Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 January 2018 to 05 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Hostacor ITD [Phosphoric acid, isotridecyl ester]

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 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. certificate)
Limit test:
yes
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 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 based on the results of Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Hostacor ITD by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2704).
These doses were selected as the test item Hostacor ITD 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 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 were did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Route of administration: Oral gavage
- Other considerations,
choice of species & strain: Rat (Rattus norvegicus) & Sprague Dawley
vehicle and number of animals: Corn oil and 12 animals per group per sex

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot No.of test material: Clariant Produkte (Deutschland) GmbH
- Expiration date of the lot: 22.02.2022
- Purity test date: 29.03.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: 6 hours at Ambient (21 to 29°C) conditions
- Solubility and stability of the test substance in the solvent/vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house miscibility test results and stable for 6 hours at Ambient (21 to 29°C) conditions


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated in corn oil


FORM AS APPLIED IN THE TEST: Liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat and Sprague-Dawley is one of the standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: Males: 239.19 g to 273.99 g; Females: 211.08 g to 244.79 g
- Housing: 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
- Water (ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: 11 January 2018 to 05 February 2018 [including Including Estrous Cycle Evaluation]
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 23.9 degree C
- Humidity (%): 40 to 69%,
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 11 January 2018; To: 12 April 2018

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The required quantity of test item was weighed into a clean beaker and there by adding little volume of corn oil (vehicle) in to the beaker, mixed well with glass rod and transferred into the measuring cylinder and the beaker was rinsed using a small volume of corn oil and the volume was transferred to measuring cylinder. The rinsing procedure was repeated until entire quantity of the test item formulation was transferred into a 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 as mentioned in the table below

VEHICLE
- Justification for use and choice of vehicle: Corn oil was used as vehicle for the preparation of formulations as evidenced by the in-house miscibility test results
- Concentration in vehicle: 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively
- Amount of vehicle: 10 mL/kg body weight
- batch no.: A1708001
- Purity: 100%
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: minimum: 1 day; maximum: 17 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 such incidence
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and dose formulation analysis was performed during week 1 and 5 of treatment period and the results were found to be within the acceptable range of
85 to 115% the nominal concentration.

The Concentrations: G1: 0 mg/mL; G2: 10 mg/mL; G3: 30 mg/mL; G4: 100 mg/mL
Duration of treatment / exposure:
Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The females were treated for 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: 13 to 14 weeks
Doses / concentrationsopen allclose all
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
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 selectedbased on the results of Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Hostacor ITD by Oral Gavage in Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2704).

These doses were selected as the test item Hostacor ITD 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 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 were did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment (if not random): 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% to +8.89% for males and -11.85% to +11.20% for females) of the mean body weight of each sex.
Positive control:
Not applicable

Examinations

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: Yes
- Time schedule: Weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once and at termination. For females 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


Oestrous cyclicity (parental animals):
Estrous cycles were monitored for two weeks after five days of acclimatization to evaluate its normal estrous cyclicity (4 to 5 days). Only females with normal estrous cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Estrous cyclicity was also monitored on the day of sacrifice.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis 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, 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 as soon as possible after the last litters in each generation were produced.]
- 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 [5, 6 and 27] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The offspring 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 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
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 to day 35 at all the tested group animals of either sex during the experimental period. However, a temporary statistical significant reduction in percent change in body weight was observed in G4 males during day 1 to 8 and day 1 to day 14 when compared with vehicle control group males.

This change is not considered treatment related but incidental and inconsistent as all animals have gained weight in absolute terms and no significant reduction in percent body weight change has been noted when considering the entire experimental period from day 1 to 35, compared to all other groups.

Additionally, no changes in feed consumption and no clinical signs of toxicity were noted during the experimental period.
Food consumption and compound intake (if feeding study):
no effects observed
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.
Description (incidence and severity):
Not applicable
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:
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
Description (incidence and severity):
Not applicable
Description (incidence and severity):
No treatment related histopathological findings noticed.

Lesions considered spontaneous and incidental were observed in treated and control rats. These lesions consisted of unilateral interstitial mononuclear cell (MNC) infiltration in epididymis.

Uterus showed yellow brown colour pigmentation. This pigmentation was observed in G4 and concurrent control group (G1) females. Pigmentation might be from decidualized myometrium because of pregnancy.

A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Not applicable
Other effects:
no effects observed
Description (incidence and severity):
Serum T4 Levels: There were no treatment related changes observed in serum T4 levels in adult males and lactation day 13 pups at any of the tested dose groups when compared with vehicle control group.

Serum Estrogen and Progesterone Levels:

There were no treatment related changes observed in serum Estrogen and Progesterone Levels of dams sacrificed on lactation day 14 at any of the tested dose groups when compared with vehicle control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes observed in the estrous 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 effect of spermatogenesis during histopathology
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect of Mating and Fertiltiy of the animals at all the tested dose groups

Details on results (P0)

The test item Hostacor ITD did not reveal any effects on reproductive performance of male and female rats.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
other: Hormone lelvels [T4, Serum Estrogen and Progesterone Levels]

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs and external anomalies observed in any of the pups of tested dose group animals during lactation period
Description (incidence and severity):
Not applicable
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were mortalities were noticed in the newborn pups from G2 and G3 groups. This mortalities not considered to the treatment related because gross pathology examination did not revealed any pathological changes
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 groups when compared with vehicle control group dams. However, statistical significant reduction in mean female pup weight on lactation day 13 in G3 group was noted when compared with vehicle control group. This change is considered as incidental but not treatment related as there were no abnormalities were noted in this group pups during lactation period and no gross pathological findings noted during necropsy
Food consumption and compound intake (if feeding 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:
not examined
Description (incidence and severity):
Not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
Not applicable
Sexual maturation:
not examined
Description (incidence and severity):
Not applicable
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
Not applicable
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological lesions noticed at necropsy of males and females as well as pups (both live and dead)
Histopathological findings:
not examined
Description (incidence and severity):
Not applicable

Details on results (F1)

No treatment related effects noted at F1 generation pups at all the tested dose groups

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
other: Srum T4 Levels of PND13 pups

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

SUMMARY OF THE STUDY


Parameters ↓

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

G1 & 0

G2 & 100

G3 & 300

G4 & 1000

Reproductive Indices

Mating Indices 

Pairs started (No.)

12

12

12

12

Males showing evidence of mating (No.) within 14 days

9

10

10

9

Male Mating Index (%) within 14 days

75.00

83.33

83.33

75.00

Females showing evidence of copulation (No.)

12

12

12

12

Female Mating Index (%)

100.00

100.00

100.00

100.00

 

Fertility Indices

Females achieving pregnancy (No.)

11

12

11

12

Female Fertility Index (%)

91.67

100.00

91.67

100.00

 

Copulatory Indices

Conceiving days 1 to 5 (No.)

4

5

7

4

Conceiving days >6 (No.)

8

7

5

8

Mean Precoital Interval (Days)

10.33

7.67

6.50

9.92

 

Gestation Indices

Pregnancy≤21 days (No.)

0

1

0

0

Pregnancy = 22 days (No.)

4

5

4

4

Pregnancy≥23 days (No.)

7

6

7

8

Mean Gestation length (Days)

22.82

22.50

22.64

22.83

Gestation Index (%)

100.00

100.00

100.00

100.00

 

Dams with live young born (No.)

11

12

11

12

Dams with live young at day 4 post-partum (No.)

11

12

11

12

Dams with live young at day 13 post-partum (No.)

11

12

11

12

 

Implantation Index and Pre and Post implantation Losses 

Implants/dam (Mean)

11.00

11.83

11.82

10.17

Corpora luetea/dam (Mean)

11.09

11.83

11.82

10.17

Implantation Index (%)

98.99

100.00

100.00

100.00

Pre-Implantation Loss (%)

1.01

0.00

0.00

0.00

Post-Implantation Loss (%)

0.00

0.64

0.76

0.00

  

Offspring Viability Indices

Live Birth Indices and Sex Ratio at Birth 

Live pups/dam at birth (mean)

11.00

11.75

11.73

10.08

Litter Size (Total No. of pups born/dam) at birth (mean)

11.00

11.75

11.73

10.08

Mean Live Birth Index/dam (%)

100.00

99.36

99.24

100.00

Male Live pups/dam at birth (mean)

5.36

6.00

6.36

5.75

Female Live pups/dam at birth (mean)

5.64

5.75

5.36

4.33

Sex Ratio (male/female)

1.00

1.17

1.27

1.54

SUMMARY OF THE STUDY (Contd..,).

Parameters ↓

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

G1 & 0

G2 & 100

G3 & 300

G4 & 1000

Pup Survival Indices and Sex Ratio during lactation  

Mean No. of Pups survived per dam ( LD1 to 4)

11.00

11.75

11.73

10.08

Mean No. of Pups dead per dam ( LD1 to 4)

0.00

0.00

0.00

0.00

Mean Pup Survival Index (%) per dam (LD1 to 4) 

100.00

100.00

100.00

100.00

Sex Ratio (male/female) per dam at LD 4

1.00

1.17

1.27

1.54

Mean No. of Pups Sacrificed for Blood Collection on LD4

1.00

1.25

1.27

0.33

Mean No. of Pups survived per dam (LD4 to 7)

10.00

10.50

10.45

9.75

Mean No. of Pups dead per dam (LD4 to 7)

0.00

0.00

0.00

0.00

Mean Pup Survival Index (%) per dam (LD4 to 7)

100.00

100.00

100.00

100.00

Sex Ratio (male/female) per dam at LD 7

1.25

1.51

1.83

1.80

Mean No. of Pups survived per dam (LD7 to 13)

10.00

10.50

10.45

9.75

Mean No. of Pups dead per dam (LD7 to 13)

0.00

0.00

0.00

0.00

Mean Pup Survival Index (%) per dam ( LD7 to 13)

100.00

100.00

100.00

100.00

Sex Ratio (male/female) per dam at LD 13

1.25

1.51

1.83

1.80

 

Pre and Postnatal loss 

Mean Pre-natal loss (implantations minus live births) (No.)

0.00

0.08

0.09

0.00

Females with 0 (No.)

11

11

10

12

Females with ≥ 1 (No.)

0

1

1

0

Post-natal loss (live births minus alive at post natal day 13)

Females with 0 (No.)

11

12

11

12

Females with ≥ 1 (No.)

0

0

0

0

 

Litter Observations 

Male Pup weight at birth (mean) in gram

7.18

7.21

7.22

7.19

Female Pup weight at birth (mean) in gram

6.86

6.70

6.84

6.61

Male Pup weight on LD4 (mean) in gram

12.02

11.95

12.05

12.27

Female Pup weight on LD4 (mean) in gram

11.27

11.26

11.36

11.49

Male Pup weight on LD7 (mean) in gram

18.00

17.62

17.56

18.29

Female Pup weight on LD7 (mean) in gram

17.35

16.93

16.44

16.94

Male Pup weight on LD13 (mean) in gram

27.83

27.89

27.68

28.16

Female Pup weight on LD13 (mean) in gram

27.03

26.32

25.93

26.46

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

8

14

21

28

35

G1, M & 0

Mean

361.58

377.04

392.59

396.99

404.48

411.57

±SD

18.23

16.73

19.62

19.39

23.07

24.96

n

12

12

12

12

12

12

G2, M & 100

Mean

357.09

372.40

384.39

387.97

398.77

407.93

±SD

24.60

26.64

27.79

28.95

32.21

31.15

n

12

12

12

12

12

12

G3, M & 300

Mean

360.53

369.07

383.84

390.53

400.83

408.85

±SD

24.29

28.11

30.11

30.99

29.43

30.63

n

12

12

12

12

12

12

G4, M & 1000

Mean

360.82

364.19

377.79

384.19

394.37

403.99

±SD

19.98

22.62

28.98

31.06

32.03

32.38

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

8

14

21#

28#

G1, F & 0

Mean

267.24

272.29

283.41

286.21

314.07

±SD

15.90

15.44

16.07

20.13

12.43

n

12

12

12

8

3

G2, F & 100

Mean

266.21

272.72

285.28

299.95

326.83

±SD

15.12

15.70

16.86

13.74

17.88

n

12

12

12

6

2

G3, F & 300

Mean

265.22

270.61

277.81

270.58

280.42

±SD

15.91

16.93

20.19

15.94

1.50

n

12

12

12

4

2

G4, F & 1000

Mean

264.11

267.45

279.09

276.09

279.41

±SD

15.27

16.69

20.22

19.01

2.13

n

12

12

12

8

3

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

#: The statistical analysis is not performed for day 22 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 (%) during Days

1 to 8

1 to 14

1 to 21

1 to 28

1 to 35

G1, M & 0

Mean

4.32

8.61

9.84

11.88

13.84

±SD

1.71

2.68

3.15

3.81

4.24

n

12

12

12

12

12

G2, M & 100

Mean

4.30

7.68

8.67

11.67

14.29

±SD

2.63

3.73

3.63

4.45

4.97

n

12

12

12

12

12

G3, M & 300

Mean

2.33

6.44

8.30

11.20

13.43

±SD

2.03

3.53

3.91

4.13

4.47

n

12

12

12

12

12

G4, M & 1000

Mean

0.91*

4.64*

6.38

9.21

11.88

±SD

1.50

4.18

4.40

4.81

4.99

n

12

12

12

12

12

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

* Statistically significant (P<0.05) change than the vehicle control group.

 

TABLE 3 (Contd..,). 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 (%) during Days

1 to 8

1 to 14

1 to 21#

1 to 28#

G1, F & 0

Mean

1.91

6.10

7.51

16.00

±SD

1.01

2.88

3.84

4.32

n

12

12

8

3

G2, F & 100

Mean

2.45

7.20

10.21

17.39

±SD

1.45

3.47

5.02

3.26

n

12

12

6

2

G3, F & 300

Mean

2.03

4.69

3.95

7.75

±SD

2.00

2.21

1.76

1.86

n

12

12

4

2

G4, F & 1000

Mean

1.24

5.67

6.72

11.85

±SD

0.98

4.29

3.70

3.60

n

12

12

8

3

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

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

 

Note: The data obtained during pre-mating period for all the females was considered for mean calculations.

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

Group, Sex & Dose

(mg/kg body weight/day)

Feed Consumption (g)

Week 1

(Day 1 to 8)

Week 2

(Day 8 to 14)

Post Mating

(Day 31 to 35)

Post Mating

(Day 35 to 37)

G1, M & 0

Mean

23.74

26.23

30.43

32.65

±SD

0.47

0.50

1.21

1.68

n

12

12

12

12

G2, M & 100

Mean

23.68

26.18

30.15

32.01

±SD

0.52

0.54

1.32

0.90

n

12

12

12

12

G3, M & 300

Mean

23.97

26.60

29.97

32.74

±SD

0.60

0.46

1.60

2.08

n

12

12

12

12

G4, M & 1000

Mean

23.91

26.21

30.26

32.89

±SD

0.51

0.61

3.59

3.13

n

12

12

12

12

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

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

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

Feed Consumption (g)

Week 1

(Day 1 to 8)

Week 2

(Day 8 to 14)

G1, F & 0

Mean

20.31

21.98

±SD

0.45

0.48

n

12

12

G2, F & 100

Mean

20.36

21.93

±SD

0.65

0.48

n

12

12

G3, F & 300

Mean

20.66

22.27

±SD

0.54

0.41

n

12

12

G4, F & 1000

Mean

20.82

22.29

±SD

0.48

0.33

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

1.5812

3.5987

3.6165

0.0300

±SD

0.0816

0.3419

0.1841

0.0022

n

12

12

12

12

G2, M & 100

Mean

1.6136

3.4643

3.4524

0.0292

±SD

0.1238

0.2595

0.2526

0.0025

n

12

12

12

12

G3, M & 300

Mean

1.5383

3.4801

3.4760

0.0295

±SD

0.1027

0.2353

0.2963

0.0032

n

12

12

12

12

G4, M & 1000

Mean

1.4543*

3.3486

3.4098

0.0294

±SD

0.0746

0.1950

0.5073

0.0023

n

12

12

12

12

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

* Statistically significant (P<0.05) change than the vehicle control group.

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

0.4658

0.0251

±SD

0.1017

0.0019

n

11

11

G2, F & 100

Mean

0.4849

0.0258

±SD

0.0867

0.0020

n

12

12

G3, F & 300

Mean

0.4393

0.0259

±SD

0.0933

0.0022

n

11

11

G4, F & 1000

Mean

0.4400

0.0253

±SD

0.0760

0.0018

n

12

12

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

#: 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

403.76

0.3925

0.8911

0.8986

0.0074

±SD

24.51

0.0256

0.0623

0.0676

0.0002

n

12

12

12

12

12

G2, M & 100

Mean

395.35

0.4101

0.8808

0.8793

0.0074

±SD

31.43

0.0418

0.0911

0.1034

0.0002

n

12

12

12

12

12

G3, M & 300

Mean

399.40

0.3868

0.8751

0.8774

0.0074

±SD

31.41

0.0329

0.0786

0.1166

0.0003

n

12

12

12

12

12

G4, M & 1000

Mean

396.20

0.3699

0.8498

0.8644

0.0074

±SD

34.11

0.0414

0.0743

0.1423

0.0003

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

308.01

0.1505

0.0082

±SD

17.08

0.0285

0.0008

n

11

11

11

G2, F & 100

Mean

306.35

0.1587

0.0084

±SD

26.63

0.0284

0.0004

n

12

12

12

G3, F & 300

Mean

317.81

0.1387

0.0082

±SD

23.75

0.0295

0.0006

n

11

11

11

G4, F & 1000

Mean

309.35

0.1425

0.0082

±SD

23.55

0.0235

0.0003

n

12

12

12

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

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

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

No. of Females

No. of Females with Regular Estrous Cyclicity during

Pre-mating and Mating

No. of Females with Regular Estrous Cyclicity

on Lactation day 14

No. of Females with Irregular Estrous Cyclicity during

Pre-mating, Mating and on Lactation day 14

G1 & 0

12

12

11

0

G2 & 100

12

12

12

0

G3 & 300

12

12

11

0

G4 & 1000

12

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 14)

G1 & 0                  

Mean

10.33

N

4

8

±SD

5.63

n

12

%

33.3

66.7

G2 & 100                  

Mean

7.67

n

5

7

±SD

5.58

n

12

%

41.7

58.3

G3 & 300                  

Mean

6.50

n

7

5

±SD

5.44

n

12

%

58.3

41.7

G4 & 1000                  

Mean

9.92

n

4

8

±SD

5.48

n

12

%

33.3

66.7

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

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

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

Body weight (g) on Gestation Days

0

7

14

20

G1 & 0                  

Mean

290.15

304.33

338.92

394.70

±SD

16.21

17.10

21.73

19.17

n

11

11

11

11

G2 & 100                  

Mean

296.25

309.37

340.03

397.88

±SD

23.35

21.84

23.69

30.89

n

12

12

12

12

G3 & 300                  

Mean

283.53

300.41

337.60

408.43

±SD

18.96

20.10

26.73

33.66

n

11

11

11

11

G4 & 1000                  

Mean

288.32

305.31

339.42

402.08

±SD

17.90

17.71

21.19

28.72

n

12

12

12

12

SD: Standard Deviation; n: Number of dams

TABLE 10.           SUMMARY RECORD OF PERENT 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

4.89

11.35

16.74

±SD

0.84

2.86

7.10

n

11

11

11

G2 & 100

Mean

4.49

9.98

16.96

±SD

1.26

3.62

2.23

n

12

12

12

G3 & 300

Mean

5.98

12.32

21.03

±SD

2.31

3.06

4.52

n

11

11

11

G4 & 1000

Mean

5.93

11.19

18.48

±SD

1.80

3.13

4.25

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

22.16

22.91

26.69

±SD

1.44

1.43

1.57

n

11

11

11

G2 & 100

Mean

22.47

23.38

27.37

±SD

1.17

1.30

1.81

n

12

12

12

G3 & 300

Mean

21.32

22.47

28.14

±SD

3.28

2.82

1.42

n

11

11

11

G4 & 1000

Mean

22.13

23.07

26.28

±SD

1.41

1.10

2.09

n

12

12

12

SD: Standard Deviation; n: Number of animals

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, F & 0

Mean

296.88

303.85

310.79

322.15

±SD

13.08

15.29

17.44

15.75

n

11

11

11

11

G2, F & 100

Mean

294.96

302.70

308.61

319.75

±SD

25.11

26.15

25.62

24.74

n

12

12

12

12

G3, F & 300

Mean

307.76

316.52

321.99

333.35

±SD

26.53

26.94

26.32

24.61

n

11

11

11

11

G4, F & 1000

Mean

299.22

304.10

311.84

323.96

±SD

22.07

22.71

22.07

22.66

n

12

12

12

12

SD: Standard Deviation; n: Number of dams

TABLE 13.           SUMMARY RECORD OF 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, F & 0

Mean

2.33

2.26

3.70

±SD

1.16

1.32

1.62

n

11

11

11

G2, F & 100

Mean

2.62

1.99

3.66

±SD

1.44

1.18

1.56

n

12

12

12

G3, F & 300

Mean

2.87

1.76

3.59

±SD

2.39

1.05

1.50

n

11

11

11

G4, F & 1000

Mean

1.64

2.58

3.93

±SD

1.52

1.30

3.26

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

22.60

26.27

26.16

±SD

3.05

2.28

4.39

n

11

11

11

G2 & 100

Mean

24.93

27.72

27.06

±SD

4.13

4.50

3.69

n

12

12

12

G3 & 300

Mean

25.80

28.46

28.03

±SD

2.21

5.32

1.40

n

11

11

11

G4 & 1000

Mean

24.41

25.53

26.31

±SD

3.81

2.45

1.24

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

22.82

11.00

11.00

5.36

5.64

0.00

0.00

0.00

1.00

100.00

±SD

0.75

1.79

1.79

1.29

1.43

0.00

0.00

0.00

0.31

0.00

n

11

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

22.50

11.83

11.75

6.00

5.75

0.08

0.00

0.08

1.17

99.36

±SD

0.80

2.69

2.67

2.00

2.26

0.29

0.00

0.29

0.48

2.22

n

12

12

12

12

12

12

12

12

12

12

G3 & 300

Mean

22.64

11.82

11.73

6.36

5.36

0.09

0.09

0.00

1.27

99.24

±SD

0.50

2.14

2.15

1.69

1.43

0.30

0.30

0.00

0.48

2.51

n

11

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

22.83

10.08

10.08

5.75

4.33

0.00

0.00

0.00

1.54

100.00

±SD

0.72

1.78

1.78

1.91

1.44

0.00

0.00

0.00

0.82

0.00

n

12

12

12

12

12

12

12

12

12

12

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

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

11.00

11.00

5.36

5.64

0.00

0.00

0.00

1.00

11.00

100.00

±SD

1.79

1.79

1.29

1.43

0.00

0.00

0.00

0.31

1.79

0.00

n

11

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

11.75

11.75

6.00

5.75

0.00

0.00

0.00

1.17

11.75

100.00

±SD

2.67

2.67

2.00

2.26

0.00

0.00

0.00

0.48

2.67

0.00

n

12

12

12

12

12

12

12

12

12

12

G3 & 300

Mean

11.73

11.73

6.36

5.36

0.00

0.00

0.00

1.27

11.73

100.00

±SD

2.15

2.15

1.69

1.43

0.00

0.00

0.00

0.48

2.15

0.00

n

11

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

10.08

10.08

5.75

4.42

0.00

0.00

0.00

1.54

10.08

100.00

±SD

1.78

1.78

1.91

1.44

0.00

0.00

0.00

0.82

1.78

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)

Animal No.

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 to 7

Pup Survival Index
(%) during
          LD 4 to 7

Live Pups (No.)

 

Dead 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

5.36

5.64

11.00

0.00

1.00

1.00

5.36

4.64

10.00

5.36

4.64

10.00

0.00

0.00

0.00

1.25

10.00

100.00

±SD

1.29

1.43

1.79

0.00

1.00

1.00

1.29

1.03

1.00

1.29

1.03

1.00

0.00

0.00

0.00

0.53

1.00

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

6.00

5.75

11.75

0.00

1.25

1.25

6.00

4.50

10.50

6.00

4.50

10.50

0.00

0.00

0.00

1.51

10.50

100.00

±SD

2.00

2.26

2.67

0.00

0.97

0.97

2.00

1.73

1.88

2.00

1.73

1.88

0.00

0.00

0.00

0.67

1.88

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

G3 & 300

Mean

6.36

5.36

11.73

0.00

1.27

1.27

6.36

4.09

10.45

6.36

4.09

10.45

0.00

0.00

0.00

1.83

10.45

100.00

±SD

1.69

1.43

2.15

0.00

0.90

0.90

1.69

1.45

1.51

1.69

1.45

1.51

0.00

0.00

0.00

1.06

1.51

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

10

10

11

11

11

11

G4 & 1000

Mean

5.75

4.33

10.08

0.00

0.33

0.33

5.75

4.00

9.75

5.75

4.00

9.75

0.00

0.00

0.00

1.80

9.75

100.00

±SD

1.91

1.44

1.78

0.00

0.78

0.78

1.91

1.48

1.14

1.91

1.48

1.14

0.00

0.00

0.00

1.26

1.14

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)

Animal No.

Live Pups (No.) on LD 7

During LD 7 to 13

 

Sex Ratio (M/F) at

LD 13

No. of Survived Pups during

LD 7 to 13

Pup Survival Index
(%) during LD 7
to 13

Live Pups (No.)

 

Dead Pups (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

Male (No.)

Female (No.)

Total (No.)

G1 & 0

Mean

5.36

4.64

10.00

5.36

4.64

10.00

0.00

0.00

0.00

1.25

10.00

100.00

±SD

1.29

1.03

1.00

1.29

1.03

1.00

0.00

0.00

0.00

0.53

1.00

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G2 & 100

Mean

6.00

4.50

10.50

6.00

4.50

10.50

0.00

0.00

0.00

1.51

10.50

100.00

±SD

2.00

1.73

1.88

2.00

1.73

1.88

0.00

0.00

0.00

0.67

1.88

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

G3 & 300

Mean

6.36

4.09

10.45

6.36

4.09

10.45

0.00

0.00

0.00

1.83

10.45

100.00

±SD

1.69

1.45

1.51

1.69

1.45

1.51

0.00

0.00

0.00

1.06

1.51

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

G4 & 1000

Mean

5.75

4.00

9.75

5.75

4.00

9.75

0.00

0.00

0.00

1.80

9.75

100.00

±SD

1.91

1.48

1.14

1.91

1.48

1.14

0.00

0.00

0.00

1.26

1.14

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

 

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

7.18

6.86

12.02

11.27

18.00

17.35

27.83

27.03

±SD

0.25

0.12

0.32

0.49

0.87

0.79

0.40

0.92

n

11

11

11

11

11

11

11

11

G2 & 100

Mean

7.21

6.70

11.95

11.26

17.62

16.93

27.89

26.32

±SD

0.51

0.56

0.75

0.86

1.16

1.22

0.99

0.98

n

12

12

12

12

12

12

12

12

G3 & 300

Mean

7.22

6.84

12.05

11.36

17.56

16.44

27.68

25.93*

±SD

0.60

0.60

0.34

0.51

1.33

1.25

0.96

0.96

n

11

11

11

11

11

11

11

11

G4 & 1000

Mean

7.19

6.61

12.27

11.49

18.37

16.94

28.16

26.46

±SD

0.28

0.37

0.28

0.54

0.46

0.68

0.43

0.81

n

12

12

12

12

12

12

12

12

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

* Statistically significant (P<0.05) change than the vehicle control group.

TABLE 19.              SUMMARY OF UTERI OBSERVATIONS RECORD

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

No. of Corpora lutea

No. of Implantations

Implantation

Index

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

11.09

11.00

98.99

1.01

0.00

0.00

0.00

0.00

0.00

0.00

±SD

1.64

1.79

3.35

3.35

0.00

0.00

0.00

0.00

0.00

0.00

n

11

11

11

11

11

11

11

11

11

11

G2 & 100  

Mean

11.83

11.83

100.00

0.00

0.64

0.08

0.00

0.00

0.00

0.00

±SD

2.69

2.69

0.00

0.00

2.22

0.29

0.00

0.00

0.00

0.00

n

12

12

12

12

12

12

12

12

12

12

G3 & 300           

Mean

11.82

11.82

100.00

0.00

0.76

0.09

0.00

0.00

0.00

0.00

±SD

2.14

2.14

0.00

0.00

2.51

0.30

0.00

0.00

0.00

0.00

n

11

11

11

11

11

11

11

11

11

11

G4 & 1000     

Mean

10.17

10.17

100.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

±SD

1.80

1.80

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

n

12

12

12

12

12

12

12

12

12

12

SD: Standard Deviation; n: Number of animals

 

TABLE 20.              SUMMARY RECORD OF ANO-GENITAL DISTANC RATIO ON LACTATION DAY 4

Group & Dose

(mg/kg body weight/day)

Mean Male AGD Ratio

Mean Female AGD Ratio

G1 & 0

Mean

2.36

1.00

±SD

0.10

0.05

n

11

11

G2 & 100

Mean

2.32

1.02

±SD

0.11

0.07

n

12

12

G3 & 300

Mean

2.37

1.02

±SD

0.13

0.04

n

11

11

G4 & 1000

Mean

2.31

1.03

±SD

0.09

0.10

n

12

12

 SD: Standard Deviation; n: Number of animals

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

11

G2 & 100

Mean

0.00

±SD

0.00

n

12

G3 & 300

Mean

0.00

±SD

0.00

n

11

G4 & 1000

Mean

0.00

±SD

0.00

n

12

SD: Standard Deviation; n: Number of animals

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

87.144

±SD

9.811

n

12

G2, M & 100

Mean

94.818

±SD

7.447

n

12

G3, M & 300

Mean

91.817

±SD

8.802

n

12

G4, M & 1000

Mean

84.652

±SD

5.674

n

12

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

TABLE 23.              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

59.709

±SD

13.496

n

11

G2 & 100

Mean

62.633

±SD

7.849

n

12

G3 & 300

Mean

56.212

±SD

5.039

n

11

G4 & 1000

Mean

58.045

±SD

9.753

n

12

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

TABLE 24.              SUMMARY OF SERUM ESTROGEN (pg/mL) RECORD - DAMS

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

 

Serum Estrogen Levels (pg/mL)

G1 & 0

Mean

237.529

±SD

25.797

n

11

G2 & 100

Mean

254.767

±SD

20.425

n

11$

G3 & 300

Mean

251.143

±SD

32.806

n

11

G4 & 1000

Mean

209.784

±SD

42.035

n

12

SD: Standard Deviation; n: Number of Dams

 $: The data of animal No. Rc6339 is excluded due to its lower obtained value.

TABLE 25.              SUMMARY OF SERUM PROGESTERONE (ng/mL) RECORD - DAMS

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

Serum Progesterone Levels (ng/mL)

G1 & 0

Mean

160.411

±SD

109.496

n

11

G2 & 100

Mean

143.256

±SD

106.684

n

12

G3 & 300

Mean

153.732

±SD

113.631

n

11

G4 & 1000

Mean

116.492

±SD

119.809

n

12

SD: Standard Deviation; n: Number of Dams

TABLE 26.     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

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 26 (Contd..,). SUMMARY OF GROSS PATHOLOGY FINDINGS OF PUPS

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

1

0

0

0

No Abnormality Detected

0

0

0

1

1

0

0

0

 

No. of pups sacrificed on Lactation Day 4

0

11

0

15

0

14

0

4

No Abnormality Detected

0

11

0

15

0

14

0

4

 

No. of pups sacrificed on Lactation Day 13

59

51

72

54

70

45

69

48

No Abnormality Detected

59

51

72

54

70

45

69

48

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

TABLE 27.     SUMMARY OF HISTOPATHOLOGY FINDINGS

Group

G1

G4

G1

G4

Dose

(mg/kg body weight)

0

1000

0

1000

No. of Animals

12

12

12

12

Sex

Male

Female

ORGANS AND OBSERVATIONS/SEVERITY

Epididymides

Interstitial MNC infiltration; unilateral

Minimal

1

1

X

X

Uterus

Yellow-brown pigmentation

Minimal

X

X

6

7

Mild

X

X

1

2

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

 

Applicant's summary and conclusion

Conclusions:
The test item Hostacor ITD was evaluated for possible adverse effects on reproductive performance following repeated oral administration to male and female rats according OECD Test Guideline 421.

Based under the experimental conditions employed, no effect on any parameter investigated was observed. Thus, the No Observed Adverse Effect Level (NOAEL) of Hostacor ITD was found to be 1000 mg/kg body weight per day for male and female rats.
Executive summary:

Hostacor ITDwas evaluated for possible adverse effects on reproductive performance such as gonadal function, mating behavior, conception, development of conceptus, parturition and early neonatal development following repeated oral administration to male and female rats. The study was conducted according to OECD Test Guideline 421 and followed the principles of GLP.

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[corn oil],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 vehicleand test item formulationswere 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 37 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 weeks 1 and 5 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 lactation day 13 pups representing each per litter was screened for T4 levels and serum collected from dams on lactation day 14 was screened for Estrogen and Progesterone levels.

Females were observed for estrous cyclicityduring pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice.The females were observed forcopulatory 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 external examinations once daily from lactation day 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 male pups were observed for retention of nipples/areolae on lactation day 13.

Gross pathology and organ weighing were performed on day 37 for males and on lactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 to day 35 and feed consumption at all the tested dose group animals of either sex during the experimental period. 

No treatment related changes were observed in organ weights (both absolute and relative) at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and lactation day 13 pups at any of the tested dose groups.

Dams did not reveal any treatment related changes in estrous cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period. There were no treatment related changes observed in serum Estrogen and Progesterone levels of dams at any of the tested dose groups.

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

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups.

There were no treatment related histopathological findings noticed during the microscopic examination.Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, which revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition to this, ovaries did not show any pathological findings/lesions.