Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Endpoint summary

Description of key information

The No Observed Adverse Effect Level (NOAEL) of the test item Acid Violet 50 was found to be 1000 mg/kg body weight when administered to the males for a total of 37 days and to the females for up to 50 days under the experimental conditions employed. No toxic effects were observed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 31 May 2017; To: 23 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, Number 422, “Combined Repeated Dose Toxicity Study
with the Reproduction/Developmental Toxicity Screening Test” adopted on 29 July 2016

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: Males: 231.73 g to 259.15 g; Females: 210.13 g to 231.58 g
- Housing: in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube
- Diet (ad libitum): Altromin maintenance diet for rats and mice; type: 1324 (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter
- Acclimation period: nineteen days (including five days of acclimatizion and 14 days of screening for oestrous cycles) From: 31 May 2017; To: 19 June 2017

DETAILS OF FOOD AND WATER QUALITY:
Altromin maintenance diet for rats and mice; type: 1324 (manufactured by Altromin Spezialfutter GmbH & Co. KG); batch no./lot no. 030118//0838

Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter with purifier (contaminant free)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.9
- Humidity (%): 40 to 69
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 31 May 2017; To: 31 May 2017;

Route of administration:

oral: gavage

Details on route of administration:

The vehicle and test item formulations were administered through oral (gavage) route as it is the most appropriate route for substances to focus on the detection of hazardous properties on reproduction

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: The test item is in-soluble in distilled water and forms uniform suspension in corn oil at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility/suspendibility test results. Hence, corn oil was selected for the test item formulation preparation
- Concentration in vehicle: Low Dose: 11 mg/mL; Mid Dose: 33 mg/mL; High Dose: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no: MR02081
- Purity: 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for homogeneity and dose concentration verification of Acid Violet 50 was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 440. Sampling and analysis of formulations was performed during first week of treatment and during week 5 of the treatment. The samples were collected in duplicates (5 mL each) from top, middle and bottom layer in duplicate sets from each concentration.

The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup purpose at established storage conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, and the results are found to be within the acceptable range of 85 to 115 % the nominal concentration and the % RSD is less than 10.0%.

Duration of treatment / exposure:

The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment).
The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
The recovery group animals were treated until the first scheduled sacrifice of dams (total of 50 days) and kept without treatment for a further 21 days observation

Frequency of treatment:

Once daily

Dose / conc.:

110 mg/kg bw/day (actual dose received)

Remarks:

Low Dose

Dose / conc.:

330 mg/kg bw/day (actual dose received)

Remarks:

330.0

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose

No. of animals per sex per dose:

Main Group: 12 males and 12 females per dose
Recoery Group: 5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 110, 330 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Acid Violet 50 when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2461).
Comparable doses were selected as the test item Acid Violet 50 when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+7.71% and -13.12% for males and +8.45% and -7.67% for females) of the mean body weight of each sex.

- Rationale for selecting satellite groups: To observe delayed effects
- Post-exposure recovery period in satellite groups: The recovery group animals were treated until the first scheduled sacrifice of dams (total of 50 days) and kept without treatment for a further 21 days observation.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals during acclimatization;

during end of the dosing period for males (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals

during lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals;

during last week for recovery group animals

- Dose groups that were examined:

vehicle control/vehicle control recovery and high dose/high dose recovery group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
five males and five females randomly selected from each main group and from all recovery group animals on the day of scheduled terminal sacrifice (for main group males after completion of 37 days of treatment; for main group females on lactation day 14 and for recovry at sacrifice).

- Anaesthetic used for blood collection: Yes (mild isoflurane)
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each main group and from all recovery group animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: five males and five females randomly selected from each main group and from all recovery group animals on the day of scheduled terminal sacrifice (for main group males after completion of 37 days of treatment; for main group females on lactation day 14 and for recovry at sacrifice).
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each main group and from all recovery group animals

URINALYSIS: Yes
- Time schedule for collection of urine: five males and five females randomly selected from each main group males and from all recovery group animals on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: five males and five females, randomly selected from each group towards the end of the dosing period for males, during lactation period for females (shortly prior to scheduled sacrifice) and from the recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice).
- Dose groups that were examined: from all the dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / hind limb foot splay

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Other examinations:

not applicable

Statistics:

The data was subjected to various statistical analyses using SPSS software version 22. All analysis
and comparisons were evaluated at the 95% level of confidence (P<0.05).
Note: Data of non-pregnant females, females mated but not littered were excluded from statistical a
nalysis.
The statistical analysis was followed to the parameters as mentioned below:
Body weight, Change in body weight,Feed consumption, Copulatory interval, Gestation length,
Hematology, Clinical chemistry, Absolute organ weights, Relative organ weights, FOB (Body t
emperature, Defecation, Urination, Rearing, Grip strength), Pup weight and Ano-genital distance: P
arametric - One-way ANOVA with Dunnett’s post test.
Pre implantation loss, Pre natal loss, Post natal loss, Total No. of early/late resorptions/dam, Corpora
lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Mean litter size, Mean pup weight: Non
Parametric - Kruskal-Wallis
Pregnancy rate, Dams with/without live pups, Dams with/without dead pups, No. of litters with/without
resorptions: Cross Tabs -Chi-square test

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity observed at any of the tested dose groups of either sex. However, the faecal matter of all the test dose group animals of either sex was found violet coloured. This observation was due to the colour of the test item.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the tested main and recovery groups of either sex

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the main and recovery group animals of either sex. However, statistical significant decrease in percent change in body weight with respect to day 1 during week 1 was noted in G4 and G4R females when compared with vehicle control group females. These changes are considered as incidental but not treatment related as there were no effects were observed in feed
consumption during this period and the changes were inconsistent.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noted in feed consumption at any of the doses of both the main and recovery group animals of either sex. However, statistical significant increase in feed consumption during week 2 was noted in G3 females when compared with vehicle control group females. This change is considered as incidental but not treatment related as there were no effects were observed in mean body weight and percent change in body weight during this period and due to lack
of dose dependency.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

no ocular changes observed in vehicle control/vehicle control recovery and high dose/high dose recovery group animals of both the sex

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in haematology parameters at any of the tested doses of either sex. However, statistically significant decrease in absolute reticulocytes and statistically significant increase in absolute neutrophils at G2 males; statistically significant increase in absolute monocytes and statistically significant increase in activated prothrombin time at G4 males;statistically significant decrease in haemoglobin at G4R males; statistically significant increase in activated prothrombin time at G4R females were noted when compared with concurrent vehicle control group animals. These changes are considered as incidental and not treatment related, due to lack of dose dependency and /or due to biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in clinical chemistry parameters at any of the tested doses of either sex. However, statistically significant increase in alkaline phosphatase at G2 and G3 males; statistically significant increase in potassium at G4 males; statistically significant increase in chloride at G3 males; statistically significant increase in potassium at G4R males; statistically significant decrease in total bilirubin at G4R females; statistically significant increase in phosphorous and statistically significant decrease in sodium at G4R females; statistically significant decrease in total protein at G4R females were noted when compared with concurrent vehicle control group animals. These changes are considered as incidental and not treatment related, due to lack of dose dependency
and /or due to biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in urinalysis parameters at any of the tested doses of main group males and either sex of recovery groups. However, statistically significant decrease in urine pH at G4R females was noted when compared with concurrent vehicle control group. This change is considered as incidental and not treatment related as the value is within historical control data.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes observed in the neurological/functional examination in both main and recovery group animals of either sex.

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in the absolute and relative organ weights at any of the doses tested of both the sex in main and recovery group animals.However, statistically significant decrease in absolute Prostate + Seminal vesicles with coagulating glands at G4R males when compared with concurrent vehicle control group. This change can be considered as incidental and not treatment related, as there were no gross and microscopic findings were noted in this organ.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes (both external and internal) observed at any of the doses tested in both the sex of adults and pups (110, 330 and 1000 mg/kg body weight). However there is slight reduction in size of testes and epididymis in one animal from G3 was observed. This observation is considered as incidental but not treatment related as there were no changes noted during microscopic examination of these organs

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related histopathological findings noticed in this study. Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations. One female in the high dose group showed MNC infiltration in liver. Similar finding was also observed in the concurrent control. Hence considered as background lesion.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

not applicable

Other effects:

not specified

Description (incidence and severity):

not applicable

Details on results:

there were no treatment related systemic and reproductive toxicity effects observed upto 1000 mg/kg body weight

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

not determinable due to absence of adverse toxic effects

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)	No. of Animals	Clinical Signs of Toxicity/Detailed Clinical Examination	Mortality (No. of Mortality / No. of Animals dosed)
G1, M & 0	12	N	0/12
G2, M & 110	12	N	0/12
G3, M & 330	12	N	0/12
G4, M & 1000	12	N	0/12

M: Male; N: Normal

TABLE 1 (Contd..,). SUMMARYOF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL

EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity/Detailed Clinical Examination	Mortality (No. of Mortality / No. of Animals dosed)
G1, F & 0	12	N	0/12
G2, F & 110	12	N	0/12
G3, F & 330	12	N	0/12
G4, F & 1000	12	N	0/12

F: Female; N: Normal

TABLE 1 (Contd..,). SUMMARYOF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL

EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity/Detailed Clinical Examination	Mortality (No. of Mortality / No. of Animals dosed)
G1R, M & 0	5	N	0/5
G4R, M & 1000	5	N	0/5
G1R, F & 0	5	N	0/5
G4R, F & 1000	5	N	0/5

M:Male; F: Female; R: Recovery; N: Normal

TABLE 2. SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	7	14	21	28	35
G1, M & 0	Mean	352.93	371.41	386.55	405.28	414.94	433.65
	±SD	21.25	23.51	31.94	36.07	35.12	39.96
	n	12	12	12	12	12	12
G2, M & 110	Mean	351.38	368.93	384.28	404.17	411.24	426.96
	±SD	18.68	20.54	31.02	35.02	36.44	41.92
	n	12	12	12	12	12	12
G3, M & 330	Mean	351.68	368.20	390.51	408.92	420.95	434.24
	±SD	18.71	17.64	24.36	27.46	30.71	35.96
	n	12	12	12	12	12	12
G4, M & 1000	Mean	353.73	372.45	389.32	406.59	416.81	430.27
	±SD	24.02	26.05	32.45	31.24	30.14	35.17
	n	12	12	12	12	12	12

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

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

Refer Appendix 2

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	7	14
G1, F & 0	Mean	250.53	261.85	267.25
	±SD	11.11	10.38	11.57
	n	12	12	12
G2, F & 110	Mean	250.20	259.54	270.27
	±SD	10.01	10.20	11.81
	n	12	12	12
G3, F & 330	Mean	250.85	260.47	270.76
	±SD	9.33	12.20	14.12
	n	12	12	12
G4, F & 1000	Mean	250.07	257.83	269.25
	±SD	9.89	9.67	11.45
	n	12	12	12

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

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	7	14	21	28	35	42	49	56	63	70
G1R, M & 0	Mean	344.42	360.82	368.75	392.73	405.74	426.68	434.53	444.52	455.90	462.71	475.86
	±SD	14.18	15.26	16.88	23.90	28.80	34.74	35.53	36.14	39.61	40.98	34.72
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	345.71	365.03	368.49	392.05	413.81	431.45	433.86	437.33	445.82	451.97	462.97
	±SD	15.98	15.44	21.31	23.33	25.04	37.54	35.63	38.45	38.41	40.84	42.91
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	245.09	256.63	259.51	267.64	272.80	280.41	282.00	284.32	296.39	298.81	300.29
	±SD	6.00	6.08	8.76	11.40	10.54	12.84	12.28	12.23	12.10	12.26	10.26
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	245.40	253.40	258.77	266.14	269.14	282.61	284.82	286.81	292.64	297.05	300.28
	±SD	6.68	7.06	12.31	11.79	16.01	17.50	18.82	18.69	19.18	18.74	18.22
	n	5	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 OF PERCENT CHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) with respect to Day 1 during Days
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35
G1, M & 0	Mean	5.23	9.42	14.68	17.45	22.71
	±SD	1.19	3.35	4.17	4.04	5.42
	n	12	12	12	12	12
G2, M & 110	Mean	4.98	9.24	14.86	16.86	21.28
	±SD	0.76	3.74	4.52	5.10	6.40
	n	12	12	12	12	12
G3, M & 330	Mean	4.73	11.02	16.25	19.69	23.45
	±SD	1.27	2.97	4.36	6.20	7.79
	n	12	12	12	12	12
G4, M & 1000	Mean	5.28	9.98	14.91	17.85	21.60
	±SD	0.76	3.05	3.00	4.01	5.06
	n	12	12	12	12	12

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

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

Group, Sex & Dose (mg/kg body weight/day)		Change in Body Weight (%) during Days
		1 to 7	1 to 14
G1, F & 0	Mean	4.54	6.71
	±SD	1.15	2.76
	n	12	12
G2, F & 110	Mean	3.74	8.02
	±SD	1.29	2.16
	n	12	12
G3, F & 330	Mean	3.81	7.92
	±SD	1.84	3.52
	n	12	12
G4, F & 1000	Mean	3.12*	7.71
	±SD	0.86	3.45
	n	12	12

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

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

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

Group, Sex & Dose (mg/kg body weight/day)		Change in Body Weight (%) during Days
		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	1 to 70
G1R, M & 0	Mean	4.76	7.08	13.99	17.73	23.75	26.03	28.93	32.21	34.17	38.09
	±SD	0.45	2.91	4.10	5.20	5.89	6.31	6.20	7.16	7.41	6.81
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	5.61	6.61	13.45	19.74	24.85	25.59	26.57	29.01	30.77	33.93
	±SD	1.70	4.54	5.75	6.05	10.06	10.13	10.62	10.26	10.50	10.80
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	4.72	5.88	9.17	11.28	14.38	15.03	15.98	20.93	21.92	22.53
	±SD	1.38	2.15	2.46	2.03	3.31	3.00	2.90	4.01	4.13	3.35
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	3.26*	5.41	8.41	9.60	15.09	15.97	16.79	19.16	20.96	22.28
	±SD	0.27	2.47	2.00	3.67	4.34	4.76	4.72	4.83	4.66	4.28
	n	5	5	5	5	5	5	5	5	5	5

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

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

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

Group & Dose (mg/kg body weight)		Feed Consumption (g)
		Week 1Feed Consumption	Week 2 Feed Consumption	Post Mating Feed Consumption
G1, M & 0	Mean	24.93	25.52	33.18
	±SD	0.77	0.58	0.51
	n	12	12	12
G2, M & 110	Mean	25.27	26.32	32.71
	±SD	0.53	0.74	1.29
	n	12	12	12
G3, M & 330	Mean	24.93	25.43	32.99
	±SD	0.84	0.84	0.94
	n	12	12	12
G4, M & 1000	Mean	25.10	26.05	32.85
	±SD	0.95	0.65	0.90
	n	12	12	12

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

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

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g)
		Week 1 Feed Consumption	Week 2 Feed Consumption
G1, F & 0	Mean	22.53	22.11
	±SD	0.52	0.69
	n	12	12
G2, F & 110	Mean	22.58	22.80
	±SD	0.80	0.40
	n	12	12
G3, F & 330	Mean	22.38	23.05*
	±SD	0.67	0.74
	n	12	12
G4, F & 1000	Mean	22.41	22.12
	±SD	1.33	0.68
	n	12	12

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

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

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

 Refer Appendix 4

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g)
		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10	Week 11
G1R, M & 0	Mean	21.05	22.89	24.48	25.46	25.16	25.36	26.54	27.75	29.12	31.75	30.20
	±SD	6.17	5.69	6.29	3.60	1.94	2.71	2.38	1.77	1.43	1.00	0.28
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	20.63	22.14	24.01	25.77	25.71	25.95	26.82	27.97	29.63	31.74	32.61
	±SD	4.37	6.29	5.86	2.45	2.11	3.07	2.53	2.51	1.34	1.46	1.36
	n	5	5	5	5	5	5	2	2	2	2	2
G1R, F & 0	Mean	17.82	18.70	20.16	23.82	24.69	23.90	24.44	25.79	26.76	28.54	27.97
	±SD	4.54	5.32	3.39	2.36	1.20	0.69	1.04	0.94	0.76	0.30	0.61
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	18.41	18.00	22.26	24.08	24.75	23.60	24.98	25.94	27.19	28.03	26.86
	±SD	4.70	5.22	6.28	2.60	1.86	1.06	1.12	0.31	0.75	0.22	0.41
	n	5	5	5	5	5	5	5	5	5	5	5

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

TABLE 5 . SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Day 37
Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0		G4, M & 1000
Number of Animals		12		12
Parameters		Observations
Eye		L	R	L	R
Adnexa	Lids	N	N	N	N
	Ducts	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	Vessels	N	N	N	N
	Macula	N	N	N	N
Optic disc		N	N	N	N
Tapetum lucidum		N	N	N	N
Tapetum nigrum		N	N	N	N

N: No Abnormality Detected; L: Left; R: Right; M: Male

TABLE 5 (Contd..,). SUMMARY OFOPHTHALMOLOGICALEXAMINATIONRECORD

During Lactation Day 13
Group, Sex & Dose (mg/kg body weight/day)		G1, F & 0		G4, F & 1000
Number of Animals		12		11
Parameters		Observations
Eye		L	R	L	R
Adnexa	Lids	N	N	N	N
	Ducts	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	Vessels	N	N	N	N
	Macula	N	N	N	N
Optic disc		N	N	N	N
Tapetum lucidum		N	N	N	N
Tapetum nigrum		N	N	N	N

N: No Abnormality Detected; L: Left; R: Right; F: Female

TABLE 5 (Contd..,). SUMMARY OFOPHTHALMOLOGICAL EXAMINATION RECORD

Day 71
Group, Sex & Dose (mg/kg body weight/day)		G1R, M & 0		G4R, M & 1000		G1R, F & 0		G4R, F & 1000
Number of Animals		5		5		5		5
Parameters		Observations
Eye		L	R	L	R	L	R	L	R
Adnexa	Lids	N	N	N	N	N	N	N	N
	Ducts	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	Vessels	N	N	N	N	N	N	N	N
	Macula	N	N	N	N	N	N	N	N
Optic disc		N	N	N	N	N	N	N	N
Tapetum lucidum		N	N	N	N	N	N	N	N
Tapetum nigrum		N	N	N	N	N	N	N	N

N: No Abnormality Detected; L: Left; R: Right; M: Male; F: Female

TABLE 6. SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERATION BATTERY (FOB) RECORD

        

Day 37
Parameters↓	Group, Sex & Dose (mg/kg body weight/day)→		G1, M & 0	G2, M & 110	G3, M & 330	G4, M & 1000
	Number of Animals (Randomly selected)→		5	5	5	5
Home cage observations
a. Convulsions			1	1	1	1
b. Tremors			1	1	1	1
c. Palpebral closure			1	1	1	1
Handling observation
a. Ease of removal from the cage			2	2	2	2
b. Ease of handling animal in hand			2	2	2	2
c. Lacrimation			1	1	1	1
d. Red deposits around		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
e. Crusty deposits around		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
f. Salivation			1	1	1	1
g. Fur appearance			1	1	1	1
h. Piloerection			1	1	1	1
i. Palpebral closure			1	1	1	1
j. Respiratory character			1	1	1	1
k. Eye prominence			1	1	1	1
l. Muscle tone			1	1	1	1
Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present, e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight 3=Severe, i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal
: Male; R: Recovery;SD: Standard Deviation

TABLE 6 (Contd..,). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

                                                                                                                                                                                                                                                                             

Day 37
Parameters↓		Group, Sex & Dose (mg/kg body weight/day)→	G1, M & 0	G2, M & 110	G3, M & 330	G4, M & 1000
		Number of Animals (Randomly selected)→	5	5	5	5
Open field Observation
a. Mobility			1	1	1	1
b. Gait			1	1	1	1
c. Arousal			3	3	3	3
d. Rearing	Mean		6.00	6.20	5.80	5.20
	±SD		0.71	1.30	0.84	1.48
e. Urination Numbers of pools of urine	Mean		4.20	3.80	4.00	4.80
	±SD		2.49	2.59	2.55	0.84
f. Defecation Number of fecal pellets	Mean		1.80	1.20	1.60	1.40
	±SD		1.30	1.30	1.14	0.89
g. Stereotypies- repetitive circling			1	1	1	1
h. Excessive grooming	Mean		2.40	2.80	2.00	2.00
	±SD		0.89	0.84	1.00	0.71
Sensory observations
a.Startle response			2	2	2	2
b. Touch response			2	2	2	2
c. Pupil response			2	2	2	2
d. Response to nociceptive stimuli			2	2	2	2
e. Righting reflex			1	1	1	1
Physiological observation Body temperature (°F)	Mean		100.24	99.94	100.00	99.84
	±SD		0.91	0.56	0.38	0.74
Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1=Absent, 2=Present,Sensory Observations:  a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -                 1=Present, 2=Slow, 3=Absent.
M: Male; R: Recovery;SD: Standard Deviation TABLE 6 (Contd..,). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD                                                                                                                                                                                                                                                                                     During Lactation Parameters↓ Group, Sex & Dose (mg/kg body weight/day)→ G1, F & 0 G2, F & 110 G3, F & 330 G4, F & 1000 Number of Animals (Randomly selected)→ 5 5 5 5 Home cage observations a. Convulsions 1 1 1 1 b. Tremors 1 1 1 1 c. Palpebral closure 1 1 1 1 Handling observation a. Ease of removal from the cage 2 2 2 2 b. Ease of handling animal in hand 2 2 2 2 c. Lacrimation 1 1 1 1 d. Red deposits around Eyes 1 1 1 1 Nose 1 1 1 1 Mouth 1 1 1 1 e. Crusty deposits around Eyes 1 1 1 1 Nose 1 1 1 1 Mouth 1 1 1 1 f. Salivation 1 1 1 1 g. Fur appearance 1 1 1 1 h. Piloerection 1 1 1 1 i. Palpebral closure 1 1 1 1 j. Respiratory character 1 1 1 1 k. Eye prominence 1 1 1 1 l. Muscle tone 1 1 1 1 Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present,  e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight, 3=Severe,            i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal F: Female; R: Recovery;SD: Standard Deviation
TABLE 6 (Contd..,). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD                                                                                                                                                                                                                                                                               During Lactation Parameters↓ Group, Sex & Dose (mg/kg body weight/day)→ G1, F & 0 G2, F & 110 G3, F & 330 G4, F & 1000 Number of Animals (Randomly selected)→ 5 5 5 5 Open field Observation a. Mobility 1 1 1 1 b. Gait 1 1 1 1 c. Arousal 3 3 3 3 d. Rearing Mean 5.80 4.80 4.80 5.80 ±SD 0.84 0.84 0.84 0.84 e. Urination Numbers of pools of urine Mean 4.20 3.00 4.00 4.20 ±SD 1.10 2.24 1.58 1.30 f. Defecation Number of fecal pellets Mean 2.20 1.80 2.00 2.20 ±SD 1.30 1.30 1.22 0.45 g. Stereotypies- repetitive circling   1 1 1 1 h. Excessive grooming Mean 3.40 2.80 3.40 3.40 ±SD 0.55 0.84 1.14 1.14 Sensory observations a.Startle response 2 2 2 2 b. Touch response 2 2 2 2 c. Pupil response 2 2 2 2 d. Response to nociceptive stimuli 2 2 2 2 e. Righting reflex 1 1 1 1 Physiological observation Body temperature (°F) Mean 100.24 100.30 100.04 100.18 ±SD 0.30 0.35 0.65 0.40 Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1=Absent, 2=Present,Sensory Observations: a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -1=Present, 2=Slow, 3=Absent M: Male; F: Female; R: Recovery;SD: Standard Deviation

TABLE 6 (Contd..,). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

                                                                                                                                                                                                                                                                                   

Day 71
Parameters↓	Group, Sex & Dose (mg/kg body weight)→		G1R, M & 0	G4R, M & 1000	G1R, F & 0	G4R, F & 1000
	Number of Animals→		5	5	5	5
Home cage observations
a. Convulsions			1	1	1	1
b. Tremors			1	1	1	1
c. Palpebral closure			1	1	1	1
Handling observation
a. Ease of removal from the cage			2	2	2	2
b. Ease of handling animal in hand			2	2	2	2
c. Lacrimation			1	1	1	1
d. Red deposits around		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
e. Crusty deposits around		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
f. Salivation			1	1	1	1
g. Fur appearance			1	1	1	1
h. Piloerection			1	1	1	1
i. Palpebral closure			1	1	1	1
j. Respiratory character			1	1	1	1
k. Eye prominence			1	1	1	1
l. Muscle tone			1	1	1	1
Home cage observations: a. Convulsions - 1=Absent, 2=Present, b. Tremors- 1=Absent, 2=Present, c. Palpebral closure - 1=Normal, Handling observation : a. Ease of removal from the cage - 2=Normal, b. Ease of handling animal in hand - 2=Normal, c. Lacrimation - 1=None, 2=Slight, 3=Severe, d. Red deposits around - 1=Absent, 2=Present, e. Crusty deposits around - 1=Absent, 2=Present , f. Salivation- 1=Normal , g. Fur Appearance - 1=Normal, h. Piloerection - 1=None, 2=Slight, 3=Severe,            i. Palpebral Closure - 1=Normal, j. Respiratory character 1=Normal, k. Eye Prominence - 1=Normal, l. Muscle tone - 1=Normal

M: Male; F: Female; R: Recovery;SD: Standard Deviation

TABLE 6 (Contd..,). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

                                                                                                                                                                                                                                                                              

Day 71
Parameters↓	Group, Sex & Dose (mg/kg body weight)→	G1R, M & 0	G4R, M & 1000	G1R, F & 0	G4R, F & 1000
	Number of Animals→	5	5	5	5
Open field Observation a. Mobility		1	1	1	1
b. Gait		1	1	1	1
c. Arousal		3	3	3	3
d. Rearing	Mean	5.80	5.40	5.60	6.00
	±SD	0.84	1.14	1.14	0.71
e. Urination Numbers of pools of urine	Mean	4.20	4.00	4.40	4.00
	±SD	2.49	2.74	3.05	2.35
f. Defecation Number of fecal pellets	Mean	1.60	1.40	1.40	1.40
	±SD	1.14	1.14	1.34	1.34
g. Stereotypies- repetitive circling		1	1	1	1
h. Excessive grooming	Mean	2.60	3.20	3.00	3.00
	±SD	0.55	1.10	0.71	0.71
Sensory observations
a.Startle response		2	2	2	2
b. Touch response		2	2	2	2
c. Pupil response		2	2	2	2
d. Response to nociceptive stimuli		2	2	2	2
e. Righting reflex		1	1	1	1
Physiological observation Body temperature (°F)	Mean	100.34	99.78	100.08	100.22
	±SD	0.77	0.71	0.47	0.53
Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling- 1=Absent, 2=Present,Sensory Observations: a.Startle Response -2=Normal,b. Touch Response -2=Normal,c. Pupil Response 2=Normal,d. Response to Nociceptive stimuli - 2=Normal,e. Righting Reflex -1=Present, 2=Slow, 3=Absent.

M: Male; F: Female; R: Recovery;SD: Standard Deviation

TABLE 7. SUMMARYOF PHOTOACTOMETER READIGS (MOVEMENTS) RECORD

Day 37
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1, M & 0	Mean	616.40
	±SD	53.43
	n	5
G2, M & 110	Mean	583.60
	±SD	82.76
	n	5
G3, M & 330	Mean	592.80
	±SD	84.49
	n	5
G4, M & 1000	Mean	608.00
	±SD	66.27
	n	5

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

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

 TABLE 7 (Contd..,). SUMMARYOF PHOTOACTOMETER READIGS (MOVEMENTS) RECORD

During Lactation
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1, F & 0	Mean	629.00
	±SD	27.62
	n	5
G2, F & 110	Mean	613.60
	±SD	32.91
	n	5
G3, F & 330	Mean	623.40
	±SD	60.31
	n	5
G4, F & 1000	Mean	620.40
	±SD	60.41
	n	5

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

 TABLE 7 (Contd..,). SUMMARYOF PHOTOACTOMETER READIGS (MOVEMENTS) RECORD

Day 71
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1R, M & 0	Mean	621.80
	±SD	21.87
	n	5
G4R, M & 1000	Mean	610.60
	±SD	15.13
	n	5
G1R, F & 0	Mean	598.20
	±SD	64.27
	n	5
G4R, F & 1000	Mean	627.40
	±SD	48.97
	n	5

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

 TABLE 8. SUMMARYOF GRIP STREGTH (kgf) MEASUREMENT RECORD

Day 37
Group, Sex & Dose (mg/kg body weight/day)		Average Fore limb Grip Strength	Average Hind limb Grip Strength
G1, M & 0	Mean	1.537	0.535
	±SD	0.023	0.039
	n	5	5
G2, M & 110	Mean	1.568	0.563
	±SD	0.020	0.041
	n	5	5
G3, M & 330	Mean	1.552	0.546
	±SD	0.043	0.028
	n	5	5
G4, M & 1000	Mean	1.541	0.561
	±SD	0.049	0.029
	n	5	5

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

 TABLE 8 (Contd..,). SUMMARYOF GRIP STREGTH (kgf) MEASUREMENT RECORD

During Lactation
Group, Sex & Dose (mg/kg body weight/day)		Average Fore limb Grip Strength	Average Hind limb Grip Strength
G1, F & 0	Mean	1.643	0.621
	±SD	0.037	0.048
	n	5	5
G2, F & 110	Mean	1.637	0.622
	±SD	0.050	0.046
	n	5	5
G3, F & 330	Mean	1.641	0.628
	±SD	0.069	0.030
	n	5	5
G4, F & 1000	Mean	1.634	0.619
	±SD	0.089	0.030
	n	5	5

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

 TABLE 8 (Contd..,). SUMMARYOF GRIP STREGTH (kgf) MEASUREMENT RECORD

Day 71
Group, Sex & Dose (mg/kg body weight/day)		Average Fore limb Grip Strength	Average Hind limb Grip Strength
G1R, M & 0	Mean	1.603	0.533
	±SD	0.019	0.042
	n	5	5
G4R, M & 1000	Mean	1.607	0.544
	±SD	0.033	0.031
	n	5	5
G1R, F & 0	Mean	1.562	0.545
	±SD	0.038	0.046
	n	5	5
G4R, F & 1000	Mean	1.558	0.548
	±SD	0.047	0.037
	n	5	5

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

 TABLE 9. SUMMARY OF HIND LIMB FOOT SPLAY (cm) RECORD

Day 37
Group, Sex & Dose (mg/kg body weight)		Average Landing Foot Splay (cm)
G1, M & 0	Mean	10.45
	±SD	1.59
	n	5
G2, M & 110	Mean	9.69
	±SD	1.02
	n	5
G3, M & 330	Mean	10.56
	±SD	1.93
	n	5
G4, M & 1000	Mean	9.52
	±SD	1.11
	n	5

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

 TABLE 9 (Contd..,).SUMMARY OF HIND LIMB FOOT SPLAY (cm) RECORD

During lactation
Group, Sex & Dose (mg/kg body weight/day)		Average Landing Foot Splay (cm)
G1, F & 0	Mean	6.65
	±SD	0.60
	n	5
G2, F & 110	Mean	7.28
	±SD	0.71
	n	5
G3, F & 330	Mean	6.52
	±SD	0.70
	n	5
G4, F & 1000	Mean	7.13
	±SD	1.08
	n	5

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

 TABLE 9 (Contd..,).SUMMARY OF HIND LIMB FOOT SPLAY (cm) RECORD

Day 71
Group, Sex & Dose (mg/kg body weight/day)		Average Landing Foot Splay (cm)
G1R, M & 0	Mean	10.36
	±SD	1.66
	n	5
G4R, M & 1000	Mean	9.60
	±SD	0.71
	n	5
G1R, F & 0	Mean	8.22
	±SD	0.76
	n	5
G4R, F & 1000	Mean	8.32
	±SD	0.55
	n	5

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

 TABLE 10. SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(10^3cells/µL)	(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	6.85	7.00	12.38	42.34	822.80	3.63	23.46	71.44	2.76	1.00	0.10
	±SD	1.31	0.80	1.91	2.13	69.12	2.39	2.54	2.95	0.46	0.47	0.07
	n	5	5	5	5	5	5	5	5	5	5	5
G2, M & 110	Mean	10.59	7.36	13.50	41.26	810.80	1.66	25.40	69.68	2.92	0.68	0.14
	±SD	2.65	0.40	0.74	1.78	36.38	0.45	4.61	4.75	1.22	0.19	0.05
	n	5	5	5	5	5	5	5	5	5	5	5
G3, M & 330	Mean	7.84	6.73	13.76	43.28	758.40	1.95	26.96	67.46	3.36	0.92	0.10
	±SD	2.33	0.51	0.22	0.51	122.36	0.36	6.11	7.40	1.26	0.36	0.07
	n	5	5	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	10.56	7.81	13.80	42.72	774.60	1.67	22.52	71.62	3.72	1.14	0.12
	±SD	2.76	0.79	1.03	2.64	110.53	0.62	2.63	2.65	0.40	0.40	0.04
	n	5	5	5	5	5	5	5	5	5	5	5

M: Male;SD: Standard Deviation; n: Number of animals (Randomly Selected)

 TABLE 10 (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	243.72	1.62	4.87	0.19	0.07	0.01	15.86	16.88
	±SD	141.28	0.45	0.74	0.06	0.05	0.01	0.90	1.55
	n	5	5	5	5	5	5	5	5
G2, M & 110	Mean	121.16*	2.66*	7.42	0.30	0.07	0.02	17.08	20.24
	±SD	29.26	0.74	2.24	0.10	0.02	0.01	1.87	3.77
	n	5	5	5	5	5	5	5	5
G3, M & 330	Mean	130.74	2.09	5.31	0.26	0.07	0.01	15.48	22.50
	±SD	19.49	0.63	1.88	0.11	0.03	0.01	1.45	4.19
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	126.56	2.33	7.60	0.40*	0.12	0.01	15.36	27.92*
	±SD	30.47	0.40	2.15	0.13	0.08	0.01	1.70	8.26
	n	5	5	5	5	5	5	5	5

M: Male;SD: Standard Deviation; n: Number of animals (Randomly Selected)

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

 TABLE 10 (Contd..,).SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haemato-crit	Platelet Count	Reticulocyte Count	Neutrophils	Lymphocytes	Mono-cytes	Eosino-phils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(10^3cells/ µL)	(%)	(%)	(%)	(%)	(%)	(%)
G1, F & 0	Mean	10.23	6.99	13.10	40.24	873.60	3.35	37.76	56.00	3.96	1.30	0.14
	±SD	4.44	0.40	0.57	2.07	192.17	0.77	3.86	4.32	0.72	0.94	0.11
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 110	Mean	13.06	6.70	12.88	39.32	886.60	2.80	38.20	54.88	4.34	1.34	0.12
	±SD	2.56	0.46	0.54	1.57	376.63	0.61	5.19	6.19	0.87	0.69	0.08
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 330	Mean	8.53	6.44	12.76	39.66	883.60	4.09	29.52	63.54	4.26	1.30	0.22
	±SD	4.19	0.66	1.35	3.51	638.76	2.21	10.75	9.44	1.58	0.83	0.16
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	11.04	6.55	12.68	39.36	784.80	2.86	29.86	62.52	4.86	1.32	0.18
	±SD	6.57	0.49	0.82	2.16	187.91	1.40	11.87	11.36	0.96	0.38	0.08
	n	5	5	5	5	5	5	5	5	5	5	5

F: Female;SD: Standard Deviation; n: Number of animals (Randomly Selected)

TABLE 10 (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	233.88	3.82	5.74	0.42	0.16	0.02	17.36	13.74
	±SD	54.79	1.53	2.65	0.19	0.16	0.02	2.01	3.40
	n	5	5	5	5	5	5	5	5
G2, F & 110	Mean	188.90	4.99	7.14	0.58	0.17	0.02	20.48	16.98
	±SD	48.05	1.27	1.51	0.23	0.08	0.01	7.73	7.54
	n	5	5	5	5	5	5	5	5
G3, F & 330	Mean	253.54	2.52	5.38	0.39	0.12	0.02	17.64	16.82
	±SD	101.81	1.76	2.48	0.26	0.12	0.01	2.58	2.40
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	183.90	3.88	6.35	0.53	0.14	0.02	20.08	19.22
	±SD	82.17	3.34	2.77	0.35	0.10	0.01	4.09	9.19
	n	5	5	5	5	5	5	5	5

F: Female;SD: Standard Deviation; n: Number of animals (Randomly Selected)

 TABLE 10 (Contd..,).SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Platelet Count	Reticulocyte Count	Neutro-phils	Lympho-cytes	Monocytes	Eosinophils	Basophils
		(WBC)	(RBC)	(HGB)	(HCT)	(PLT)	(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(10^3cells/µL)	(%)	(%)	(%)	(%)	(%)	(%)
G1R, M & 0	Mean	9.19	7.92	14.50	42.76	845.20	1.26	20.16	74.30	3.02	1.68	0.16
	±SD	1.08	0.40	0.35	3.03	239.96	0.21	2.01	2.04	0.59	0.42	0.05
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	9.61	7.70	13.20*	40.48	897.80	1.60	25.96	67.56	3.74	1.64	0.16
	±SD	1.86	0.26	0.22	0.73	46.69	0.35	7.13	6.85	0.61	0.27	0.05
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	7.11	7.50	13.48	40.22	875.20	1.46	25.08	68.30	3.96	1.62	0.10
	±SD	1.94	0.37	0.47	1.24	133.85	0.41	6.76	8.28	1.81	0.38	0.00
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	8.63	7.59	13.62	41.38	815.60	1.47	28.02	66.64	2.86	1.52	0.14
	±SD	2.04	0.67	0.66	2.91	135.75	0.50	10.17	10.11	0.36	0.33	0.05
	n	5	5	5	5	5	5	5	5	5	5	5

F: Female;SD: Standard Deviation; n: Number of animals (Randomly Selected)

 TABLE 10 (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	99.06	1.86	6.83	0.28	0.15	0.02	19.02	23.38
	±SD	12.65	0.32	0.82	0.06	0.03	0.01	2.79	5.66
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	122.94	2.54	6.45	0.35	0.16	0.01	16.82	23.36
	±SD	26.24	1.02	1.22	0.05	0.05	0.00	0.69	1.27
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	108.98	1.75	4.91	0.26	0.12	0.01	16.28	11.62
	±SD	28.05	0.59	1.61	0.07	0.04	0.01	0.70	3.70
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	112.48	2.51	5.66	0.25	0.13	0.01	16.26	25.76*
	±SD	43.28	1.38	1.15	0.07	0.02	0.00	0.65	5.22
	n	5	5	5	5	5	5	5	5

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

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

 TABLE 11.SUMMARY OF CLIICAL CHEMISRTY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(GLU)	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		mg/dL	mg/dL	mg/dL	mg/dL	g/dL	g/dL	U/L	U/L	U/L
G1, M & 0	Mean	94.00	0.54	47.20	59.80	6.32	3.13	56.80	106.20	136.40
	±SD	12.73	0.05	6.38	24.73	0.24	0.16	11.37	23.16	42.09
	n	5	5	5	5	5	5	5	5	5
G2, M & 110	Mean	96.00	0.53	53.40	81.20	6.40	3.06	60.00	102.00	338.40*
	±SD	7.25	0.03	6.35	35.70	0.31	0.13	4.85	20.45	130.16
	n	5	5	5	5	5	5	5	5	5
G3, M & 330	Mean	103.60	0.59	50.80	70.80	6.32	3.12	66.20	119.60	342.60*
	±SD	13.69	0.03	7.22	27.64	0.38	0.19	16.08	19.48	165.35
	n	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	101.40	0.56	47.20	77.20	6.38	3.00	68.60	123.80	268.60
	±SD	22.21	0.02	6.98	26.70	0.24	0.33	10.45	29.83	69.88
	n	5	5	5	5	5	5	5	5	5

M: Male;SD: Standard Deviation; n: Number of Animals (Randomly Selected)     

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

 TABLE 11 (Contd..,).SUMMARY OF CLIICAL CHEMISRTY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(BIT)	(CAL)	(PHO)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		mg/dL	mg/dL	mg/dL	g/dL	mg/dL	mmol/L	mmol/L	mmol/L
G1, M & 0	Mean	0.03	9.04	5.26	3.19	10.13	143.84	3.36	104.36
	±SD	0.04	0.23	0.58	0.21	2.14	0.38	0.16	0.50
	n	5	5	5	5	5	5	5	5
G2, M & 110	Mean	0.01	8.96	4.74	3.34	7.44	144.12	3.51	105.72
	±SD	0.03	0.22	0.31	0.29	1.92	0.65	0.16	1.88
	n	5	5	5	5	5	5	5	5
G3, M & 330	Mean	0.02	9.04	5.74	3.20	11.73	144.46	3.62	106.40*
	±SD	0.04	0.25	0.29	0.29	3.71	0.71	0.28	1.05
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	0.00	9.00	5.20	3.38	8.48	144.48	3.99*	106.28
	±SD	0.01	0.46	0.85	0.35	2.20	1.66	0.48	0.89
	n	5	5	5	5	5	5	5	5

M: Male;SD: Standard Deviation; n: Number of Animals (Randomly Selected)

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

 TABLE 11 (Contd..,).SUMMARY OF CLIICAL CHEMISRTY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(GLU)	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		mg/dL	mg/dL	mg/dL	mg/dL	g/dL	g/dL	U/L	U/L	U/L
G1, F & 0	Mean	94.40	0.58	73.00	96.20	6.16	3.19	100.20	105.40	323.20
	±SD	9.61	0.07	14.23	54.83	0.27	0.23	18.19	14.94	130.02
	n	5	5	5	5	5	5	5	5	5
G2, F & 110	Mean	95.80	0.57	75.00	114.00	6.33	3.29	116.40	116.80	305.80
	±SD	5.67	0.06	7.35	27.16	0.46	0.17	13.09	14.92	93.31
	n	5	5	5	5	5	5	5	5	5
G3, F & 330	Mean	99.80	0.56	65.40	120.00	5.88	3.08	113.20	121.00	318.00
	±SD	24.35	0.10	20.67	55.03	0.36	0.26	26.83	19.60	116.12
	n	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	116.60	0.52	68.40	92.80	6.14	3.24	104.20	123.60	315.40
	±SD	25.30	0.05	4.51	23.12	0.30	0.11	33.73	31.83	64.40
	n	5	5	5	5	5	5	5	5	5

 F: Female;SD: Standard Deviation; n: Number of Animals (Randomly Selected)

 TABLE 11 (Contd..,).SUMMARY OF CLIICAL CHEMISRTY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(BIT)	(CAL)	(PHO)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		mg/dL	mg/dL	mg/dL	g/dL	mg/dL	mmol/L	mmol/L	mmol/L
G1, F & 0	Mean	0.00	9.94	8.32	2.97	24.16	139.10	3.88	102.38
	±SD	0.00	0.17	0.58	0.17	2.78	2.09	0.25	1.25
	n	5	5	5	5	5	5	5	5
G2, F & 110	Mean	0.00	10.46	8.24	3.03	21.34	140.36	3.74	103.08
	±SD	0.00	0.34	1.88	0.29	3.14	2.14	0.13	1.20
	n	5	5	5	5	5	5	5	5
G3, F & 330	Mean	0.00	9.74	8.90	2.80	23.26	140.80	4.08	102.36
	±SD	0.00	0.76	1.54	0.40	9.80	2.99	0.74	2.77
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	0.00	10.14	9.14	2.90	19.73	140.50	4.22	101.86
	±SD	0.00	0.77	1.26	0.28	4.97	1.79	0.46	1.25
	n	5	5	5	5	5	5	5	5

F: Female;SD: Standard Deviation; n: Number of Animals (Randomly Selected)

 TABLE 11 (Contd..,).SUMMARY OF CLIICAL CHEMISRTY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase
		(GLU)	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)	(ALP)
		mg/dL	mg/dL	mg/dL	mg/dL	g/dL	g/dL	U/L	U/L	U/L
G1R, M & 0	Mean	107.40	0.59	48.00	43.20	6.48	3.18	58.60	105.40	98.00
	±SD	5.18	0.03	6.48	11.26	0.30	0.21	13.59	18.99	15.30
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	103.20	0.58	50.80	56.40	6.66	3.18	57.60	111.00	92.60
	±SD	2.77	0.04	3.03	16.82	0.15	0.17	5.59	19.86	17.53
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	106.00	0.64	64.80	42.20	7.48	3.78	49.80	94.00	86.00
	±SD	13.96	0.03	16.84	8.56	0.27	0.15	11.69	12.69	32.53
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	107.00	0.60	69.60	45.60	7.16*	3.52	63.60	116.40	74.80
	±SD	6.67	0.03	11.61	18.04	0.09	0.21	33.35	41.45	31.92
	n	5	5	5	5	5	5	5	5	5

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

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

TABLE 11 (Contd..,).SUMMARY OF CLIICAL CHEMISRTY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(BIT)	(CAL)	(PHO)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		mg/dL	mg/dL	mg/dL	g/dL	mg/dL	mmol/L	mmol/L	mmol/L
G1R, M & 0	Mean	0.01	9.88	4.60	3.30	13.37	144.20	3.48	105.74
	±SD	0.02	0.51	0.22	0.18	1.68	0.68	0.15	1.30
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.00	10.28	4.62	3.48	11.41	144.66	3.82*	105.84
	±SD	0.00	0.36	0.64	0.09	1.60	0.67	0.19	0.83
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.05	10.72	3.46	3.70	13.43	144.54	3.50	107.18
	±SD	0.03	0.31	0.50	0.19	1.71	0.79	0.29	0.76
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	0.01*	10.78	4.24*	3.64	14.77	143.00*	3.74	106.16
	±SD	0.01	0.23	0.23	0.21	2.06	1.15	0.18	0.95
	n	5	5	5	5	5	5	5	5

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

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

TABLE 12.SUMMARY OF URINALSIS RECORD

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0	G2, M & 110	G3, M & 330	G4, M & 1000
	Number of Animals (Randomly selected)		5	5	5	5
Physical Examination	Colour	Pale Yellow	5	5	5	4
		Yellow	-	-	-	1
	Appearance	Clear	4	4	5	5
		Turbidity	1	1	-	-
	Volume (mL)	Mean	4.80	5.90	6.10	5.90
		±SD	1.89	1.08	1.29	2.16
Chemical Examination	pH	Mean	7.20	7.10	7.30	7.20
		±SD	0.45	0.96	0.76	0.67
	Specific Gravity (SG)	Mean	1.015	1.011	1.015	1.017
		±SD	0.006	0.004	0.009	0.006
	Urobilinogen (UBG) mg/dL	Mean	0.20	0.20	0.36	0.36
		±SD	0.00	0.00	0.36	0.36
	Microalbumin (mg/dL)	>15	5	5	5	5
	Bilirubin (BIL) mg/dL	Neg	5	5	5	5
	Ketones (KET) mg/dL	15	-	-	-	2
		5	4	3	4	2
		Neg	1	2	1	1
	Blood (BLD) Ery/µL	Neg	1	-	1	2
		Ca10	1	1	1	1
		Ca25	1	-	2	1
		Ca80	1	3	1	1
		>=Ca200	1	1	-	-
	Protein (PRO) mg/dL	Trace	1	2	-	-
		30	-	1	3	1
		100	4	2	1	2
		>=300	-	-	1	2
	Nitrite (NIT)	Neg	3	5	4	2
		Pos	2	-	1	3
	Leucocytes (LEU) Leu/µL	Neg	3	3	3	2
		Ca15	2	1	1	3
		Ca70	-	1	1	-
	Glucose (GLU) mg/dL	250	-	1	-	-
		Neg	5	4	5	5
Microscopic Examination	Epi Cells	0	1	1	2	-
		0-1	3	2	3	4
		1-2	1	2	-	1
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

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

Pos: Positive; Neg: Negative; Ca: Calculated; >=: Greater than or equal to.

TABLE 12 (Contd..,).SUMMARY OF URINALSIS 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	5	5	5	5
	Appearance	Clear	5	5	4	4
		Turbidity	-	-	1	1
	Volume (mL)	Mean	8.60	8.50	8.70	8.40
		±SD	1.34	0.79	1.35	1.02
Chemical Examination	pH	Mean	7.20	7.40	7.10	6.50*
		±SD	0.57	0.22	0.42	0.35
	Specific gravity (SG)	Mean	1.014	1.011	1.015	1.015
		±SD	0.004	0.002	0.000	0.000
	Urobilinogen (UBG)              mg/dL	Mean	0.20	0.20	0.20	0.20
		±SD	0.00	0.00	0.00	0.00
	Microalbumin (mg/dL)	>15	5	5	4	-
		Neg	-	-	1	5
	Bilirubin (BIL) mg/dL	Neg	5	5	5	5
	Ketones (KET) mg/dL	5	4	4	-	-
		Neg	1	1	5	5
	Blood (BLD) Ery/µL	Ca10	-	1	4	-
		Ca80	2	3	1	1
		>=Ca200	3	1	-	4
	Protein (PRO) mg/dL	Trace	2	5	4	-
		30	3	-	-	-
		Neg	-	-	1	5
	Nitrite (NIT)	Neg	5	5	5	4
		Pos	-	-	-	1
	Leucocytes (LEU) Leu/µL	Neg	5	5	5	5
	Glucose (GLU) mg/dL	Neg	5	5	5	5
Microscopic Examination	Epi Cells	0	2	3	2	1
		0-1	2	2	1	3
		1-2	1	-	2	1
		2-3	-	-	-	-
	Casts	Absent	5	5	5	5
	Crystals	Present	5	5	5	5

M: Male; F: Female;SD: Standard Deviation; n: Number of Animals; Pos: Positive; Neg: Negative; Ca: Calculated

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

TABLE 13 (Contd..,).SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Epididymes	Testes	Heart	Kidneys	Brain	Liver	PSC	Thyroid along with parathyroid^#
G1, M & 0	Mean	0.0748	0.4083	1.4875	1.4644	3.2042	1.5285	2.9538	2.2363	12.4668	4.7458	0.0230
	±SD	0.0112	0.0686	0.7760	0.1628	0.3175	0.1467	0.4860	0.1587	2.4600	0.6170	0.0040
	n	5	5	5	12	12	5	5	5	5	12	12
G2, M & 110	Mean	0.0693	0.4236	1.3154	1.5012	3.3500	1.5301	3.0747	2.2646	11.6021	4.3578	0.0236
	±SD	0.0103	0.0489	0.4042	0.1499	0.2902	0.1276	0.4144	0.1684	1.4523	0.6411	0.0054
	n	5	5	5	12	12	5	5	5	5	12	12
G3, M & 330	Mean	0.0787	0.3932	1.4196	1.4830	3.1904	1.5830	3.4092	2.3726	12.6911	4.5579	0.0218
	±SD	0.0103	0.0854	0.2984	0.2442	0.7644	0.2104	0.1248	0.1261	1.3841	0.8952	0.0023
	n	5	5	5	12	12	5	5	5	5	12	12
G4, M & 1000	Mean	0.0710	0.3928	1.1159	1.5932	3.4803	1.4070	3.5326	2.4086	11.2318	5.0252	0.0221
	±SD	0.0093	0.0908	0.4592	0.2333	0.3318	0.1488	0.6437	0.0552	1.4246	0.7548	0.0054
	n	5	5	5	12	12	5	5	5	5	12	12

M: Male;SD: Standard Deviation; n: Number of animals: #: Weighed post fixation: PSC:Prostate + Seminal vesicles with coagulating glands

TABLE 13 (Contd..,).SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid^#
G1, F & 0	Mean	0.0966	0.2964	0.5788	1.2573	2.1996	2.1245	11.8333	0.0219
	±SD	0.0199	0.0293	0.0401	0.0411	0.1216	0.1900	0.8625	0.0046
	n	5	5	5	5	5	5	5	12
G2, F & 110	Mean	0.0937	0.2753	0.9697	1.2310	2.5220	2.3099	12.3020	0.0240
	±SD	0.0086	0.0152	0.2926	0.1081	0.3362	0.1754	1.4895	0.0068
	n	5	5	5	5	5	5	5	11
G3, F & 330	Mean	0.0937	0.2864	0.8288	1.1555	2.1549	2.1199	11.5027	0.0206
	±SD	0.0136	0.0407	0.4319	0.0899	0.2358	0.0991	1.2154	0.0024
	n	5	5	5	5	5	5	5	11
G4, F & 1000	Mean	0.2680	0.3007	0.6806	1.3033	2.1556	2.1361	12.0178	0.0211
	±SD	0.4172	0.0308	0.0499	0.0607	0.0912	0.1073	1.0290	0.0026
	n	5	5	5	5	5	5	5	11

 F: Female;SD: Standard Deviation; n: Number of animals; #: Weighed post fixation (excluding non-pregnant animals)

TABLE 13 (Contd..,).SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Epididymes	Testes	Heart	Kidneys	Brain	Liver	PSC	Thyroid along with parathyroid^#
G1R, M & 0	Mean	0.0540	0.2383	1.1859	1.4440	3.2241	1.4134	2.5498	2.0356	11.5613	3.4105	0.0260
	±SD	0.0071	0.0382	0.1382	0.0625	0.1891	0.1147	0.1590	0.1122	1.1807	0.3936	0.0044
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.0585	0.2403	1.0514	1.3748	3.2331	1.3274	2.5014	2.0688	12.1786	2.9304	0.0252*
	±SD	0.0068	0.0268	0.1085	0.0244	0.1568	0.0511	0.1411	0.0768	1.0689	0.2115	0.0033
	n	5	5	5	5	5	5	5	5	5	5	5

M: Male; R: Recovery;SD: Standard Deviation; n: Number of animals; PSC:Prostate + Seminal vesicles with coagulating glands; #: Weighed post fixation

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

TABLE 13 (Contd..,).SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid^#
G1R, F & 0	Mean	0.0627	0.2513	0.6045	0.9485	1.6102	1.9874	7.3470	0.0272
	±SD	0.0072	0.0190	0.0830	0.0625	0.0802	0.1058	0.5912	0.0030
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	0.0585	0.2312	0.6047	0.9361	1.6242	2.0209	7.7516	0.0276
	±SD	0.0035	0.0308	0.0664	0.0616	0.0614	0.0831	0.5586	0.0029
	n	5	5	5	5	5	5	5	5

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

TABLE 14.SUMMARY OF FASTING BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Epididymes	Testes	Heart	Kidneys	Brain	Liver	PSC	Thyroid along with parathyroid
G1, M & 0	Mean	410.03	0.0183	0.1005	0.3675	0.3583	0.7834	0.3711	0.7155	0.5481	3.0055	1.1692	0.0056
	±SD	37.56	0.0034	0.0226	0.2037	0.0398	0.0687	0.0099	0.0821	0.0780	0.3359	0.1983	0.0009
	n	12	5	5	5	12	12	5	5	5	5	12	12
G2, M & 110	Mean	402.30	0.0176	0.1064	0.3240	0.3760	0.8390	0.3846	0.7789	0.5750	2.9049	1.0904	0.0059
	±SD	43.50	0.0038	0.0101	0.0732	0.0450	0.0901	0.0301	0.1573	0.1010	0.1895	0.1795	0.0016
	n	12	5	5	5	12	12	5	5	5	5	12	12
G3, M & 330	Mean	410.88	0.0192	0.0969	0.3501	0.3663	0.7832	0.3823	0.8280	0.5779	3.0611	1.1252	0.0053
	±SD	34.87	0.0034	0.0300	0.1115	0.0854	0.1962	0.0355	0.0725	0.0746	0.1318	0.2860	0.0006
	n	12	5	5	5	12	12	5	5	5	5	12	12
G4, M & 1000	Mean	403.88	0.0184	0.1020	0.2935	0.3956	0.8654	0.3633	0.9094	0.6240	2.9117	1.2476	0.0055
	±SD	35.72	0.0039	0.0296	0.1454	0.0534	0.0882	0.0566	0.1664	0.0899	0.5896	0.1804	0.0015
	n	12	5	5	5	12	12	5	5	5	5	12	12

M: Male;SD: Standard Deviation; n: Number of animals; PSC:Prostate + Seminal vesicles with coagulating glands

TABLE 14 (Contd..,).SUMMARY OF FASTING BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid
G1, F & 0	Mean	293.81	0.0330	0.1012	0.1972	0.4288	0.7507	0.7254	4.0356	0.0074
	±SD	19.27	0.0072	0.0104	0.0076	0.0046	0.0456	0.0703	0.2574	0.0014
	n	12	5	5	5	5	5	5	5	12
G2, F & 110	Mean	301.74	0.0307	0.0904	0.3183	0.4020	0.8219	0.7573	4.0189	0.0079
	±SD	15.26	0.0034	0.0091	0.0977	0.0165	0.0609	0.0726	0.3752	0.0021
	n	11	5	5	5	5	5	5	5	11
G3, F & 330	Mean	296.91	0.0330	0.1015	0.3007	0.4078	0.7594	0.7513	4.0753	0.0070
	±SD	25.78	0.0037	0.0164	0.1590	0.0200	0.0515	0.0669	0.5067	0.0009
	n	11	5	5	5	5	5	5	5	11
G4, F & 1000	Mean	302.66	0.0932	0.1029	0.2328	0.4457	0.7370	0.7293	4.1049	0.0070
	±SD	19.33	0.1467	0.0128	0.0229	0.0367	0.0557	0.0415	0.3896	0.0010
	n	11	5	5	5	5	5	5	5	11

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

TABLE 14 (Contd..,).SUMMARY OF FASTING BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Epididymes	Testes	Heart	Kidneys	Brain	Liver	PSC	Thyroid along with parathyroid
G1R, M & 0	Mean	453.53	0.0119	0.0525	0.2612	0.3210	0.7146	0.3141	0.5650	0.4505	2.5559	0.7586	0.0058
	±SD	36.36	0.0013	0.0067	0.0186	0.0404	0.0716	0.0462	0.0560	0.0339	0.2477	0.1313	0.0011
	n	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	434.34	0.0135	0.0555	0.2428	0.3188	0.7513	0.3088	0.5801	0.4795	2.8212	0.6797	0.0059
	±SD	43.79	0.0018	0.0056	0.0207	0.0284	0.0949	0.0411	0.0598	0.0418	0.3202	0.0769	0.0012
	n	5	5	5	5	5	5	5	5	5	5	5	5

M: Male; R: Recovery;SD: Standard Deviation; n: Number of animals; PSC:Prostate + Seminal vesicles with coagulating glands

TABLE 14 (Contd..,).SUMMARY OF FASTING BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO FASTING BODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Heart	Kidneys	Brain	Liver	Thyroid along with parathyroid
G1R, F & 0	Mean	277.89	0.0225	0.0906	0.2176	0.3414	0.5798	0.7151	2.6452	0.0098
	±SD	8.40	0.0022	0.0086	0.0290	0.0208	0.0326	0.0297	0.2147	0.0010
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	275.98	0.0212	0.0838	0.2197	0.3397	0.5899	0.7336	2.8066	0.0100
	±SD	13.81	0.0013	0.0103	0.0274	0.0246	0.0405	0.0459	0.0818	0.0013
	n	5	5	5	5	5	5	5	5	5

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

TABLE 15.SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATON OF OESTRUS CYCLICITY

Group & Dose (mg/kg body weight/day)	No. of Females	No. of Females with Regular Oestrus Cyclicity during Pre-mating, mating and on Lactation day 14	No. of Females with Irregular Oestrus Cyclicity during Pre-mating, mating and on Lactation day 14
G1 & 0	12	12	0
G2 & 110	12	12	0
G3 & 330	12	12	0
G4 & 1000	12	12	0

TABLE 16. SUMMARY OF COHABITATION RECORD

Group & Dose (mg/kg body weight/day)		Copulatory Interval (Days)		Conceiving Days (1 to 5)	Conceiving Days (6 to 20)
G1 & 0	Mean	5.17	n	10	2
	±SD	4.97
	n	12	%	83.3	16.7
G2 & 110	Mean	3.83	n	11	1
	±SD	3.79
	n	11	%	91.7	8.3
G3 & 330	Mean	8.17	n	6	6
	±SD	5.77
	n	11	%	50.0	50.0
G4 & 1000	Mean	6.33	n	7	5
	±SD	5.16
	n	11	%	58.3	41.7

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

TABLE 17. SUMMARY RECORD OF BODY WEIGHT (g) DURING GESTATION PERIOD

Group & Dose (mg/kg body weight/day)		Body weight (g) on Gestation Days
		0	7	14	20
G1 & 0	Mean	271.36	283.93	312.15	370.46
	±SD	12.65	12.11	17.64	24.09
	n	12	12	12	12
G2 & 110	Mean	273.59	287.73	314.80	369.77
	±SD	13.40	18.89	20.86	32.89
	n	11	11	11	11
G3 & 330	Mean	275.14	289.37	315.43	377.64
	±SD	14.90	17.01	19.06	32.03
	n	11	11	11	11
G4 & 1000	Mean	276.17	291.09	319.92	377.45
	±SD	16.34	19.83	28.07	33.43
	n	11	11	11	11

SD: Standard Deviation; n: Number of dams

TABLE 18. SUMMARY RECORD OF CHANGE IN BODY WEIGHT (%) DURING GESTATION PERIOD

Group & Dose (mg/kg body weight/day)		Change in body weight (%) during Gestation
		0 to 7	7 to 14	14 to 20
G1 & 0	Mean	4.67	9.93	18.70
	±SD	1.92	3.89	4.69
	n	12	12	12
G2 & 110	Mean	5.11	9.42	17.36
	±SD	2.68	2.43	4.85
	n	11	11	11
G3 & 330	Mean	5.17	9.04	19.73
	±SD	2.23	3.57	7.02
	n	11	11	11
G4 & 1000	Mean	5.39	9.83	18.18
	±SD	3.17	4.16	7.36
	n	11	11	11

SD: Standard Deviation; n: Number of dams

TABLE 19. SUMMARY RECORD OF CHANGE IN BODY WEIGHT (%) DURING GESTATION PERIOD

Group, Sex & Dose (mg/kg body weight)		Feed Consumption (g) during Gestation
		GD 0 to 7	GD 7 to 14	GD 14 to 20
G1 & 0	Mean	20.86	22.19	25.03
	±SD	1.23	1.93	2.43
	n	12	12	12
G2 & 110	Mean	20.60	21.25	23.76
	±SD	2.65	1.80	1.58
	n	11	11	11
G3 & 330	Mean	20.86	22.38	24.60
	±SD	1.84	2.34	3.16
	n	11	11	11
G4 & 1000	Mean	20.13	20.87	23.14
	±SD	1.76	1.31	1.89
	n	11	11	11

SD: Standard Deviation; n: Number of dams

TABLE 20. SUMMARY RECORD OF BODY WEIGHT (g) DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day
		1	4	7	13
G1 & 0	Mean	278.44	294.08	298.24	314.94
	±SD	16.31	16.65	18.10	17.65
	n	12	12	12	12
G2 & 110	Mean	281.82	292.91	300.97	322.16
	±SD	21.53	23.35	19.32	18.01
	n	11	11	11	11
G3 & 330	Mean	281.39	288.04	298.27	316.03
	±SD	33.43	30.09	26.65	24.33
	n	11	11	11	11
G4 & 1000	Mean	293.33	296.65	303.91	322.55
	±SD	17.02	19.39	21.63	17.91
	n	11	11	11	11

SD: Standard Deviation; n: Number of dams

TABLE 21. SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT (%) DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Change in body weight (%) during Lactation
		1 to 4	4 to 7	7 to 13
G1 & 0	Mean	5.65	1.43	5.68
	±SD	2.27	2.95	3.32
	n	12	12	12
G2 & 110	Mean	3.98	2.90	7.15
	±SD	4.23	3.16	4.00
	n	11	11	11
G3 & 330	Mean	2.55*	3.75	6.07
	±SD	2.79	4.13	2.69
	n	11	11	11
G4 & 1000	Mean	1.10*	2.43	6.25
	±SD	1.20	2.21	2.55
	n	11	11	11

SD: Standard Deviation; n: Number of dams

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

TABLE 22. SUMMARY RECORD OF FEED CONSUMPTION (g/dam/day) DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		Feed Consumption (g) during Lactation
		LD 1 to 4	LD 4 to 7	LD 7 to 14
G1 & 0	Mean	25.34	35.64	36.01
	±SD	2.71	5.41	4.19
	n	12	12	12
G2 & 110	Mean	25.22	38.92	37.93
	±SD	2.61	3.95	4.15
	n	11	11	11
G3 & 330	Mean	26.02	32.38	35.29
	±SD	4.05	5.19	4.84
	n	11	11	11
G4 & 1000	Mean	26.04	35.80	35.73
	±SD	4.47	6.61	5.59
	n	11	11	11

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

TABLE 23. SUMMARY RECORD OF GESTATION LENGTH (DAYS) AND DELIVERY DATA

Group & Dose (mg/kg body weight/day)		Gestation Length (Days)		Litter Size (No.)	Live Pups (No.)				Dead Pups (No.)				Sex Ratio (M/F) at Birth	Live Birth Index (%)
					Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	22.08		11.08	10.75	4.67	6.08		0.33	0.33	0.08		0.83	97.34
	±SD	0.79		1.93	1.71	1.72	1.51		0.65	0.65	0.29		0.44	5.03
	n	12		12	12	12	12		12	12	12		12	12
G2 & 110	Mean	22.36		11.55	11.55	5.82	5.73		0.00*	0.00	0.00		1.17	100.00
	±SD	0.81		1.37	1.37	1.66	1.49		0.00	0.00	0.00		0.66	0.00
	n	11		11	11	11	11		11	11	11		11	11
G3 & 330	Mean	22.55		10.73	10.73	5.55	5.18		0.00*	0.00	0.00		1.31	100.00
	±SD	0.69		1.79	1.79	1.97	2.04		0.00	0.00	0.00		0.82	0.00
	n	11		11	11	11	11		11	11	11		11	11
G4 & 1000	Mean	22.55		10.91	10.64	5.45	5.18		0.27	0.18	0.09		1.29	97.27
	±SD	0.82		2.43	4.00	2.34	2.09		0.90	0.60	0.30		0.82	9.05
	n	11		11	11	11	11		11	11	11		11	11

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

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

TABLE 24. SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		No. of Live Pups At Birth	During LD 1-4							Sex Ratio (M/F) at LD 4	No. of Survived Pups during LD 1-4	Pup Survival Index (%)
			Live Pups (No.)				Dead Pups (No.)
			Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	10.75	10.75	4.67	6.08		0.00	0.00	0.00	0.83	10.75	100.00
	±SD	1.71	1.71	1.72	1.51		0.00	0.00	0.00	0.44	1.71	0.00
	n	12	12	12	12		12	12	12	12	12	12
G2 & 110	Mean	11.55	11.55	5.82	5.73		0.00	0.00	0.00	1.17	11.55	100.00
	±SD	1.37	1.37	1.66	1.49		0.00	0.00	0.00	0.66	1.37	0.00
	n	11	11	11	11		11	11	11	11	11	11
G3 & 330	Mean	10.73	10.55	5.45	5.09		0.18	0.09	0.09	1.31	10.55	97.73
	±SD	1.79	2.16	2.07	2.12		0.60	0.30	0.30	0.82	2.16	7.54
	n	11	11	11	11		11	11	11	11	11	11
G4 & 1000	Mean	10.64	10.64	5.45	5.18		0.00	0.00	0.00	1.29	10.64	100.00
	±SD	2.69	2.69	2.34	2.09		0.00	0.00	0.00	0.82	2.69	0.00
	n	11	11	11	11		11	11	11	11	11	11

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

TABLE 24 (Contd..,). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		Pups Sacrificed for Blood Collection on LD 4 (No.)		Pups Sacrificed for Blood Collection on LD 4 (No.)	No. of Survived Pups during LD 1-4	No. of Live Pups on LD 4	During LD 4-7							Sex Ratio (M/F) at LD 7	No. of Survived Pups during LD 4-7	Pup Survival Index (%) during LD 4-7
							Live Pups (No.)				Dead Pups (No.)
		Male (No.)	Female (No.)				Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	0.00	0.92	0.92	10.75	9.83	9.83	4.67	5.17		0.00	0.00	0.00	1.12	9.83	100.00
	±SD	0.00	1.00	1.00	1.71	0.94	0.94	1.72	1.40		0.00	0.00	0.00	0.98	0.94	0.00
	n	12	12	12	12	12	12	12	12		12	12	12	12	12	12
G2 & 110	Mean	0.00	1.27	1.27	11.55	10.27	10.27	5.82	4.45		0.00	0.00	0.00	1.51	10.27	100.00
	±SD	0.00	1.01	1.01	1.37	0.65	0.65	1.66	1.44		0.00	0.00	0.00	0.72	0.65	0.00
	n	11	11	11	11	11	11	11	11		11	11	11	11	11	11
G3 & 330	Mean	0.00	1.18	1.18	10.55	9.36	9.36	5.45	3.91		0.00	0.00	0.00	2.14	9.36	100.00
	±SD	0.00	0.98	0.98	2.16	1.43	1.43	2.07	1.87		0.00	0.00	0.00	2.40	1.43	0.00
	n	11	11	11	11	11	11	11	11		11	11	11	11	11	11
G4 & 1000	Mean	0.00	0.82	0.82	10.64	9.82	9.82	5.45	4.36		0.00	0.00	0.00	1.63	9.82	100.00
	±SD	0.00	0.98	0.98	2.69	1.89	1.89	2.34	1.69		0.00	0.00	0.00	1.35	1.89	0.00
	n	11	11	11	11	11	11	11	11		11	11	11	11	11	11

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

TABLE 24 (Contd..,). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)		No. of Live Pups on LD 7	During LD 7-13							Sex Ratio (M/F) at LD 13	No. of Survived Pups during LD 7-13	Pup Survival Index (%) during LD 7-13
			Live Pups (No.)				Dead Pups (No.)
			Total (No.)	Male (No.)	Female (No.)		Total (No.)	Male (No.)	Female (No.)
G1 & 0	Mean	9.83	9.83	4.67	5.17		0.00	0.00	0.00	1.12	9.83	100.00
	±SD	0.94	0.94	1.72	1.40		0.00	0.00	0.00	0.98	0.94	0.00
	n	12	12	12	12		12	12	12	12	12	12
G2 & 110	Mean	10.27	10.27	5.82	4.45		0.00	0.00	0.00	1.51	10.27	100.00
	±SD	0.65	0.65	1.66	1.44		0.00	0.00	0.00	0.72	0.65	0.00
	n	11	11	11	11		11	11	11	11	11	11
G3 & 330	Mean	9.36	9.36	5.45	3.91		0.00	0.00	0.00	2.14	9.36	100.00
	±SD	1.43	1.43	2.07	1.87		0.00	0.00	0.00	2.40	1.43	0.00
	n	11	11	11	11		11	11	11	11	11	11
G4 & 1000	Mean	9.82	9.82	5.45	4.36		0.00	0.00	0.00	1.63	9.82	100.00
	±SD	1.89	1.89	2.34	1.69		0.00	0.00	0.00	1.35	1.89	0.00
	n	11	11	11	11		11	11	11	11	11	11

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

TABLE 25. SUMMARY OF PUP OBSERVATION RECORD DURING LACTATION PERIOD

Group & Dose (mg/kg body weight/day)	Pup Observation on LD 1	Pup Observation on LD 4	Pup Observation on LD 7	Pup Observation on LD 13
G1 & 0	N	N	N	N
G2 & 110	N	N	N	N
G3 & 330	N	N	N	N
G4 & 1000	N	N	N	N

LD: Lactation Day

TABLE 26. SUMMARY OF PUP WEIGHT (g) DURING LACTATION PERIOD

Group& Dose (mg/kg body weight/day)		Pup weight (g) on LD 1			Pup weight (g) on LD 4			Pup Weight (g) on LD 7			Pup weight (g) on LD 13
		Male	Female		Male	Female		Male	Female		Male	Female
G1 & 0	Mean	7.24	6.54		10.29	9.10		15.17	13.35		26.87	22.96
	±SD	0.55	0.55		1.67	1.02		2.33	1.07		2.44	0.71
	n	12	12		12	12		12	12		12	12
G2 & 110	Mean	6.83	6.47		9.57	9.11		14.26	13.69		25.79	22.98
	±SD	0.76	0.68		1.75	1.49		1.48	1.57		2.21	1.12
	n	11	11		11	11		11	11		11	11
G3 & 330	Mean	6.83	6.28		9.82	9.41		15.42	13.65		27.17	22.96
	±SD	0.66	0.47		1.87	1.11		1.63	1.77		0.74	3.27
	n	11	11		11	11		11	11		11	11
G4 & 1000	Mean	7.47	7.20		11.17	10.26		16.05	14.47		28.03	23.91
	±SD	1.05	1.30		1.59	2.12		2.07	2.71		1.64	3.82
	n	11	11		11	11		11	11		11	11

LD: Lactation Day

TABLE 27. SUMMARY OF UTERI OBSERVATIONS RECORD

Group & Dose (mg/kg body weight/day)		No. of Corpo raluetea	No. of Implantations	Implantation index (%)	Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Pre-natal Loss (No.)	Pre-natal Loss (%)	Post-natal Loss (No.)	Post-natal Loss (%)	No. of Early Resorptions	No. of Late Resorptions
G1 & 0	Mean	11.08	11.08	100.00	0.00	2.66	0.33	97.34	0.00	0.00	0.00	0.00
	±SD	1.93	1.93	0.00	0.00	5.03	0.65	5.03	0.00	0.00	0.00	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G2 & 110	Mean	11.55	11.55	100.00	0.00	0.00	0.00	100.00	0.00	0.00	0.00	0.00
	±SD	1.37	1.37	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G3 & 330	Mean	10.73	10.73	100.00	0.00	0.00	0.00	100.00	0.18	2.27	0.00	0.00
	±SD	1.79	1.79	0.00	0.00	0.00	0.00	0.00	0.60	7.54	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4 & 1000	Mean	10.91	10.91	100.00	0.00	2.73	0.27	97.27	0.00	0.00	0.00	0.00
	±SD	2.43	2.43	0.00	0.00	9.05	0.90	9.05	0.00	0.00	0.00	0.00
	n	11	11	11	11	11	11	11	11	11	11	11

SD: Standard Deviation; n: Number of animals

TABLE 28. SUMMARY RECORD OF ANO-GENITAL DISTANCE (mm) OF PUPS ON LACTATION DAY 4

Group & Dose (mg/kg body weight/day)		Male Mean AGD (mm)	Female Mean AGD (mm)
G1 & 0	Mean	6.97	2.93
	±SD	0.76	0.36
	n	12	12
G2 & 110	Mean	6.98	2.86
	±SD	0.97	0.29
	n	11	11
G3 & 330	Mean	6.85	2.94
	±SD	0.53	0.27
	n	11	11
G4 & 1000	Mean	7.42	2.95
	±SD	0.69	0.37
	n	11	11

SD: Standard Deviation; n: Number of animals; mm: milli meter

TABLE 29. SUMMARY RECORD OF MALE PUP NIPPLE RETENTION (No.) ON LACTATION DAY 13

Group & Dose (mg/kg body weight/day)		No. of Nipples/Areolae
G1 & 0	Mean	0.00
	±SD	0.00
	n	12
G2 & 110	Mean	0.00
	±SD	0.00
	n	11
G3 & 330	Mean	0.00
	±SD	0.00
	n	11
G4 & 1000	Mean	0.00
	±SD	0.00
	n	11

SD: Standard Deviation; n: Number of animals

TABLE 30. SUMMARY RECORD OF SERUM T4 LEVELS (ng/mL)

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	81.773
	±SD	8.886
	n	12
G2, M & 110	Mean	74.769
	±SD	5.861
	n	12
G3, M & 330	Mean	74.183
	±SD	5.778
	n	12
G4, M & 1000	Mean	81.428
	±SD	10.239
	n	12

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

TABLE 31. SUMMARY RECORD OF SERUM T4 LEVELS (ng/mL) IN LACATION DAY 13 PUPS

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1 & 0	Mean	58.407
	±SD	4.208
	n	12
G2 & 110	Mean	53.282
	±SD	6.297
	n	11
G3 & 330	Mean	53.750
	±SD	4.964
	n	11
G4 & 1000	Mean	49.324*
	±SD	5.452
	n	11

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

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

 

TABLE 32. SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD

Organs/ Lesions	Group	G1		G2		G3		G4
	Dose (mg/kg body weight/day)	0		110		330		1000
	Sex	M	F	M	F	M	F	M	F
	No. of Animals	12	12	12	12	12	12	12	12
No Abnormality Detected		12	12	12	12	11	12	12	12
Testes and Epididymis - reduced in size (bilateral)		-	-	-	-	1	-	-	-

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

TABLE 32 (Contd..,). SUMMARY OF GROSS PATHOLOGY FINDINGS RECORD

Organs/ Lesions	Group	G1R		G4R
	Dose (mg/kg body weight/day)	0		1000
	Sex	M	F	M	F
	No. of Animals	5	5	5	5
No Abnormality Detected		5	5	5	5

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

TABLE 33. SUMMARY OF PUP GROSS PATHOLOGY FINDINGS RECORD

Organs/ Lesions	Group	G1		G2		G3		G4
	Dose (mg/kg body weight/day)	0		110		330		1000
	Sex	M	F	M	F	M	F	M	F
	No. of Dead pups (At birth)	4	-	-	-	-	-	2	1
No Abnormality Detected		4	-	-	-	-	-	2	1
	No. of Dead pups (Lactation Day 2)	-	-	-	-	1	1	-	-
No Abnormality Detected		-	-	-	-	1	1	-	-
	No. of pups sacrificed on Lactation Day 4	-	11	-	14	-	13	-	9
No Abnormality Detected		-	11	-	14	-	13	-	9
	No. of pups sacrificed on Lactation Day 13	56	62	64	48	60	43	60	48
No Abnormality Detected		56	62	64	48	60	43	60	48

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

TABLE 34. SUMMARY OF HISTOPATHOLOGY FINDINGS RECORD

Group No.	G1		G4
Dose (mg/kg body weight)	0		1000
Sex	M	F	M	F
No. of Animals (randomly selected)	5	5	5	5
No. of Animals (not randomly selected)	7	7	7	7
ORGANS AND OBSERVATIONS
LIVER
MNC infiltration	0	1	0	1
SPLEEN
Red pulp; increased golden-brown pigment	0	1	0	0
PROSTATE GLAND
Neutrophils infiltration	1	X	0	X

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

 

Conclusions:

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Acid Violet 50 was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and recovery group animals for 50 days, with a recovery period of 21 days observation under the experimental conditions employed in this study.

Executive summary:

The test item, Acid Violet 50 was evaluated for possible adverse effects following repeated oral dosing to males for 37 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups. Each main group (G1, G2, G3and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle[Corn oil], theanimals in G2, G3 and G4/G4Rgroups were administered

withtest itemat the dose levels of110, 330 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation

day 14 after overnight fasting (water allowed). The recovery group males and females were treated until the first scheduled sacrifice of dams (total of 50 days) and kept without treatment for a further 21 days observation.

The dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results are found to be within the acceptable range of 85 to 115 % the nominal concentration and the % RSD is less than 10.0%.

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Ophthalmological examination was carried out once before treatment for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice) for vehicle control and high dose group animals andduring last weekfor recovery group animals.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), during lactation period for females (shortly prior to scheduled sacrifice) and from both the recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice).

The clinical pathology (hematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) andfor all recovery group animals. Urinalysis was performed from the five randomly selected males of each main group during the last week of the treatment period and for all recovery animals at termination. The serum from adult males of all the main

groups and from lactation day 13 pups representing two pups per litter was screened for T4 levels.

The main group females were observed for oestrus cyclicityduring pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice.The main group females were observed forcopulatory interval and all the adult animals of main groups were observed for mating and fertility index.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index,number of pups, sex ratio and pup survival indexthroughout the lactation period. The pups were observed forclinical signs and external anomalies once daily from PND 1 to 13. The both male and female pupweights were recorded separately on lactation days 1, 4, 7 and 13.The anogenital distance of each pup was measured on lactation day 4. The numbers of nipples/areolae in male pups were counted on lactation day 13.

Gross pathology and organ weighing were performed on day 38 for males, on lactation day 13 for pups, lactation day 14 for dams and on day 72 for recovery group animals. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the animals did not reveal any clinical signs of toxicity except, violet colored fecal matter after dose administration from all the tested dose group animals of either sex which can be due to the colour of the test item. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic

examination, neurological/functional examination, haematology, clinical chemistry, urine analysis nor in organ weights (both absolute and relative) were observed in both the sex of main and recovery group animals. There were no treatment related changes observed in serum T4 levels of males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.

The dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period, uteri observations.

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

The animals did not reveal any treatment related gross pathological and histopathological findings.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

No toxic effects were noticed in a short term oral repeated dose study in rats according to OECD TG 422.