Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The data is from Study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29th July, 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
EC Number:
226-999-5
EC Name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
Cas Number:
5590-18-1
Molecular formula:
C22H6Cl8N4O2
IUPAC Name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
Test material form:
solid
Details on test material:
- Name of test material : C.I. Pigment Yellow 110
- Molecular formula : C22H6Cl8N4O2
- Molecular weight : 641.939 g/mol
- Smiles notation (if other than submission substance): C=1(c2c(c(c(Cl)c(Cl)c2C(=O)N1)Cl)Cl)Nc1ccc(cc1)NC1=NC(c2c1c(c(c(c2Cl)Cl)Cl)Cl)=O
- InChl (if other than submission substance): 1S/C22H6Cl8N4O2/c23-11-7-9(13(25)17(29)15(11)27)21(35)33-19(7)31-5-1-2-6(4-3-5)32-20-8-10(22(36)34-20)14(26)18(30)16(28)12(8)24/h1-4H,(H,31,33,35)(H,32,34,36)
- Substance type: Organic
- Physical state: Solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: PO190512; Shaanxi Pioneer Biotech Co., Ltd, Hengxianhe Town, Hanzhang City, Shaanxi Province, China
- Expiration date of the lot/batch: 12th May, 2021
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: NA
- Stability under storage conditions: Ambient (21 to 29°C).
- Stability under test conditions: NA
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: NA
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): NA

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment.
In-house bred animals
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals.
- Animals: 12 males + 12 females (total of 96 rats, 46 males + 46 females)
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study receipt: 10 weeks
- Weight at study initiation: Males: 240.18 to 285.03 g; Females: 200.76 to 249.63 g
- Fasting period before study: No
- 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.
- Bedding: Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, two animals of same sex were housed.
- Pre-mating - Per cage, two animals of same sex and group were housed.
- Cohabitation Period (mating) - Per cage, two animals (one male and one female) of same group were housed.
- Post-mating - After confirmation of 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 nesting material for all females from gestation day 20 onwards.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice was available ad libitum.
- Water (e.g. ad libitum): Water was provided ad libitum. Deep bore-well water was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Five days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 to 22.9°C
- Humidity (%): 49 to 65%
- 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared before dose administration and administered within the stability period. The following stock solutions were prepared: of 25, 50, and 100 mg/mL of test item for low, mid and high dose groups, respectively. The test formulations were maintained under stirring conditions using a magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and not suspended uniformly in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL. The test item was suspended uniformly in corn oil at the concentration of 100 mg/mL.
- Stock solutions of test item: 0 mg/mL, 25 mg/mL, 50 mg/mL and 100 mg/mL.
- Dose volume (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): N12009002
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Re-mating: Re-mating of females was not considered as all the females were confirmed with mating with the first male.
Non-pregnant females: Non-pregnant females were sacrificed after 24 days from the last day of confirmation of mating.
- After successful mating each pregnant female was caged (how): pregnant females were housed individually
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males - 29 days
Females - 63 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
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 dose levels were selected based on the dose-range finding study where animals were orally gavaged. No treatment related effects were observed at any of the dose levels in dose
- 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% (Males: -8.21 to 11.55%; Females: -10.72 to 8.51%) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment by randomization based on the body weights. Body weights of the animals were analyzed statistically for mean body weight and there were no statistically significant differences noted between groups within each sex.
- Fasting period before blood sampling for clinical biochemistry: No data available.
- Other: No data available.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at receipt, on the fist day of dosing, weekly thereafter and at termination. Females were weighed on GD 0, 7, 14 and 20 during pregnancy and on lactation day 1, 4, 7 and 13 during lactation period and on day 14.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Feed consumption was measured for all animals once a week during premating period. Feed consumption was not measured during the mating period for either male or female rats. After mating, feed consumption for females was recorded during gestation day (GD) 0 to 7, 7 to 14 and 14 to 20 and during lactation day 1 to 4, 4 to 7 and 7 to 13.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data available

OTHER: No data available
Oestrous cyclicity (parental animals):
Oestrous cyclicity was monitored for two weeks after five days of acclimatization. Vaginal smears were monitored daily from the beginning of treatment period until evidence of mating. The status of oestrus cyclicity of females was determined on termination day (lactation day 14).
Sperm parameters (parental animals):
A detailed histopathological examination was performed on the ovaries, testes, and epididymides. Particular emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure of the animals was given.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS: Yes.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No data available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No data available
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after 29 days of treatment
- Maternal animals: All surviving animals were sacrificed on Lactation day 14 (after 63 days of treatment)

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively are indicated in Table 1
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at PND 4 and or PND 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) are indicated in Table 2.

GROSS NECROPSY
- The pups, which were sacrificed on PND 4 or 13 were examined for gross abnormalities with particular attention to the external reproductive genitals.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.
Statistics:
The statistical analysis was followed to the parameters in the table 3 given below.
Reproductive indices:
Mating and Fertility Indexes (both genders), Gestation Index, Parturition Index, and Pregnancy Index.
Offspring viability indices:
Litter Index and Pup Viability Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related clinical signs of toxicity in all the groups during the experimental period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals were survived to planned death.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant changes noted in mean body weight or mean body weight gain with respect to day 1 in any of the groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no significant changes noted in the mean feed consumption measured during pre-mating period (first 2 weeks) in treatment groups when compared to control groups.

There were no test item-related changes noted in mean gestational feed consumption in treatment groups when compared to control groups.

There were no significant changes noted in mean lactation feed consumption in treatment groups when compared to control groups.
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:
no effects observed
Description (incidence and severity):
There were no changes observed in serum Thyroxine (T4) hormone levels in treatment groups males when compared to control group males. As there were no changes noted in T4 levels of adult males, the assessment was not extended to adult females (dams).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related histopthological changes observed in treatment group related to test item.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic lesions were observed at 0 and 1000 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
There were no significant changes in mean gestational body weight or mean gestational body weight gain in the treatment groups when compared to control group.

There were no significant changes in mean lactation body weight or mean lactation body weight gain in the treatment groups when compared to control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no cases of irregular oestrus cyclicity during pre-mating or mating. No significant changes in mean oestrus length were observed in treatment groups when compared to control group during pre-mating or mating periods.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related adverse effects on the stages of spermatogenesis were observed during the microscopic examination of interstitial testicular cell structure.
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment related adverse effects on male or female mating index, male or female fertility index, pre-coital interval, gestational length, gestation index, parturition index, pregnancy index, live birth index, post implantation loss, mean number of Implantations were observed.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
<= 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
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
All pups showed normal behaviour during the daily inspections.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant changes in live birth index (%) were observed and no cases of unscheduled mortality were observed during the post-natal period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related changes noted in mean pup weight on PND 1, 4, 7 or 13 in treatment groups when compared to control group.
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:
no effects observed
Description (incidence and severity):
There were no significant changes in serum Thyroxine (T4) hormone levels on PND 13 pups in treatment groups when compared to control group.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No significant changes were observed.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no test item-related effects noted on mean pup AGD (M/F) or AGD ratio (M/F) measured on PND 4 in treatment groups compared to control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no cases of male nipple retention on PND 13 in all the dose groups.
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 findings in any of the groups.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
<= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology
other: Anogenital Distance and Nipple Retention in male pups

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Treatment related:
no

Any other information on results incl. tables

Table 1. Table for CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD (Males)

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Mortalityb:

No. of Mortalities
(Total No. of Animals)

G1, M & 0

12

Day 1 to termination:N (12)

0 (12)

G2, M & 250

12

Day 1 to termination:N (12)

0 (12)

G3, M & 500

12

Day 1 to termination:N* (12)

0 (12)

G4, M & 1000

12

Day 1 to termination:N* (12)

0 (12)

M: Male; N: Normal.; a: observed daily once; b: observed twice daily.

Contd. CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD (Females)

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Mortalityb

No. of Mortalities
(Total No. of Animals)

G1, F & 0

12

Day 1 to termination:N (12)

0 (12)

G2, F & 250

12

Day 1 to termination:N (12)

0 (12)

G3, F & 500

12

Day 1 to termination:N* (12)

0 (12)

G4, F & 1000

12

Day 1 to termination:N* (12)

0 (12)

F: Female; N: Normal.; a: observed daily once; b: observed twice daily.

Table 2. Table for BODY WEIGHT (g) RECORD (Males)

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Day

1

7

14

21

28

G1, M & 0

Mean

344.92

364.74

380.43

401.18

413.79

±SD

22.32

26.88

28.74

35.61

36.70

n

12

12

12

12

12

G2, M & 250

Mean

346.79

363.64

382.83

397.60

411.86

±SD

22.18

23.83

22.16

25.73

27.90

n

12

12

12

12

12

G3, M & 500

Mean

348.51

369.31

387.54

399.54

413.68

±SD

20.30

26.17

28.75

30.67

31.73

n

12

12

12

12

12

G4, M & 1000

Mean

348.93

365.83

387.13

405.21

420.46

±SD

19.36

21.24

23.17

26.89

30.10

n

12

12

12

12

12

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

Contd. Table for BODY WEIGHT (g) RECORD (Females)

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Day

1

7

14

21#

G1, F & 0

Mean

253.80

260.79

268.54

272.30

±SD

16.30

16.43

15.99

15.79

n

12

12

12

7

G2, F & 250

Mean

253.72

261.62

267.91

284.49

±SD

17.75

17.79

17.86

23.30

n

12

12

12

3

G3, F & 500

Mean

253.98

262.91

268.04

284.09

±SD

16.19

16.48

15.51

21.72

n

12

12

12

4

G4, F & 1000

Mean

254.95

264.13

269.90

277.58

±SD

17.65

17.20

16.74

20.86

n

12

12

12

4

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

#: The data obtained from females in cohabitation only considered for mean calculations. The data of Day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

Table 3. Table for GESTATION BODY WEIGHT (g)

Group, Sex & Dose

(mg/kg body weight/day)

 

Body Weight (g) on Gestation Day (GD)

0

7

14

20

G1, F & 0

Mean

278.53

288.75

311.73

377.30

±SD

20.06

20.06

20.97

20.61

n

11

11

11

11

G2, F & 250

Mean

273.15

285.52

308.59

372.18

±SD

18.85

20.76

20.58

19.75

n

12

12

12

12

G3, F & 500

Mean

275.89

287.04

309.71

370.99

±SD

18.68

18.93

19.81

20.26

n

11

11

11

11

G4, F & 1000

Mean

276.32

289.94

312.84

375.40

±SD

17.58

18.19

19.20

21.30

n

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams.

Table 4. Table for LACTATION BODY WEIGHT (g)

Group, Sex & Dose

(mg/kg body weight/day)

Body Weight (g) on Lactation Day (LD)

1

4

7

13

G1, F & 0

Mean

287.29

292.32

300.80

312.73

±SD

19.07

18.20

17.69

16.16

n

11

11

11

11

G2, F & 250

Mean

285.03

289.58

297.82

310.76

±SD

19.88

19.39

18.64

19.42

n

12

12

12

12

G3, F & 500

Mean

284.79

289.38

297.30

310.79

±SD

17.71

17.41

16.90

16.58

n

11

11

11

11

G4, F & 1000

Mean

288.68

293.09

302.35

315.11

±SD

16.28

16.61

16.26

16.35

n

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams.

Table 5. Table for PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD (Males)

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) Gain during Day

1 to 7

1 to 14

1 to 21

1 to 28

G1, M & 0

Mean

5.70

10.23

16.16

19.81

±SD

1.53

1.82

3.75

3.88

n

12

12

12

12

G2, M & 250

Mean

4.87

10.46

14.67

18.76

±SD

1.68

2.67

2.37

2.60

n

12

12

12

12

G3, M & 500

Mean

5.91

11.13

14.56

18.63

±SD

2.27

3.05

3.51

3.94

n

12

12

12

12

G4, M & 1000

Mean

4.84

10.94

16.09

20.43

±SD

1.35

2.06

2.67

3.34

n

12

12

12

12

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

Contd. Table for PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD (Females)

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight (%) Gain during Day

1 to 7

1 to 14

1 to 21#

G1, F & 0

Mean

2.77

5.85

8.27

±SD

1.05

1.56

1.34

n

12

12

7

G2, F & 250

Mean

3.13

5.62

7.97

±SD

0.88

1.05

1.45

n

12

12

3

G3, F & 500

Mean

3.53

5.58

9.87

±SD

1.01

1.58

1.70

n

12

12

4

G4, F & 1000

Mean

3.63

5.92

9.42

±SD

0.91

1.47

2.41

n

12

12

4

F: Female; SD: Standard Deviation.

n: Number of Animals; #: The data obtained from females in cohabitation only considered for mean calculations. The data of Day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

Table 6. Table for PERCENT CHANGE IN GESTATION BODY WEIGHT GAIN (%) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight Gain (%)

during Gestation Day (GD)

0 to 7

7 to 14

14 to 20

G1, F & 0

Mean

3.69

7.98

21.13

±SD

0.74

0.90

1.76

n

11

11

11

G2, F & 250

Mean

4.50

8.12

20.72

±SD

0.81

0.89

2.38

n

12

12

12

G3, F & 500

Mean

4.05

7.91

19.87

±SD

0.95

0.63

2.57

n

11

11

11

G4, F & 1000

Mean

4.94*

7.91

20.04

±SD

0.79

0.66

2.22

n

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams.*: Statistically significant (P<0.05) change than the vehicle control group.

Table 7. Table for PERCENT CHANGE IN LACTATION BODY WEIGHT GAIN (%) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Percent Change in Body Weight Gain (%)

during Lactation Day (LD)

1 to 4

4 to 7

7 to 13

G1, F & 0

Mean

1.78

2.93

4.01

±SD

0.60

0.78

0.97

n

11

11

11

G2, F & 250

Mean

1.62

2.88

4.35

±SD

0.70

1.04

1.21

n

12

12

12

G3, F & 500

Mean

1.63

2.75

4.56

±SD

0.59

0.93

1.31

n

11

11

11

G4, F & 1000

Mean

1.53

3.17

4.23

±SD

0.44

0.67

1.09

n

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams.

Table 8. Table for FEED CONSUMPTION (g/animal/day) RECORD (Males)

Group, Sex & Dose

(mg/kg body weight/day)

Feed Consumption (g/animal/day) during Pre-mating Period

Week 1

Week 2

G1, M & 0

Mean

20.71

24.73

±SD

1.68

1.27

n

12

12

G2, M & 250

Mean

21.29

23.50

±SD

1.49

1.53

n

12

12

G3, M & 500

Mean

20.23

24.05

±SD

2.49

1.12

n

12

12

G4, M & 1000

Mean

22.35

24.45

±SD

1.99

1.06

n

12

12

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

Contd. Table for FEED CONSUMPTION (g/animal/day) RECORD (Females)

Group, Sex & Dose

(mg/kg body weight/day)

Feed Consumption (g/animal/day) during Pre-mating Period

Week 1

Week 2

G1, F & 0

Mean

17.03

18.98

±SD

0.73

1.41

n

6

6

G2, F & 250

Mean

16.31

18.27

±SD

1.67

1.44

n

6

6

G3, F & 500

Mean

16.62

18.46

±SD

0.60

1.56

n

6

6

G4, F & 1000

Mean

17.18

19.39

±SD

2.85

1.57

n

6

6

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

Table 9. Table for FEED CONSUMPTION (g/animal/day) DURING GESTATION PERIOD

Group, Sex & Dose

(mg/kg body weight/day)

Feed Consumption (g/animal/day) during Gestation Day (GD)

0 to 7

7 to 14

14 to 20

G1, F & 0

Mean

19.09

22.90

26.29

±SD

0.53

0.40

0.51

n

11

11

11

G2, F & 250

Mean

19.30

22.57

26.43

±SD

0.58

0.38

0.57

n

12

12

12

G3, F & 500

Mean

19.15

22.64

26.71

±SD

0.55

0.29

0.49

n

11

11

11

G4, F & 1000

Mean

17.90

23.08

26.84*

±SD

4.44

0.57

0.47

n

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams.*: Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Table for FEED CONSUMTION (g/animal/day) DURING LACTATION PERIOD

Group, Sex & Dose

(mg/kg body weight/day)

 

Feed Consumption (g/animal/day) during Lactation Day (LD)

1 to 4

4 to 7

7 to 13

G1, F & 0

Mean

27.78

31.61

35.24

±SD

0.96

0.59

1.45

n

11

11

11

G2, F & 250

Mean

28.34

31.71

36.47

±SD

1.23

0.40

1.26

n

12

12

12

G3, F & 500

Mean

28.25

31.75

35.82

±SD

0.86

0.99

1.17

n

11

11

11

G4, F & 1000

Mean

28.19

31.59

36.09

±SD

0.93

0.93

1.23

n

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams.

Table 11. Table for VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Determination of Oestrus Cyclicity during Pre-Mating Treatment Period

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

Total No. of

Females Evaluated

No. of Females with Complete Regular Oestrus Cycle

No. of Females with at least one Irregular Oestrus Cycle

Average Length of

Oestrus Cycle (Days)

G1, F & 0

12

n

12

0

Mean

4.45

%

100.0

0.0

±SD

0.23

n

12

G2, F & 250

12

n

12

0

Mean

4.56

%

100.0

0.0

±SD

0.35

n

12

G3, F & 500

12

n

12

0

Mean

4.44

%

100.0

0.0

±SD

0.26

n

12

G4, F & 1000

12

n

12

0

Mean

4.47

%

100.0

0.0

±SD

0.27

n

12

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

Table 12. Table for DELIVERY DATA (AT BIRTH) RECORD PER LITTER

Group, Sex

& Dose
(mg/kg body

weight/day)

Litter Delivered (No.)

Total Litter

Size (No.)

Live Pups (No.)

Dead Pups (No.)

Cannibalized Pups (No.)

Sex Ratio

(m/f)

at Birth

Live Birth

Index
(%)

Male

Female

Total

Male

Female

Total

Undetermined

Male

Female

Total

G1, F & 0

Mean

12.73

6.00

6.55

12.55

0.00

0.00

0.00

0.18

0.00

0.00

0.18

0.97

98.70

±SD

1.19

1.67

0.93

1.04

0.00

0.00

0.00

0.40

0.00

0.00

0.40

0.43

2.89

n

11

11

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

12.25

6.50

5.67

12.17

0.00

0.08

0.08

0.00

0.00

0.00

0.00

1.30

99.36

±SD

1.82

0.90

1.97

1.80

0.00

0.29

0.29

0.00

0.00

0.00

0.00

0.59

2.22

n

12

12

12

12

12

12

12

12

12

12

12

12

12

G3, F & 500

Mean

12.18

5.82

6.27

12.09

0.00

0.09

0.09

0.00

0.00

0.00

0.00

1.01

99.35

±SD

1.17

1.40

1.42

1.04

0.00

0.30

0.30

0.00

0.00

0.00

0.00

0.49

2.15

n

11

11

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

11.64

4.55*

6.64

11.18

0.00

0.45

0.45

0.00

0.00

0.00

0.00

0.80

96.39

±SD

1.69

1.44

2.06

1.72

0.00

1.04

1.04

0.00

0.00

0.00

0.00

0.49

8.17

n

11

11

11

11

11

11

11

11

11

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: male/female.*: Statistically significant (P<0.05) change than the vehicle control group.

Table 13. Table for LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group, Sex

& Dose
(mg/kg body

weight/day)

During LD 1 to 4

Sex Ratio

(m/f) on LD 4

Pup Survival

Index (%)

during LD 1 to 4

Live Pups (No.)

Dead Pups (No.)

Cannibalized Pups (No.)

Male

Female

Total

Male

Female

Total

Male

Female

Total

G1, F & 0

Mean

6.00

6.55

12.55

0.00

0.00

0.00

0.00

0.00

0.00

0.97

100.00

±SD

1.67

0.93

1.04

0.00

0.00

0.00

0.00

0.00

0.00

0.43

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

6.50

5.67

12.17

0.00

0.00

0.00

0.00

0.00

0.00

1.30

100.00

±SD

0.90

1.97

1.80

0.00

0.00

0.00

0.00

0.00

0.00

0.59

0.00

n

12

12

12

12

12

12

12

12

12

12

12

G3, F & 500

Mean

5.82

6.27

12.09

0.00

0.00

0.00

0.00

0.00

0.00

1.01

100.00

±SD

1.40

1.42

1.04

0.00

0.00

0.00

0.00

0.00

0.00

0.49

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

4.55*

6.64

11.18

0.00

0.00

0.00

0.00

0.00

0.00

0.80

100.00

±SD

1.44

2.06

1.72

0.00

0.00

0.00

0.00

0.00

0.00

0.49

0.00

n

11

11

11

11

11

11

11

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: male/female; LD: Lactation Day.

Contd. LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group, Sex

& Dose
(mg/kg body

weight/day)

Pups Sacrificed for

Litter Standardization (No.)

Live Pups (No.) on LD 4 after 

Litter Standardization (No.)

During LD 5 to 7

Sex Ratio

(m/f)

on LD 7

Pup Survival

Index (%)

during

LD 5 to 7

Live Pups (No.)

Dead Pups (No.)

Cannibalized (No.)

Male

Female

Total

Male

Female

Total

Male

Female

Total

Male

Female

Total

Male

Female

Total

G1, F & 0

Mean

0.00

1.91

1.91

6.00

4.64

10.64

6.00

4.64

10.64

0.00

0.00

0.00

0.00

0.00

0.00

1.44

100.00

±SD

0.00

0.30

0.30

1.67

1.03

0.92

1.67

1.03

0.92

0.00

0.00

0.00

0.00

0.00

0.00

0.79

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

0.00

1.50

1.50

6.50

4.17

10.67

6.50

4.17

10.67

0.00

0.00

0.00

0.00

0.00

0.00

1.90

100.00

±SD

0.00

0.80

0.80

0.90

1.70

1.37

0.90

1.70

1.37

0.00

0.00

0.00

0.00

0.00

0.00

1.09

0.00

n

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

12

G3, F & 500

Mean

0.00

1.73

1.73

5.82

4.55

10.36

5.82

4.55

10.36

0.00

0.00

0.00

0.00

0.00

0.00

1.55

100.00

±SD

0.00

0.65

0.65

1.40

1.44

0.67

1.40

1.44

0.67

0.00

0.00

0.00

0.00

0.00

0.00

1.09

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

0.00

1.00

1.00

4.55

5.64

10.18

4.55*

5.64

10.18

0.00

0.00

0.00

0.00

0.00

0.00

0.97*

100.00

±SD

0.00

1.00

1.00

1.44

1.69

0.87

1.44

1.69

0.87

0.00

0.00

0.00

0.00

0.00

0.00

0.65

0.00

n

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; m/f: male/female.*: Statistically significant (P<0.05) change than the vehicle control group.

Contd. LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group, Sex

& Dose
(mg/kg body

weight/day)

During LD 8 to 13

Sex Ratio

(m/f)

on LD 13

Pup Survival

Index (%)

during LD 8 to 13

Live Pups (No.)

Dead Pups (No.)

Cannibalized (No.)

Male

Female

Total

Male

Female

Total

Male

Female

Total

G1, F & 0

Mean

6.00

4.64

10.64

0.00

0.00

0.00

0.00

0.00

0.00

1.44

100.00

±SD

1.67

1.03

0.92

0.00

0.00

0.00

0.00

0.00

0.00

0.79

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G2, F & 250

Mean

6.50

4.17

10.67

0.00

0.00

0.00

0.00

0.00

0.00

1.90

100.00

±SD

0.90

1.70

1.37

0.00

0.00

0.00

0.00

0.00

0.00

1.09

0.00

n

12

12

12

12

12

12

12

12

12

12

12

G3, F & 500

Mean

5.82

4.55

10.36

0.00

0.00

0.00

0.00

0.00

0.00

1.55

100.00

±SD

1.40

1.44

0.67

0.00

0.00

0.00

0.00

0.00

0.00

1.09

0.00

n

11

11

11

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

4.55*

5.64

10.18

0.00

0.00

0.00

0.00

0.00

0.00

0.97*

100.00

±SD

1.44

1.69

0.87

0.00

0.00

0.00

0.00

0.00

0.00

0.65

0.00

n

11

11

11

11

11

11

11

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; m/f: male/female.*: Statistically significant (P<0.05) change than the vehicle control group.

Table 14. Table for REPRODUCTIVE PERFORMANCE RECORD (Males)

Group, Sex & Dose

(mg/kg body weight/day)

No. of Males with Evidence of Mating
(Total No. of Males used for Mating)

Male Mating Index (%)

No. of Males Capable of Impregnating a Female
(No. of males used for Mating)

Male Fertility Index (%)

G1, M & 0

12 (12)

100.0

11 (12)

91.7

G2, M & 250

12 (12)

100.0

12 (12)

100.0

G3, M & 500

12 (12)

100.0

11 (12)

91.7

G4, M & 1000

12 (12)

100.0

11 (12)

91.7

M: Male.

Contd. Table for REPRODUCTIVE PERFORMANCE RECORD (Females)

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Pre-coital Interval / Mean Time to Mating (Days)

 Gestation Length /

Duration of Pregnancy (Days)

Pre-Coital Interval /

Copulatory Interval

Conceiving Days

(1 to 5)

Conceiving Days

(5 to More)

Gestation Length (Days)

G1, F & 0

Mean

8.08

n

3

9

22.45

±SD

3.82

%

27.27

81.82

0.52

n

12

11

G2, F & 250

Mean

4.42

n

9

3

22.33

±SD

2.87

%

75.00

25.00

0.49

n

12

12

G3, F & 500

Mean

5.67

n

7

5

22.45

±SD

3.73

%

63.64

45.45

0.52

n

12

11

G4, F & 1000

Mean

5.25

n

7

5

22.55

±SD

4.86

%

63.64

45.45

0.52

n

12

11

F: Female; SD: Standard Deviation.

n: Number of Females confirmed with mating (in case ofPre-Coital Interval) /Number of Females confirmed with pregnancy (in case ofgestation length).

Contd. Table for REPRODUCTIVE PERFORMANCE RECORD (Females)

Group, Sex

& Dose

(mg/kg body weight/day)

Female Mating Index (%)

Female Fertility Index (%)

Female Fecundity or Pregnancy Index (%)

No. of

Females with Evidence

of Mating

No. of

Females used for Mating

Female Mating Index (%)

No. of

Females confirmed as Fertile

No. of

Females used for Mating

Female

Fertility Index (%)

No. of

Pregnant Females

No. of

Females with confirmed Mating

Female Fecundity or Pregnancy

Index (%)

G1, F & 0

12

12

100.0

11

12

91.7

11

12

91.7

G2, F & 250

12

12

100.0

12

12

100.0

12

12

100.0

G3, F & 500

12

12

100.0

11

12

91.7

11

12

91.7

G4, F & 1000

12

12

100.0

11

12

91.7

11

12

91.7

Contd. Table for REPRODUCTIVE PERFORMANCE RECORD (Females)

 Group, Sex

& Dose

(mg/kg body

weight/day)

Gestation index (%)

 

Parturition index (%)

With Live born

Pups at Parturition

No. of Females with evidence of pregnancy

Gestation index

(%)

 

No. of Females Littered

No. of Females with evidence of pregnancy

Parturition index

(%)

G1, F & 0

11

11

100.0

 

11

11

100.0

G2, F & 250

12

12

100.0

 

12

12

100.0

G3, F & 500

11

11

100.0

 

11

11

100.0

G4, F & 1000

11

11

100.0

 

11

11

100.0

Contd.Table for REPRODUCTIVE PERFORMANCE RECORD (Females)

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Post-implantation Loss (%)

 

Post-natal Loss (%)

No. of Implantations

No. of Viable

(Live) Pups

Post-implantation Loss (No.)

Post-implantation Loss (%)

 

Total No. of Pups Found Dead/

Cannibalized during lactation period

Post-natal Loss (%)

G1, F & 0

Mean

12.91

12.55

0.36

2.65

 

0.00

0.00

±SD

1.22

1.04

0.50

3.68

 

0.00

0.00

n

11

11

11

11

 

11

11

G2, F & 250

Mean

12.42

12.17

0.25

1.93

 

0.00

0.00

±SD

1.83

1.80

0.45

3.50

 

0.00

0.00

n

12

12

12

12

 

12

12

G3, F & 500

Mean

12.36

12.09

0.27

1.86

 

0.00

0.00

±SD

1.43

1.04

0.65

4.37

 

0.00

0.00

n

11

11

11

11

 

11

11

G4, F & 1000

Mean

11.73

11.18

0.55

4.11

 

0.00

0.00

±SD

1.79

1.72

1.29

9.53

 

0.00

0.00

n

11

11

11

11

 

11

11

F: Female; n: Number of Dams.

Table 15. Table for ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Testes

Epididymides

Prostate

Seminal vesicles

with coagulating glands

Thyroid along

with parathyroid

G1, M & 0

Mean

3.2784

1.3315

1.1175

1.4823

0.0284

±SD

0.2789

0.0716

0.1440

0.2196

0.0036

n

12

12

12

12

12

G2, M & 250

Mean

3.2778

1.3408

1.1698

1.6503

0.0277

±SD

0.2537

0.0947

0.1620

0.1872

0.0023

n

12

12

12

12

12

G3, M & 500

Mean

3.2840

1.3214

1.1262

1.4692

0.0275

±SD

0.2547

0.0952

0.1147

0.1836

0.0033

n

12

12

12

12

12

G4, M & 1000

Mean

3.2368

1.3370

1.2206

1.4698

0.0283

±SD

0.3159

0.1511

0.2052

0.1959

0.0029

n

12

12

12

12

12

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

Contd. ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Ovaries

Thyroid along
with parathyroid#

G1, F & 0

Mean

0.1429

0.0255

±SD

0.0077

0.0017

n

12

12

G2, F & 250

Mean

0.1446

0.0244

±SD

0.0082

0.0012

n

12

12

G3, F & 500

Mean

0.1465

0.0259

±SD

0.0050

0.0019

n

12

12

G4, F & 1000

Mean

0.1412

0.0265

±SD

0.0108

0.0016

n

12

12

F: Female; SD: Standard Deviation; n: Number of Dams; #: Weighed post fixation.

Table 16. Table for TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Terminal

Body Weight (g)

Testes

Epididymides

Prostate

Seminal vesicles with coagulating glands

Thyroid along

with parathyroid

G1, M & 0

Mean

391.35

0.8413

0.3429

0.2875

0.3788

0.0073

±SD

37.78

0.0692

0.0343

0.0444

0.0408

0.0009

n

12

12

12

12

12

12

G2, M & 250

Mean

391.42

0.8402

0.3434

0.3006

0.4235

0.0071

±SD

27.39

0.0743

0.0266

0.0480

0.0548

0.0007

n

12

12

12

12

12

12

G3, M & 500

Mean

396.15

0.8339

0.3352

0.2880

0.3738

0.0070

±SD

40.24

0.0787

0.0250

0.0485

0.0555

0.0008

n

12

12

12

12

12

12

G4, M & 1000

Mean

402.50

0.8046

0.3322

0.3045

0.3653

0.0071

±SD

30.92

0.0533

0.0275

0.0527

0.0416

0.0008

n

12

12

12

12

12

12

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

Contd. Table for TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO TERMINAL BODY WEIGHT (%) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

 

Terminal

Body Weight (g)

Ovaries

Thyroid along
with parathyroid

G1, F & 0

Mean

302.15

0.0474

0.0085

±SD

16.19

0.0029

0.0006

n

12

12

12

G2, F & 250

Mean

301.04

0.0482

0.0081

±SD

17.62

0.0040

0.0005

n

12

12

12

G3, F & 500

Mean

300.99

0.0488

0.0086

±SD

16.17

0.0035

0.0007

n

12

12

12

G4, F & 1000

Mean

304.73

0.0463

0.0087

±SD

16.39

0.0027

0.0008

n

12

12

12

F: Female; SD: Standard Deviation; n: Number of Dams.

Table 17. Table for SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - MALES

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

Serum T4 Levels (ng/mL)

G1, M & 0

Mean

82.133

±SD

9.622

n

12

G2, M & 250

Mean

82.175

±SD

7.976

n

12

G3, M & 500

Mean

80.692

±SD

8.452

n

12

G4, M &1000

Mean

81.318

±SD

9.291

n

12

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

Table 18. Table for PUP OBSERVATIONS RECORD DURING POSTNATAL PERIOD

Group, Sex

& Dose

(mg/kg body

weight/day)

At Birth (PND 1)

PND

1 to 4

Pups Sacrificed for Blood Collection

on PND 4 (No.)*

PND

5 to 7$

PND

8 to 13$

G1, F & 0

No. of Dams / Litters#

11

11

11

11

11

No. of Live Pups

138

138

21

117

117

Pup Observation/

No. of Pups observed

N/138

N/138

-

N/117

N/117

G2, F & 250

No. of Dams / Litters#

12

12

10

12

12

No. of Live Pups

146

146

18

128

128

Pup Observation/

No. of Pups observed

N/146

N/146

-

N/128

N/128

G3, F & 500

No. of Dams / Litters#

11

11

10

11

11

No. of Live Pups

133

133

19

114

114

Pup Observation/

No. of Pups observed

N/133

N/133

-

N/114

N/114

G4, F & 1000

No. of Dams / Litters#

11

11

6

11

11

No. of Live Pups

123

123

11

112

112

Pup Observation/

No. of Pups observed

N/123

N/123

-

N/112

N/112

F: Female; N: Normal; PND: Postnatal Day;#: confirmed with live pups.*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded.

Table 19. Table for MEAN PUP WEIGHT (g) PER LITTER RECORD DURING LACTATION PERIOD

Group, Sex

& Dose

(mg/kg body weight/day)

PND 1

PND 4

PND 7

PND 14

Mean Pup Weight (g)

Mean Pup Weight (g)

Mean Pup Weight (g)

Mean Pup Weight (g)

Male

Female

 

Male

Female

 

Male

Female

 

Male

Female

G1, F & 0

Mean

7.19

6.78

11.20

10.88

15.12

14.97

25.75

24.86

±SD

0.23

0.24

0.25

0.26

0.32

0.19

0.42

0.36

n

11

11

11

11

11

11

11

11

G2, F & 250

Mean

7.01

6.64

11.03

10.51*

15.13

14.95

25.55

24.77

±SD

0.44

0.46

0.39

0.31

0.25

0.34

0.82

0.38

n

12

12

12

12

12

12

12

12

G3, F & 500

Mean

7.13

6.81

11.07

10.73

15.01

15.05

25.57

24.77

±SD

0.19

0.22

0.22

0.31

0.20

0.32

0.63

0.52

n

11

11

11

11

11

11

11

11

G4, F & 1000

Mean

7.12

6.75

11.05

10.84

15.19

15.10

26.15

24.95

±SD

0.42

0.34

0.34

0.23

0.26

0.20

0.69

0.43

n

11

11

11

11

11

11

11

11

F: Female; SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day.

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

Table 20. Table for MEAN PUP ANOGENITAL DISTANCE (AGD) MEASUREMENT (mm) AND ANOGENITAL DISTANCE (AGD) RATIO PER LITTER RECORD ON POSTNATAL DAY 4

Group

& Dose

(mg/kg body

weight/day)

 

Mean Anogenital Distance Measurement (mm)

 

Mean Anogenital Distance Ratio

Male

Female

 

Male

Female

G1 & 0

Mean

4.98

2.47

2.23

1.11

±SD

0.12

0.06

0.06

0.03

n

11

11

 

11

11

G2 & 250

Mean

4.83*

2.52

2.17

1.15

±SD

0.15

0.11

0.08

0.05

n

12

12

 

12

12

G3 & 500

Mean

4.94

2.50

2.22

1.13

±SD

0.11

0.11

0.05

0.06

n

11

11

 

11

11

G4 & 1000

Mean

4.97

2.59*

2.23

1.17*

±SD

0.17

0.10

0.09

0.05

n

11

11

 

11

11

F: Female; SD: Standard Deviation; n: Number of Litters; AGD: Anogenital Distance.

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

Table 21. Table for MALE PUP NIPPLE/AREOLAE RETENTION (no.) RECORD PER LITTER

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

Mean No. of Pups with Retention of Nipples/

Areolae on Postnatal Day 13

G1 & 0

Mean

0.00

±SD

0.00

n

11

G2 & 250

Mean

0.00

±SD

0.00

n

12

G3 & 500

Mean

0.00

±SD

0.00

n

11

G4 & 1000

Mean

0.00

±SD

0.00

n

11

SD: Standard Deviation; n: Number of Litters.

Table 22. Table for SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY 13 PUPS (PND)

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

Serum T4 Levels (ng/mL)

G1 & 0

Mean

68.196

±SD

6.763

n

11

G2 & 250

Mean

71.669

±SD

2.452

n

12

G3 & 500

Mean

72.121

±SD

2.791

n

11

G4 &1000

Mean

65.087

±SD

1.589

n

11

SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day.

Applicant's summary and conclusion

Conclusions:
The study-derived NOAEL for systemic, reproductive, and developmental toxicity was considered at 1000 mg/kg bw/day.
Executive summary:

The study was performed according to OECD 421 (2016) and according to GLP. The substance was given to 12 rats per sex per dose level at 0 (corn oil), 250, 500 and 1000 mg/kg bw/day. Male rats were treated before mating and during mating for a total of 29 days. Female rats were treated before mating, during mating, during pregnancy, and up to day 13 of lactation for a total of approx. 63 days.Results (Parent generation):All animals survived to planned death and there were no clinical signs of toxicity. No test item-related changes noted in body weight or body weight gain and feed consumption of both sexes from any of the dose levels during the experimental period. There were no significant changes noted in serum thyroxine hormone (T4) levels in all treatment groups compared to control groups. No significant changes in absolute or relative organ weights of both sexes in any of the dose levels were observed. There were no gross pathological changes observed during necropsy in all of the adult animals except in groups G3 and G4 wherein, both gastric and intestinal contents were found with yellow discoloration. This noted coloration was due to colored nature of test-item. Histopathological examinations of G1 (males and females) and G4 (males and females) animals did not reveal any treatment-related effects. There were no irregularities observed in the oestrus cyclicity in any of the female groups during the pre-mating or mating period. No significant changes in mean oestrus cycle length were observed between the groups during the pre-mating or mating period. No treatment related adverse effects on male or female mating index, male or female fertility index, pre-coital interval, gestational length, gestation index, parturition index, pregnancy index, live birth index, post implantation loss, mean number of Implantations were observed.Results (F1 generation):All pups showed normal behaviour during the daily inspections. No significant changes in live birth index (%) were observed and no cases of unscheduled mortality were observed during the post-natal period. No significant changes in pup weight were observed at all the tested dose levels. No significant changes in AGD measurement/ratio were observed at all the tested dose levels. No significant changes in pup weight or AGD measurement/ratio were observed at all the tested dose levels. No cases of nipple retention were observed on PND 13 among male pups. Serum thyroxine (T4) hormone levels measured on PND 13 were unaffected by treatment. No significant changes in sex ratio were observed. There were no gross pathological findings in any of the groups.Conclusion:NOAEL for systemic, reproductive, and developmental toxicity was considered at 1000 mg/kg bw/day.

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