2,9-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Endpoint summary

Description of key information

No adverse effects observed in a subacute combined oral and reproductive toxicity study in rats using a cloese analogue substance at doses including 1000 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

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

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 Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: 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 Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: 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 Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: 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)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/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)
		(10^9cells/L)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/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)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/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)
		(10^9cells/L)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/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)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/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)
		(10^9cells/L)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/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 both sexes and organ weight relative to terminal body weight was calculated. All the animals (main and recovery groups) of both sexes were observed for both external and internal gross pathological changes during conduct of necropsy. Histopathological examination was conducted on all the