

13.79

26.12

22.21

 

 

 

 

 

 

Entire gestation period (Days 0-20)

Mean

89.95

93.29

74.30*

12.00*

SD

12.17

14.97

27.86

23.56

 

 

 

 

 

 

TABLE 3. contd. Summary of Maternal Body Weight and Weight Gain

 

Table3D: Summary of Corrected Body Weight and Body Weight Gain (g)

Group No.

Dose

(mg/kg/day)

No. of

Dams

 

Gestation Day

5

Terminal body wt on GD 20

Uterine Wt

Carcass weight

(corrected body weight on GD 20)

Corrected Bwt gain.

G1

0

23

Mean

226.12

302.53

60.73

241.80

15.68

 

 

 

SD

15.59

22.59

11.10

19.28

9.62

 

 

 

 

 

 

 

 

 

G2

100

24

Mean

226.59

305.70

62.17

243.53

16.94

 

 

 

SD

14.88

22.76

16.71

21.35

14.09

 

 

 

 

 

 

 

 

 

G3

300

24

Mean

226.92

286.72

47.09*

239.63

12.71

 

 

 

SD

16.51

33.15

21.50

22.88

13.85

 

 

 

 

 

 

 

 

 

G4

1000

24

Mean

225.97

224.38*

9.67*

214.70*

-11.26*

 

 

 

SD

15.77

29.24

10.09

26.50

19.48

 

 

 

 

 

 

 

 

 

GD: Gestation day;

Corrected body weight on gestation day 20 (Carcass weight) = Terminal body weight on
day 20 - unopened uterine weight.

 

Corrected body weight gain = carcass weight - body weight on day 5.

*: Significantly different from vehicle control group

TABLE 4.           Summary of Food Intake (g/rat /day)

Period of treatment

(days of gestation)

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of Dams

23

24

24

24

Intermittent food intake

 

 

 

 

 

 

 

 

 

 

 

 

0-3

 

Mean

15.77

16.51

16.14

16.16

 

 

SD

2.23

1.62

1.89

1.74

 

 

 

 

 

 

 

3-5

 

Mean

16.77

17.54

17.58

16.76

 

 

SD

2.19

1.60

2.85

2.08

 

 

 

 

 

 

 

5-8

 

Mean

13.14

13.33

12.78

9.83*

 

 

SD

1.44

1.89

2.45

2.22

 

 

 

 

 

 

 

8-11

 

Mean

13.78

14.37

13.20

8.07*

 

 

SD

1.56

1.30

2.16

3.59

 

 

 

 

 

 

 

11-14

 

Mean

14.68

14.84

14.49

7.68*

 

 

SD

1.49

1.37

2.79

4.21

 

 

 

 

 

 

 

14-17

 

Mean

15.79

16.41

15.05

10.96*

 

 

SD

1.68

1.35

3.78

4.11

 

 

 

 

 

 

 

17-20

 

Mean

14.13

15.03

13.77

9.39*

 

 

SD

2.08

3.89

4.78

4.23

 

 

 

 

 

 

 

TABLE 4 contd. Summary of Food Intake (g/rat /day)

Group No.

G1

G2

G3

G4

Period of treatment Dose (mg/kg/day) 

0

100

300

1000

                                   No. of Dams

23

24

24

24

 

 

 

 

 

 

Pre-treatment period (Days 0-5)

Mean

16.17

16.92

16.72

16.40

SD

1.52

1.53

2.14

1.81

 

 

 

 

 

 

Treatment Period (Days 5-20)

Mean

14.31

14.80

13.86

9.19*

SD

1.31

1.59

2.78

2.60

 

 

 

 

 

 

Entire gestation period (Days 0-20)

Mean

14.77

15.33

14.57

10.99*

 

SD

1.26

1.49

2.47

2.13

 

 

 

 

 

 

*: Significantly different from vehicle control group

TABLE 5.           Summary of Maternal Data

 

Group No.

G1

G2

G3

G4

Parameters

Dose (mg/kg/day)

0

100

300

1000

 

No. of Dams

23#

24

24

24

Gravid uterine weight (g)

Mean

60.73

62.17

47.09*

9.67*

 

SD

11.10

16.71

21.50

10.09

Number of Corpora lutea

Total

303

329

325

319

Mean

13.17

13.71

13.54

13.29

SD

2.15

1.68

1.53

1.92

Number of Implantations

Total

264

279

277

270

Mean

11.48

11.63

11.54

11.25

SD

2.54

2.93

2.26

2.40

Early Resorptions

Total

9

10

30

21

Mean

0.39

0.42

1.25

0.88

SD

0.89

0.78

2.44

1.26

Late Resorptions

Total

3

2

44

225

Mean

0.13

0.08

1.83*

9.38*

SD

0.63

0.28

3.58

2.87

Pre-implantation Loss

Total number

39

50

48

49

Mean

1.70

2.08

2.00

2.04

SD

1.46

2.52

2.06

1.90

Post-implantation Loss

Total number

12

12

74

246

Mean

0.52

0.50

3.08*

10.25*

SD

7.70

9.50

30.79

15.55

Dams with any Resorption

Total

7

9

19*

7

Dams with all Resorption

Total

0

0

2

17*

*: Significantly different from vehicle control group;#: one animal not pregnant;

All mean values were calculated with numbers of dams mentioned in table

TABLE 5. contd. Summary of Maternal Data (% per Litter)

 

Group No.

G1

G2

G3

G4

Parameters

Dose (mg/kg/day)

0

100

300

1000

 

No. of Dams

23

24

24

24

 

 

 

Early Resorptions

Mean 

2.94

4.78

10.79

7.89

 

SD 

6.22

9.59

20.31

11.75

 

 

 

 

 

 

Late Resorptions

Mean 

1.09

0.62

15.89

83.92

 

SD 

5.21

2.10

28.05

19.39

 

 

 

 

 

 

Pre-implantation Loss

Mean 

13.27

15.30

14.59

15.15

 

SD 

12.61

18.41

14.67

14.15

 

 

 

 

 

 

Post-implantation Loss

Mean 

4.02

5.39

26.67

91.81

 

SD 

7.70

9.50

30.79

15.55

 

 

 

 

 

 

Implantation Index

Mean

86.73

84.70

85.41

84.85

 

SD

12.61

18.41

14.67

14.15

 

 

 

 

 

 

Note: Values are calculated according Indices (see above "Examination", subpoint "indices") and not rounded.

TABLE 6.           Summary of Litter Data

 

Group No.

G1

G2

G3

G4

Parameters

Dose (mg/kg/day)

0

100

300

1000

 

No. of Dams

23

24

24

24

 

 

 

 

 

 

No. of litters

 

23

24

22

7

 

 

 

 

 

 

Total no. of fetuses

 

252

267

203

24*

 

 

 

 

 

 

Mean litter size 

 

11.0

11.1

9.2

3.4

 

 

 

 

 

 

Dead fetuses

Total

0

0

0

0

 

%

0

0

0

0

 

 

 

 

 

 

Total live fetuses

 

 

 

 

 

a.  Number

 

252

267

203

24*

 

 %

 100

 100

 100

 100

b.  Weight (g)

Mean

3.54

3.59

3.38

2.60*

 

SD

0.25

0.24

0.36

0.40

 

 

 

 

 

 

Live male fetuses

 

 

 

 

 

a.  Number

 

127

136

97

9*

 

 %

 50

 51

 48

 38

b.  Weight (g)

Mean

3.65

3.67

3.50

2.59*

 

SD

0.29

0.25

0.35

0.52

 

 

 

 

 

 

Live female fetuses

 

 

 

 

 

a.  Number

 

125

131

106

15*

 

 %

 50

 49

 52

 62

b.  Weight (g)

Mean

3.40

3.49

3.29

2.78*

 

SD

0.25

0.26

0.38

0.23

 

 

 

 

 

 

Sex Ratio - Male : Female

 

1:0.98

1:0.96

1:1.09

1:1.67

 

 

 

 

 

 

*: Significantly different from vehicle control group

TABLE 7.           Summary of Gross Pathological Findings

Group No.

G1

G2

G3

G4

Parameters Dose (mg/kg/day)

0

100

300

1000

 

1.  No. of rats subjected to caesarean section

 

24

24

24

24

 

2.  No. of rats pregnant at caesarean section

 

23

24

24

24

3.  No. of rats showing gross pathology

 

 

 

 

 

 

 

 

 

     Adrenal enlarged – Bilateral

0

0

1

12

     Thymus: Small

0

0

0

10

     Thymus: Not prominent

0

0

0

9

 

 

 

 

 

Fetal External Observation (see Table 8 below)

At 1000 mg/kg/day anasarca was found in three foetuses which was considered as secondary to the maternal toxicity.

At 300 mg/kg/day anasarca was found in 4 fetuses of one dam (Rs4625). This dam exhibited remarkable findings prior to treatment, such as reduction in body weight, body weight gain and food intake (Appendix 3 and 4) indicating a bad health condition from begin on. The bad health condition of this dam is seen in smaller body weight gain and poor food intake during gestation days 0-3 and 3-5 (time prior to treatment) compared to other females of this dosing group. At termination the corrected body weight and corrected body weight gain was clearly decreased compared to all other dams of this dose group. Further,  poorly formed faeces containing mostly water was described on GD 20 for this dam. No abnormality were detected at clinical and physical examination at any other dam of this dosing group. Thus, the appearance of anasarca in fetals of this dam (Rs4625) of the 300 mg/kg/day group is considered as secondary to maternal toxicity. There were no incidences of major external malformations in any of the treatment groups.

Table 8.                    Summary of Fetal external Observations

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

252

267

203

24

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

 

 

 

 

 

 

 

 

 

 

 

 

 

Normal Variant

None

 

 

 

 

 

 

 

 

 

 

 

 

 

Minor Anomalies

 

 

Anasarca

 

0

 

0.00

 

0.00

 

0

 

0.00

 

0.00

 

4*

 

1.97

 

3.00

 

3*

 

12.50*

 

17.86*

 

 

 

 

 

 

 

 

 

 

 

 

 

Major Malformations

None

 

 

 

 

 

 

 

 

 

 

 

 

 

*: Significantly different from vehicle control group

 

 

Fetal Visceral Observation (See Table 9 below)

There was an incidence of a fetus with slight dilatation of renal pelvis of kidney in a fetus from control (1/126) which is indicated as anormal as well as a minor variant. There were no observations in any of the other treated groups and there were no incidences of major malformations in any of the treatment groups.

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

134

102

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Normal Variant

 

 

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

 

1

 

0.79

 

1.00

 

1

 

0.75

 

1.00

 

0

 

0.00

 

0.00

 

0

 

0.00

 

0.00

Minor Anomalies

 

 

 

 

 

 

 

 

 

 

 

 

 

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

 

1

 

0.79

 

1.00

 

0

 

0.00

 

0.00

 

0

 

0.00

 

0.00

 

0

 

0.00

 

0.00

Major Malformations

None

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fetal Skeletal Observation (see Table 10 below)

The incidences/percentage of normal variants [delayed skeletal ossification (DSO) and incomplete/poor ossification (INO/PO)] and minor anomalies were comparable between the vehicle control and 100 mg/kg/day dose.

The statistically significant changes in normal variants [delayed skeletal ossification (DSO) and incomplete/poor ossification (INO/PO)] and minor anomalies noted at 300 and 1000 mg/kg/day are expected changes mainly due to the poor development of the fetuses at these doses. There were major malformations in any of the treatment groups.

 

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Normal Variant

 

DSO:  CV:centra:7/7

6

4.76

5.04

3

2.26

3.27

16*

15.84*

18.69*

9*

75.00*

82.14

            TV:centra:1/13

1

0.79

0.87

0

0.00

0.00

2

1.98

3.03

2*

16.67*

28.57

            SV:centra:1/4

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

            CdV:centra:1/4

11

8.73

8.54

31*

23.31*

22.61*

32*

31.68*

30.94*

3

25.00*

25.00

            CdV:centra:2/4

3

2.38

2.46

8

6.02

6.88

4

3.96

5.19

2*

16.67*

7.14*

            CdV:centra:3/4

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

2*

16.67*

28.57*

            CdV:centra:4/4

0

0.00

0.00

0

0.00

0.00

3

2.97

4.55*

0

0.00

0.00

            CdV:arch:1/2

0

0.00

0.00

7*

5.26*

6.05*

2

1.98

2.16

2*

16.67*

28.57*

            CdV:arch:2/2

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

            F limb:Metacarp.:1/4

46

36.51

38.24

63

47.37

47.25

64*

63.37*

63.65

11*

91.67*

92.86

*: Significantly different from vehicle control group

 

 

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Normal Variant

 

DSO:  F limb:Pr. Phal:2/2

118

93.65

93.91

131*

98.50

98.61

101*

100.00*

100.00

12

100.00*

100.00

            F limb:Dt. Phal:1/4

1

0.79

0.87

8*

6.02*

7.09*

6*

5.94*

6.26*

0

0.00

0.00

            F limb:Dt. Phal:4/4

2

1.59

1.96

5

3.76

6.29

19*

18.81*

22.62*

5*

41.67*

39.29

            H limb:Metatar.:1/4

2

1.59

1.74

0

0.00

0.00

3

2.97

4.55

1

8.33*

14.29*

            H limb:Pr. Phal:1/4

0

0.00

0.00

1

0.75

2.08

0

0.00

0.00

0

0.00

0.00

            H limb:Dt. Phal:1/5

1

0.79

0.87

1

0.75

0.69

0

0.00

0.00

0

0.00

0.00

            H limb:Dt. Phal:5/5

3

2.38

2.83

7

5.26

8.03

25*

24.75*

28.65*

6*

50.00*

42.86

INO/PO: Skull bones

0

0.00

0.00

0

0.00

0.00

3

2.97

4.55*

5*

41.67*

57.14

                Parietal

0

0.00

0.00

1

0.75

0.69

1

0.99

0.91

0

0.00

0.00

                Pa, Ipa

1

0.79

0.87

7*

5.26

6.91*

11*

10.89*

11.98*

1*

8.33*

3.57*

*: Significantly different from vehicle control group

 

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Normal Variant

 

INO/PO: Inter pariet.

0

0.00

0.00

1

0.75

1.04

1

0.99

0.91

0

0.00

0.00

                Fr, Pa, Ipa

1

0.79

0.72

2

1.50

2.68

3

2.97

3.94

6*

50.00*

39.29

                Supraocci

1

0.79

0.72

2

1.50

2.78

2

1.98

2.42

2*

16.67*

17.86*

                Stern:# 1-4

1

0.79

0.72

1

0.75

1.04

2

1.98

3.03

0

0.00

0.00

                Stern:# 1

1

0.79

0.87

3

2.26

3.37

13*

12.87*

14.18*

2*

16.67*

21.43*

                Stern:# 2

14

11.11

10.37

24

18.05

17.82*

56*

55.45*

54.48

1

8.33

7.14*

                Stern:# 3

1

0.79

0.87

2

1.50

2.78

8*

7.92*

9.44*

3*

25.00*

21.43*

                Stern:# 4

2

1.59

1.74

3

2.26

3.47

14*

13.86*

15.19*

3*

25.00*

21.43*

                Stern:# 5

50

39.68

41.05

52

39.10

37.55

62*

61.39*

57.67

1*

8.33*

7.14

                Stern:# 6

34

26.98

24.46

35

26.32

27.68

27

26.73

25.93

0*

0.00*

0.00*

*: Significantly different from vehicle control group

 

 

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Normal Variant

 

INO/PO: CV:centra:1/7

3

2.38

2.43

9

6.77

6.03

13*

12.87*

10.63*

1

8.33

7.14*

                CV:centra:2/7

9

7.14

6.68

7

5.26

5.01

8

7.92

13.18*

0

0.00*

0.00

                CV:centra:3/7

7

5.56

5.33

4

3.01

2.64

7

6.93

6.41

1

8.33

7.14*

                CV:centra:4/7

2

1.59

1.35

0

0.00

0.00

5

4.95

4.59

1

8.33*

3.57*

                CV:centra:5/7

3

2.38

2.22

4

3.01

2.72

0

0.00

0.00

0

0.00

0.00

                CV:centra:6/7

2

1.59

1.24

3

2.26

1.98

8*

7.92*

6.86*

0

0.00

0.00

                CV:centra:7/7

1

0.79

0.72

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

                TV:centra:1/13

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

3*

25.00*

17.86*

                TV:centra:2/13

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

                SV:centra:2/4

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

*: Significantly different from vehicle control group

 

  

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Normal Variant

 

INO/PO:   SV:arch:2/4

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

                  Ileum

0

0.00

0.00

0

0.00

0.00

3

2.97

4.55*

0

0.00

0.00

                  Pubis

2

1.59

1.59

0

0.00

0.00

5

4.95

7.58*

5*

41.67*

50.00

                  F limb:Metacarp.:1/4

0

0.00

0.00

0

0.00

0.00

1

0.99

0.65

0

0.00

0.00

                  SV:centra&arch:3,4

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

14.29*

                  SV:centra:3,4

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

14.29*

                  SV:arch:3,4

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

14.29*

*: Significantly different from vehicle control group

 

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Minor Anomalies

 

HYPOPLASTIC:Stern:# 1

1

0.79

0.72

1

0.75

1.04

6*

5.94*

7.62*

1*

8.33*

7.14*

                             Stern:# 2

1

0.79

0.72

3

2.26

3.82

18*

17.82*

16.94*

1*

8.33*

3.57*

                             Stern:# 3

1

0.79

0.72

1

0.75

1.04

5

4.95

6.71*

0

0.00

0.00

                             Stern:# 4

1

0.79

0.72

1

0.75

1.04

6*

5.94*

7.62*

0

0.00

0.00

                             Stern:# 5

17

13.49

13.23

18

13.53

14.10

20

19.80

17.94

1

8.33

3.57

                             Stern:# 6

2

1.59

1.24

1

0.75

0.83

3

2.97

6.36*

0

0.00

0.00

                             TV:centra:1/13

0

0.00

0.00

0

0.00

0.00

2

1.98

3.03

1*

8.33*

14.29*

DB: TV:centra:1/13

8

6.35

7.97

6

4.51

7.29

20*

19.80*

21.45*

5*

41.67*

42.86

        TV:centra:2/13

1

0.79

1.09

2

1.50

1.19

9*

8.91*

9.03*

2*

16.67*

21.43*

        TV:centra:3/13

0

0.00

0.00

1

0.75

0.69

0

0.00

0.00

0

0.00

0.00

  *: Significantly different from vehicle control group

 

 

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Minor Anomalies

 

DB: TV:centra:6/13

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

3.57*

        :LV:centra:1/6

0

0.00

0.00

0

0.00

0.00

1

0.99

0.91

0

0.00

0.00

ASY OSSI:  stern:# 1

0

0.00

0.00

0

0.00

0.00

3

2.97

3.94*

3*

25.00*

25.00*

                     stern:# 3

0

0.00

0.00

0

0.00

0.00

2

1.98

2.42

3*

25.00*

25.00*

                     stern:# 4

1

0.79

0.87

0

0.00

0.00

2

1.98

2.42

4*

33.33*

28.57*

                     stern:# 3,4

0

0.00

0.00

4*

3.01

2.33

2

1.98

2.65

0

0.00

0.00

                     LV:centra:2/6

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

14.29*

ASY DB:TV:centra:1/13

0

0.00

0.00

1

0.75

2.08

1

0.99

0.65

0

0.00

0.00

SPLIT:  Stern:# 1

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

10*

83.33*

85.71

              Stern:# 2

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

3.57*

*: Significantly different from vehicle control group

 

  

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Minor Anomalies

 

SPLIT:  Stern:# 3

0

0.00

0.00

0

0.00

0.00

2

1.98

3.03

10*

83.33*

78.57

              Stern:# 4

0

0.00

0.00

0

0.00

0.00

2

1.98

3.03

10*

83.33*

78.57

              Stern:# 5

0

0.00

0.00

0

0.00

0.00

0

0.00

0.00

1*

8.33*

3.57*

              Stern:1-4

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

              Stern:1,3,4

0

0.00

0.00

0

0.00

0.00

4*

3.96*

6.06*

0

0.00

0.00

              TV:centra:1/13

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

1*

8.33*

7.14*

EXTRA:LV:centra,arch:# 7

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

0

0.00

0.00

              RIB(Rt/Lt/B):# 14

0

0.00

0.00

2

1.50

1.39

1

0.99

1.52

0

0.00

0.00

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

4

3.17

3.20

5

3.76

3.61

6

5.94

7.12

1

8.33

14.29*

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

44

34.92

34.63

60

45.11

49.36

59*

58.42*

55.02

8*

66.67*

67.86

*: Significantly different from vehicle control group

 

  

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

Group No.

G1

G2

G3

G4

Dose (mg/kg/day)

0

100

300

1000

No. of litters examined

23

24

22

7

No. of fetuses examined

126

133

101

12

 

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Inc.

Fetus %

Litter %

Minor Anomalies

 

UNILAT OSSI:TV:centra:1/13

0

0.00

0.00

0

0.00

0.00

1

0.99

1.52

1*

8.33*

14.29*

F.limb(Rt/Lt/B(+/++)) flexed at wrist

0

0.00

0.00

0

0.00

0.00

1

0.99

0.65

0

0.00

0.00

WAVY:RIB(Rt/Lt/B)(+/++):3/13

0

0.00

0.00

1

0.75

2.08

0

0.00

0.00

0

0.00

0.00

 

 

 

 

 

 

 

 

 

 

 

 

 

Major Malformations

None

 

 

 

 

 

 

 

 

 

 

 

 

 

*: Significantly different from vehicle control group

Applicant's summary and conclusion

Conclusions:
Based on the above findings, it is concluded that the No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and Fetal developmental toxicity is 100 mg/kg/day in Wistar rats when Hostanox O 3 P was administered orally by gavage during GD 5-19 under the test conditions and doses employed in this study due to:
Treatment related clinical sign of reddish vaginal discharge at 1000 mg/kg /day, significant reduction in maternal body weight gains at 300 and 1000 mg/kg/day and food consumption at 1000 mg/kg/day. Due to the before mentioned effects the following observations are considered of secondary nature: Decreased gravid uterine weights and significant increase in late resorptions and post implantation loss at 300 and 1000 mg/kg/day along with significant increase in dams with any resorption and complete resorption.
Executive summary:

The objective of this study was to evaluate the embryo-fetal developmental toxicity of test item Hostanox O 3 P when administered to pregnant Wistar rats by oral route during gestation days (GD) 5 to GD19.

Ninety six presumed pregnant Wistar rats were assigned to four groups by body weight stratification (Group 1 to Group 4) and each group (G1: control, G2: low dose, G3: mid dose and G4: high dose) consisted of 24 presumed pregnant rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

The test item, Hostanox O 3 P was suspended in sunflower oil and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 100, 300 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. Rats in the vehicle control (G1) group received the vehicle (sunflower oil) alone. A constant dose volume of 10 mL/kg body weight was administered to all groups. The mated females were observed twice daily for clinical signs (during treatment period), mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. Food consumption was determined on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation.

Caesarean section was performed for all rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animal were 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 dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were 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.

·      There was no mortality at any of the doses tested.

·      At 1000 mg/kg/day, treatment-related clinical sign of reddish discharge from the vagina was observed between GD 13 - 20. This finding was associated with dams with resorptions at necropsy.

- At terminal sacrifice, there were gross pathological changes of enlarged adrenals in dams treated at 300 (1/24) and 1000 mg/kg/day (10/24) indicative of maternal stress at these doses. In addition, gross finding of small /not prominent thymus at 1000 mg/kg/day dose level was also indicative of maternal stress at this dose.

·     At 1000 mg/kg/day final body weight (including uterus) was 26 % reduced compared to control group. At 300 and 1000 mg/kg/day, there were treatment-related significant reduction in maternal body weight gains during the treatment period GD5 to 20 (-22% and -102%, respectively) and entire gestation period 0 to 20 (-17% and -87%, respectively). The body weight change corrected for gravid uterine weight at 1000 mg/kg/day was significantly lower (-172%) indicating maternal toxicity

·      At 1000 mg/kg/day, the food consumption during the treatment period GD5 to 20 (-39%) and entire gestation period 0 to 20 (-28%) was significantly lower as compared to vehicle control group indicating maternal toxicity.

·      At 300 and 1000 mg/kg/day, the gravid uterine weights were significantly lower (-17% and -45%, respectively) and there was significant increase in late resorptions and post implantation loss. There was significant increase in dams with any resorption and complete resorptions at 300 and 1000 mg/kg/day, respectively indicating developmental toxicity. Gross evaluation of placenta revealed no findings.

·      At 1000 mg/kg/day, the litter data parameters such as the total number of fetuses and weight of male and female fetuses were significantly lower and were consequently considered to be treatment-related developmental toxic effects.

·      Fetal visceral observations revealed no signs of fetal malformations or developmental toxicity at the doses tested.

·      Fetal skeletal examination revealed no signs of teratogenicity / malformations in the tested dose levels. The statistically significant increase in the incidence and percentage of normal variants and minor anomalies in some of the bone components at 300 and 1000 mg/kg/day doses are expected changes mainly due to the poor development of the fetuses at these doses.

Based on the above findings, it is concluded that the No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and Fetal developmental toxicity is 100 mg/kg/day in Wistar rats when Hostanox O 3 P was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study due to:

Treatment related clinical sign of reddish vaginal discharge at 1000 mg/kg /day, significant reduction in maternal body weight gains at 300 and 1000 mg/kg/day and food consumption at 1000 mg/kg/day. Due to the before mentioned effects the following observations are considered of secundary nature: Decreased gravid uterine weights and significant increase in late resorptions and post implantation loss at 300 and 1000 mg/kg/day along with significant increase in dams with any resorption and complete resorption.