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Diss Factsheets

Administrative data

Description of key information

The subacute oral toxicity of the test item was assessed in a study acording to OECD TG 422. No adverse effects occured. The NOAEL was determiend as 1000 mg/kgbw/day.


Regarding repeated dose toxicity via inhalation route, the information will be provided later based on the final decision Nr.: CCH-D-2114498202-49-01/F, according to analogue approaches.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Sprague Dawley Rats
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June 2021 to 22 December 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29ºC)
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.
0.5% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
The test item was stable for 6 hours and 48 hours at room temperature in 0.5% w/v Carboxymethyl cellulose with the concentrations of 5 mg/mL and 100 mg/mL.

FORM AS APPLIED IN THE TEST: The test item or vehicle were administered to the animals through the oral (gavage) route once daily
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: Males: 254.36 to 291.64 g; Females: 200.54 to 211.92 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
During acclimatization, two animals of same sex were housed.
i. Main Group Animals:
- 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 on 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 for main group females from gestation day 20 onwards.
ii. Recovery Group Animals:
- Maximum of two animals per cage per group of same sex were housed, tunnels were provided as enrichment for the animals which were housed individually.
- Diet: Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water: Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: Analysis of food and water qaulity included as annexures in the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 to 22.9 Degree Centigrade
- Humidity (%): 48 to 63%,
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 24 June 2021; To: 16 September 2021
Route of administration:
oral: gavage
Details on route of administration:
The test item or vehicle were administered to the animals through the oral (gavage) route once daily.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% w/v CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.
0.5% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies
- Concentration in vehicle: The concentrations of of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively
- Amount of vehicle (if gavage): 10mL/Kg body weight
- Batch no.: SLCH1520, expiry date: September 2026; Make Sigma Aldrich
- Purity: Medium viscocity grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Dose formulation samples of the test item were analyzed for homogeneity and dose concentration by UV-Vis Method and the results are found within the range of 85 to 115% of the nominal concentration and the %RSD is <15%

Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and week 5 of the treatment. The samples were collected in duplicates (5 mL each) from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control.
The prepared test item formulations were stirred using magnetic stirrer during sampling.

The mean obtained results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is less than 15%.
Duration of treatment / exposure:
Males: 31 Days
Females: The females were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 50 to 62 days).
The females with positive mating signal, but no-evidence of pregnancy / littering were treated during two-week pre-mating period, during mating and further 24 days from the day of positive mating signal (group G1 - one female until experimental day 39; group G2 - one female until experimental day 52, one female until experimental day 46; G3 - two females until experimental day 49; group G4 - one female until experimental day 39).
Recovery group animals: 52 Days
Frequency of treatment:
Once Daily
Dose / conc.:
0 other: mg/kg body weight/day
Remarks:
Vehicle Control
Dose / conc.:
111 other: mg/kg body weight/day
Remarks:
Low Dose
Dose / conc.:
333 other: mg/kg body weight/day
Remarks:
Mid Dose
Dose / conc.:
1 000 other: mg/kg body weight/day
Remarks:
High Dose
No. of animals per sex per dose:
Main Group: 24 (12 Males + 12 Females)
Recovery Group: 10 (5 Males + 5 Females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item.

The doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.

There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).

Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for the present study in consultation with the sponsor.

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the main and recovery groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study does not exceed ±20% (Males: -6.80 to 11.14%; Females: -12.58 to 8.29%) of the mean body weight of each sex. The grouping was done one day prior to initiation of treatment. Body weight of the animals were analyzed statistically for mean body weight to rule out statistical significant differences between groups within each sex.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for general clinical signs of toxicity. These observations were made approximately at the same time on each day after dose administration. All the animals were observed twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and approximately at the same time.
BODY WEIGHT: Yes
- Time schedule for examinations: The main group males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.
The main group females were weighed at receipt, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating. All the pregnant animals were weighed on gestation days (GD) 0, 7, 14 and 20 and on lactation days (LD) 1, 4, 7, 13 and 14 (terminal body weight). The non-pregnant animals were weighed weekly once until termination and the terminal body weights were recorded before scheduled sacrifice.
The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD EFFICIENCY:
- Cage wise feed consumption was measured for all main group males and females, once in a week during premating period coinciding with body weight recording.
Cage wise feed consumption was measured for all pregnant animals during gestation day (GD) 0 to 7, 7 to 14 and 14 to 20, during lactation day (LD) 1 to 4, 4 to 7 and
7 to 13 coinciding with body weight recording.
Cage wise feed consumption was measured for all recovery group animals once in a week throughout the experimental period coinciding with body weight recording.
Average feed intake per animal (g/animal/day) was calculated using the amount of feed offered and left over in each cage and the number of rats per cage.
Feed consumption was not measured during following occasions, but offered ad libitum of feed:
- cohabitation period for both sexes of main groups;
- after GD 20 and until parturition for pregnant females;
- from day 20 after confirmation of mating until termination for non-pregnant females.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was carried out for all animals before initiation of treatment i.e. after grouping and randomization (last day of acclimatization). Ophthalmological examination was also carried out at the end of dosing period for all males (shortly prior to scheduled sacrifice, i.e. day 30) and during lactation period for females (shortly prior to scheduled sacrifice, i.e. on lactation day 13) and during last week (i.e. day 65) for the recovery group animals.
- Dose groups that were examined: G1/G1R, G2, G3 and G4/G4R.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples of both males and females were collected on the day of scheduled terminal sacrifice (for main group males after completion of 31 days of dose administration, for main group females on lactation day 14 and for recovery group animals on the day of scheduled sacrifice.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 12 Males and 12 Femles from all the main groups; 5 Males and 5 Females from all the recovery groups.
- Parameters checked in table [No.16] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples of both males and females were collected on the day of scheduled terminal sacrifice (for main group males after completion of 31 days of dose administration, for main group females on lactation day 14 and for recovery group animals on the day of scheduled sacrifice.
- Animals fasted: Yes
- How many animals: 12 Males and 12 Femles from all the main groups; 5 Males and 5 Females from all the recovery groups.
- Parameters checked in table [No.17] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected from five randomly selected males of each main group and for all recovery animals at termination. The selected animals were placed in urine collection cages overnight and not given access to feed but water was provided ad libitum during their stay in the urine collection cages. The overnight urine volume (mL) collected from these animals were measured.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.18] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was performed for five males and five females, randomly selected from each group, towards the end of the dosing period for males (shortly prior to scheduled sacrifice, i.e. on day 30) and during the lactation period for females (shortly prior to scheduled sacrifice, i.e. on lactation day 13). Neurological/Functional examination was performed for all recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice, i.e.
on day 65.
- Dose groups that were examined: G1/G1R, G2, G3 and G4/G4R.
- Battery of functions tested: sensory activity, grip strength, motor activity and Hindlimb Footsplay.

Thyroid Hormone Estimations: Blood collection for Thyroxine (T4) Level estimation: Blood samples were collected from all the main group animals for measurement of serum T4 levels on the following schedule:
- All dams at termination (lactation day 14).
- All adult males, at termination (after completion of 31 days of treatment).
The assessment of serum T4 levels was performed for adult males. Further assessment of T4 levels in serum samples from the dams was not assessed as there were no treatment-related effects observed in the T4 levels of adult males.
The collected blood samples were centrifuged at 5000 rpm for 10 minutes; the serum was separated and stored at freezing conditions (-80ºC ± 10ºC) till estimation.
The serum samples were estimated for thyroid hormonal levels (Thyroxine - T4) by the ELISA method using commercial assay kits.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The statistical analysis tests followed for the interpretation of data are as mentioned below:
• Body weight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Hematology
• Clinical chemistry
• Urinalysis
• Absolute/relative organ weights
• Functional Observation parameters
• Thyroid hormonal levels
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity recorded in any of the animals of both sexes from all the tested dose groups throughout the experimental period. However, dark purple colored faeces were noted from day 2 observation throughout the experiment period till termination of treatment in group G3 & G4/G4R animals. This coloration was due to colored nature of the test item but not any adverse signs.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
other: THyroid hormonal lelves
Key result
Critical effects observed:
no

TABLE 1.     SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND
MORTALITY RECORD

Group, Sex & Dose

(mg/kg body weight/day)

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Detailed Clinical Examinationb:

Observation
(No. of Animals revealed)

Mortalityc:

No. of Mortalities
(Total No. of Animals)

G1, M & 0

12

N (12)

NAD (12)

0 (12)

G2, M & 111

12

N (12)

NAD (12)

0 (12)

G3, M & 333

12

N (12)

NAD (12)

0 (12)

G4, M & 1000

12

N (12)

NAD (12)

0 (12)

M: Male; N: Normal; NAD: No Abnormality Detected.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note:Dark purple colored faeces were noted from day 2 observation throughout the experiment period till termination in group G3 & G4 animals.

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION
AND MORTALITY RECORD

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

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Detailed Clinical Examinationb:

Observation
(No. of Animals revealed)

Mortalityc:

No. of Mortalities
(Total No. of Animals)

G1, F & 0

12

N (12)

NAD (12)

0 (12)

G2, F & 111

12

N (12)

NAD (12)

0 (12)

G3, F & 333

12

N (12)

NAD (12)

0 (12)

G4, F & 1000

12

N (12)

NAD (12)

0 (12)

F: Female; N: Normal; NAD: No Abnormality Detected.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note:Dark purple colored faeces were noted from day 2 observation throughout the experiment period till termination in group G3 & G4 animals.

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION
AND MORTALITY RECORD

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

Total No. of Animals

Clinical Signs of Toxicitya:

Observation
(No. of Animals revealed)

Detailed Clinical Examinationb:

Observation
(No. of Animals revealed)

Mortalityc:

No. of Mortalities
(Total No. of Animals)

G1R, M & 0

5

N (5)

NAD (5)

0 (5)

G4R, M & 1000

5

N (5)

NAD (5)

0 (5)

G1R, F & 0

5

N (5)

NAD (5)

0 (5)

G4R, F & 1000

5

N (5)

NAD (5)

0 (5)

M: Male; F: Female; R: Recovery; N: Normal; NAD: No abnormality detected.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note:Dark purple colored faeces were noted from day 2 throughout the experiment period till termination in group G4R animals.

TABLE 2.     SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Body Weight (g) on Day

1

7

14

21

28

G1, M & 0

Mean

400.75

421.20

438.19

446.01

458.77

±SD

22.07

32.34

36.49

37.08

38.53

n

12

12

12

12

12

G2, M & 111

Mean

401.74

417.26

433.41

442.75

452.56

±SD

20.39

24.35

30.70

31.29

30.16

n

12

12

12

12

12

G3, M & 333

Mean

402.72

419.51

438.54

444.84

456.36

±SD

19.35

23.54

30.11

27.23

24.95

n

12

12

12

12

12

G4, M & 1000

Mean

401.99

422.76

443.74

453.94

463.38

±SD

21.50

27.21

34.32

36.83

39.17

n

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 Day

1

7

14

21#

G1, F & 0

Mean

250.88

257.35

267.91

280.34

±SD

14.86

17.17

18.25

26.26

n

12

12

12

3

G2, F & 111

Mean

251.05

256.43

266.53

272.56

±SD

12.78

15.62

15.30

25.98

n

12

12

12

3

G3, F & 333

Mean

251.13

256.32

261.08

260.46

±SD

13.59

14.00

15.94

17.45

n

12

12

12

4

G4, F & 1000

Mean

251.24

261.46

265.51

269.05

±SD

15.56

18.91

20.71

24.37

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 2 (Contd…). SUMMARY OF BODY WEIGHT (g) RECORD

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

 

Body Weight (g) on Day

1

7

14

21

28

35

42

49

56

63

G1R, M & 0

Mean

395.45

407.17

422.63

436.11

448.75

458.35

473.23

489.33

502.87

514.37

±SD

21.72

21.10

31.29

32.45

32.78

34.62

33.67

33.58

34.92

37.99

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

396.36

412.35

430.15

444.31

454.98

464.82

475.04

493.68

508.20

519.69

±SD

20.86

20.17

12.53

16.96

17.27

14.72

15.50

9.28

11.65

11.69

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

245.24

250.97

262.44

268.72

277.56

284.92

295.96

306.72

315.86

324.75

±SD

10.97

10.11

13.68

15.80

15.60

15.72

19.40

19.59

17.41

15.21

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

245.66

248.99

264.41

270.43

278.43

286.33

298.35

308.32

318.51

327.87

±SD

12.82

12.43

13.84

12.16

11.19

12.10

12.79

13.72

10.97

12.44

n

5

5

5

5

5

5

5

5

5

5

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 3.     SUMMARY RECORD OF 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

272.42

287.38

319.92

375.62

±SD

18.33

21.41

28.89

35.87

n

11

11

11

11

G2, F & 111

Mean

269.81

286.87

328.45

391.71

±SD

11.98

12.37

15.11

22.46

n

10

10

10

10

G3, F & 333

Mean

264.91

282.42

317.51

369.85

±SD

15.69

14.58

20.68

32.90

n

10

10

10

10

G4, F & 1000

Mean

273.49

291.35

327.07

382.89

±SD

17.37

17.37

23.26

39.30

n

11

11

11

11

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

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.

TABLE 4.     SUMMARY RECORD OF 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

294.60

300.48

308.18

323.25

±SD

22.07

22.28

22.06

20.39

n

11

11

11

11

G2, F & 111

Mean

306.24

309.48

314.78

330.57

±SD

22.46

21.39

20.72

19.26

n

10

10

10

10

G3, F & 333

Mean

293.35

297.31

304.48

319.65

±SD

19.47

20.23

20.46

22.22

n

10

10

10

10

G4, F & 1000

Mean

303.31

306.82

313.01

325.64

±SD

22.77

24.10

23.73

23.27

n

11

11

11

11

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

TABLE 5.     SUMMARY OF PERCENT CHANGE IN BODY WEIGHT GAIN (%)
WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21

1 to 28

G1, M & 0

Mean

5.01

9.25

11.19

14.36

±SD

3.10

4.96

4.86

4.98

n

12

12

12

12

G2, M & 111

Mean

3.84

7.80

10.13

12.59

±SD

2.04

3.09

3.48

3.20

n

12

12

12

12

G3, M & 333

Mean

4.15

8.89

10.50

13.42

±SD

2.15

5.14

5.14

5.61

n

12

12

12

12

G4, M & 1000

Mean

5.12

10.29

12.82

15.16

±SD

1.91

3.83

4.55

5.27

n

12

12

12

12

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

TABLE 5 (Contd…). SUMMARY OF PERCENTCHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21#

G1, F & 0

Mean

2.56

6.78

10.07

±SD

2.21

3.23

3.01

n

12

12

3

G2, F & 111

Mean

2.12

6.21

9.94

±SD

2.46

4.16

1.49

n

12

12

3

G3, F & 333

Mean

2.08

3.94

5.98

±SD

1.81

2.08

1.64

n

12

12

4

G4, F & 1000

Mean

4.05

5.61

9.37

±SD

3.24

3.20

3.75

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 1 to 21 body weight was not subjected to statistical analysis due to uneven number of variables.

TABLE 5 (Contd…). SUMMARY OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY1 RECORD

Group, Sex & Dose

(mg/kg body weight/day)

 

Percent Change in Body Weight (%) during Day

1 to 7

1 to 14

1 to 21

1 to 28

1 to 35

1 to 42

1 to 49

1 to 56

1 to 63

G1R, M & 0

Mean

2.98

6.83

10.25

13.47

15.92

19.69

23.73

27.13

30.00

±SD

0.92

4.09

4.67

5.29

6.27

5.81

4.88

4.25

4.30

n

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

4.06

8.66

12.20

14.91

17.44

20.09

24.77

28.45

31.32

±SD

1.73

3.60

3.33

3.84

4.90

6.78

5.51

6.10

5.14

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.35

7.00

9.55

13.20

16.19

20.62

25.02

28.77

32.44

±SD

0.72

2.36

3.15

4.61

4.45

4.14

4.54

3.55

3.44

n

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

1.37

7.66

10.20

13.45

16.65

21.52

25.60

29.78

33.57

±SD

1.07

2.71

4.80

4.07

3.73

3.02

4.10

3.75

3.31

n

5

5

5

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 6.     SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING GESTATION PERIOD

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

5.46

11.22

17.44

±SD

1.77

2.82

4.18

n

11

11

11

G2, F & 111

Mean

6.35

14.52

19.23

±SD

2.12

3.20

3.16

n

10

10

10

G3, F & 333

Mean

6.67

12.40

16.35

±SD

1.92

3.55

3.43

n

10

10

10

G4, F & 1000

Mean

6.56

12.23

16.88

±SD

1.60

3.35

4.81

n

11

11

11

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

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.

TABLE 7.     SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING LACTATION PERIOD

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

2.01

2.58

4.96

±SD

0.96

0.73

1.51

n

11

11

11

G2, F & 111

Mean

1.08

1.74

5.07

±SD

0.86

0.95

1.60

n

10

10

10

G3, F & 333

Mean

1.35

2.42

4.98

±SD

0.95

1.29

1.32

n

10

10

10

G4, F & 1000

Mean

1.14

2.04

4.06

±SD

1.00

0.93

0.83

n

11

11

11

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

TABLE 8.     SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

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

21.31

24.01

±SD

2.38

1.81

n

12 (6)

12 (6)

G2, M & 111

Mean

21.39

23.12

±SD

1.76

0.75

n

12 (6)

12 (6)

G3, M & 333

Mean

22.25

23.93

±SD

2.39

1.85

n

12 (6)

12 (6)

G4, M & 1000

Mean

22.28

24.17

±SD

1.23

0.67

n

12 (6)

12 (6)

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

TABLE 8 (Contd…). SUMMARY OF FEED CONSUMPTION (g/animal/day)RECORD

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

18.14

19.17

±SD

1.56

1.22

n

12 (6)

12 (6)

G2, F & 111

Mean

17.79

20.11

±SD

1.85

1.70

n

12 (6)

12 (6)

G3, F & 333

Mean

18.26

19.22

±SD

1.99

1.31

n

12 (6)

12 (6)

G4, F & 1000

Mean

18.43

20.07

±SD

2.28

1.22

n

12 (6)

12 (6)

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

TABLE 8 (Contd…). SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Feed Consumption (g/animal/day)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

G1R, M & 0

Mean

23.52

24.34

25.11

26.35

27.86

29.05

30.75

31.83

32.47

±SD

3.71

1.67

1.55

1.27

0.97

1.11

0.47

3.66

3.01

n

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

G4R, M & 1000

Mean

22.84

23.75

24.26

27.17

28.86

30.05

31.07

32.19

33.03

±SD

0.77

1.04

1.34

2.09

2.61

2.18

2.12

1.59

1.67

n

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

G1R, F & 0

Mean

17.44

18.36

19.45

10.21

23.17

24.20

25.35

26.19

27.04

±SD

1.79

0.40

0.40

9.72

0.99

0.82

0.62

0.41

0.99

n

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

G4R, F & 1000

Mean

17.21

19.05

20.42

11.29

24.18

25.37

26.19

27.14

27.89

±SD

0.69

1.77

0.91

10.38

0.03

0.80

0.84

0.69

0.68

n

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

5 (3)

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals(Number of cages).

TABLE 9.     SUMMARY RECORD OF 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

19.02

21.07

25.81

SD

0.70

1.95

1.63

n

11

11

11

G2, F & 111

Mean

19.23

22.20

26.84

SD

0.88

1.81

1.78

n

10

10

10

G3, F & 333

Mean

19.00

21.49

26.11

SD

0.74

1.39

1.26

n

10

10

10

G4, F & 1000

Mean

19.43

22.01

26.67

SD

0.58

1.70

1.97

n

11

11

11

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

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis but presented in individual animal data.

TABLE 10.     SUMMARY RECORD OF 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

25.21

35.80

40.88

SD

1.20

7.26

1.43

n

11

11

11

G2, F & 111

Mean

24.58

32.72

41.09

SD

1.34

0.99

0.98

n

10

10

10

G3, F & 333

Mean

25.11

32.75

40.54

SD

1.42

1.16

0.94

n

10

10

10

G4, F & 1000

Mean

23.94

33.14

39.84

SD

5.99

1.28

1.03

n

11

11

11

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

TABLE 11.     SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)

Group, Sex

& Dose
(mg/kg body
     

weight/day) →

G1, M & 0

G2, M & 111

G3, M & 333

G4, M & 1000

Number of Animals

12

12

12

12

Observations↓

Eye

Region↓

LE

RE

LE

RE

LE

RE

LE

RE

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

M: Male; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)

Group, Sex

& Dose
(mg/kg body

weight/day) →

G1, F & 0

G2, F & 111

G3, F & 333

G4, F & 1000

Number of Animals

12

12

12

12

Observations↓

Eye

Region↓

LE

RE

LE

RE

LE

RE

LE

RE

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

F: Female; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)

Group, Sex

& Dose
(mg/kg body
     

weight/day) →

G1R, M & 0

G4R, M & 1000

Number of Animals

5

5

Observations↓

Eye

Region↓

LE

RE

LE

RE

Eye Lids

N

N

N

N

Cornea

N

N

N

N

Iris

N

N

N

N

Aqueous Humour

N

N

N

N

Lens

N

N

N

N

Vitreous Humour

N

N

N

N

Retina/Optic disc

N

N

N

N

M: Male; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)

Group, Sex

& Dose
(mg/kg body

weight/day) →

G1R, F & 0

G4R, F & 1000

Number of Animals

5

5

Observations↓

Eye

Region↓

LE

RE

LE

RE

Eye Lids

N

N

N

N

Cornea

N

N

N

N

Iris

N

N

N

N

Aqueous Humour

N

N

N

N

Lens

N

N

N

N

Vitreous Humour

N

N

N

N

Retina/Optic disc

N

N

N

N

F: Female; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day 30

Group, Sex

& Dose
(mg/kg body
 

weight/day) →

G1, M & 0

G2, M & 111

G3, M & 333

G4, M & 1000

Number of Animals

12

12

12

12

Observations↓

Eye

Region↓

LE

RE

LE

RE

LE

RE

LE

RE

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

Other findings:

N

N

N

N

N

N

N

N

M: Male; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Pregnant/Littered females: Lactation day 13;

Non-pregnant females: (Experimental day 39 to 52)

Group, Sex

& Dose
(mg/kg body

weight/day) →

G1, F & 0

G2, F & 111

G3, F & 333

G4, F & 1000

Number of Animals

12

12

12

12

Observations↓

Eye

Region↓

LE

RE

LE

RE

LE

RE

LE

RE

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

Other findings:

N

N

N

N

N

N

N

N

F: Female; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day 65

Group, Sex

& Dose
(mg/kg body

weight/day) →

G1R, M & 0

G4R, M & 1000

Number of Animals

5

5

Observations↓

Eye

Region↓

LE

RE

LE

RE

Eye Lids

N

N

N

N

Cornea

N

N

N

N

Iris

N

N

N

N

Aqueous Humour

N

N

N

N

Lens

N

N

N

N

Vitreous Humour

N

N

N

N

Retina/Optic disc

N

N

N

N

Other findings:

N

N

N

N

M: Male; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day 65

Group, Sex

& Dose
(mg/kg body
 

weight/day) →

G1R, F & 0

G4R, F & 1000

Number of Animals

5

5

Observations↓

Eye

Region↓

LE

RE

LE

RE

Eye Lids

N

N

N

N

Cornea

N

N

N

N

Iris

N

N

N

N

Aqueous Humour

N

N

N

N

Lens

N

N

N

N

Vitreous Humour

N

N

N

N

Retina/Optic disc

N

N

N

N

Other findings:

N

N

N

N

F: Female; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 12.     SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Day 30

Parameters↓

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

Dose (mg/kg body weight/day)

0

111

333

1000

Number of Animals (randomly selected)

5

5

5

5

Home Cage Observations

Home cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral closure

1

1

1

1

Handling Observations

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur appearance

1

1

1

1

Piloerection

1

1

1

1

Eye prominence

1

1

1

1

Muscle tone

1

1

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Day 30

Parameters↓

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

Dose (mg/kg body weight/day)

0

111

333

1000

Number of Animals (randomly selected)

5

5

5

5

Open Field Observations

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of rearing

Mean

4.4

4.8

4.8

4.8

±SD

1.1

0.8

0.8

0.8

Number of urine pools

Mean

3.4

3.8

3.8

3.8

±SD

0.5

0.8

0.8

0.8

Number of defecations

Mean

3.4

3.2

3.4

3.2

±SD

0.5

0.8

0.5

0.4

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype behaviour

1

1

1

1

Number of grooming

Mean

3.4

3.2

3.4

3.2

±SD

0.5

0.8

0.5

0.8

Sensory Observations

Approach response

2

2

2

2

Auditory response

2

2

2

2

Touch response

2

2

2

2

Pupil reflex

2

2

2

2

Tail pinch response

2

2

2

2

Righting reflex

1

1

1

1

Physiological Observation

Body temperature (°F)

Mean

98.4

98.4

98.1

98.4

±SD

0.5

0.4

0.3

0.5

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Lactation Day 13

Parameters↓

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

Dose (mg/kg body weight/day)

0

111

333

1000

Number of Animals (randomly selected)

5

5

5

5

Home Cage Observations

Home cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral closure

1

1

1

1

Handling Observations

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur appearance

1

1

1

1

Piloerection

1

1

1

1

Eye prominence

1

1

1

1

Muscle tone

1

1

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Lactation Day 13

Parameters↓

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

Dose (mg/kg body weight/day)

0

111

333

1000

Number of Animals (randomly selected)

5

5

5

5

Open Field Observations

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of rearing

Mean

4.8

4.8

4.8

4.8

±SD

0.8

0.8

0.8

0.8

Numbers of urine pools

Mean

3.6

3.8

3.6

3.8

±SD

0.5

0.8

0.5

0.8

Number of defecations

Mean

3.4

3.0

2.8

3.2

±SD

0.9

0.7

0.8

0.4

Clonic involuntary movement

 1

 1

 1

 1

Tonic involuntary movement

 1

 1

 1

 1

Stereotype behaviour

 1

 1

 1

 1

Number of grooming

Mean

3.2

3.2

2.6

3.0

±SD

0.4

0.8

0.5

0.7

Sensory Observations

Approach response

2

2

2

2

Auditory response

2

2

2

2

Touch response  

2

2

2

2

Pupil reflex

2

2

2

2

Tail pinch response

2

2

2

2

Righting reflex

1

1

1

1

Physiological Observation

Body temperature (°F)

Mean

98.4

98.4

98.8

98.7

±SD

0.6

0.5

0.5

0.8

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65

Parameters↓

Group & Sex

G1R & M

G4R & M

Dose (mg/kg body weight/day)

0

1000

Number of Animals

5

5

Home Cage Observations

Home cage posture

1

1

Respiratory pattern

1

1

Clonic involuntary movements

1

1

Tonic involuntary movements

1

1

Vocalization

1

1

Palpebral closure

1

1

Handling Observations

Ease of removal from the cage

2

2

Ease of handling animal in hand

2

2

Red or crusty deposits

Eyes

1

1

Nose

1

1

Mouth

1

1

Lacrimation

1

1

Salivation

1

1

Fur appearance

1

1

Piloerection

1

1

Eye prominence

1

1

Muscle tone

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65

Parameters↓

Group & Sex

G1R & M

G4R & M

Dose (mg/kg body weight/day)

0

1000

Number of Animals

5

5

Open Field Observations

Mobility

1

1

Gait

1

1

Arousal

3

3

Number of rearing

Mean

5.0

4.4

±SD

1.0

1.1

Numbers of urine pools

Mean

3.6

3.4

±SD

0.5

0.5

Number of defecations

Mean

3.6

3.4

±SD

0.5

0.5

Clonic involuntary movement

 1

 1

Tonic involuntary movement

 1

 1

Stereotype behaviour

 1

 1

Number of grooming

Mean

3.6

3.2

±SD

0.5

0.4

Sensory Observations

Approach response

2

2

Auditory response

2

2

Touch response  

2

2

Pupil reflex

2

2

Tail pinch response

2

2

Righting reflex

1

1

Physiological Observation

Body temperature (°F)

Mean

98.5

98.6

±SD

0.5

0.5

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65

Parameters↓

Group & Sex

G1R & F

G4R & F

Dose (mg/kg body weight/day)

0

1000

Number of Animals

5

5

Home Cage Observations

Home cage posture

1

1

Respiratory pattern

1

1

Clonic involuntary movements

1

1

Tonic involuntary movements

1

1

Vocalization

1

1

Palpebral closure

1

1

Handling Observations

Ease of removal from the cage

2

2

Ease of handling animal in hand

2

2

Red or crusty deposits

Eyes

1

1

Nose

1

1

Mouth

1

1

Lacrimation

1

1

Salivation

1

1

Fur appearance

1

1

Piloerection

1

1

Eye prominence

1

1

Muscle tone

1

1

Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65

Parameters↓

Group & Sex

G1R & F

G4R & F

Dose (mg/kg body weight/day)

0

1000

Number of Animals

5

5

Open Field Observations

Mobility

1

1

Gait

1

1

Arousal

3

3

Number of rearing

Mean

5.0

4.8

±SD

1.0

0.8

Numbers of urine pools

Mean

3.6

3.8

±SD

0.5

0.8

Number of defecations

Mean

3.4

3.0

±SD

0.9

0.7

Clonic involuntary movement

 1

 1

Tonic involuntary movement

 1

 1

Stereotype behaviour

 1

 1

Number of grooming

Mean

3.2

3.2

±SD

0.4

0.8

Sensory Observations

Approach response

2

2

Auditory response

2

2

Touch response  

2

2

Pupil reflex

2

2

Tail pinch response

2

2

Righting reflex

1

1

Physiological Observation

Body temperature (°F)

Mean

98.4

98.3

±SD

0.5

0.7

Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 13.     SUMMARY OF LOCOMOTOR ACTIVITY / MOVEMENT COUNTS RECORD

Day 30

Group, Sex & Dose

(mg/kg body weight/day)

Movement Counts (no.)

G1, M & 0

Mean

1911.4

±SD

108.9

n

5

G2, M & 111

Mean

1961.8

±SD

138.9

n

5

G3, M & 333

Mean

1954.2

±SD

126.1

n

5

G4, M & 1000

Mean

1931.4

±SD

104.0

n

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 13 (Contd…). SUMMARY OF LOCOMOTOR ACTIVITY / MOVEMENT COUNTS RECORD

Lactation Day 13

Group, Sex & Dose

(mg/kg body weight/day)

Movement Counts (no.)

G1, F & 0

Mean

1923.4

±SD

102.7

n

5

G2, F & 111

Mean

1941.4

±SD

125.1

n

5

G3, F & 333

Mean

1989.0

±SD

103.1

n

5

G4, F & 1000

Mean

1973.2

±SD

84.7

n

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 13 (Contd…). SUMMARY OF LOCOMOTOR ACTIVITY / MOVEMENT COUNTS RECORD

Day 65

Group, Sex & Dose

(mg/kg body weight/day)

Movement Counts (no.)

G1R, M & 0

Mean

2014.2

±SD

210.7

n

5

G4R, M & 1000

Mean

1980.6

±SD

197.8

n

5

G1R, F & 0

Mean

1943.4

±SD

87.8

n

5

G4R, F & 1000

Mean

1956.2

±SD

91.6

n

5

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

TABLE 14.     SUMMARY OF AVERAGE GRIP STRENGTH (kgf) MEASUREMENT RECORD

Day 30

Group, Sex & Dose

(mg/kg body weight/day)

Grip Strength (kgf)

Forelimb

Hindlimb

G1, M & 0

Mean

1.529

0.510

±SD

0.023

0.025

n

5

5

G2, M & 111

Mean

1.501

0.539

±SD

0.020

0.021

n

5

5

G3, M & 333

Mean

1.521

0.552*

±SD

0.048

0.008

n

5

5

G4, M & 1000

Mean

1.539

0.544*

±SD

0.040

0.015

n

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 14 (Contd…). SUMMARY OF AVERAGE GRIP STRENGTH (kgf) MEASUREMENT RECORD

Lactation Day 13

Group, Sex & Dose

(mg/kg body weight/day)

Grip Strength (kgf)

Forelimb

Hindlimb

G1, F & 0

Mean

1.496

0.508

±SD

0.009

0.008

n

5

5

G2, F & 111

Mean

1.493

0.488

±SD

0.010

0.015

n

5

5

G3, F & 333

Mean

1.496

0.493

±SD

0.009

0.021

n

5

5

G4, F & 1000

Mean

1.498

0.499

±SD

0.011

0.020

n

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 14 (Contd…). SUMMARY OF AVERAGE GRIP STRENGTH (kgf) MEASUREMENT RECORD

Day 65

Group, Sex & Dose

(mg/kg body weight/day)

Grip Strength (kgf)

Forelimb

Hindlimb

G1R, M & 0

Mean

1.515

0.534

±SD

0.020

0.035

n

5

5

G4R, M & 1000

Mean

1.528

0.533

±SD

0.029

0.009

n

5

5

G1R, F & 0

Mean

1.516

0.499

±SD

0.031

0.021

n

5

5

G4R, F & 1000

Mean

1.503

0.481

±SD

0.012

0.012

n

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 15.     SUMMARY OF AVERAGE HINDLIMB FOOT SPLAY (cm) RECORD

Day 30

Group, Sex & Dose

(mg/kg body weight/day)

Hindlimb Foot Splay (cm)

G1, M & 0

Mean

9.0

±SD

1.0

n

5

G2, M & 111

Mean

8.8

±SD

1.3

n

5

G3, M & 333

Mean

9.3

±SD

0.6

n

5

G4, M & 1000

Mean

8.6

±SD

0.6

n

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 15 (Contd…). SUMMARY OF AVERAGE HINDLIMB FOOT SPLAY (cm) RECORD

Lactation Day 13

Group, Sex & Dose

(mg/kg body weight/day)

Hindlimb Foot Splay (cm)

G1, F & 0

Mean

8.0

±SD

0.5

n

5

G2, F & 111

Mean

7.9

±SD

0.5

n

5

G3, F & 333

Mean

8.1

±SD

1.0

n

5

G4, F & 1000

Mean

8.5

±SD

0.9

n

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 15 (Contd…). SUMMARY OF AVERAGE HINDLIMB FOOT SPLAY (cm) RECORD

Day 65

Group, Sex & Dose

(mg/kg body weight/day)

Hindlimb Foot Splay (cm)

G1R, M & 0

Mean

8.6

±SD

0.3

n

5

G4R, M & 1000

Mean

9.0

±SD

0.4

n

5

G1R, F & 0

Mean

8.4

±SD

0.4

n

5

G4R, F & 1000

Mean

7.6*

±SD

0.2

n

5

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

TABLE 16.     SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Total

Leucocyte

Count

Total

Erythrocyte

Count

Haemoglobin

Haematocrit

Mean

Corpuscular

Volume

Mean

Corpuscular

Haemoglobin

Mean

Corpuscular

Haemoglobin

Concentration

Platelet

Count

Mean

Platelet

Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, M & 0

Mean

8.26

9.64

17.56

52.80

55.00

18.30

33.26

776.40

7.72

±SD

1.95

1.09

1.34

4.49

3.09

1.14

0.53

330.86

0.52

n

5

5

5

5

5

5

5

5

5

G2, M & 111

Mean

9.68

9.37

16.82

51.32

54.72

17.94

32.80

1135.20

7.46

±SD

2.59

0.15

0.33

0.89

1.12

0.29

0.58

184.30

0.19

n

5

5

5

5

5

5

5

5

5

G3, M & 333

Mean

8.67

9.40

16.58

50.78

54.12

17.64

32.62

962.60

7.72

±SD

3.67

0.62

0.87

1.91

1.72

0.59

0.70

256.00

0.52

n

5

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

8.14

9.35

16.88

52.02

55.74

18.06

32.46

1017.60

7.50

±SD

2.42

0.54

0.55

1.58

2.71

0.74

0.53

93.02

0.38

n

5

5

5

5

5

5

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

(%)

G1, M & 0

Mean

2.25

23.48

72.02

2.38

0.44

0.80

±SD

1.24

9.09

9.80

1.15

0.17

0.49

n

5

5

5

5

5

5

G2, M & 111

Mean

1.87

19.66

75.74

2.28

1.04

0.54

±SD

0.64

3.44

3.55

0.74

0.59

0.23

n

5

5

5

5

5

5

G3, M & 333

Mean

1.87

21.98

70.58

2.40

1.04

0.62

±SD

0.60

5.35

7.57

2.37

0.34

0.61

n

5

5

5

5

5

5

G4, M & 1000

Mean

2.01

20.86

75.78

1.42

0.72

0.54

±SD

0.79

3.34

2.92

0.23

0.25

0.15

n

5

5

5

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Absolute

Reticulocyte Count

Absolute Neutrophils

Absolute

Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated

Prothrombin Time

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

212.96

1.83

6.07

0.19

0.04

0.07

15.54

22.26

±SD

101.87

0.42

2.01

0.08

0.02

0.06

0.78

6.12

n

5

5

5

5

5

5

5

5

G2, M & 111

Mean

174.50

1.91

7.33

0.23

0.10*

0.05

15.72

25.28

±SD

56.93

0.64

1.96

0.10

0.04

0.02

1.03

1.56

n

5

5

5

5

5

5

5

5

G3, M & 333

Mean

175.20

1.80

5.98

0.27

0.09*

0.07

16.72

24.30

±SD

59.25

0.52

1.97

0.39

0.03

0.11

1.49

1.59

n

5

5

5

5

5

5

5

5

G4, M & 1000

Mean

184.78

1.70

6.17

0.11

0.06

0.04

15.98

26.78

±SD

67.09

0.55

1.89

0.04

0.02

0.02

0.89

1.29

n

5

5

5

5

5

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Total

Leucocyte

Count

Total

Erythrocyte

Count

Haemoglobin

Haematocrit

Mean

Corpuscular Volume

Mean

Corpuscular Haemoglobin

Mean

Corpuscular Haemoglobin Concentration

Platelet

Count

Mean

Platelet

Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, F & 0

Mean

10.56

7.85

15.74

46.26

59.02

20.04

34.04

752.60

6.02

±SD

3.78

0.45

0.65

3.16

4.16

1.08

0.91

361.58

0.64

n

5

5

5

5

5

5

5

5

5

G2, F & 111

Mean

11.38

7.41

14.66

44.60

60.26

19.74

32.82

1130.00

6.72

±SD

3.45

0.68

1.58

3.35

1.37

0.42

1.34

672.21

0.54

n

5

5

5

5

5

5

5

5

5

G3, F & 333

Mean

9.62

7.95

15.56

46.34

58.34

19.60

33.60

1030.20

6.42

±SD

3.95

0.28

0.72

2.05

3.12

0.81

1.07

104.22

0.57

n

5

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

12.38

7.39

14.68

43.46

59.06

19.98

33.80

1003.00

6.92

±SD

3.58

0.76

0.89

2.75

3.76

1.58

0.63

227.17

0.68

n

5

5

5

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

(%)

G1, F & 0

Mean

2.28

32.72

60.66

3.68

1.46

0.60

±SD

0.87

15.77

16.88

1.06

0.98

0.23

n

5

5

5

5

5

5

G2, F & 111

Mean

4.52

30.00

62.56

4.60

1.18

0.42

±SD

4.56

11.00

11.24

1.35

0.63

0.19

n

5

5

5

5

5

5

G3, F & 333

Mean

1.49

25.26

66.92

4.46

1.22

0.80

±SD

0.67

9.73

12.89

4.23

0.33

1.12

n

5

5

5

5

5

5

G4, F & 1000

Mean

4.16

37.00

57.76

3.10

0.88

0.28

±SD

2.72

20.56

20.80

0.97

0.47

0.11

n

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Absolute

Reticulocyte Count  

Absolute

Neutrophils   

Absolute

Lymphocytes  

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils       

Prothrombin Time  

Activated

Prothrombin Time       

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

177.36

3.64

6.21

0.40

0.15

0.06

17.60

23.34

±SD

60.94

2.11

2.30

0.19

0.09

0.03

1.02

2.05

n

5

5

5

5

5

5

5

5

G2, F & 111

Mean

313.86

3.59

6.91

0.56

0.12

0.05

22.06

27.48

±SD

276.22

2.17

1.72

0.29

0.04

0.02

8.41

10.47

n

5

5

5

5

5

5

5

5

G3, F & 333

Mean

117.60

2.64

6.29

0.39

0.12

0.06

16.88

22.08

±SD

50.69

2.06

2.26

0.31

0.07

0.08

3.84

7.81

n

5

5

5

5

5

5

5

5

G4, F & 1000

Mean

291.70

4.81

6.94

0.38

0.10

0.04

15.66

19.92

±SD

145.98

3.66

3.06

0.17

0.03

0.02

3.85

5.41

n

5

5

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Total

Leucocyte

Count

Total

Erythrocyte

Count

Haemoglobin

Haematocrit

Mean

Corpuscular Volume

Mean

Corpuscular Haemoglobin

Mean

Corpuscular Haemoglobin Concentration

Platelet

Count

Mean

Platelet

Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1R, M & 0

Mean

9.13

9.21

16.04

48.76

52.94

17.42

32.92

894.80

6.50

±SD

0.78

0.14

0.43

1.79

1.78

0.39

0.42

134.10

0.31

n

5

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

9.55

9.14

15.80

48.54

53.08

17.30

32.62

1071.80

6.42

±SD

1.05

0.32

0.65

2.25

0.91

0.30

0.57

268.87

0.37

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

6.97

7.65

14.26

42.16

55.20

18.64

33.78

873.60

6.32

±SD

2.14

0.43

0.65

2.05

1.99

0.70

0.29

80.79

0.19

n

5

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

7.59

7.73

14.32

42.78

55.34

18.48

33.46

739.60

6.52

±SD

3.51

0.22

0.57

1.63

2.15

0.70

0.25

253.21

0.13

n

5

5

5

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(%)

(%)

(%)

(%)

(%)

(%)

G1R, M & 0

Mean

1.63

25.28

69.00

1.76

2.30

0.88

±SD

0.26

6.62

7.82

0.49

0.89

0.26

n

5

5

5

5

5

5

G4R, M & 1000

Mean

2.03

21.70

73.32

1.48

2.02

0.60

±SD

0.50

5.52

5.80

0.37

0.36

0.29

n

5

5

5

5

5

5

G1R, F & 0

Mean

1.84

26.12

68.68

2.30

1.34

0.68

±SD

0.36

9.06

8.57

1.19

0.45

0.15

n

5

5

5

5

5

5

G4R, F & 1000

Mean

2.48*

29.02

64.16

2.40

2.86*

0.64

±SD

0.39

7.89

6.96

0.87

1.30

0.13

n

5

5

5

5

5

5

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

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Absolute

Reticulocyte Count  

Absolute

Neutrophils   

Absolute

Lymphocytes  

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils      

Prothrombin Time  

Activated

Prothrombin Time       

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1R, M & 0

Mean

150.40

2.27

6.35

0.16

0.21

0.08

16.00

21.56

±SD

23.36

0.40

1.23

0.04

0.07

0.02

2.04

2.05

n

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

186.92

2.06

7.02

0.14

0.19

0.05

15.50

22.48

±SD

52.68

0.53

1.09

0.04

0.04

0.03

1.17

3.12

n

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

140.08

1.78

4.83

0.15

0.09

0.05

14.98

24.56

±SD

23.05

0.73

1.73

0.05

0.02

0.01

0.53

1.98

n

5

5

5

5

5

5

5

5

G4R, F & 1000

Mean

192.10*

2.06

4.98

0.17

0.25

0.05

15.92

19.34*

±SD

30.17

0.60

2.77

0.07

0.24

0.02

1.17

2.34

n

5

5

5

5

5

5

5

5

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

TABLE 17.     SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

(GLU)

-

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

G1, M & 0

Mean

126.60

30.52

0.58

51.60

25.60

7.74

3.47

±SD

15.47

2.56

0.03

3.36

2.30

0.26

0.19

n

5

5

5

5

5

5

5

G2, M & 111

Mean

123.60

33.30

0.57

60.60

30.40

7.24*

3.55

±SD

5.55

3.91

0.04

4.83

6.07

0.33

0.15

n

5

5

5

5

5

5

5

G3, M & 333

Mean

122.60

33.78

0.60

58.00

29.40

7.44

3.65

±SD

12.92

2.83

0.03

6.71

7.73

0.22

0.29

n

5

5

5

5

5

5

5

G4, M & 1000

Mean

131.00

36.04

0.59

56.00

27.00

7.58

3.68

±SD

19.79

4.26

0.02

9.57

6.16

0.29

0.24

n

5

5

5

5

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Alanine
aminotransferase

Aspartate
aminotransferase

Alkaline
phosphatase

Total Bilirubin

Calcium

Phosphorous

(ALT)

(AST)

(ALP)

(BIT)

(CAL)

(PHO)

(U/L)

(U/L)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

G1, M & 0

Mean

61.20

111.20

125.80

0.05

9.36

6.48

±SD

16.93

12.34

21.11

0.01

0.11

0.58

n

5

5

5

5

5

5

G2, M & 111

Mean

47.40

93.20

120.00

0.06

9.64

6.54

±SD

8.96

13.46

31.92

0.03

0.47

0.62

n

5

5

5

5

5

5

G3, M & 333

Mean

40.00

81.60*

127.20

0.06

10.40*

6.82

±SD

5.96

12.30

38.01

0.03

1.05

0.62

n

5

5

5

5

5

5

G4, M & 1000

Mean

52.20

91.80

114.80

0.06

9.94

6.62

±SD

24.52

18.69

28.70

0.02

0.30

0.49

n

5

5

5

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected) ; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Globulin

Albumin/

Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(GLO)

(A/G Ratio)

(BUN)

(Na)

(K)

(CLO)

(g/dL)

-

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

4.27

0.82

14.24

141.64

3.96

104.10

±SD

0.30

0.09

1.19

0.94

0.25

2.51

n

5

5

5

5

5

5

G2, M & 111

Mean

3.69*

0.97

15.54

142.16

3.71

105.38

±SD

0.38

0.12

1.83

0.53

0.15

1.40

n

5

5

5

5

5

5

G3, M & 333

Mean

3.79*

0.97

15.77

142.44

3.86

105.78

±SD

0.12

0.10

1.32

0.93

0.36

1.73

n

5

5

5

5

5

5

G4, M & 1000

Mean

3.90

0.95

16.82

142.76

3.85

106.40

±SD

0.29

0.11

1.99

0.66

0.09

0.86

n

5

5

5

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

(GLU)

-

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

G1, F & 0

Mean

102.20

48.68

0.54

76.60

78.60

7.32

3.63

±SD

26.22

8.11

0.05

22.67

26.83

1.08

0.67

n

5

5

5

5

5

5

5

G2, F & 111

Mean

121.20

51.14

0.52

67.80

59.40

7.34

3.41

±SD

29.64

13.28

0.03

17.67

23.65

0.75

0.30

n

5

5

5

5

5

5

5

G3, F & 333

Mean

103.80

44.78

0.57

75.60

43.20

7.56

3.58

±SD

13.81

19.81

0.04

14.91

21.48

0.29

0.32

n

5

5

5

5

5

5

5

G4, F & 1000

Mean

114.60

51.66

0.57

75.80

105.20

7.76

3.70

±SD

16.44

16.16

0.07

20.44

68.28

0.58

0.11

n

5

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Alanine
aminotransferase

Aspartate
aminotransferase

Alkaline
phosphatase

Total Bilirubin

Calcium

Phosphorous

(ALT)

(AST)

(ALP)

(BIT)

(CAL)

(PHO)

(U/L)

(U/L)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

G1, F & 0

Mean

84.60

119.40

164.40

0.03

10.06

5.92

±SD

14.43

14.31

75.62

0.04

1.06

0.95

n

5

5

5

5

5

5

G2, F & 111

Mean

113.80

143.80

420.00

0.02

9.42

5.50

±SD

66.08

71.78

367.29

0.02

0.64

1.64

n

5

5

5

5

5

5

G3, F & 333

Mean

96.20

99.40

305.80

0.06

10.04

6.06

±SD

82.35

16.01

412.83

0.02

0.85

1.93

n

5

5

5

5

5

5

G4, F & 1000

Mean

88.60

101.80

212.40

0.03

10.38

6.38

±SD

46.69

22.65

81.17

0.04

0.91

3.11

n

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Globulin

Albumin/Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(GLO)

(A/G Ratio)

(BUN)

(Na)

(K)

(CLO)

(g/dL)

-

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

3.69

0.98

22.72

143.04

4.02

106.18

±SD

0.50

0.11

3.79

1.34

0.81

2.96

n

5

5

5

5

5

5

G2, F & 111

Mean

3.93

0.88

23.87

142.18

3.57

107.22

±SD

0.66

0.14

6.20

1.47

0.28

3.01

n

5

5

5

5

5

5

G3, F & 333

Mean

3.98

0.91

20.90

142.36

3.60

107.94

±SD

0.40

0.16

9.25

1.61

0.33

2.63

n

5

5

5

5

5

5

G4, F & 1000

Mean

4.06

0.92

24.11

142.62

3.86

107.06

±SD

0.50

0.09

7.55

1.62

0.40

3.20

n

5

5

5

5

5

5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

(GLU)

-

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

G1R, M & 0

Mean

99.80

27.36

0.55

59.60

23.00

6.98

3.10

±SD

6.53

3.92

0.02

16.21

5.52

0.26

0.07

n

5

5

5

5

5

5

5

G4R, M & 1000

Mean

99.40

25.74

0.56

68.80

31.60

7.12

3.05

±SD

10.53

1.19

0.03

8.76

10.78

0.11

0.04

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

120.40

35.68

0.61

106.40

49.60

8.44

4.10

±SD

7.16

7.57

0.02

17.07

34.09

0.56

0.18

n

5

5

5

5

5

5

5

G4R, F & 1000

Mean

104.00*

31.42

0.59

89.20

41.60

8.04

3.70

±SD

13.49

2.25

0.01

16.98

11.87

0.42

0.44

n

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Alanine aminotransferase

Aspartate aminotransferase

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

(ALT)

(AST)

(ALP)

(BIT)

(CAL)

(PHO)

(U/L)

(U/L)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

G1R, M & 0

Mean

52.00

101.80

99.00

0.02

10.42

5.72

±SD

40.34

53.17

23.40

0.02

0.37

0.34

n

5

5

5

5

5

5

G4R, M & 1000

Mean

42.60

98.40

105.20

0.02

10.02

5.26

±SD

9.86

18.49

29.84

0.01

0.15

0.50

n

5

5

5

5

5

5

G1R, F & 0

Mean

54.00

94.00

70.80

0.02

11.20

4.20

±SD

28.82

36.89

40.92

0.02

0.45

0.38

n

5

5

5

5

5

5

G4R, F & 1000

Mean

53.80

103.20

69.00

0.04

10.90

4.42

±SD

21.53

39.30

60.10

0.02

0.37

0.18

n

5

5

5

5

5

5

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

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Globulin

Albumin/

Globulin ratio

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(GLO)

(A/G Ratio)

(BUN)

(Na)

(K)

(CLO)

(g/dL)

-

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1R, M & 0

Mean

3.88

0.80

12.77

142.82

3.56

105.48

±SD

0.27

0.06

1.83

0.36

0.41

0.92

n

5

5

5

5

5

5

G4R, M & 1000

Mean

4.07

0.75

12.01

143.16

3.59

108.06*

±SD

0.15

0.04

0.56

0.68

0.41

1.65

n

5

5

5

5

5

5

G1R, F & 0

Mean

4.34

0.95

16.65

142.62

3.45

105.76

±SD

0.42

0.08

3.53

0.94

0.17

1.52

n

5

5

5

5

5

5

G4R, F & 1000

Mean

4.34

0.85

14.66

142.26

3.62

106.40

±SD

0.21

0.12

1.05

1.41

0.48

2.88

n

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 18.     SUMMARY OF URINALYSIS RECORD

Examination

Group, Sex & Dose

(mg/kg body weight/day)

G1,

M & 0

G2,

M & 111

G3,

M & 333

G4,

M & 1000

Number of Animals

(randomly selected)

5

5

5

5

Physical Examination

Color

Pale Yellow

5

5

5

5

Appearance

Clear

5

5

5

5

Volume (mL)

Mean

10.6

10.2

11.0

11.4

±SD

0.7

1.4

0.8

0.4

Chemical Examination

pH

Mean

7.5

7.4

7.4

7.5

±SD

0.0

0.4

0.5

0.0

Specific Gravity

Mean

1.008

1.011

1.009

1.009

±SD

0.003

0.002

0.002

0.002

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

Bilirubin (mg/dL)

Neg

5

5

5

5

Ketones (mg/dL)

Neg

2

-

2

1

5

3

5

3

4

Blood

(Ery/µL)

Neg

5

4

5

5

Ca80

-

1

-

-

Protein

(mg/dL)

Neg

3

-

3

1

Trace

2

4

1

4

30

-

1

1

-

Nitrite

Neg

4

4

5

1

Pos

1

1

-

4

Leucocytes

Neg

3

-

3

1

Ca15

1

1

1

2

Ca70

1

3

1

2

Ca125

-

1

-

-

Glucose

(mg/dL)

Neg

5

5

5

5

Microalbumin

(mg/dL)

Neg

-

-

2

1

>15

2

5

2

4

15

3

-

1

-

Microscopic Examination

Epi Cells

0

3

4

2

4

0-1

1

1

2

1

1-2

1

-

1

-

Casts

Absent

4

4

4

5

Present

1

1

1

-

Crystals

Present

5

5

5

5

M: Male; SD: Standard Deviation; Pos: Positive; Neg: Negative; Ca: Calculated.
 >=: Greater than or equal to; >: Greater than.

TABLE 18 (Contd…). SUMMARY OF URINALYSIS RECORD

Examination

Group, Sex & Dose

(mg/kg body weight/day)

G1R,

M & 0

G4R,

M &1000

G1R,

F & 0

G4R,

F & 1000

Number of Animals

(randomly selected)

5

5

5

5

Physical Examination

Color

Pale Yellow

2

5

3

3

Yellow

3

-

2

2

Appearance

Clear

3

2

4

4

Turbid

2

3

1

1

Volume (mL)

Mean

6.4

8.6

7.9

7.8

±SD

1.7

3.1

2.2

0.8

Chemical Examination

pH

Mean

6.7

7.3

6.8

7.6

±SD

0.9

0.4

1.0

1.0

Specific Gravity

Mean

1.020

1.014

1.014

1.012

±SD

0.007

0.002

0.007

0.008

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.6

±SD

0.0

0.0

0.0

0.8

Bilirubin (mg/dL)

Neg

2

5

5

2

1

3

-

-

3

Ketones (mg/dL)

Neg

1

2

5

5

5

4

3

-

-

Blood

(Ery/µL)

Neg

5

5

5

4

Ca10

-

-

-

1

Protein

(mg/dL)

Neg

2

5

4

2

Trace

2

-

1

-

30

1

-

-

-

100

-

-

-

1

>=300

-

-

-

2

Nitrite

Neg

3

5

5

5

Pos

2

-

-

-

Leucocytes

Neg

-

1

3

2

Ca15

1

4

2

-

Ca70

4

-

-

3

Glucose

(mg/dL)

Neg

5

5

5

5

Microalbumin

(mg/dL)

Neg

-

1

3

1

>15

3

-

1

3

15

2

4

1

1

Microscopic Examination\

Epi Cells

0

3

3

2

2

0-1

1

2

2

1

1-2

1

-

1

-

2-3

-

-

-

2

Casts

Absent

5

2

5

4

Present

-

3

-

1

Crystals

Absent

-

2

1

2

Present

5

3

4

3

M: Male; F: Female; R: Recovery; SD: Standard Deviation; Pos: Positive; Neg: Negative.                              
Ca: Calculated; >=: Greater than or equal to; >: Greater than.

TABLE 19.     SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Testes

Epididymides

Heart

Kidneys

Brain

G1, M & 0

Mean

0.0667

0.2891

0.7915

3.3315

1.4198

1.3943

2.9537

2.1193

±SD

0.0045

0.0466

0.2701

0.3035

0.1420

0.2048

0.2510

0.0321

n

5

5

5

12

12

5

5

5

G2, M & 111

Mean

0.0719

0.3589

0.8023

3.4284

1.4112

1.5033

2.8196

2.1336

±SD

0.0094

0.0292

0.0928

0.1782

0.1365

0.1061

0.1723

0.0957

n

5

5

5

12

12

5

5

5

G3, M & 333

Mean

0.0709

0.4450*

0.8041

3.3728

1.4195

1.4754

2.8317

2.1467

±SD

0.0060

0.0665

0.1455

0.2771

0.1776

0.1560

0.1507

0.0446

n

5

5

5

12

12

5

5

5

G4, M & 1000

Mean

0.0716

0.4129*

0.8155

3.3454

1.5221

1.5218

3.0082

2.2033

±SD

0.0115

0.1013

0.1404

0.2101

0.1812

0.3979

0.7565

0.1355

n

5

5

5

12

12

5

5

5

M: Male; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Liver

Prostate

Seminal vesicles with coagulating glands

Lungs

Pituitary

Thyroid along with parathyroid#

G1, M & 0

Mean

10.0491

1.1499

1.6300

1.9467

0.0208

0.0296

±SD

1.6192

0.1975

0.4468

0.1258

0.0014

0.0017

n

5

12

12

5

5

12

G2, M & 111

Mean

10.3930

1.1988

1.6027

2.0659

0.0197

0.0290

±SD

0.4389

0.2552

0.3454

0.1175

0.0013

0.0020

n

5

12

12

5

5

12

G3, M & 333

Mean

10.6576

1.3430

1.7846

2.0478

0.0203

0.0301

±SD

0.7376

0.3501

0.3700

0.1024

0.0009

0.0013

n

5

12

12

5

5

12

G4, M & 1000

Mean

10.8065

1.2209

1.9124

2.0837

0.0205

0.0305

±SD

2.8155

0.3931

0.5906

0.1492

0.0015

0.0023

n

5

12

12

5

5

12

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

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Adrenals

Thymus

Spleen

Ovaries

Uterus with Cervix

Heart

G1, F & 0

Mean

0.0741

0.2781

0.7048

0.1738

0.5901

1.1848

±SD

0.0083

0.0317

0.0646

0.0299

0.1167

0.1262

n

5

5

5

12

11@

5

G2, F & 111

Mean

0.0884

0.2632

0.7503

0.1432*

0.5097

1.1940

±SD

0.0110

0.0345

0.1289

0.0332

0.1138

0.0729

n

5

5

5

12

11@

5

G3, F & 333

Mean

0.0728

0.2267*

0.6584

0.1291*

0.5413

1.2001

±SD

0.0094

0.0282

0.1051

0.0193

0.0886

0.0513

n

5

5

5

12

12

5

G4, F & 1000

Mean

0.0775

0.2179*

0.6109

0.1466*

0.5283

1.3318

±SD

0.0093

0.0063

0.0413

0.0190

0.1008

0.3482

n

5

5

5

12

12

5

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

@: One animal data from group G1 and G2 is excluded for mean calculations as the weight varies from normal uterus due to presence of dead foetus during necropsy, but the data is presented in individual animal data. (Appendix 22).

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

Kidneys

Brain

Liver

Lungs

Pituitary

Thyroid along

with parathyroid#

G1, F & 0

Mean

2.2668

2.0179

11.5641

1.2626

0.0179

0.0266

±SD

0.1164

0.0746

0.5339

0.1185

0.0011

0.0015

n

5

5

5

5

5

12

G2, F & 111

Mean

2.0463*

2.0056

12.5985

1.5260

0.0183

0.0272

±SD

0.1081

0.1131

2.0359

0.2954

0.0014

0.0015

n

5

5

5

5

5

12

G3, F & 333

Mean

2.0269*

1.9917

10.1148

1.3551

0.0176

0.0265

±SD

0.0937

0.0552

1.8164

0.1082

0.0008

0.0015

n

5

5

5

5

5

12

G4, F & 1000

Mean

1.9230*

2.0216

10.2708

1.3780

0.0180

0.0275

±SD

0.0912

0.0446

1.3180

0.1188

0.0010

0.0014

n

5

5

5

5

5

12

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

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

Adrenals

Thymus

Spleen

Testes

Epididymides

Heart

Kidneys

Brain

G1R, M & 0

Mean

0.0671

0.2367

0.8804

3.3055

1.6954

1.6355

2.2619

2.0448

±SD

0.0080

0.0348

0.0907

0.1930

0.0763

0.1477

0.3680

0.1172

n

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

0.0638

0.2709

0.7442*

3.4656

1.6882

1.6050

2.3506

2.0483

±SD

0.0051

0.0738

0.0528

0.1133

0.0690

0.3581

0.3464

0.1621

n

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Liver

Prostate

Seminal vesicles with

coagulating glands

Lungs

Pituitary

Thyroid along

with parathyroid#

G1R, M & 0

Mean

12.6730

1.3824

1.8300

2.0437

0.0207

0.0299

±SD

1.2027

0.2475

0.4858

0.5420

0.0009

0.0015

n

5

5

5

5

5

5

G4R, M &

1000

Mean

12.5717

1.6038

1.8342

1.9398

0.0210

0.0306

±SD

1.2099

0.1130

0.4887

0.0584

0.0011

0.0008

n

5

5

5

5

5

5

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

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

 

Adrenals

Thymus

Spleen

Ovaries

Uterus with Cervix

Heart

G1R, F & 0

Mean

0.0706

0.2511

0.6464

0.1265

0.6499

1.0512

±SD

0.0064

0.0443

0.0886

0.0164

0.0920

0.0882

n

5

5

5

5

5

5

G4R, F & 1000

Mean

0.0702

0.2467

0.6261

0.1543*

0.6334

1.0135

±SD

0.0121

0.0337

0.1113

0.0105

0.0853

0.0746

n

5

5

5

5

5

5

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

TABLE 19 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

Kidneys

Brain

Liver

Lungs

Pituitary

Thyroid along

with parathyroid#

G1R, F & 0

Mean

1.9138

2.0075

9.6308

1.8480

0.0185

0.0278

±SD

0.0978

0.0986

0.9760

0.0946

0.0009

0.0014

n

5

5

5

5

5

5

G4R, F & 1000

Mean

1.8218

1.9767

8.6614

2.0609

0.0193

0.0271

±SD

0.1551

0.0844

1.7719

0.4014

0.0014

0.0006

n

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation.

TABLE 20.     SUMMARY OF 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)

Adrenals

Thymus

Spleen

Testes

Epididymides

Heart

G1, M & 0

Mean

440.14

0.0150

0.0648

0.1748

0.7637

0.3257

0.3099

±SD

38.09

0.0018

0.0115

0.0484

0.1095

0.0493

0.0251

n

12

5

5

5

12

12

5

G2, M & 111

Mean

433.53

0.0162

0.0812

0.1809

0.7928

0.3262

0.3390

±SD

29.89

0.0021

0.0090

0.0188

0.0447

0.0319

0.0185

n

12

5

5

5

12

12

5

G3, M & 333

Mean

437.73

0.0159

0.0995*

0.1797

0.7734

0.3250

0.3314

±SD

26.65

0.0016

0.0135

0.0302

0.0792

0.0413

0.0427

n

12

5

5

5

12

12

5

G4, M & 1000

Mean

443.94

0.0156

0.0891*

0.1779

0.7570

0.3432

0.3304

±SD

38.78

0.0013

0.0124

0.0187

0.0592

0.0298

0.0626

n

12

5

5

5

12

12

5

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

TABLE 20 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

Kidneys

Brain

Liver

Prostate

Seminal vesicles with coagulating glands

Lungs

Pituitary

Thyroid along

with parathyroid#

G1, M & 0

Mean

0.6591

0.4745

2.2336

0.2614

0.3768

0.4360

0.0047

0.0067

±SD

0.0371

0.0334

0.2239

0.0389

0.1233

0.0428

0.0003

0.0004

n

5

5

5

12

12

5

5

12

G2, M & 111

Mean

0.6380

0.4818

2.3449

0.2758

0.3681

0.4668

0.0044

0.0067

±SD

0.0606

0.0272

0.0585

0.0505

0.0681

0.0350

0.0003

0.0006

n

5

5

5

12

12

5

5

12

G3, M & 333

Mean

0.6350

0.4812

2.3924

0.3052

0.4081

0.4594

0.0046

0.0069

±SD

0.0467

0.0237

0.2415

0.0710

0.0824

0.0376

0.0001

0.0004

n

5

5

5

12

12

5

5

12

G4, M & 1000

Mean

0.6545

0.4865

2.3426

0.2767

0.4271

0.4575

0.0045

0.0069

±SD

0.1286

0.0618

0.4199

0.0940

0.1161

0.0285

0.0002

0.0004

n

5

5

5

12

12

5

5

12

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

TABLE 20 (Contd…). SUMMARY OF 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)

Adrenals

Thymus

Spleen

Ovaries

Uterus

with Cervix

Heart

G1, F & 0

Mean

305.54

0.0242

0.0912

0.2307

0.0571

0.1938

0.3872

±SD

21.99

0.0035

0.0163

0.0360

0.0100

0.0422

0.0618

n

12

5

5

5

12

11@

5

G2, F & 111

Mean

314.85

0.0286

0.0853

0.2428

0.0458*

0.1635

0.3854

±SD

19.21

0.0044

0.0142

0.0457

0.0115

0.0448

0.0220

n

12

5

5

5

12

11@

5

G3, F & 333

Mean

302.78

0.0236

0.0730

0.2142

0.0431*

0.1801

0.3879

±SD

22.49

0.0038

0.0045

0.0429

0.0092

0.0352

0.0151

n

12

5

5

5

12

12

5

G4, F & 1000

Mean

306.64

0.0245

0.0690*

0.1938

0.0483

0.1749

0.4241

±SD

25.44

0.0033

0.0062

0.0238

0.0080

0.0430

0.1222

n

12

5

5

5

12

12

5

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

@: One animal data from group G1 and G2 is excluded for mean calculations as the weight varies from normal uterus due to presence of dead foetus during necropsy, but the data is presented in individual animal data. (Appendix 23).

TABLE 20 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex

& Dose

(mg/kg body weight/day)

Kidneys

Brain

Liver

Lungs

Pituitary

Thyroid along

with parathyroid#

G1, F & 0

Mean

0.7409

0.6583

3.7703

0.4138

0.0058

0.0087

±SD

0.0909

0.0663

0.3600

0.0696

0.0003

0.0005

n

5

5

5

5

5

12

G2, F & 111

Mean

0.6618

0.6488

4.0668

0.4903

0.0059

0.0087

±SD

0.0563

0.0584

0.6665

0.0770

0.0002

0.0009

n

5

5

5

5

5

12

G3, F & 333

Mean

0.6560

0.6443

3.2646

0.4388

0.0057

0.0088

±SD

0.0476

0.0330

0.5652

0.0452

0.0002

0.0007

n

5

5

5

5

5

12

G4, F & 1000

Mean

0.6097*

0.6402

3.2355

0.4353

0.0057

0.0090

±SD

0.0642

0.0516

0.3329

0.0373

0.0008

0.0007

n

5

5

5

5

5

12

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

TABLE 20 (Contd…). SUMMARY OF 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)

Adrenals

Thymus

Spleen

Testes

Epididymides

Heart

G1R, M & 0

Mean

496.58

0.0136

0.0476

0.1785

0.6684

0.3430

0.3320

±SD

40.88

0.0022

0.0051

0.0257

0.0554

0.0286

0.0478

n

5

5

5

5

5

5

5

G4R, M & 1000

Mean

502.58

0.0127

0.0538

0.1482*

0.6902

0.3358

0.3184

±SD

12.09

0.0010

0.0137

0.0126

0.0356

0.0088

0.0643

n

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 20 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex

& Dose

(mg/kg body

weight/day)

Kidneys

Brain

Liver

Prostate

Seminal vesicles with coagulating glands

Lungs

Pituitary

Thyroid along

with parathyroid#

G1R, M & 0

Mean

0.4554

0.4147

2.5665

0.2783

0.3746

0.4088

0.0042

0.0060

±SD

0.0586

0.0488

0.3239

0.0415

0.1185

0.0840

0.0002

0.0003

n

5

5

5

5

5

5

5

5

G4R, M & 1000

Mean

0.4682

0.4080

2.4984

0.3193

0.3643

0.3862

0.0042

0.0061

±SD

0.0730

0.0361

0.1858

0.0248

0.0941

0.0153

0.0002

0.0003

n

5

5

5

5

5

5

5

5

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

TABLE 20 (Contd…). SUMMARY OF 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)

Adrenals

Thymus

Spleen

Ovaries

Uterus

with Cervix

Heart

G1R, F & 0

Mean

308.17

0.0229

0.0822

0.2106

0.0410

0.2112

0.3432

±SD

16.88

0.0016

0.0185

0.0336

0.0043

0.0292

0.0498

n

5

5

5

5

5

5

5

G4R, F & 1000

Mean

311.82

0.0225

0.0791

0.2015

0.0495*

0.2026

0.3256

±SD

12.71

0.0037

0.0102

0.0405

0.0029

0.0205

0.0293

n

5

5

5

5

5

5

5

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

Conclusions:
In conclusion, the oral administration of test item over the time of the study, carried out according to the OECD guideline 422 (for main group males with a total of 31 days, for main group females ranging from 50 to 62 days and for recovery group animals with a total period of 52 days) did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.
Executive summary:

The objective of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of the test item by oral gavage in Sprague Dawley Rats was to determine the possible health hazards likely to arise from repeated exposure to the test item over a relatively limited period of time. This study was also conducted to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition and an estimate of the No Observed Adverse Effect Level (NOAEL).


 


A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group consisted of 12 males and 12 females and each recovery group consisted of 5 males and 5 females. The animals in group G1/G1R were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], animals in groups G2, G3 and G4/G4R were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight/day for low, mid and high/high dose recovery, respectively. Vehicle and test item formulations were administered orally by gavage with the dose volume of
10 mL/kg body weight/day once daily at similar times on each day for 7 days a week.


 


All the main group males were administered with test item during pre-mating (14-days), during mating and during post-mating period (total of 31 days). The pregnant females from main groups were administered with test item during pre-mating (14-days), during mating, pregnancy (gestation) and up to lactation day 13.The females with positive mating signal, but no-evidence of pregnancy / litteringwere administered with test itemduringpre-mating (14-days),during mating, and further 24 days from the day of positive mating signal. All the recovery group animals were administered with test item for 50 days followed by a 14-day observation period without treatment.


 


The test item formulations werestable for 6 hours and 48 hours at room temperature in 0.5% w/v Carboxymethyl Cellulose with the concentrations of 5 mg/mL and 100 mg/mL.The homogeneity and dose form concentration analysis for preparedtest item formulations during treatment periodwas performed during week 1 and 5 and the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is less than 15%.


 


For systemic toxicity assessment, all the animals (main and recovery groups) of both sexes were observed once daily for clinical signs, twice daily for mortality and morbidity and once in a week for detailed clinical examination. Body weight of all the animals (main and recovery groups) of both sexes were recorded once in a week until termination. Further, the body weight of all pregnant females was recorded on gestation day (GD) 0, 7, 14 and 20 during pregnancy and on lactation day (LD) 1, 4, 7, 13 and 14 during lactation.Body weight of all non-pregnant females was recorded once in a week until termination. Feed consumption for all the animals of both sexes were recorded once in a week during pre-mating for main groups and throughout the experimental period for recovery groups. Further, feed consumption of all pregnant females was recorded during GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13.Ophthalmological examination was carried out once before treatment and at the end of the dosing period for all males and females (main and recovery groups). Neurological/functional observations were performed for five randomly selected males and females from each main group, for all animals from recovery groups towards end of the dosing period. The investigations of haematology and clinical chemistry for all animals (main and recovery groups), investigations of urinalysis for five randomly selected males from each main group and for all recovery groups animals was conducted at termination. Serum thyroxine hormone (T4) levels were estimated for all main group males by ELISA. The organs were collected and weighed on the day of termination for all animals (main and recovery groups) of