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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In order to assess the fertility properties of the test substance Hostacerin DGSB after repeated oral treatment for a subchronic period, one dose-range-finding and one main OECD 421 screening study is available.

A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[0.5% w/v Carboxy Methyl Cellulose],animals in G2, G3 and G4groups were administered withtest itemat dose levels of100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively. The vehicle and test item formulations were administered orally by gavageat the dose volume of 10 mL/kg body weight. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). 

All animals did not reveal any clinical signs of toxicity, no mortality or morbidity observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, organ weights (both absolute and relative) were observed in both the sex of all tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult animals and in serum T4 levels of pups at any of the tested dose groups.

There were no treatment-related effects on the mean pre-coital time, mating and fertility indices of sires and dams at all the doses tested.Females did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, mean gestation length, implantation index, pre-implantation loss, post-implantation loss and number of pups delivered when compared with vehicle control group. No effects were observed for live birth index of each litter, sex ratio and pup survival index at all the tested dose groups throughout the lactation periodwhen compared with vehicle control group animals.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, anogenital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

All animals of all dose groups did not reveal any treatment related gross pathological and histopathological findings compared to vehicle control group.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19 May 2017 to 13 October 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is a dose range finding study.
Remarks:
6 animals /sex/dose were used
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
yes
Remarks:
Study is a dose-range finding study. 6 rats /sex and dose were used.
Principles of method if other than guideline:
Study is performed as dose-range-finding study. Thus, only 6 rats per sex and dose are used.
GLP compliance:
no
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN:

- Premating exposure duration for parental (P0) animals: 14 days
- Basis for dose level selection: The doses of 100, 300 and 1000 mg/kg body weight were selected as low (G2), mid (G3) and high dose (G4) levels based on the NOAEL of test item Hostacerin DGSB was found to be 1000 mg/kg/day when administered for a period of 14 consecutive days by oral (gavage) to Sprague Dawley rats obtained from the dose range finding study (Bioneeds Study Number: BIO-TX 2503). I

- Route of administration: Oral gavage

- Other considerations:
choice of species, strain: Rat & Sprague Dawley

vehicle and number of animals: 0.5 % Carboxy methyl cellulose and 6 per group per sex
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material : Clariant Produkte(Deutschland) GmbH84504 Burgkirchen, Germany.
- Batch No.of test material: DEG4338635
- Expiration date of thebatch:04 Oct 2018
- Purity test date:04 Oct 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29 oC)
- Stability under test conditions: not established (freshly prepared test item formulations were administered to the animals and homogeneity was achieved thorough stirring).
- Solubility in the solvent/vehicle: forms uniform suspension in 0.5% w/v carboxy methyl cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by thein-house solubility/suspendibilty test results.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: ground and made in to suspension
- Final preparation of a solid: Suspension

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:In-house bred
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 225.13 g to 254.32 g; Females: 201.03 g to 221.19 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.

i. Pre mating
Two animals of same sex and group per cage were housed.

ii. Mating
During mating, two animals (one male and one female) of same group were housed.

iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.

- Diet (ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum.

- Water (ad libitum):Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter with purifier was provided in plastic water bottles with stainless steel sipper tubes.

- Acclimation period:19 May 2017 to 06 June 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19.1 to 24.5
- Humidity (%):41 to 69
- Air changes (per hr):12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 19 May 2017 To:05 August 2017
Route of administration:
oral: gavage
Remarks on MMAD:
not applicable
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item grind well in a mortar and in a mortar and pestle by adding small quantity of 0.5% w/v Carboxy Methyl Cellulose untilaclear suspension was formed. Thereafter, the entire quantity of the formulation was transferred intoa measuring cylinder. Again,a small quantity of the vehicle was added to rinse the mortar and pestleand this was transferred into the measuring cylinder. The rinsing of mortar and pestle was repeated to ensure the complete transfer of the contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged : individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test substance in dose formulation were not established under this dose range findiong study. However, test item formulations were prepared daily fresh.
Duration of treatment / exposure:
Males: 30 days (premating, mating and post mating periods).

Females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Frequency of treatment:
Once daily
Details on study schedule:
Age at mating of the mated animals in the study: 14 to 15 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
6 animals per sex per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The doses of 100, 300 and 1000 mg/kg body weight were selected as low (G2), mid (G3) and high dose (G4) levels for test item Hostacerin DGSB based on the No Observed Adverse Effect Level (NOAEL) of the test item Hostacerin DGSB was found to be 1000 mg/kg/day when administered for a period of 14 consecutive days by oral (gavage) to Sprague Dawley rats obtained from the dose range finding study (Bioneeds Study Number: BIO-TX 2503).

- Rationale for animal assignment :The body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+5.07 and -7.14 for males and +5.59 and -4.96 for females) of the mean body weight of each sex.
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period.

FOOD CONSUMPTION : Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; weekly once; gestation days 0 to 7, 7 to 14 and 14 to 20 duringpregnancy and day 1 to 4, 4 to 7, 7 to 13 during lactation period.

Oestrous cyclicity (parental animals):
two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days), daily from the beginning of the treatment period until evidence of mating and on the day of sacrifice for females.
Sperm parameters (parental animals):
Parameters examined Epididymis, Prostate+ Seminal vesicles weight and Testes weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death for pups born or found dead.



Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals [after completion of 30 days of treatment]
- Maternal animals: All animals [on lactation day 14]

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

ORGAN WEIGHTS
In Table [5 and 6] weighed and preserved.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed at [table 20] 13 days of age (lactation day 13)
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

Statistics:
The data was subjected to various statistical analysis using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05)
The statistical analysis was followed to the parameters as mentioned below:
Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ
weights, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal
loss, Post implantation loss:- Parametric - One-way ANOVA with Dunnett’s post test.

Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight:- Non
Parametric - Kruskal-Wallis followed by the Mann-Whitney test.

Pregnancy rate. Dams with live pups, Dams with dead pups, No. of litters with/without dead pups, No.
of litters with/without resorptions:- Cross Tabs - Chi-square test/ Fischer's Exact Test
Reproductive indices:
Reproductive indices
Male Fertility Index (%) = Number of Males Impregnating Females
------------------------------------------------- x 100
Number of Males used for Mating
Female Fertility Index (%) = Number of Females confirmed with Pregnancy
--------------------------------------------------------- x 100
Number of Females used for Mating
Parturition (%) = Number Of Parturitions
------------------------------- x 100
Number Of Pregnancies
Copulatory Interval (Days) = Date of Initiation of Cohabitation - Date of Confirmation of Mating
Gestation Length (days) = Date of Delivery - Date of Confirmation of Mating
Offspring viability indices:
Offspring viability indices
Male/Female Sex Ratio = Number Male Pups Per Dam
-----------------------------------
Number Female Pups Per Dam
Live Birth Index (%) = Number Of Viable Pups Born
------------------------------------ x 100
Number Of Pups Born
Lactation Day 4
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 4
----------------------------------------------------- x 100
Number Of Pups Born
Lactation Day 7
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 7
------------------------------------------------------ x 100
Number Of Viable Pups on Lactation Day 4
Lactation Day 13 Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 13
------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 7
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity observed at any of the tested dose group animals and vehicle control group animals of either sex during premating period and mating period; during post mating period in males and during gestation/lactation period in females.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any of the tested dose group animals and vehicle control group animals of either sex during premating period and mating period; during post mating period in males and during gestation/lactation period in females.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose group animals of either sex when compared with vehicle control group animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes noted in feed consumption at any of the tested dose group animals of either sex when compared with vehicle control group animals. However, statistical significant increase during week 2 in G2 and G3 males; increase during post mating in G2 males; increase during week 1 and 2 in G4 females was noted when compared with vehicle control group animals. This can be considered as incidental due to lack of dose dependency and no changes were noted in body weight during these periods.
There were no changes observed in the feed consumption during gestation period at any of the tested dose group animals when compared with vehicle control group animals. However, statistical significant increase in mean feed consumption during gestation day 0 to 7 was noted at low dose group animals when compared with vehicle control group animals. This change is considered as incidental and not treatment related as there were no effects noted in mean body weight at this duration and the change is inconsistent.
There were no changes observed in the feed consumption during lactation period at any of the tested dose group animals when compared with vehicle control group animals
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related changes were observed in serum T4 levels of adult animals.
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes observed in the oestrus cyclicity of females at any of the tested doses during pre-mating treatment, mating treatment and on lactation day 14.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
Reproductive performance:
no effects observed
Description (incidence and severity):
No effects were noted in mating index and fertility index of both the males and females at all the tested dose groups
No effects were noted up to 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 30 days) and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13.
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: No adverse effects observed
Key result
Critical effects observed:
no
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significant increase in mean pup weight at high dose group (G4) male mean pup weight on lactation day 4 and 7 was noted when compared with vehicle control group. This change is considered as incidental but not treatment related as there were no effects noted in other parameters. No effects were noted in female mean pup weight at this group. The obtained weights are within historical control range.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in serum T4 levels on lactation day 4 and 13 pups at any of the tested dose groups when compared with vehicle control group
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No post natal death observed (at birth until lactation day 13)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose group litters when compared with vehicle control group litters.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes (both external and internal) observed at any of the tested dose group animals of either sex of pups.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Description (incidence and severity):
No effects were noted in offsprings at all the tested dose groups.
No effects were noted in offsprings at all the tested dose groups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

TABLE 1.        SUMMARY OF CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

                                                                                                                

Group, Sex & Dose

(mg/kg body weight)

No. of Animals

 Clinical Signs of Toxicity

Mortality

(No. of Mortality/

No. of Animals dosed)

G1, M & 0

6

N

0/6

G2, M & 100

6

N

0/6

G3, M & 300

6

N

0/6

G4, M & 1000

6

N

0/6

M: Male; N: Normal

TABLE 1 (Contd..,). SUMMARYOF CLINICAL SIGNS OF TOXICITYAND MORTALITY RECORD

      

Group, Sex & Dose

(mg/kg body weight)

No. of Animals

Clinical Signs of Toxicity

Mortality

(No. of Mortality/

No. of Animals dosed)

G1, F & 0

6

N

0/6

G2, F & 100

6

N

0/6

G3, F & 300

6

N

0/6

G4, F & 1000

6

N

0/6

F: Female; N: Normal

TABLE 2.        SUMMARY OF BODY WEIGHTS (g) RECORD

  

Group, Sex & Dose

(mg/kg body weight)

 

Body Weight (g) on Days

1

8

15

22

29

G1, M & 0

Mean

331.25

362.71

386.31

409.37

421.80

±SD

13.90

10.59

7.59

9.44

8.63

n

6

6

6

6

6

G2, M & 100

Mean

331.77

357.98

381.55

405.92

418.53

±SD

14.34

10.81

10.70

10.11

8.89

n

6

6

6

6

6

G3, M & 300

Mean

332.10

361.95

380.97

402.04

415.77

±SD

16.49

9.19

8.89

7.83

6.15

n

6

6

6

6

6

G4, M & 1000

Mean

334.17

362.38

382.72

406.87

422.84

±SD

16.95

13.77

10.38

10.84

12.60

n

6

6

6

6

6

  M: Male; SD: Standard Deviation; n: Number of Animals

TABLE 2 (Contd..,). SUMMARY OF BODY WEIGHTS (g) RECORD

 

Group, Sex & Dose

(mg/kg body weight)

 

Body Weight (g) on Days

1

8

15

22#

G1, F & 0

Mean

240.69

252.13

260.78

267.33

±SD

8.35

7.06

2.96

-

n

6

6

6

1

G2, F & 100

Mean

241.04

253.47

263.23

267.77

±SD

7.44

6.97

7.49

2.46

n

6

6

6

2

G3, F & 300

Mean

240.83

253.07

262.61

269.33

±SD

7.53

7.63

7.60

-

n

6

6

6

1

G4, F & 1000

Mean

242.16

255.14

264.16

260.79

±SD

7.12

6.55

6.73

-

n

6

6

6

1

F: Female; SD: Standard Deviation; n: Number of Animals; -: Not applicable

#:The body weight of females only in cohabitation was consideredfor mean calculation.

TABLE 3.  SUMMARY OF PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

                                       

Group, Sex & Dose (mg/kg body weight)

 

Percent Change in Body Weight (%) during Days

1 to 8

1 to 14

1 to 21

1 to 29

G1, M & 0

Mean

9.60

16.74

23.68

27.44

±SD

4.14

3.88

3.24

3.12

n

6

6

6

6

G2, M & 100

Mean

7.99

15.13

22.55

26.34

±SD

3.53

4.71

6.33

6.00

n

6

6

6

6

G3, M & 300

Mean

9.11

14.85

21.22

25.38

±SD

3.05

3.60

3.81

4.49

n

6

6

6

6

G4, M & 1000

Mean

8.51

14.65

21.88

26.65

±SD

2.00

2.90

2.92

2.94

n

6

6

6

6

  M: Male; SD: Standard Deviation; n: Number of Animals

TABLE 3 (Contd..,). SUMMARY OF CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

 

Group, Sex & Dose

(mg/kg body weight)

Percent Change in Body Weight (%) during Days

1 to 8

1 to 14

1 to 21#

G1, F & 0

Mean

4.78

8.45

8.96

±SD

1.17

3.52

-

n

6

6

1

G2, F & 100

Mean

5.17

9.22

13.13

±SD

1.05

1.19

1.35

n

6

6

2

G3, F & 300

Mean

5.09

9.06

15.52

±SD

1.11

1.68

-

n

6

6

1

G4, F & 1000

Mean

5.37

9.10

11.71

±SD

0.70

1.33

-

n

6

6

1

   F: Female; SD: Standard Deviation; n: Number of Animals; -: Not applicable

   #:The percent change in body weight of females only in cohabitation was consideredfor mean calculation.

TABLE 4.  SUMMARY OF FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose
(mg/kg body weight)

Week 1

Feed Consumption (Day 1 to 8)

Week 2

Feed Consumption (Day 8 to 14)

Post mating

Feed Consumption (Day 25 to 29)

Post mating

Feed Consumption (Day 29 to 30)

       

G1, M & 0

          

Mean

24.38

27.37

28.83

28.18

±SD

1.10

0.71

0.63

1.16

n

6

6

6

6

G2, M & 100

Mean

25.20

28.29*

29.72*

28.07

±SD

0.49

0.47

0.57

0.71

n

6

6

6

6

G3, M & 300

Mean

25.19

28.42*

29.63

29.00

±SD

0.73

0.58

0.82

0.53

n

6

6

6

6

G4, M & 1000

Mean

25.07

28.00

29.08

28.00

±SD

0.72

0.60

0.71

1.13

n

6

6

6

6

 M: Male;SD: Standard Deviation; n: Number ofanimals

    *: Statistically significant than the control group (p<0.05)

TABLE 4 (Contd..,). SUMMARY OF FFED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose
(mg/kg body weight)

Feed Consumption (g) during Premating

Week 1

Week 2

G1, F & 0

Mean

19.97

21.26

±SD

0.55

0.53

n

6

6

G1, F & 100

Mean

19.86

21.35

±SD

0.64

0.53

n

6

6

G3, F & 300

Mean

20.38

21.91

±SD

0.62

0.41

n

6

6

G4, F & 1000

Mean

21.81*

23.19*

±SD

1.68

1.30

n

6

6

F: Female; SD: Standard Deviation;Number of animals

   *: Statistically significant than the control group (p<0.05)

TABLE 5.           SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight)

Epididymis

Testes

Prostate + Seminal vesicles with coagulating glands (PSC)

G1, M & 0

Mean

1.7403

3.5235

3.8015

±SD

0.0339

0.2507

0.4641

n

6

6

6

G2, M & 100

Mean

1.5891

3.4216

3.4161

±SD

0.0593

0.2754

0.2124

n

6

6

6

G3, M & 300

Mean

1.6754

3.4561

3.5618

±SD

0.1192

0.2128

0.3601

n

6

6

6

G4, M & 1000

Mean

1.6969

3.4100

3.7384

±SD

0.1082

0.2617

0.1575

n

6

6

6

M: Male;SD: Standard Deviation; n: Number of animals

TABLE 5 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

                                                                                                      

Group, Sex & Dose

(mg/kg body weight)

Uterus

Ovaries

G1, F & 0

0.4807

0.1691

0.0440

0.0339

6

6

G2, F & 100

0.5102

0.1695

0.0627

0.0335

6

6

G3, F & 300

0.4604

0.1868

0.0367

0.0255

6

6

G4, F & 1000

0.4749

0.1799

0.0424

0.0206

6

6

F: Female;SD: Standard Deviation; n: Number of animals

TABLE 6.           SUMMARY OF TERMINAL BODY WEIGHT (g) AND RELATIVE ORGAN WEIGHT (%) RECORD

Group, Sex & Dose

(mg/kg body weight)

Fasting Body Weight

(g)

Epididymis

Testes

Prostate +

Seminal vesicles with coagulating glands (PSC)

G1, M & 0

Mean

402.18

0.4329

0.8762

0.9471

±SD

8.40

0.0153

0.0609

0.1324

n

6

6

6

6

G2, M & 100

Mean

397.70

0.3996

0.8602

0.8591

±SD

5.44

0.0136

0.0650

0.0537

n

6

6

6

6

G3, M & 300

Mean

394.75

0.4243

0.8753

0.9027

±SD

6.35

0.0273

0.0483

0.0944

n

6

6

6

6

G4, M & 1000

Mean

400.77

0.4233

0.8522

0.9331

±SD

12.55

0.0220

0.0797

0.0373

n

6

6

6

6

M: Male;SD: Standard Deviation; n: Number of animals

TABLE 6 (Contd..,). SUMMARY OF RELATIVE ORGAN WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight)

Fasting Body Weight (g)

Uterus

Ovaries

G1, F & 0

Mean

283.56

0.1693

0.0596

±SD

12.27

0.0106

0.0117

n

6

6

6

G2, F & 100

Mean

298.89

0.1722

0.0572

±SD

29.01

0.0277

0.0127

n

6

6

6

G3, F & 300

Mean

279.78

0.1653

0.0668

±SD

13.38

0.0197

0.0093

n

6

6

6

G4, F & 1000

Mean

278.56

0.1716

0.0650

±SD

23.11

0.0223

0.0096

n

6

6

6

F: Female;SD: Standard Deviation; n: Number of animals

TABLE 7.           SUMMARY OF VAGINAL SMEAR EXAMINATION RECORD FOR DETERMINATION OF OESTRUS CYCLICITY

Group & Dose
(mg/kg body weight)

No. of Females

No. of Females with Regular Oestrus Cyclicity during

Pre-mating, mating and on Lactation day 14

No. of Females with Irregular Oestrus Cyclicity during

Pre-mating, mating and on Lactation day 14

G1 & 0

6

6

0

G2 & 100

6

6

0

G3 & 300

6

6

0

G4 & 1000

6

6

0

TABLE 8.           SUMMARY OF COHABITATION RECORD

Group & Dose
(mg/kg body weight)

Copulatory Interval

(Days)

 

Conceiving Days

(1 to 5)

Conceiving Days

(6 to 20)

G1 & 0

Mean

6.50

n

2

4

±SD

3.33

n

6

%

33.3

66.7

G2 & 100

Mean

5.17

n

4

2

±SD

3.97

n

6

%

66.7

33.3

G3 & 300

Mean

4.00

n

4

2

±SD

3.41

n

6

%

66.7

33.3

G4 & 1000

Mean

5.33

n

4

2

±SD

2.80

n

6

%

66.7

33.3

SD: Standard Deviation; n: Number of animals; %: Percent

TABLE 9.           SUMMARY OF BODY WEIGHT (g) RECORD DURING GESTATION

Group & Dose
(mg/kg body weight)

Body weight (g) on Gestation Days

0

7

14

20

G1 & 0

Mean

265.73

277.18

312.04

378.43

±SD

3.75

6.04

16.21

20.04

n

6

6

6

6

G2 & 100

Mean

268.66

281.41

328.45

398.77

±SD

10.15

10.55

11.72

22.33

n

6

6

6

6

G3 & 300

Mean

264.01

275.66

311.19

380.17

±SD

7.22

3.86

5.59

25.44

n

6

6

6

6

G4 & 1000

Mean

266.89

278.45

313.20

377.31

±SD

5.22

5.19

10.25

24.42

n

6

6

6

6

SD: Standard Deviation; n: Number of animals

TABLE 10.           SUMMARY OF CHANGE IN BODY WEIGHT (%) RECORD DURING GESTATION

Group & Dose
(mg/kg body weight)

Change in body weight (%) during Gestation

0 to 7

7 to 14

14 to 20

0 to 20

G1 & 0

Mean

4.30

12.53

21.31

42.39

±SD

0.98

3.97

3.40

6.73

n

6

6

6

6

G2 & 100

Mean

4.77

16.79

21.36

48.65

±SD

2.17

4.20

3.91

10.80

n

6

6

6

6

G3 & 300

Mean

4.45

12.91

22.10

44.25

±SD

1.90

2.89

6.40

12.93

n

6

6

6

6

G4 & 1000

Mean

4.34

12.50

20.41

41.45

±SD

1.57

3.71

5.16

10.04

n

6

6

6

6

 SD: Standard Deviation; n: Number of animals

TABLE 11.           SUMMARY OF FEED CONSUMPTION (g/dam/day) RECORD DURING GESTATION

Group & Dose
(mg/kg body weight)

Feed Consumption (g) during Gestation

GD 0 to 7

GD 7 to 14

GD 14 to 20

G1 & 0

Mean

22.25

25.64

27.24

±SD

1.53

1.41

1.90

n

6

6

6

G2 & 100

Mean

22.60

25.44

25.49

±SD

1.14

1.93

2.44

n

6

6

6

G3 & 300

Mean

22.73

24.57

25.98

±SD

0.60

2.00

3.02

n

6

6

6

G4 & 1000

Mean

22.33

26.08

25.96

±SD

1.41

2.16

0.89

n

6

6

6

 SD: Standard Deviation; n: Number of animals

TABLE 12.           SUMMARY OF BODY WEIGHT (g) RECORD DURING LACTATION

                                                                                                           

Group & Dose
(mg/kg body weight)

Body Weight (g) on Lactation Day

1

4

7

13

G1 & 0

Mean

279.76

287.68

294.42

304.97

±SD

19.67

21.31

18.89

15.00

n

6

6

6

6

G2 & 100

Mean

292.41

299.66

307.01

320.33

±SD

34.67

35.09

34.00

30.14

n

6

6

6

6

G3 & 300

Mean

276.63

283.55

290.71

301.02

±SD

16.51

16.40

16.14

14.71

n

6

6

6

6

G4 & 1000

Mean

273.45

279.79

286.38

296.82

±SD

23.69

23.94

24.64

24.15

n

6

6

6

6

 SD: Standard Deviation; n: Number of animals

TABLE 13.           SUMMARY OF CHANGE IN BODY WEIGHT (%) RECORD DURING LACTATION

Group & Dose
(mg/kg body weight)

Change in body weight (%) during Lactation

1 to 4

4 to 7

7 to 13

G1 & 0

Mean

2.82

2.41

3.68

±SD

1.67

1.12

2.21

n

6

6

6

G2 & 100

Mean

2.49

2.52

4.52

±SD

1.15

1.02

2.74

n

6

6

6

G3 & 300

Mean

2.51

2.54

3.59

±SD

0.57

0.66

1.59

n

6

6

6

G4 & 1000

Mean

2.33

2.35

3.69

±SD

1.13

0.59

1.51

n

6

6

6

 SD: Standard Deviation; n: Number of animals

TABLE 14.           SUMMARY OF FEED CONSUMTION (g/dam/day) RECORD DURING LACTATION

Group & Dose
(mg/kg body weight)

Feed Consumption (g) during Lactation

LD 1 to 4

LD 4 to 7

LD 7 to 14

G1 & 0

Mean

32.09

40.27

40.91

±SD

2.46

3.65

6.25

n

6

6

6

G2 & 100

Mean

31.75

40.42

43.24

±SD

3.79

5.40

5.29

n

6

6

6

G3 & 300

Mean

29.77

39.58

44.18

±SD

3.34

4.87

5.27

n

6

6

6

G4 & 1000

Mean

32.98

45.05

41.47

±SD

5.93

6.65

6.34

n

6

6

6

 SD: Standard Deviation; n: Number of animals; LD: Lactation Day

TABLE 15.           SUMMARY OF GESTATION LENGTH AND DELIVERY RECORD

Group & Dose
(mg/kg body weight)

 

Gestation Length (Days)

 

Delivery Record at Birth

Litter Size (No.)

Live Pups (No.)

Dead Pups (No.)

Sex Ratio (M/F) at Birth

Live Birth Index
(%)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

22.17

12.33

12.33

6.50

5.83

0.00

0.00

0.00

1.30

100.00

±SD

0.41

2.94

2.94

0.84

2.56

0.00

0.00

0.00

0.59

0.00

n

6

6

6

6

6

6

6

6

6

6

G2 & 100

Mean

21.83

11.50

11.33

4.67*

6.67

0.17

0.00

0.17

0.80*

98.33

±SD

0.41

1.22

1.51

1.75

1.75

0.41

0.00

0.41

0.60

4.08

n

6

6

6

6

6

6

6

6

6

6

G3 & 300

Mean

22.17

12.17

11.50

3.83*

7.67

0.67

0.33

0.33

0.53*

91.82

±SD

0.41

3.54

4.32

1.47

3.27

1.21

0.82

0.52

0.25

17.17

n

6

6

6

6

6

6

6

6

6

6

G4 & 1000

Mean

22.33

12.17

12.00

4.83*

7.17

0.17

0.00

0.17

0.78*

98.72

±SD

1.03

2.32

2.28

0.75

2.23

0.41

0.00

0.41

0.45

3.14

n

6

6

6

6

6

6

6

6

6

6

 M: Male: F: Female;SD: Standard Deviation; n: Number of animals; LD: Lactation Day

    *: Statistically significant than the control group (p<0.05)

TABLE 16.           SUMMARY OF LITTER OBSERVATION DATA DURING LACTATION 

Group & Dose
(mg/kg body weight)

 

During LD 1 to 4

No. of Pups

Sex Ratio (M/F) at LD 4

No. of Survived Pups during

LD 1-4

Pup Survival Index
(%)

Live Pups (No.)

 

 

 

 

 

 

 

 

Dead Pups (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0                  

Mean

12.33

6.50

5.83

0.00

0.00

0.00

1.30

12.33

100.00

±SD

2.94

0.84

2.56

0.00

0.00

0.00

0.59

2.94

0.00

n

6

6

6

6

6

6

6

6

6

G2 & 100                  

Mean

11.33

4.67*

6.67

0.00

0.00

0.00

0.80*

11.33

100.00

±SD

1.51

1.75

1.75

0.00

0.00

0.00

0.60

1.51

0.00

n

6

6

6

6

6

6

6

6

6

G3 & 300                  

Mean

11.50

3.83*

7.67

0.00

0.00

0.00

0.53*

11.50

100.00

±SD

4.32

1.47

3.27

0.00

0.00

0.00

0.25

4.32

0.00

n

6

6

6

6

6

6

6

6

6

G4 & 1000                  

Mean

12.00

4.83*

7.17

0.00

0.00

0.00

0.78*

12.00

100.00

±SD

2.28

0.75

2.23

0.00

0.00

0.00

0.45

2.28

0.00

n

6

6

6

6

6

6

6

6

6

M: Male: F: Female;SD: Standard Deviation; n: Number of animals; LD: Lactation Day

    *: Statistically significant than the control group (p<0.05)

TABLE 16 (Contd..,). SUMMARY OF LITTER OBSERVATION DATA DURING LACTATION

Group & Dose
(mg/kg body weight)

 

During LD 4 to 7

No. of Pups

Sex Ratio

(M/F)

at

LD 7

No. of Survived Pups during

LD 4-7

Pup Survival Index
(%) during

LD 4-7

Live Pups (No.)

Dead Pups (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

12.33

6.50

5.83

0.00

0.00

0.00

1.30

12.33

100.00

±SD

2.94

0.84

2.56

0.00

0.00

0.00

0.59

2.94

0.00

n

6

6

6

6

6

6

6

6

6

G2 & 100

Mean

11.33

4.67*

6.67

0.00

0.00

0.00

0.80*

11.33

100.00

±SD

1.51

1.75

1.75

0.00

0.00

0.00

0.60

1.51

0.00

n

6

6

6

6

6

6

6

6

6

G3 & 300

Mean

11.50

3.83*

7.67

0.00

0.00

0.00

0.53*

11.50

100.00

±SD

4.32

1.47

3.27

0.00

0.00

0.00

0.25

4.32

0.00

n

6

6

6

6

6

6

6

6

6

G4 & 1000

Mean

12.00

4.83*

7.17

0.00

0.00

0.00

0.78*

12.00

100.00

±SD

2.28

0.75

2.23

0.00

0.00

0.00

0.45

2.28

0.00

n

6

6

6

6

6

6

6

6

6

M: Male: F: Female;SD: Standard Deviation; n: Number of animals; LD: Lactation Day

    *: Statistically significant than the control group (p<0.05)

TABLE 16 (Contd..,). SUMMARY OF LITTER OBSERVATION DATA DURING LACTATION

Group & Dose
(mg/kg body weight)

 

During LD 7 to 14

No. of Pups

Sex Ratio (M/F)

at LD 13

No. of Survived Pups during LD 7-13

Pup Survival Index
(%) during

LD 7-13

Live Pups (No.)

Dead Pups (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

12.33

6.50

5.83

0.00

0.00

0.00

1.30

12.33

100.00

±SD

2.94

0.84

2.56

0.00

0.00

0.00

0.59

2.94

0.00

n

6

6

6

6

6

6

6

6

6

G2 & 100

Mean

11.33

4.67*

6.67

0.00

0.00

0.00

0.80*

11.33

100.00

±SD

1.51

1.75

1.75

0.00

0.00

0.00

0.60

1.51

0.00

n

6

6

6

6

6

6

6

6

6

G3 & 300

Mean

11.50

3.83*

7.67

0.00

0.00

0.00

0.53*

11.50

100.00

±SD

4.32

1.47

3.27

0.00

0.00

0.00

0.25

4.32

0.00

n

6

6

6

6

6

6

6

6

6

G4 & 1000

Mean

12.00

4.83*

7.17

0.00

0.00

0.00

0.78*

12.00

100.00

±SD

2.28

0.75

2.23

0.00

0.00

0.00

0.45

2.28

0.00

n

6

6

6

6

6

6

6

6

6

M: Male: F: Female;SD: Standard Deviation; n: Number of animals; LD: Lactation Day

    *: Statistically significant than the control group (p<0.05)

TABLE 17.           SUMMARY OF UTERI OBSERVATION RECORD

Group & Dose
(mg/kg body weight)

No. of Corpora luetea

No. of Implantations

Implantation Index (%)

Pre-Implantation Loss (%)

Post-Implantation Loss (%)

Pre-natal Loss (No.)

Post-natal Loss (At birth to

LD 13)

(%)

Post-natal Loss (At birth to

 LD 13)

(No.)

No. of Early Resorptions (No.)

No. of Late Resorptions (No.)

G1 & 0

Mean

12.83

12.33

95.69

4.31

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.64

2.94

4.75

4.75

0.00

0.00

0.00

0.00

0.00

0.00

n

6

6

6

6

6

6

6

6

6

6

G2 & 100

Mean

11.83

11.50

96.97

3.03

1.67

0.17

0.00

0.00

0.00

0.00

±SD

0.75

1.22

4.69

4.69

4.08

0.41

0.00

0.00

0.00

0.00

n

6

6

6

6

6

6

6

6

6

6

G3 & 300

Mean

12.67

12.17

96.52

3.48

8.18

0.67

0.00

0.00

0.00

0.00

±SD

3.88

3.54

3.98

3.98

17.17

1.21

0.00

0.00

0.00

0.00

n

6

6

6

6

6

6

6

6

6

6

G4 & 1000

Mean

12.50

12.17

97.53

2.47

1.28

0.17

0.00

0.00

0.00

0.00

±SD

2.43

2.32

3.83

3.83

3.14

0.41

0.00

0.00

0.00

0.00

n

6

6

6

6

6

6

6

6

6

6

SD: Standard Deviation; n: Number of animals; LD: Lactation Day

TABLE 18.           SUMMARY OF PUP OBSERVATION RECORD DURING LACTATION

Group & Dose
(mg/kg body weight)

Pup Observation on LD 1

Pup Observation on LD 4

Pup Observation on LD 7

Pup Observation on LD 13

G1 & 0

N

N

N

N

G2 & 100

N

N

N

N

G3 & 300

N

N

N

N

G4 & 1000

N

N

N

N

N: Normal

TABLE 19.           SUMMARY OF PUP WEIGHT (g) RECORD

Group & Dose
(mg/kg body weight)

Pup Weight (g) on

Lactation Day 1

Pup Weight (g) on

Lactation Day 4

Pup Weight (g) on

Lactation Day 7

Pup Weight (g) on

Lactation Day 14

Male

Female

Male

Female

Male

Female

Male

Female

G1 & 0

Mean

6.68

6.24

9.84

8.55

15.22

11.90

26.83

20.61

±SD

0.50

0.17

0.53

0.33

0.49

0.43

0.70

0.61

n

6

6

6

6

6

6

6

6

G2 & 100

Mean

6.86

6.15

10.27

8.81

15.40

12.33

27.18

19.22

±SD

0.40

0.41

0.98

0.45

0.72

0.84

1.08

4.12

n

6

6

6

6

6

6

6

6

G3 & 300

Mean

7.22

6.16

9.67

7.19

15.43

12.36

27.40

21.55

±SD

0.61

0.29

1.58

3.52

0.26

0.57

1.19

0.68

n

6

6

6

6

6

6

6

6

G4 & 1000

Mean

7.01

6.43

10.51

9.07

15.19

12.34

26.48

21.09

±SD

0.75

0.41

0.51

0.46

0.57

0.37

0.50

0.67

n

6

6

6

6

6

6

6

6

SD: Standard Deviation; n: Number of dams

TABLE 20.           SUMMARY OF GROSS PATHOLOGY FINDINGS

Organs/ Lesions

Group

G1

G2

G3

G4

Dose

(mg/kg body weight)

0

100

300

1000

Sex

M

F

M

F

M

F

M

F

No. of Animals

6

6

6

6

6

6

6

6

No Abnormality Detected

6

6

6

6

6

6

6

6

 M: Male; F: Female 

TABLE 20 (Contd..,). SUMMARY OF GROSS PATHOLOGY FINDINGS

Organs/ Lesions

Group

G1

G2

G3

G4

Dose

(mg/kg body weight)

0

100

300

1000

Sex

M

F

M

F

M

F

M

F

No. of Dead pups

(At birth)

0

0

0

1

2

2

0

1

No Abnormality Detected

0

0

0

1

2

2

0

1

 

No. of pups sacrificed on Lactation Day 13

39

35

28

40

23

46

29

43

No Abnormality Detected

39

35

28

40

23

46

29

43

Note:. M: Male; F: Female  

 

 

Conclusions:
Based on the results of the dose-range-finding study discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hostacerin DGSB was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 30 days) and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 under the experimental conditions employed in this study. Based on the results form the dose range finding study, dose levels of 0, 100, 300 and 1000 mg/kg bw/day are recommended for the main OECD 421 study.
Executive summary:

The test item, Hostacerin DGSB was evaluated to generate information on health hazards likely to arise from repeated exposure of test item Hostacerin DGSB when administered to male and female Sprague Dawley rats through oral route over a relatively limited period of time and to select the doses for subsequent Reproduction/Developmental Toxicity Screening Test.

A total of 48 (24 males + 24 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 6 males and 6 females. The animals in G1 group were administered with vehicle [0.5% w/v Carboxy Methyl Cellulose], the animals in G2, G3 and G4groups were administered with test item at the dose levels of100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively. The vehicleand test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The males were treated for two weeks pre-mating, during variable period of mating and up to the day before sacrifice during post-mating period (total of 30 days of treatment). The females were treated for two weeks pre-mating period, during variable period of mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).  

All animals were observed for clinical signs, mortality and morbidity, body weight and feed consumption.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), dead pups born on day 1 and live births were recorded. Body weights of male and female pups were recorded separately on lactation days 1, 4, 7 and 13. 

Gross pathology and organ weighing were performed on day 31 for males and on lactation day 14 for dams. The gross pathology was performed on lactation day 13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

The dams were observed for body weight and feed consumption during gestation and lactation period.

The animals did not reveal any clinical signs of toxicity. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption and in organ weights (both absolute and relative) were observed in both the sex of all the group animals.

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations and pup weights.

The adults and lactation day 13 pups did not reveal any treatment related gross pathological findings.

Based on the results form the dose range finding study, dose levels of 0, 100, 300 and 1000 mg/kg bw/day are recommended for the main OECD 421 study.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-16 to 2018-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN:
- Premating exposure duration for parental (P0) animals: Yes (14 days)
- Basis for dose level selection: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected in consultation with the sponsor based on the results of Dose Range Finding Study for Reproduction/Developmental Toxicity Screening Test of Hostacerin DGSB by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2516).
These doses were selected as the test item Hostacerin DGSB when administered orally to Sprague Dawley males for two weeks prior to mating, during mating and up to the day before sacrifice during post-mating period (total of at 30 days treatment) and females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13, females (dams) were sacrificed on lactation day 14.
- Route of administration: oral (gavage) route
- Other considerations:
choice of species and strain: Rat (Rattus norvegicus) and Sprague Dawley (standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities)
vehicle: The test item was in-soluble in distilled water and forms clear homogenous suspension in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility and suspendibility test results. Hence, 0.5 % w/v Carboxy Methyl Cellulose was selected for the test item formulation preparation.
number of animals: 96 (48 Males + 48 Females); 12 Males + 12 Females/group as per OECD 421 guideline specification
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant Produkte (Deutschland) GmbH
84504 Burgkirchen, Germany and DEG4338635

- Expiration date of the batch: 04.10.2018
- Purity test date: 04.10.2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The stability of the test item in dose formulations was established under Bioneeds Study No.: BIO-ANM 450. The test item formulations were stable for 6 hours at room temperature and for 24 hours at 2 to 8 oC at the concentrations of 10 mg/mL and 100 mg/mL.
- Solubility of the test substance in the solvent/vehicle: 0.5 % w/v Carboxy Methyl Cellulose was used as vehicle for the preparation of formulations as the test item formed clear homogenous suspension in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility and suspendibility test results.

FORM AS APPLIED IN THE TEST: Liquid (Suspension)
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 220.17 g to 255.97 g; Females: 201.51 g to 231.96 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
i. Pre mating
Two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- Diet: Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co.
- Water: Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period (Including Oestrus Cycle Evaluation): 18 September 2017 to 06 October 2017.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 23.4°C
- Humidity (%): 43 to 69%
- Air changes (per hr): 12 to 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

IN-LIFE DATES: From: To: 18 September 2017 to 10 December 2017 (till end of last female necropsy)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % w/v Carboxy Methyl Cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle : The test item was in-soluble in distilled water and formed clear homogenous suspension in 0.5 % w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility and suspendibility test results. Hence, 0.5 % w/v Carboxy Methyl Cellulose was selected for the test item formulation preparation.
- Concentration in vehicle: G1 (vehicle control): 0 mg/mL; G2 (low dose): 10 mg/mL; G3 (mid dose): 30 mg/mL; G4 (high dose): 100 mg/mL
- Amount of vehicle: 10 mL/kg body weight
- Batch no.: BCBN1690V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited.
The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 450. Sampling and analysis of formulations was performed during first week of treatment and during week 4 of the treatment. The samples were collected in duplicates (20 mL each) from top, middle and bottom layer from low and mid dose concentrations, in duplicates (5 mL each) from top, middle and bottom layer from high dose concentrations and from middle layer in duplicate sets from vehicle control.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup purpose at 2 to 8°C conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Duration of treatment / exposure:
Males: two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days of treatment).
Females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 14 to 15 weeks
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
12 animals per sex per dose (group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control, low dose, mid dose and high dose respectively were selected in consultation with the sponsor based on the results of Dose Range Finding Study for Reproduction/Developmental Toxicity Screening Test of Hostacerin DGSB by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2516).
These doses were selected as the test item Hostacerin DGSB when administered orally to Sprague Dawley males for two weeks prior to mating, during mating and up to the day before sacrifice during post-mating period (total of at 30 days treatment) and females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13, females (dams) were sacrificed on lactation day 14 did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).
- Rationale for animal assignment:
The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+12.02% and -11.65% for males and +12.09% and -10.47% for females) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment. Body weight of the animals was analyzed statistically for mean body weight to rule out the statistical significant difference between groups within each sex.
Positive control:
not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table 1 were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once were included in (table 1)

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing, weekly thereafter and at termination. The
females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 1
3 during lactation period. (table 2, 3, 9, 10, 12 & 13)

FOOD CONSUMPTION: Food consumption for each animal determined and mean daily diet consumption calculated as
g food/kg body weight/day: Yes; weekly once; gestation days 0 to 7, 7 to 14 and 14 to 20 during
pregnancy and day 1 to 4, 4 to 7, 7 to 13 during lactation period. (table 4, 11 & 14)
Oestrous cyclicity (parental animals):
Two weeks after five days of acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days), daily from the beginning of the treatment period until evidence of mating and on the day of sacrifice for females.
Sperm parameters (parental animals):
Parameters examined: testis weight, epididymis weight
Special emphasis on stages of spermatogenesis and histology of interstitial testicular cell structure.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals [after completion of mating period (34 days of treatment)]
- Maternal animals: All animals [on lactation day 14]

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [26 and 27] (i.e. Epidymides, Prostate and Seminal vesicles and coagulating glands, Testes Uterus with cervix Thyroid along with parathyroid) were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed on lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid along with parathyroid was collected from one male and one female pup per litter on lactation day 13 and were prepared for microscopic examination
Statistics:
The data was subjected to various statistical analysis using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05)
The statistical analysis was followed to the parameters as mentioned below table: Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Organ
weights, Anogenital distance, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal loss, Post implantation loss:- Parametric - One-way ANOVA with Dunnett’s post test.
Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight:- Non Parametric - Kruskal-Wallis followed by the Mann-Whitney test. Pregnancy rate. Dams with live pups, Dams with dead pups, No. of litters with/without dead pups, No.of litters with/without resorptions:- Cross Tabs - Chi-square test/ Fischer's Exact Test.
Reproductive indices:
Male Fertility Index (%) = Number of Males Impregnating Females
----------------------------------------------------------- x 100
Number of Males used for Mating

Female Fertility Index (%) = Number of Females confirmed with Pregnancy
------------------------------------------------------------------ x 100
Number of Females used for Mating

Parturition (%) = Number of Parturitions
--------------------------------- x 100
Number of Pregnancies

Copulatory Interval (Days) = Date of Initiation of Cohabitation - Date of Confirmation of Mating
Gestation Length (days) = Date of Delivery - Date of Confirmation of Mating
Offspring viability indices:
Male/Female Sex Ratio = Number Male Pups Per Dam
------------------------------------------
Number Female Pups Per Dam

Live Birth Index (%) = Number of Viable Pups Born
----------------------------------------- x 100
Number of Pups Born
Lactation Day 4
Pup Survival Index (%) = Number of Viable Pups on Lactation Day 4
---------------------------------------------------------------x 100
Number of Pups Born
Lactation Day 7
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 7
--------------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 4

Lactation Day 13
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 13
---------------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 7
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity observed at any of the tested dose group animals of either sex during the experimental period.
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes observed in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose group of either sex during the experimental period.
There were no treatment related changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals. However, statistical significant increase in mean body weight on gestation day 14 at G3 group animals was observed when compared with vehicle control group animals. This change is considered as incidental as there were no changes noted during the other gestation periods and there were no effects noted in feed consumption at this period.
There were no treatment related changes observed in the lactation body weight and percent change in lactation body weight at any of the tested dose group animals when compared with vehicle control group animals.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes observed in feed consumption at any of the tested dose group animals of either sex during the experimental period.
There were no changes observed in the feed consumption during gestation period at any of the tested dose group animals when compared with vehicle control group animals. However, statistical significant increase in mean feed consumption during gestation day 0 to 7 was noted at low and high dose group animals when compared with vehicle control group animals. This change is considered as incidental and not treatment related as there were no effects noted in mean body weight at this duration and the change is inconsistent.
There were no changes observed in the feed consumption during lactation period at any of the tested dose group animals when compared with vehicle control group animals.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in serum T4 levels in males, females (pregnant), lactation day 4 and 13 pups at any of the tested dose groups when compared with vehicle control group.
However, statistically significant decrease in T4 levels at all the tested dose group males when compared with vehicle control group males and statistically significant decrease in T4 levels at mid and high dose group females when compared with vehicle control group females was noted.
This significant variation could be due to two males in G1 group (Animal No. Rc3917 and Rc3922) with a serum T4 level of 139.88 and 134.97 respectively which are significantly more than other animals of same group. In case of females the variation is not dose dependant. Hence, the variations observed in the T4 levels are considered to be incidental.
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related histopathological findings noticed in ovaries, testes and epididymides (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) of the animals from the high dose group during histopathological examination.
Minimal grade and focally distributed interstitial mononuclear cell [MNC] infiltration, unilateral was observed in epididymis of one male animal of G4 group. This lesion was considered as incidental finding because of lack of consistency and similar finding was observed in concurrent control animal.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes observed in the oestrus cyclicity at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
no effects were noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
No effects were noted. There were no treatment related changes observed in the copulatory interval and number of conceiving days at any of the tested dose group animals when compared with vehicle control group animals. There were no changes observed in the gestation length, implantation index, pre-implantation loss, post-implantation loss, number of pups delivered, sex ratio and live birth index of each litter at any of the tested dose group animals when compared with vehicle control group animals.
no treatment elated effects were noted in reproductive and developmental end points at all the tested dose groups
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Mean live birth index is 99.3, 87.85, 98.52 and 100 % for dose groups G1, G2, G3, and G4, respectively. Total number of dead pups at birth were 1, 3, 2 and 0 for dose groups G1, G2, G3, and G4, respectively.
Mean pup survival index (LD 1-4) is 98.48, 100, 100, and 100 % for dose groups G1, G2, G3, and G4, respectively. Total number of dead pups were 2, 0, 0 and 0 for dose groups G1, G2, G3, and G4, respectively.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no changes observed in anogenital distance ratio of both male and female pups, recorded on lactation day 4 at any dose group litters when compared with vehicle control group litters.
There were no occurrences of nipples in male pups of dams of the tested dose group litters when compared with vehicle control group litters.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment related changes observed in serum T4 levels in lactation day 4 and 13 pups at any of tested dose groups when compared with vehicle control group.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect s observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

TABLE 1.        SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD

                                                                                                          

Group, Sex & Dose

(mg/kg body weight/day)

No. of Animals

Clinical Signs of Toxicity/

Detailed Clinical Examination

Mortality

(No. of Mortality/

No. of Animals dosed)

G1, M & 0

12

N

0/12

G2, M & 100

12

N

0/12

G3, M & 300

12

N

0/12

G4, M & 1000

12

N

0/12

M: Male; N: Normal

TABLE 1 (Contd..,). SUMMARYOF CLINICAL SIGNSOF TOXICITY, DETAILED CLINICAL EXAMINATIONAND

MORTALITY RECORD

      

Group, Sex & Dose

(mg/kg body weight/day)

No. of Animals

Clinical Signs of Toxicity/

Detailed Clinical Examination

Mortality

(No. of Mortality/

No. of Animals dosed)

G1, F & 0

12

N

0/12

G2, F & 100

12

N

0/12

G3, F & 300

12

N

0/12

G4, F & 1000

12

N

0/12

F: Female; N: Normal

TABLE 2.        SUMMARY OF BODY WEIGHT (g)RECORD

    

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Days

1

7

14

21

28

34

G1, M & 0

Mean

331.48

355.57

372.35

392.61

408.21

429.21

±SD

21.09

25.46

26.61

30.62

31.33

35.64

n

12

12

12

12

12

12

G2, M & 100

Mean

333.49

348.67

366.07

382.71

394.48

414.61

±SD

20.91

24.45

30.62

31.50

33.73

39.05

n

12

12

12

12

12

12

G3, M & 300

Mean

333.86

349.97

361.35

384.41

399.83

423.79

±SD

20.50

18.44

22.25

23.24

26.15

27.88

n

12

12

12

12

12

12

G4, M & 1000

Mean

331.99

357.47

378.38

396.73

411.93

426.47

±SD

18.56

18.42

19.85

21.46

21.62

23.80

n

12

12

12

12

12

12

  M: Male; SD: Standard Deviation; n: Number of Animals

 

TABLE 2 (Contd..,). SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Days

1

7

14

21#

G1, F & 0

Mean

263.38

269.72

279.18

292.40

±SD

16.71

16.23

19.53

22.58

n

12

12

12

4

G2, F & 100

Mean

263.21

269.81

281.42

292.27

±SD

16.04

13.15

13.93

21.16

n

12

12

12

5

G3, F & 300

Mean

263.38

269.54

281.01

281.16

±SD

15.85

13.80

16.37

21.01

n

12

12

12

6

G4, F & 1000

Mean

262.96

269.34

286.13

284.89

±SD

14.76

14.63

15.25

19.99

n

12

12

12

5

F: Female; SD: Standard Deviation; n: Number of Animals

#: The statistical analysis is not performed for day 21 body weight as the presented mean and standard deviations were calculated only for the females in cohabitation.


TABLE 3.  SUMMARY OF PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

                                      

Group, Sex & Dose (mg/kg body weight/day)

Percent Change in Body Weight (%) with respect to Day 1 during Days

1 to 7

1 to 14

1 to 21

1 to 28

1 to 34

G1, M & 0

Mean

7.23

12.33

18.43

23.16

29.51

±SD

2.62

4.24

5.26

5.83

7.60

n

12

12

12

12

12

G2, M & 100

Mean

4.55

9.74

14.78

18.31

24.26

±SD

2.94

5.40

6.29

6.98

7.56

n

12

12

12

12

12

G3, M & 300

Mean

4.94

8.42

15.37

19.94

27.21

±SD

3.94

6.65

7.45

7.38

9.22

n

12

12

12

12

12

G4, M & 1000

Mean

7.79

14.23

19.91

24.49

28.83

±SD

4.61

7.57

9.96

10.01

9.93

n

12

12

12

12

12

  M: Male; SD: Standard Deviation; n: Number of Animals

 

TABLE 3 (Contd..,). SUMMARY OF PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1RECORD

 

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) with respect to Day 1 during Days

1 to 7

1 to 14

1 to 21#

G1, F & 0

Mean

2.45

6.02

9.31

±SD

1.97

3.71

5.11

n

12

12

4

G2, F & 100

Mean

2.59

7.10

11.07

±SD

2.35

5.26

4.99

n

12

12

5

G3, F & 300

Mean

2.42

6.86

8.90

±SD

3.00

6.13

6.84

n

12

12

6

G4, F & 1000

Mean

2.45

8.87

11.94

±SD

1.38

3.28

8.06

n

12

12

5

   F: Female; SD: Standard Deviation; n: Number of Animals

  #: The statistical analysis is not performed for day 21 body weight as the presented mean and standard deviations were calculated only for the females in cohabitation.


TABLE 4.  SUMMARY OFFEED CONSUMPTION (g/rat/day)RECORD

Group, Sex & Dose
(mg/kg body weight/day)

Feed Consumption (g)

Week 1

(Day 1 to 7)

Week 2

(Day 7 to 14)

Post Mating

(Day 28 to 34)

G1, M & 0

Mean

23.12

22.81

29.41

±SD

1.34

2.28

0.67

n

12

12

12

G2, M & 100

Mean

22.76

23.08

28.95

±SD

1.71

1.95

0.76

n

12

12

12

G3, M & 300

Mean

22.66

23.23

29.49

±SD

1.48

2.35

0.66

n

12

12

12

G4, M & 1000

Mean

24.16

24.26

29.68

±SD

1.12

1.73

0.66

n

12

12

12

M: Male;SD: Standard Deviation; n: Number ofanimals

 

TABLE 4 (Contd..,). SUMMARY OF FEED CONSUMPTION (g/rat/day)RECORD

Group, Sex & Dose
(mg/kg body weight/day)

Feed Consumption (g)

Week 1 (Day 1 to 7)

Week 2 (Day 7 to 14)

G1, F & 0

Mean

18.19

18.43

±SD

1.13

1.55

n

12

12

G2, F & 100

Mean

18.85

18.65

±SD

1.93

1.58

n

12

12

G3, F & 300

Mean

17.63

18.21

±SD

1.98

0.86

n

12

12

G4, F & 1000

Mean

18.33

18.60

±SD

1.18

0.80

n

12

12

F: Female; SD: Standard Deviation; n: Number of animals


TABLE 5.           SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose
(mg/kg body weight/day)

Epididymes

Testes

Prostate+Seminal vesicles with coagulating glands (PSC)

Thyroid along with parathyroid#

G1, M & 0

Mean

1.7248

3.3488

3.8053

0.0357

±SD

0.1932

0.2725

0.5705

0.0033

n

12

12

12

12

G2, M & 100

Mean

1.7994

3.3460

3.7837

0.0370

±SD

0.1389

0.2603

0.3461

0.0022

n

12

12

12

12

G3, M & 300

Mean

1.6893

3.2506

4.1135

0.0359

±SD

0.1685

0.2056

0.2480

0.0027

n

12

12

12

12

G4, M & 1000

Mean

1.7526

3.3840

4.1498

0.0363

±SD

0.1925

0.2029

0.4138

0.0020

n

12

12

12

12

M: Male;SD: Standard Deviation; n: Number of animals: #: Weighed post fixation


TABLE 5 (Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

                                                                                                       

Group, Sex & Dose
(mg/kg body weight/day)

 

Uterus

Thyroid along with parathyroid#

G1, F & 0

Mean

0.6154

0.0291

±SD

0.0395

0.0031

n

11

11

G2, F & 100

Mean

0.5932

0.0253

±SD

0.0270

0.0033

n

11

11

G3, F & 300

Mean

0.6264

0.0279

±SD

0.0764

0.0056

n

11

11

G4, F & 1000

Mean

0.5864

0.0306

±SD

0.0420

0.0048

n

12

12

 F: Female;SD: Standard Deviation; n: Number of dams;

#: Weighed post fixation (excluding non-pregnant animals)


TABLE 6.           SUMMARY OF FASTING BODY WEIGHT (g) AND
ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Fasting Body Weight (g)

Epididymes

Testes

Prostate+Seminal vesicles with coagulating glands (PSC)

Thyroid along with parathyroid

G1, M & 0

Mean

403.62

0.4300

0.8341

0.9467

0.0089

±SD

34.33

0.0578

0.0859

0.1466

0.0011

n

12

12

12

12

12

G2, M & 100

Mean

391.26

0.4644

0.8645

0.9795

0.0095

±SD

37.96

0.0638

0.1260

0.1606

0.0010

n

12

12

12

12

12

G3, M & 300

Mean

397.31

0.4275

0.8208

1.0396

0.0091

±SD

26.57

0.0570

0.0674

0.0932

0.0008

n

12

12

12

12

12

G4, M & 1000

Mean

399.57

0.4393

0.8503

1.0407

0.0091

±SD

24.64

0.0473

0.0805

0.1104

0.0008

n

12

12

12

12

12

M: Male;SD: Standard Deviation; n: Number of animals

TABLE 6 (Contd..,). SUMMARY OF FASTING BODY WEIGHT (g) AND
ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Fasting Body Weight
(g)

Uterus

Thyroid along with parathyroid

G1, F & 0

Mean

304.24

0.2035

0.0096

±SD

21.64

0.0224

0.0013

n

11

11

11

G2, F & 100

Mean

306.66

0.1943

0.0083

±SD

23.92

0.0148

0.0012

n

11

11

11

G3, F & 300

Mean

324.21

0.1933

0.0087

±SD

28.55

0.0177

0.0022

n

11

11

11

G4, F & 1000

Mean

306.69

0.1921

0.0101

±SD

24.23

0.0182

0.0020

n

12

12

12

 F: Female;SD: Standard Deviation; n: Number of animals

TABLE 7.           SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Group & Dose
(mg/kg body weight/day)

No. of Females

No. of Females with Regular Oestrus Cyclicity during

Pre-mating, mating and on Lactation day 14

No. of Females with Irregular Oestrus Cyclicity during

Pre-mating, mating and on Lactation day 14

G1 & 0

12

12

0

G2 & 100

12

12

0

G3 & 300

12

12

0

G4 & 1000

12

12

0

TABLE 8.           SUMMARY OF COHABITATION RECORD

Group & Dose
(mg/kg body weight/day)

Copulatory Interval

(Days)

 

Conceiving Days

(1 to 5)

Conceiving Days

(6 to 20)

G1 & 0

Mean

5.83

n

8

4

±SD

4.63

n

12

%

66.7

33.3

G2 & 100

Mean

7.00

n

6

6

±SD

4.24

n

12

%

50.0

50.0

G3 & 300

Mean

7.42

n

5

7

±SD

4.60

n

12

%

41.7

58.3

G4 & 1000

Mean

7.58

n

6

6

±SD

4.80

n

12

%

50.0

50.0

SD: Standard Deviation; n: Number of animals; %: Percent

TABLE 9.           SUMMARY RECORD OF BODY WEIGHT (g) DURINGGESTATION PERIOD

Group & Dose
(mg/kg body weight/day)

Body weight (g) on Gestation Days

0

7

14

20

G1 & 0                  

Mean

283.86

297.35

342.70

421.08

±SD

21.63

21.53

28.88

20.97

n

11

11

11

11

G2 & 100                  

Mean

291.15

304.38

349.07

422.35

±SD

15.19

13.65

19.69

17.65

n

11

11

11

11

G3 & 300                  

Mean

291.93

307.30

364.18*

440.73

±SD

11.96

11.79

12.56

22.28

n

11

11

11

11

G4 & 1000                  

Mean

292.90

305.82

358.55

429.48

±SD

14.68

15.66

17.10

24.61

n

12

12

12

12

SD: Standard Deviation; n: Number of dams

*: Statistically significant than the control group (p<0.05) 

TABLE 10.       SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT (%) DURING GESTATION PERIOD

Group & Dose
(mg/kg body weight/day)

Percent Change in body weight (%) during Gestation

0 to 7

7 to 14

14 to 20

G1 & 0                    

Mean

4.78

15.24

23.31

±SD

0.82

4.95

6.98

n

11

11

11

G2 & 100       

Mean

4.59

14.70

21.17

±SD

1.38

4.36

5.18

n

11

11

11

G3 & 300       

Mean

5.28

18.57

21.02

±SD

1.78

3.46

4.51

n

11

11

11

G4 & 1000       

Mean

4.41

17.30

19.82

±SD

0.92

2.71

4.74

n

12

12

12

SD: Standard Deviation; n: Number of dams

TABLE 11.       SUMMARY RECORD OF FEED CONSUMPTION (g/dam/day) DURING GESTATION PERIOD

Group, Sex & Dose
(mg/kg body weight/day)

Feed Consumption (g) during Gestation

GD 0 to 7

GD 7 to 14

GD 14 to 20

G1 & 0

Mean

22.64

26.85

30.28

±SD

1.35

1.46

1.12

n

11

11

11

G2 & 100

Mean

23.92*

27.62

30.43

±SD

0.79

0.96

0.91

n

11

11

11

G3 & 300

Mean

23.19

27.05

30.00

±SD

0.89

1.49

1.06

n

11

11

11

G4 & 1000

Mean

23.92*

26.79

30.47

±SD

0.78

1.12

0.69

n

12

12

12

SD: Standard Deviation; n: Number of dams
*: Statistically significant than the control group (p<0.05)

TABLE 12.       SUMMARY RECORD OF BODY WEIGHT (g) DURING LACTATION PERIOD

Group, Sex & Dose
(mg/kg body weight/day)

Body Weight (g) on Lactation Day

1

4

7

13

G1 & 0                    

Mean

309.57

313.98

320.23

327.06

±SD

21.76

22.19

24.05

21.94

n

11

11

11

11

G2 & 100       

Mean

307.28

313.82

318.42

329.52

±SD

30.13

28.65

28.85

23.41

n

11

11

11

11

G3 & 300       

Mean

327.46

335.18

341.73

346.58

±SD

35.21

31.80

31.26

28.52

n

11

11

11

11

G4 & 1000       

Mean

306.28

313.07

318.52

327.51

±SD

33.02

28.48

27.29

24.06

n

12

12

12

12

SD: Standard Deviation; n: Number of animals

TABLE 13.       SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION PERIOD

Group, Sex & Dose
(mg/kg body weight/day)

Percent Change in body weight (%) during Lactation

1 to 4

4 to 7

7 to 13

G1 & 0                    

Mean

1.42

1.97

2.20

±SD

0.46

1.22

1.28

n

11

11

11

G2 & 100       

Mean

2.20

1.48

3.70

±SD

1.85

0.49

3.39

n

11

11

11

G3 & 300       

Mean

2.50

1.98

1.50

±SD

2.26

0.89

1.15

n

11

11

11

G4 & 1000       

Mean

2.42

1.79

2.93

±SD

2.73

0.64

1.70

n

12

12

12

SD: Standard Deviation; n: Number of dams    

                                                                      

TABLE 14.       SUMMARY RECORD OF FEED CONSUMTION (g/dam/day) DURING LACTATION PERIOD

Group & Dose
(mg/kg body weight/day)

Feed Consumption (g) during Lactation

LD 1 to 4

LD 4 to 7

LD 7 to 13

G1 & 0                     

Mean

23.34

27.53

32.34

±SD

1.26

1.55

1.22

n

11

11

11

G2 & 100                     

Mean

24.65

29.18

33.01

±SD

2.32

2.43

1.04

n

11

11

11

G3 & 300                     

Mean

22.80

28.77

33.44

±SD

1.89

3.14

2.17

n

11

11

11

G4 & 1000                     

Mean

24.67

28.77

33.64

±SD

3.09

2.52

1.33

n

12

12

12

SD: Standard Deviation; n: Number of dams; LD: Lactation Day

 TABLE 15.       SUMMARY RECORD OF GESTATION LENGTH (DAYS) AND DELIVERY DATA

Group & Dose
(mg/kg body weight/day)

 

Gestation Length (Days)

 

Delivery Data

Litter Size (No.)

Live Pups (No.)

Dead Pups (No.)

 

Sex Ratio (M/F)

at Birth

Live Birth Index
(%)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0

Mean

22.64

12.64

12.55

5.91

6.64

0.09

0.00

0.09

1.13

99.30

±SD

1.03

2.06

2.07

1.76

2.84

0.30

0.00

0.30

0.71

2.32

n

11

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

22.55

13.73

13.45

7.00

6.45

0.27

0.09

0.18

1.14

97.85

±SD

1.13

2.15

2.30

1.95

1.51

0.47

0.30

0.40

0.42

3.74

n

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

22.73

13.00

12.82

6.36

6.45

0.18

0.00

0.18

1.16

98.52

±SD

1.42

1.95

2.04

1.86

2.21

0.40

0.00

0.40

0.69

3.31

n

11

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

22.83

11.83

11.83

5.58

6.25

0.00

0.00

0.00

0.92

100.00

±SD

1.27

2.17

2.17

2.15

0.87

0.00

0.00

0.00

0.38

0.00

n

12

12

12

12

12

12

12

12

12

12

  M: Male; F: Female;SD: Standard Deviation; n: Number of dams; NAD: No abnormality detected; LD: Lactation Day

 

TABLE 16.       SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose
(mg/kg body weight/day)

 

No. of Live Pups At Birth

During LD 1 to 4

Sex Ratio (M/F)
at

LD 4

No. of Survived Pups during LD 1 to 4

Pup Survival Index
(%)

LD 1 to 4

Live Pups (No.)

 

 

 

 

 

Dead Pups (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

G1 & 0                  

Mean

12.55

12.36

5.91

6.45

0.18

0.00

0.18

1.15

11.33

98.48

±SD

2.07

2.20

1.76

2.81

0.60

0.00

0.60

0.70

4.14

5.03

n

11

11

11

11

11

11

11

11

12

11

G2 & 100                  

Mean

13.45

13.45

7.00

6.45

0.00

0.00

0.00

1.14

12.33

100.00

±SD

2.30

2.30

1.95

1.51

0.00

0.00

0.00

0.42

4.46

0.00

n

11

11

11

11

11

11

11

11

12

11

G3 & 300                  

Mean

12.82

12.82

6.36

6.45

0.00

0.00

0.00

1.16

11.75

100.00

±SD

2.04

2.04

1.86

2.21

0.00

0.00

0.00

0.69

4.18

0.00

n

11

11

11

11

11

11

11

11

12

11

G4 & 1000                  

Mean

11.83

11.83

5.58

6.25

0.00

0.00

0.00

0.92

11.83

100.00

±SD

2.17

2.17

2.15

0.87

0.00

0.00

0.00

0.38

2.17

0.00

n

12

12

12

12

12

12

12

12

12

12

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

TABLE 16 (Contd..,). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose
(mg/kg body weight/

day)

Live Pups (No.) on LD 4

 

 

 

 

 

 

 

 

Pups Sacrificed for Blood Collection on LD 4 (No.)

 

 

 

 

 

 

 

 

Live Pups (No.) on LD 4 after Sacrificed for Blood Collection

During LD 4 to 7

 

 

 

 

 

 

 

 

Sex Ratio (M/F) at
LD 7

No. of Survived Pups during LD 4-7

Pup Survival Index
(%) during LD 4-7

Live Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

 

 

 

 

 

 

 

Male (No.)

Female (No.)

Total (No.)

G1 &
0

Mean

5.91

6.45

12.36

0.00

1.36

1.36

5.91

5.09

11.00

5.91

5.09

11.00

0.00

0.00

0.00

1.62

11.00

100.00

±SD

1.76

2.81

2.20

0.00

0.81

0.81

1.76

2.47

1.55

1.76

2.47

1.55

0.00

0.00

0.00

1.32

1.55

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G2 &
 100

Mean

7.00

6.45

13.45

0.00

1.64

1.64

7.00

4.82

11.82

7.00

4.82

11.82

0.00

0.00

0.00

1.65

11.82

100.00

±SD

1.95

1.51

2.30

0.00

0.67

0.67

1.95

1.47

1.83

1.95

1.47

1.83

0.00

0.00

0.00

0.81

1.83

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G3 &
 300

Mean

6.36

6.45

12.82

0.00

1.55

1.55

6.36

4.91

11.27

6.36

4.91

11.27

0.00

0.00

0.00

1.75

11.27

100.00

±SD

1.86

2.21

2.04

0.00

0.82

0.82

1.86

2.12

1.49

1.86

2.12

1.49

0.00

0.00

0.00

1.29

1.49

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

5.58

6.25

11.83

0.00

1.00

1.00

5.58

5.25

10.83

5.58

5.25

10.83

0.00

0.00

0.00

1.17

10.83

100.00

±SD

2.15

0.87

2.17

0.00

0.95

0.95

2.15

1.06

1.40

2.15

1.06

1.40

0.00

0.00

0.00

0.64

1.40

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

TABLE 16 (Contd..,). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose
(mg/kg body weight/day)

Live Pups (No.) on LD 7

During LD 7 to 13

 

Sex Ratio (M/F) at

LD 7

No. of Survived Pups during LD
7 to 13

Pup Survival Index
(%) during LD
7 to 13

Live Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

G1 & 0

Mean

5.91

5.09

11.00

5.91

5.09

11.00

0.00

0.00

0.00

1.62

11.00

100.00

±SD

1.76

2.47

1.55

1.76

2.47

1.55

0.00

0.00

0.00

1.32

1.55

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

7.00

4.82

11.82

7.00

4.82

11.82

0.00

0.00

0.00

1.65

11.82

100.00

±SD

1.95

1.47

1.83

1.95

1.47

1.83

0.00

0.00

0.00

0.81

1.83

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

6.36

4.91

11.27

6.36

4.91

11.27

0.00

0.00

0.00

1.75

11.27

100.00

±SD

1.86

2.12

1.49

1.86

2.12

1.49

0.00

0.00

0.00

1.29

1.49

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

5.58

5.25

10.83

5.58

5.25

10.83

0.00

0.00

0.00

1.17

10.83

100.00

±SD

2.15

1.06

1.40

2.15

1.06

1.40

0.00

0.00

0.00

0.64

1.40

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

M: Male: F: Female;SD: Standard Deviation; n: Number of dams; LD: Lactation Day

 

TABLE 17.       SUMMARY OF PUP OBSERVATIONS RECORD DURING LACTATION PERIOD

Group & Dose

(mg/kg body weight/day)

Pup Observation on LD 1

Pup Observation on LD 4

Pup Observation on LD 7

Pup Observation on LD 13

G1 & 0

N

N

N

N

G2 & 100

N

N

N

N

G3 & 300

N

N

N

N

G4 & 1000

N

N

N

N

LD: Lactation Day; N: Normal

 

 TABLE 18.          SUMMARY RECORD OF MEAN PUP WEIGHT (g) DURING LACTATION PERIOD

Group& Dose
(mg/kg body weight/day)

Mean Pup weight (g) on LD 1

 

Mean Pup weight (g) on LD 4

Mean Pup Weight (g) on
LD 7

Mean Pup weight (g) on LD 13

Male

Female

Male

Female

Male

Female

Male

Female

G1 & 0

Mean

6.68

5.98

9.99

8.52

14.55

12.15

25.65

21.90

±SD

0.49

0.66

0.76

0.74

1.12

1.26

1.63

2.36

n

11

11

11

11

11

11

11

11

G2 & 100

Mean

6.24

5.30

10.26

8.89

14.99

13.13

27.22

22.26

±SD

0.68

0.83

1.15

1.11

1.54

1.82

2.26

2.16

n

11

11

11

11

11

11

11

11

G3 & 300

Mean

6.39

5.76

10.22

8.65

15.63

13.13

27.11

21.93

±SD

0.59

0.66

1.41

0.83

1.14

1.62

1.48

3.26

n

11

11

11

11

11

11

11

11

G4 & 1000

Mean

6.52

5.60

11.19*

9.06

16.03*

13.22

27.29

21.68

±SD

0.64

0.44

1.22

1.36

1.19

1.61

1.90

3.61

n

12

12

12

12

12

12

12

12

SD: Standard Deviation; n: Number of dams;LD: Lactation Day

*: Statistically significant than the control group (p<0.05)

 

 TABLE 19.          SUMMARY OF UTERI OBSERVATIONS RECORD

Group & Dose
(mg/kg body weight/day)

No. of Corporaluetea

No. of Implantations

Pre-Implantation Loss
(%)

Post-Implantation Loss
(%)

Pre-natal Loss
(No.)

Post-natal Loss
(%)

Post-natal Loss
(No.)

No. of Early Resorptions

No. of
Late Resorptions

G1 & 0

Mean

12.82

12.64

1.22

0.70

0.09

1.52

0.18

0.00

0.00

±SD

2.23

2.06

2.72

2.32

0.30

5.03

0.60

0.00

0.00

n

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

13.82

13.73

0.57

2.15

0.27

0.00

0.00

0.00

0.00

±SD

2.23

2.15

1.88

3.74

0.47

0.00

0.00

0.00

0.00

n

11

11

11

11

11

11

11

11

11

G3 & 300

Mean

13.18

13.00

1.36

1.48

0.18

0.00

0.00

0.00

0.00

±SD

1.99

1.95

3.11

3.31

0.40

0.00

0.00

0.00

0.00

n

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

12.00

11.83

1.28

0.00

0.00

0.00

0.00

0.00

0.00

±SD

2.26

2.17

3.05

0.00

0.00

0.00

0.00

0.00

0.00

n

12

12

12

12

12

12

12

12

12

SD: Standard Deviation; n: Number of dams

 

 TABLE 20.          SUMMARY RECORD OF ANO-GENITAL DISTANCE [AGD] RATIO ON LACTATION DAY 4

Group & Dose
(mg/kg body weight/day)

 

Mean Male AGD Ratio

Mean Female AGD Ratio

G1 & 0

Mean

2.53

1.17

±SD

0.18

0.07

n

11

11

G2 & 100

Mean

2.66

1.17

±SD

0.16

0.09

n

11

11

G3 & 300

Mean

2.65

1.18

±SD

0.11

0.07

n

11

11

G4 & 1000

Mean

2.69

1.23

±SD

0.21

0.17

n

12

12

 SD: Standard Deviation; n: Number of dams

 

 TABLE 21.          SUMMARY RECORD OF MALE PUP NIPPLE/AREOLAE RETENTION (no.) ON LACTATION DAY 13

Group & Dose
(mg/kg body weight/day)

No. of Nipples/Areolae

G1 & 0

Mean

0.00

±SD

0.00

n

12

G2 & 100

Mean

0.00

±SD

0.00

n

11

G3 & 300

Mean

0.00

±SD

0.00

n

11

G4 & 1000

Mean

0.00

±SD

0.00

n

11

SD: Standard Deviation; n: Number of dams

TABLE 22.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - MALES

Group, Sex & Dose

(mg/kg body weight/day)

Serum T4 Levels

(ng/mL)

G1, M & 0

Mean

101.492

±SD

18.172

n

12

G2, M & 100

Mean

84.759*

±SD

5.744

n

12

G3, M & 300

Mean

82.176*

±SD

8.380

n

12

G4, M & 1000

Mean

73.659*

±SD

5.815

n

12

M: Male; SD: Standard Deviation; n: Number of animals

 *: Statistically significant than the control group (p<0.05)

                   

TABLE 23.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - FEMALES

Group, Sex & Dose

(mg/kg body weight/day)

Serum T4 Levels

(ng/mL)

G1, F & 0

Mean

78.821

±SD

11.933

n

11

G2, F & 100

Mean

73.067

±SD

7.682

n

11

G3, F & 300

Mean

64.148*

±SD

5.037

n

11

G4, F & 1000

Mean

67.385*

±SD

8.707

n

12

SD: Standard Deviation; n: Number of dams

       *: Statistically significant than the control group (p<0.05)

 

TABLE 24.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - LACTATION DAY 4 PUPS

Group & Dose
(mg/kg body weight/day)

Serum T4 Levels (ng/mL)

G1 & 0

Mean

70.985

±SD

5.708

n

9

G2 & 100

Mean

69.599

±SD

3.939

n

10

G3 & 300

Mean

69.114

±SD

3.762

n

9

G4 & 1000

Mean

70.672

±SD

5.016

n

7

SD: Standard Deviation; n: Number of dams (Serum collected from pups with litter size of more than ten per dam pooled for analysis)

TABLE 25.          SUMMARY OF SERUM T4 LEVELS (ng/mL) RECORD - LACTATION DAY 13 PUPS

Group & Dose
(mg/kg body weight/day)

Serum T4 Levels (ng/mL)

G1 & 0

Mean

73.460

±SD

8.044

n

11

G2 & 100

Mean

71.633

±SD

7.751

n

11

G3 & 300

Mean

71.633

±SD

7.751

n

11

G4 & 1000

Mean

75.088

±SD

7.169

n

12

SD: Standard Deviation; n: Number of dams (Serum collected from pups per dam pooled for analysis 

 

 TABLE 26.          SUMMARY OF GROSS PATHOLOGY RECORD

Organs/Lesions

Group

G1

G2

G3

G4

Dose

(mg/kg body weight/day)

0

100

300

1000

Sex

M

F

M

F

M

F

M

F

No. of Animals

12

12

12

12

12

12

12

12

No Abnormality Detected

12

12

12

12

12

12

12

12

Note: The numeral indicates number of animal/s without lesion. M: Male; F: Female  

TABLE 27.          SUMMARY OF HISTOPATHOLOGY RECORD

 

Group No.

G1

G4

Dose (mg/kg body weight/day)

0

1000

Sex

M

F

M

F

No. of Animals

12

12

12

12

ORGANS AND OBSERVATIONS

EPIDIDYMIDES

 

 

 

 

Interstitial MNC infiltration ; unilateral

1

X

1

X

Note: The numeral indicates number of animal/s with lesion; M: Male; F: Female; X: Organs not subjected for histopathological examination due to sex difference.

 

Conclusions:
Based on the results of the OECD 421 screening study in rats discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hostacerin DGSB was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 observation under the experimental conditions employed in the study.
Executive summary:

The test item, Hostacerin DGSB was evaluated for possible adverse effects following repeated oral dosing to males for 34 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development.

A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle[0.5% w/v Carboxy Methyl Cellulose],animals in G2, G3 and G4groups were administered withtest itemat dose levels of100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 34 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). Dose formulation analysis for homogeneity and concentration verification was performed during week 1 and 4 of the treatment period and the results are found to be within the acceptable range of 85 to 115 % the nominal concentration and the % RSD is less than 10.0%.All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once.The serum collected from adult males and females and from lactation day 4 and 13 pups representing each per litter was screened for T4 levels. Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival indexthroughout the lactation period. The pups were observed forclinical signs and once daily from PND 1 to 13. The both male and female pupweights were recorded separately on lactation days 1, 4, 7 and 13.The anogenital distance of each pup was measured on lactation day 4. The numbers of nipples/areolae in male pups were counted on lactation day 13.Gross pathology and organ weighing were performed on day 35 for males. The gross pathology was performed on lactation day 13 for pups, lactation day 14 for dams. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All animals did not reveal any clinical signs of toxicity, no mortality or morbidity observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, organ weights (both absolute and relative) were observed in both the sex of all tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult animals and in serum T4 levels of pups at any of the tested dose groups.

Females did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, fertility index, body weights and feed consumption during gestation and lactation periods, gestation length, implantation index, pre-implantation loss, post-impalntation loss, number of pups delivered, live birth index of each litter, sex ratio and pup survival index at all the tested dose groups throughout the lactation periodwhen compared with vehicle control group animals.

All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, anogenital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

All animals of all dose groups did not reveal any treatment related gross pathological and histopathological findings compared to vehicle control group.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study is an OECD 421 guideline conform GLP study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

There is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference to the OECD 421 studies performed with the registration substance, it is concluded that the test item is not subject to classification and labelling according to Regulation 1272/2008/EC regarding reproductive toxicity.

Additional information