Ethyltriphenylfosfonium bromide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from a study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

According to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylfosfonium bromide

EC Number:

216-223-3

EC Name:

Ethyltriphenylfosfonium bromide

Cas Number:

1530-32-1

Molecular formula:

C20H20P.Br

IUPAC Name:

bromo(ethyl)triphenylphosphorane

Test material form:

solid: crystalline

Details on test material:

Name: Ethyltriphenylfosfonium bromide
IUPAC Name: Phosphonium, ethyltriphenyl-, bromide (1:1)
CAS No.: 1530-32-1
Molecular Weight: 371.25 g/mol
Molecular Formula: C20-H20-P.Br
SMILES: [Br-].CC[P+](c1ccccc1)(c2ccccc2)c3ccccc3

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 Weeks
- Weight at study initiation: Male: 148.4g -153.8 g
Female: 147.1 g -158.6 g
- Fasting period before study: No Data Available
- Housing: Animals were housed in an autoclaved polycarbonate cages with stainless steel top grill having facilities for holding pellet feed and drinking water in polycarbonate bottle. Autoclaved corn cob was used as bedding material and it was changed at least once in a week.
- Diet (e.g. ad libitum): Rodent feed from a certified vendor was provided as ad libitum.
- Water (e.g. ad libitum): Aquaguard purified water was provided ad libitum by a water dispensing bottle.
- Acclimation period: Nineteen days under laboratory conditions, after veterinary examination. Only animals without any visible signs of illness were used for the study.

DETAILS OF FOOD AND WATER QUALITY: No Data Available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C – 23.4°C.
- Humidity (%): 51 – 61 %
- Air changes (per hr): 10 Air Changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle per day

IN-LIFE DATES:
From: 30-06-2020
To: 10-09-2020

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Administration of the test chemical by gavage is a common and accepted route of exposure for this type of studies.

Vehicle:

water

Remarks:

Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was weighed and transferred to a labelled beaker. 10 mL of distilled water was added and mixed with a glass rod. Final volume was made up to 50 ml to get a concentration of 3 mg/ml for Low Dose, 6 mg/ml for Intermediate dose and 12 mg/ml for High dose by mixing with magnetic stirrer. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Distilled water was used as a vehicle.
- Concentration in vehicle: 10 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of dose formulation was checked before treatment start day at 0 and 6 hours. The test chemical was checked for its stability in the dose formulation with samples collected immediately (0 hours) and 6 hours post dose formulation preparation. Samples of the dose formulations were taken immediately after dilution of the concentrates with the diluents (vehicle) on treatment start date and treatment end date for homogeneity (mean of homogeneity was given as dose concentration) analyses. On week 3, samples of all dose formulations were taken for dose concentration analysis.Analysis was performed based on the validated analytical method.

Linearity: Linearity was carried out by injecting a minimum of 5 different test concentrations (three replications from each concentration).The correlation coefficient and regression was calculated (r=1.00).
Accuracy: Accuracy was performed at three levels ie., 50, 100, 150%. The mean recovery values of samples for 50% level is 99.82% , for 100% level is 99.58% and for 150% level is 98.99%.
Precision: The relative standard deviation of analysis of five independent fortifications samples were not exceed ±20% of the mean value.
Specificity: No interference was observed above 30 % of total peak area for target analyte.

Duration of treatment / exposure:

28 days for main group and 14 days of recoery period.

Frequency of treatment:

Once Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control Group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low Dose Group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Mid Dose Group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High Dose Group

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses for the main study were selected on the basis of a dose range finding (DRF) study. The doses for the DRF study were 0, 50, 100 and 200 mg/kg bw/day. During the treatment 03 Males and 04 Females showed Mortality in High dose (200 mg/kg Body weight). There was no mortality in Control, Low Dose and Mid Dose. Piloerection and dullness were observed in High dose group. Hence, based on the Dose Range Finding Study, the doses of 30, 60 and 120 mg/kg body weight were confirmed for main study.
- Rationale for animal assignment (if not random): Animals were selected and grouped based on stratified randomization by using body weights taken before start of treatment. Computerized statistical analysis was used for randomization (MS Excel programme).
- Fasting period before blood sampling for clinical biochemistry: The animals were placed individually in metabolic cages and fasted overnight before blood sampling but allowed access to water ad libitum.
- Rationale for selecting satellite groups: The satellite groups were selected to observe the reversibility of the effects that are observed during and after the dosing of the animals for 28 days.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): No Data Available
- Other: No Data Available

Positive control:

No Data Available

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals was observed twice daily for morbidity and mortality while for Clinical Signs, Once daily during acclimatization phase and twice daily on first 3 days of treatment; once daily thereafter. During Recovery period, animals were observed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and weekly after.

BODY WEIGHT: Yes
- Time schedule for examinations: Once during Acclimatization and before randomization and Weekly Once during Treatment Period and Recovery Period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Once weekly during treatment and recovery period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY: No Data Available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during acclimatization, during week 4 in all animals of groups 1 and 4.
- Dose groups that were examined: All animals from all the dose groups were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 (Main Groups) and Day 43 (Recovery Group), before sacrifice
- Anaesthetic used for blood collection: Yes (Isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: All animals from all the dose groups.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (Main Groups) and Day 43 (Recovery Group), before sacrifice
- Animals fasted: Yes
- How many animals: All animals from all the dose groups.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected in the morning before the blood collection.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: End of week 4 for main groups and by the end of Week 6 for recovery groups.
- Dose groups that were examined: All animals from all the dose groups.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Not specified
- Time schedule for examinations: No Data Available
- How many animals: No Data Available
- Dose groups that were examined: No Data Available

OTHER: No Data Available

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: Yes, All organ and tissue samples, as defined under Necropsy were processed, embedded and cut at an approximate thickness of 3 to 5 micrometers, stained with hematoxylin and eosin and examined under light microscope. Full histopathology was performed on the preserved organs and tissues of all animals in the control and high dose groups. Histopathology examination was extended to the organs such as liver, testes and epididymides in low dose and intermediate dose group.

Other examinations:

No Data Available

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, organ weights as well as clinical pathology data.
• Data was summarized in tabular form. Statistical analysis was performed using SAS/STAT® Software (Version 9.3).
• All the data were analysed for homogeneity using Levene’s test
• The difference between the means was analyzed using Dunnet’s t- test
• Significant differences between control and treated groups analyzed using one-way ANOVA statistics
• Values were summarized as mean ± standard deviation (SD)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in all the dose groups examined including the recovery groups.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in all the dose groups examined including the recovery groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicological relevant changes in th body weight between treated and control group were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No signficance in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No effects were observed after opthalmological examinations in both the control groups and treatment groups.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related effects were observed in the both male and female animals with respect to hematological parameters viz RBC,WBC Hb, MCV, MCHC, PCV, PT and APTT when compared with control group. However, in the treatment group, Platelets count was increased in Low Dose (G2), Intermediate Dose (G3) and high dose (G4) group male rats when compared with control group was observed and in high dose recovery group females Monocytes count was decresed comparable with control. This signficance may not be attributed to treatment since all the haematological values fall with in the normal biological range and no biological significance was noted.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was no dose related effect on few biochemical parameters. There was a significant decrese in both Asparate aminotransferase (AST) and Alanine aminotransferase (ALT) levels in males. Significant changes were observed indicating the test chemical's effect on liver. In males, Urea levels, Bilirubin levels decreased, while Potassium levels increased in low dose group. In Mid Dose Group, Bilirubin levels decreased, while alkaline phosphatase levels and Potassium levels increased, when compared with the control group. In females, Glucose and Sodium levels increased in High dose group, while cholesterol and triglyceride levels decreased, when compared with control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant changes were observed in any of the treated groups and recovery group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test chemical related effects on the grip strength, motor activity, sensory activity was noted in both treated and recovery group animals.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Both in males and females organ weights of Heart increased High dose group (G4) compared with control group. In Recovery group Females, organ weights of Brain decreased High dose group (G4), when compared with control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

During necropsy, macroscopic lesions were observed on liver and lungs where hemorrhagic spots were observed in both the organs in high dose group. Apart from these observations, no other treatment related macroscopic finding in any of the animals in all groups including Systemic and Reproductive organs.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In microscopic observations, sinusoidal hemorrhages were observed in liver in control males, but such effect was observed in both the sexes in high dose group. Also, Foci of necrosis/inflammation of hepatocytes were observed in centri lobular of liver in three male and two female animals from G4 group. Alveolar wall thickening or alveolar inflammation noticed in the lungs of two male and two female animals from G4 group.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No clinical signs were observed in all the dose groups examined including the recovery groups. No mortality was observed in all the dose groups examined including the recovery groups. No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicological relevant changes in the body weight between treated and control group were observed. No significance in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control group. No effects were observed after ophthalmological examinations in both the control groups and treatment groups. No treatment related effects were observed in the both male and female animals with respect to hematological parameters viz RBC, WBC, Hb, MCV, MCHC, PCV, PT and APTT when compared with control group. However, in the treatment group, Platelets count was increased in Low Dose (G2), Intermediate Dose (G3) and high dose (G4) group male rats when compared with control group was observed and in high dose recovery group females Monocytes count was decreased comparable with control. This significance may not be attributed to treatment since all the hematological values fall within the normal biological range and no biological significance was noted. There was no dose related effect on few biochemical parameters. There was a significant decrease in both Asparate aminotransferase (AST) and Alanine aminotransferase (ALT) levels in males. Significant changes were observed indicating the test chemical's effect on liver. In males, Urea levels, Bilirubin levels decreased, while Potassium levels increased in low dose group. In Mid Dose Group, Bilirubin levels decreased, while alkaline phosphatase levels and Potassium levels increased, when compared with the control group. In females, Glucose and Sodium levels increased in High dose group, while cholesterol and triglyceride levels decreased, when compared with control group. No significant changes were observed in any of the treated groups and recovery group. No test chemical related effects on the grip strength, motor activity, sensory activity was noted in both treated and recovery group animals. Both in males and females organ weights of Heart increased High dose group (G4) compared with control group. In Recovery group Females, organ weights of Brain decreased High dose group (G4), when compared with control group. During necropsy, macroscopic lesions were observed on liver and lungs where hemorrhagic spots were observed in both the organs in high dose group. Apart from these observations, no other treatment related macroscopic finding in any of the animals in all groups including Systemic and Reproductive organs. In microscopic observations, sinusoidal hemorrhages were observed in liver in control males, but such effect was observed in both the sexes in high dose group. Also, Foci of necrosis/inflammation of hepatocytes were observed in centri lobular of liver in three male and two female animals from G4 group. Alveolar wall thickening or alveolar inflammation noticed in the lungs of two male and two female animals from G4 group.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

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

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Adverse effects were observed in liver and lungs.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Table for Clinical Signs

TREATMENT

Day of observation	Control (G1)		Low dose (G2)		Middle dose (G3)		High dose (G4)
	M	F	M	F	M	F	M	F
1	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
2	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
3	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
4	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
5	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
6	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
7	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
8	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
9	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
10	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
11	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
12	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
13	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
14	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
15	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
16	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
17	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
18	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
19	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
20	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
21	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
22	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
23	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
24	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
25	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
26	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
27	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5
28	01/5	01/5	01/5	01/5	01/5	01/5	01/5	01/5

Key: M – Male; F – Female; Observation/No. of animals; 01 – Normal.

 

Recovery (R)

Day of observation	Control Recovery (G1R)		High dose Recovery (G4R)
	M	F	M	F
1	01/5	01/5	01/5	01/5
2	01/5	01/5	01/5	01/5
3	01/5	01/5	01/5	01/5
4	01/5	01/5	01/5	01/5
5	01/5	01/5	01/5	01/5
6	01/5	01/5	01/5	01/5
7	01/5	01/5	01/5	01/5
8	01/5	01/5	01/5	01/5
9	01/5	01/5	01/5	01/5
10	01/5	01/5	01/5	01/5
11	01/5	01/5	01/5	01/5
12	01/5	01/5	01/5	01/5
13	01/5	01/5	01/5	01/5
14	01/5	01/5	01/5	01/5
15	01/5	01/5	01/5	01/5
16	01/5	01/5	01/5	01/5
17	01/5	01/5	01/5	01/5
18	01/5	01/5	01/5	01/5
19	01/5	01/5	01/5	01/5
20	01/5	01/5	01/5	01/5
21	01/5	01/5	01/5	01/5
22	01/5	01/5	01/5	01/5
23	01/5	01/5	01/5	01/5
24	01/5	01/5	01/5	01/5
25	01/5	01/5	01/5	01/5
26	01/5	01/5	01/5	01/5
27	01/5	01/5	01/5	01/5
28	01/5	01/5	01/5	01/5

Recovery (R) Contd.

Day of observation	Control Recovery (G1R)		High dose Recovery (G4R)
	M	F	M	F
29	01/5	01/5	01/5	01/5
30	01/5	01/5	01/5	01/5
31	01/5	01/5	01/5	01/5
32	01/5	01/5	01/5	01/5
33	01/5	01/5	01/5	01/5
34	01/5	01/5	01/5	01/5
35	01/5	01/5	01/5	01/5
36	01/5	01/5	01/5	01/5
37	01/5	01/5	01/5	01/5
38	01/5	01/5	01/5	01/5
39	01/5	01/5	01/5	01/5
40	01/5	01/5	01/5	01/5
41	01/5	01/5	01/5	01/5
42	01/5	01/5	01/5	01/5

Key: M – Male; F – Female; Observation/No. of animals; 01 – Normal.

 

Table for Mortality

TREATMENT

Days of observation	G1		G2		G3		G4
	M	F	M	F	M	F	M	F
1	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
2	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
3	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
4	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
5	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
6	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
7	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
8	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
9	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
10	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
11	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
12	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
13	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
14	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
15	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
16	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
17	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
18	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
19	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
20	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
21	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
22	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
23	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
24	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
25	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
26	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
27	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
28	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5

Key: M – Male; F – Female; No. of animals found dead/Total no. of animals.

Recovery (R)

Days of observation	G1R		G4R
	M	F	M	F
29	0/5	0/5	0/5	0/5
30	0/5	0/5	0/5	0/5
31	0/5	0/5	0/5	0/5
32	0/5	0/5	0/5	0/5
33	0/5	0/5	0/5	0/5
34	0/5	0/5	0/5	0/5
35	0/5	0/5	0/5	0/5
36	0/5	0/5	0/5	0/5
37	0/5	0/5	0/5	0/5
38	0/5	0/5	0/5	0/5
39	0/5	0/5	0/5	0/5
40	0/5	0/5	0/5	0/5
41	0/5	0/5	0/5	0/5
42	0/5	0/5	0/5	0/5

Key: M – Male; F – Female; No. of animals found dead/Total no. of animals.

 

Table for Body Weights (G) (Males)

TREATMENT

		Group 1	Group 2	Group 3	Group 4
Day 1	Mean	151.8	151.3	151.5	151.6
	SD	2.05	1.55	1.67	1.76
	Min	148.4	149.3	149.2	149.3
	Max	153.8	153.2	153.2	153.8
	N	5	5	5	5
Day 8	Mean	166.1	163.9	164.8	164.9
	SD	1.85	1.88	1.87	1.43
	Min	163.7	161.2	162.4	163.2
	Max	168.7	166.4	166.8	166.8
	N	5	5	5	5
Day 15	Mean	177.8	178.1	178.6	179.4
	SD	1.69	1.90	0.80	1.00
	Min	175.8	175.4	177.4	178.1
	Max	179.8	180.3	179.4	180.7
	N	5	5	5	5

Day 22	Mean	190.3	192.9	192.4	193.8
	SD	1.95	2.19	1.16	0.65
	Min	187.4	190.2	191.2	193.1
	Max	192.5	195.5	193.7	194.7
	N	5	5	5	5
Day 28	Mean	203.2	208.1	207.5	207.7
	SD	1.98	2.20	1.26	0.82
	Min	200.4	205.4	206.3	206.9
	Max	205.5	210.3	209.2	208.7
	N	5	5	5	5

Table for Body Weights (G) (Females)

TREATMENT

		Group 1	Group 2	Group 3	Group 4
Day 1	Mean	151.5	151.4	151.7	151.6
	SD	2.87	3.18	3.40	4.05
	Min	147.9	147.1	148.4	148.5
	Max	155.7	155.4	156.9	158.6
	N	5	5	5	5
Day 8	Mean	164.4	164.2	164.2	165.1
	SD	2.97	2.29	2.29	3.80
	Min	160.3	161.7	161.7	161.2
	Max	168.3	167.3	167.3	171.3
	N	5	5	5	5
Day 15	Mean	176.3	178.2	180.3	180.2
	SD	3.11	2.01	3.46	3.82
	Min	172.9	175.2	177.2	176.5
	Max	180.5	180.5	185.3	186.5
	N	5	5	5	5

Day 22	Mean	192.7	193.6	194.6	194.5
	SD	1.60	1.75	4.15	4.37
	Min	190.9	191.4	191.2	190.3
	Max	194.8	195.9	200.3	201.6
	N	5	5	5	5
Day 28	Mean	206.8	208.4	209.7	208.6
	SD	2.05	1.87	3.13	4.25
	Min	204.1	206.2	207.1	204.3
	Max	208.9	211.1	214.5	215.3
	N	5	5	5	5

Table for Body Weights (G) (Males)

RECOVERY

		Group 1R	Group 4R
Day 1	Mean	151.2	151.6
	SD	1.42	1.65
	Min	149.2	149.2
	Max	152.5	153.7
	N	5	5
Day 8	Mean	164.9	164.7
	SD	1.19	1.79
	Min	163.2	163.2
	Max	166.3	167.4
	N	5	5
Day 15	Mean	179.3	180.2
	SD	1.49	2.12
	Min	177.5	178.1
	Max	181.4	183.3
	N	5	5

Day 22	Mean	193.5	193.6
	SD	1.75	2.04
	Min	191.3	191.4
	Max	195.6	196.2
	N	5	5
Day 29	Mean	207.5	207.9
	SD	1.71	1.94
	Min	205.5	205.2
	Max	209.9	210.3
	N	5	5
Day 36	Mean	221.5	222.1
	SD	1.70	2.65
	Min	219.3	219.3
	Max	223.4	225.5
	N	5	5
Day 42	Mean	235.6	235.6
	SD	1.73	2.48
	Min	233.1	232.6
	Max	237.5	239.3
	N	5	5

 

Table for Body Weights (G) (Females)

RECOVERY

		Group 1R	Group 4R
Day 1	Mean	151.6	151.3
	SD	1.78	2.54
	Min	149.6	149.5
	Max	154.2	155.4
	N	5	5
Day 8	Mean	164.4	165.2
	SD	1.87	3.02
	Min	162.3	162.6
	Max	167.1	169.7
	N	5	5
Day 15	Mean	178.8	179.5
	SD	2.07	3.18
	Min	176.2	176.2
	Max	181.9	184.3
	N	5	5

		Group 1R	Group 4R
Day 22	Mean	194.1	193.7
	SD	1.60	3.35
	Min	192.7	191.2
	Max	196.7	199.3
	N	5	5
Day 29	Mean	208.8	207.5
	SD	1.85	3.58
	Min	207.3	204.3
	Max	211.8	213.4
	N	5	5
Day 36	Mean	223.7	222.2
	SD	1.46	3.84
	Min	221.9	218.6
	Max	225.6	228.2
	N	5	5
Day 42	Mean	236.5	235.9
	SD	1.97	4.89
	Min	234.1	231.2
	Max	238.6	243.2
	N	5	5

 

BODY WEIGHT GAIN (%) (MALES)

TREATMENT

		Group 1	Group 2	Group 3	Group 4
Day 1	Mean	0.0	0.0	0.0	0.0
	SD	0.00	0.00	0.00	0.00
	Min	0.0	0.0	0.0	0.0
	Max	0.0	0.0	0.0	0.0
	N	5	5	5	5
Day 8	Mean	9.4	8.4	8.8	8.8
	SD	0.58	1.78	0.45	1.10
	Min	8.9	5.2	8.2	6.8
	Max	10.3	9.6	9.2	9.4
	N	5	5	5	5
Day 15	Mean	17.1	17.7	17.9	18.4
	SD	0.97	1.81	0.91	1.29
	Min	15.8	14.5	16.8	16.3
	Max	18.5	18.8	18.9	19.5
	N	5	5	5	5

Day 22	Mean	25.3	27.5	27.1	27.9
	SD	1.08	1.88	1.28	1.48
	Min	23.6	24.2	25.2	25.8
	Max	26.3	28.6	28.2	29.5
	N	5	5	5	5
Day 28	Mean	33.9	37.5	37.0	37.1
	SD	1.02	1.95	1.30	1.59
	Min	32.5	34.1	35.0	34.9
	Max	35.0	38.8	38.3	38.7
	N	5	5	5	5

 

BODY WEIGHT GAIN (%) (FEMALES)

TREATMENT

		Group 1	Group 2	Group 3	Group 4
Day 1	Mean	0.0	0.0	0.0	0.0
	SD	0.00	0.00	0.00	0.00
	Min	0.0	0.0	0.0	0.0
	Max	0.0	0.0	0.0	0.0
	N	5	5	5	5
Day 8	Mean	8.5	8.4	9.1	8.9
	SD	0.28	1.08	0.50	0.60
	Min	8.1	7.7	8.4	8.0
	Max	8.8	10.3	9.8	9.4
	N	5	5	5	5
Day 15	Mean	16.3	17.7	18.8	18.9
	SD	0.65	1.74	0.54	0.78
	Min	15.6	16.2	18.1	17.6
	Max	17.1	20.7	19.4	19.5
	N	5	5	5	5

Day 22	Mean	27.2	27.9	28.3	28.3
	SD	2.28	1.86	0.78	1.00
	Min	24.2	26.1	27.2	27.1
	Max	29.3	31.0	29.0	29.4
	N	5	5	5	5
Day 28	Mean	36.5	37.7	38.2	37.6
	SD	2.51	1.95	1.23	1.29
	Min	34.0	35.8	36.7	35.8
	Max	39.1	40.9	39.6	38.9
	N	5	5	5	5

 

BODY WEIGHT GAIN (%) (MALES)

RECOVERY

		Group 1R	Group 4R
Day 1	Mean	0.0	0.0
	SD	0.00	0.00
	Min	0.0	0.0
	Max	0.0	0.0
	N	5	5
Day 8	Mean	9.1	8.6
	SD	0.39	0.57
	Min	8.5	7.9
	Max	9.4	9.4
	N	5	5
Day 15	Mean	18.6	18.9
	SD	0.74	0.52
	Min	17.8	18.1
	Max	19.5	19.4
	N	5	5

Day 22	Mean	28.0	27.7
	SD	0.46	0.85
	Min	27.3	26.5
	Max	28.5	28.8
	N	5	5
Day 29	Mean	37.3	37.1
	SD	0.75	1.19
	Min	36.4	35.6
	Max	38.1	38.9
	N	5	5
Day 36	Mean	46.5	46.5
	SD	0.89	1.35
	Min	45.6	44.9
	Max	47.7	48.3
	N	5	5
Day 42	Mean	55.9	55.4
	SD	0.90	1.43
	Min	55.0	53.7
	Max	57.2	57.6
	N	5	5

 

BODY WEIGHT GAIN (%) (FEMALES)

RECOVERY

		Group 1R	Group 4R
Day 1	Mean	0.0	0.0
	SD	0.00	0.00
	Min	0.0	0.0
	Max	0.0	0.0
	N	5	5
Day 8	Mean	8.4	9.1
	SD	0.44	0.30
	Min	7.8	8.8
	Max	9.0	9.6
	N	5	5
Day 15	Mean	18.0	18.6
	SD	0.43	1.13
	Min	17.3	17.9
	Max	18.4	20.6
	N	5	5

Day 22	Mean	28.0	28.0
	SD	0.63	0.47
	Min	27.4	27.5
	Max	28.8	28.6
	N	5	5
Day 29	Mean	37.7	37.1
	SD	0.76	0.65
	Min	36.8	36.3
	Max	38.6	38.0
	N	5	5
Day 36	Mean	47.6	46.8
	SD	1.47	0.66
	Min	46.3	45.8
	Max	50.1	47.7
	N	5	5
Day 42	Mean	56.0	55.9
	SD	2.24	0.98
	Min	53.7	54.2
	Max	59.4	56.7
	N	5	5

 

FEED CONSUMPTION (G/ANIMAL/DAY) (MALES)

TREATMENT

		Group 1	Group 2	Group 3	Group 4
Week 1	Mean	16.25	16.47	16.49	16.30
	SD	2.76	2.98	2.63	2.73
Week 2	Mean	16.25	16.47	16.49	16.30
	SD	2.76	2.98	2.63	2.73
Week 3	Mean	16.25	16.47	16.49	16.30
	SD	2.76	2.98	2.63	2.73
Week 4	Mean	16.25	16.47	16.49	16.30
	SD	2.76	2.98	2.63	2.73

 

FEED CONSUMPTION (G/ANIMAL/DAY) (MALES)

Recovery (R)

		Group 1R	Group 4R
Week 1	Mean	16.43	16.31
	SD	2.34	2.32
Week 2	Mean	16.43	16.31
	SD	2.34	2.32
Week 3	Mean	16.43	16.31
	SD	2.34	2.32
Week 4	Mean	16.43	16.31
	SD	2.34	2.32
Week 5	Mean	16.43	16.31
	SD	2.34	2.32
Week 6	Mean	16.43	16.31
	SD	2.34	2.32

 

FEED CONSUMPTION (G/ANIMAL/DAY) (FEMALES)

TREATMENT

		Group 1	Group 2	Group 3	Group 4
Week 1	Mean	16.58	16.22	16.21	16.27
	SD	3.05	2.93	2.80	2.89
Week 2	Mean	16.58	16.22	16.21	16.27
	SD	3.05	2.93	2.80	2.89
Week 3	Mean	16.58	16.22	16.21	16.27
	SD	3.05	2.93	2.80	2.89
Week 4	Mean	16.58	16.22	16.21	16.27
	SD	3.05	2.93	2.80	2.89

 

FEED CONSUMPTION (G/ANIMAL/DAY) (FEMALES)

RECOVERY (R)

		Group 1R	Group 4R
Week 1	Mean	16.25	16.30
	SD	2.76	2.57
Week 2	Mean	16.25	16.30
	SD	2.76	2.57
Week 3	Mean	16.25	16.30
	SD	2.76	2.57
Week 4	Mean	16.25	16.30
	SD	2.76	2.57
Week 5	Mean	16.25	16.30
	SD	2.76	2.57
Week 6	Mean	16.25	16.30
	SD	2.76	2.57

 

DETAILED CLINICAL EXAMINATIONS

Group CONTROL(G1)

S.NO	Parameter	Pre Dose	Day 8	Day 15	Day 22	Day 28
1	Body Posture	1(10)	1(10)	1(10)	1(10)	1(10)
2	Gait	1(10)	1(10)	1(10)	1(10)	1(10)
3	Skin	1(10)	1(10)	1(10)	1(10)	1(10)
4	Fur	1(10)	1(10)	1(10)	1(10)	1(10)
5	Eyes	1(10)	1(10)	1(10)	1(10)	1(10)
6	Mucous membrane	1(10)	1(10)	1(10)	1(10)	1(10)
7	Respiratory pattern	1(10)	1(10)	1(10)	1(10)	1(10)
8	Salivation	1(10)	1(10)	1(10)	1(10)	1(10)
9	Response to handling	1(10)	1(10)	1(10)	1(10)	1(10)
10	Convulsions	1(10)	1(10)	1(10)	1(10)	1(10)
11	Stereotypic behaviour	1(10)	1(10)	1(10)	1(10)	1(10)
12	Bizzare Behaviour	1(10)	1(10)	1(10)	1(10)	1(10)

Key: 1(10) – Observation(No of animals), 1-Normal

 

Group Low Dose(G2)

S.NO	Parameter	Pre Dose	Day 8	Day 15	Day 22	Day 28
1	Body Posture	1(10)	1(10)	1(10)	1(10)	1(10)
2	Gait	1(10)	1(10)	1(10)	1(10)	1(10)
3	Skin	1(10)	1(10)	1(10)	1(10)	1(10)
4	Fur	1(10)	1(10)	1(10)	1(10)	1(10)
5	Eyes	1(10)	1(10)	1(10)	1(10)	1(10)
6	Mucous membrane	1(10)	1(10)	1(10)	1(10)	1(10)
7	Respiratory pattern	1(10)	1(10)	1(10)	1(10)	1(10)
8	Salivation	1(10)	1(10)	1(10)	1(10)	1(10)
9	Response to handling	1(10)	1(10)	1(10)	1(10)	1(10)
10	Convulsions	1(10)	1(10)	1(10)	1(10)	1(10)
11	Stereotypic behaviour	1(10)	1(10)	1(10)	1(10)	1(10)
12	Bizzare Behaviour	1(10)	1(10)	1(10)	1(10)	1(10)

Key: 1(10) – Observation(No of animals), 1-Normal

 

Group Intermediate Dose(G3)

S.NO	Parameter	Pre Dose	Day 8	Day 15	Day 22	Day 28
1	Body Posture	1(10)	1(10)	1(10)	1(10)	1(10)
2	Gait	1(10)	1(10)	1(10)	1(10)	1(10)
3	Skin	1(10)	1(10)	1(10)	1(10)	1(10)
4	Fur	1(10)	1(10)	1(10)	1(10)	1(10)
5	Eyes	1(10)	1(10)	1(10)	1(10)	1(10)
6	Mucous membrane	1(10)	1(10)	1(10)	1(10)	1(10)
7	Respiratory pattern	1(10)	1(10)	1(10)	1(10)	1(10)
8	Salivation	1(10)	1(10)	1(10)	1(10)	1(10)
9	Response to handling	1(10)	1(10)	1(10)	1(10)	1(10)
10	Convulsions	1(10)	1(10)	1(10)	1(10)	1(10)
11	Stereotypic behaviour	1(10)	1(10)	1(10)	1(10)	1(10)
12	Bizzare Behaviour	1(10)	1(10)	1(10)	1(10)	1(10)

Key: 1(10) – Observation(No of animals), 1-Normal

 

Group High Dose(G4)

S.NO	Parameter	Pre Dose	Day 8	Day 15	Day 22	Day 28
1	Body Posture	1(10)	1(10)	1(10)	1(10)	1(10)
2	Gait	1(10)	1(10)	1(10)	1(10)	1(10)
3	Skin	1(10)	1(10)	1(10)	1(10)	1(10)
4	Fur	1(10)	1(10)	1(10)	1(10)	1(10)
5	Eyes	1(10)	1(10)	1(10)	1(10)	1(10)
6	Mucous membrane	1(10)	1(10)	1(10)	1(10)	1(10)
7	Respiratory pattern	1(10)	1(10)	1(10)	1(10)	1(10)
8	Salivation	1(10)	1(10)	1(10)	1(10)	1(10)
9	Response to handling	1(10)	1(10)	1(10)	1(10)	1(10)
10	Convulsions	1(10)	1(10)	1(10)	1(10)	1(10)
11	Stereotypic behaviour	1(10)	1(10)	1(10)	1(10)	1(10)
12	Bizzare Behaviour	1(10)	1(10)	1(10)	1(10)	1(10)

Key: 1(10) – Observation(No of animals), 1-Normal

 

Group Vehicle Control Recovery(G1R)

S.NO	Parameter	Pre Dose	Day 8	Day 15	Day 22	Day 28	Day 36	Day 42
1	Body Posture	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
2	Gait	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
3	Skin	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
4	Fur	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
5	Eyes	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
6	Mucous membrane	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
7	Respiratory pattern	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
8	Salivation	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
9	Response to handling	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
10	Convulsions	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
11	Stereotypic behaviour	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
12	Bizzare Behaviour	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)

Key: 1(10) – Observation(No of animals), 1-Normal

 

Group High Dose Recovery(G4R)

S.NO	Parameter	Pre Dose	Day 8	Day 15	Day 22	Day 28	Day 36	Day 42
1	Body Posture	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
2	Gait	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
3	Skin	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
4	Fur	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
5	Eyes	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
6	Mucous membrane	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
7	Respiratory pattern	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
8	Salivation	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
9	Response to handling	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
10	Convulsions	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
11	Stereotypic behaviour	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)
12	Bizzare Behaviour	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)	1(10)

Key: 1(10) – Observation(No of animals), 1-Normal

 

HEMATOLOGY – MALES

Week -04

		RBC	Hb	PCV	MCV	MCH	MCHC	Reticulocyte
		x10^6Cells/µL	g/dL	%	fL	pg	g/dL	%
Group 1	Mean	9.21	16.28	52.30	57.54	17.98	31.26	1.87
	SD	2.493	3.269	11.744	4.510	1.692	0.750	0.111
Group 2	Mean	8.75	15.46	49.30	56.66	17.78	31.36	1.87
	SD	0.759	0.456	1.754	4.697	1.632	0.462	0.041
Group 3	Mean	8.46	14.10	45.54	54.40	16.88	30.98	1.85
	SD	1.134	0.875	3.035	6.120	2.202	0.701	0.069
Group 4	Mean	8.19	14.74	46.80	57.30	18.04	31.50	1.84
	SD	0.624	0.503	1.726	2.664	0.783	0.374	0.045

		PLT	WBC	DC (%)					PT	APTT
		X10^3cells/µl	X10^3cells/ µl	Neut	Lymph	Mono	Eos	Baso	Sec	Sec
Group 1	Mean	622.80	17.54	20.60	74.60	3.20	1.60	0.00	16.20	42.20
	SD	97.983	3.211	13.428	14.346	0.447	0.894	0.000	1.095	2.950
Group 2	Mean	911.60!	18.44	10.20	86.00	2.40	1.40	0.00	16.40	44.20
	SD	154.936	5.244	2.168	2.345	0.548	0.548	0.000	1.140	2.950
Group 3	Mean	930.40*	23.54	13.20	82.00	3.00	1.80	0.00	17.60	43.20
	SD	95.340	7.606	3.271	3.674	0.707	0.447	0.000	1.140	3.421
Group 4	Mean	947.40#	25.76	12.40	83.00	2.80	1.80	0.00	17.40	41.40
	SD	126.211	6.158	3.647	3.674	0.447	0.447	0.000	1.140	3.209

! Significant at p ≥ 0.05 level with group 1, * Significant at p ≥ 0.05 level with group 1, # Significant at p ≥ 0.05 level with group 1.

 

HEMATOLOGY – MALES

Week -06

		RBC	Hb	PCV	MCV	MCH	MCHC	Reticulocyte
		x10^6Cells/µL	g/dL	%	fL	pg	g/dL	%
Group 1R	Mean	8.90	14.92	47.74	53.76	16.78	31.24	1.84
	SD	0.528	0.646	2.056	3.704	1.121	0.261	0.073
Group 4R	Mean	9.48	16.52	53.84	57.02	17.50	30.68	1.84
	SD	1.336	1.925	6.953	3.666	1.382	0.705	0.072

 

		PLT	WBC	DC (%)					PT	APTT
		x10^3cells/µL	x10^3cells/ µL	Neut	Lymph	Mono	Eos	Baso	Sec	Sec
Group 1R	Mean	811.40	18.46	14.00	81.20	3.20	1.60	0.00	16.00	41.80
	SD	106.129	3.129	5.612	7.596	1.304	1.140	0.000	1.225	2.683
Group 4R	Mean	696.80	22.00	23.40	71.00	4.00	1.60	0.00	16.40	41.20
	SD	165.194	15.086	14.893	17.263	1.581	1.140	0.000	1.140	2.387

 

HEMATOLOGY – FEMALES

Week -04

		RBC	Hb	PCV	MCV	MCH	MCHC	Reticulocyte
		x10^6Cells/µL	g/dL	%	fL	pg	g/dL	%
Group 1	Mean	7.71	14.16	43.32	56.60	18.54	32.72	1.85
	SD	0.985	0.792	2.729	4.243	1.598	0.370	0.080
Group 2	Mean	8.11	13.86	42.74	52.66	17.08	32.42	1.86
	SD	0.656	1.307	3.960	1.352	0.676	0.653	0.076
Group 3	Mean	8.26	14.44	45.32	55.08	17.58	31.90	1.89
	SD	0.780	0.336	1.932	3.241	1.438	0.682	0.046
Group 4	Mean	7.44	14.92	44.22	59.94	20.18	33.66	1.86
	SD	0.802	0.814	2.758	4.620	1.760	0.994	0.068

 

		PLT	WBC	DC (%)					PT	APTT
		X10^3cells/µl	X10^3cells/ µl	Neut	Lymph	Mono	Eos	Baso	Sec	Sec
Group 1	Mean	787.60	21.58	11.20	84.00	3.40	1.40	0.00	16.40	40.80
	SD	186.871	4.871	2.280	2.236	0.894	0.548	0.000	1.140	1.789
Group 2	Mean	921.60	22.86	13.80	81.80	2.60	1.80	0.00	15.60	42.20
	SD	29.246	7.730	3.421	3.768	0.548	0.447	0.000	1.140	2.168
Group 3	Mean	933.80	19.36	15.20	79.60	3.60	1.60	0.00	16.60	42.80
	SD	111.237	5.647	5.891	7.503	0.894	0.894	0.000	1.342	2.864
Group 4	Mean	962.80	16.34	10.40	85.40	3.00	1.20	0.00	16.60	40.40
	SD	119.242	5.671	2.608	2.510	0.000	0.447	0.000	1.673	2.881

 

HEMATOLOGY – FEMALES

Week -06

		RBC	Hb	PCV	MCV	MCH	MCHC	Reticulocytes
		x10^6Cells/µL	g/dL	%	fL	pg	g/dL	%
Group 1R	Mean	8.31	14.48	44.52	53.62	17.44	32.56	1.90
	SD	0.350	0.460	1.711	0.887	0.344	0.329	0.091
Group 4R	Mean	7.90	13.12	43.60	55.50	16.74	30.06	1.89
	SD	0.868	1.796	3.055	4.435	2.745	3.479	0.044

 

		PLT	WBC	DC (%)					PT	APTT
		x10^3cells/µL	x10^3cells/ µL	Neut	Lymph	Mono	Eos	Baso	Sec	Sec
Group 1R	Mean	866.40	14.92	12.80	81.80	3.60	1.80	0.00	15.60	43.60
	SD	93.055	3.104	3.768	3.347	0.894	0.447	0.000	1.140	3.435
Group 4R	Mean	758.80	17.36	13.00	83.60	2.40#	1.00	0.00	16.40	41.80
	SD	266.210	6.579	1.581	2.302	0.548	0.707	0.000	1.140	3.347

#Significant at p ≤ 0.05 level with group 1

 

CLINICAL BIOCHEMISTRY – MALES

WEEK -04

		GLU	Urea	CREA	BLI	ALT	AST	ALP	TPO
		mmol /L	mmol /L	µmol /L	µmol /L	U/L	U/L	U/L	g/L
Group 1	Mean	99.00	54.20	0.90	0.13	73.80	168.80	232.00	7.04
	SD	16.90	16.18	0.34	0.02	45.16	48.40	52.53	0.33
Group 2	Mean	100.40	29.20!	0.72	0.09!	65.80	151.60	272.80	7.26
	SD	18.34	5.54	0.04	0.01	10.08	8.56	69.55	0.24
Group 3	Mean	73.60	41.60	0.70	0.10*	59.60	167.40	377.20*	7.48
	SD	15.95	5.18	0.07	0.01	8.96	26.94	88.09	0.47
Group 4	Mean	92.20	42.00	0.74	0.12	59.60	146.80	289.80	7.42
	SD	21.72	3.81	0.05	0.02	6.07	23.45	105.77	0.24

^! Significant at p ≤ 0.05 level with group 1, ! Significant at p ≤ 0.05 level with group 1, * Significant at p ≤ 0.05 level with group 1

* Significant at p ≥ 0.05 level with group 1.

		ALB	GLB	CHOL	TRIGL	Na	K	Cl	GGT	A/G
		g/L	g/L	mmol /L	mmol /L	mmol /L	mmol /L	mmol /L	U/L	_
Group 1	Mean	2.26	4.76	62.40	66.20	142.86	4.01	102.58	1.92	0.47
	SD	0.35	0.29	42.91	23.92	5.37	0.28	5.00	1.34	0.08
Group 2	Mean	2.41	4.86	46.00	88.40	138.54	4.82!	98.62	1.34	0.49
	SD	0.18	0.21	9.67	32.25	0.90	0.28	0.63	0.96	0.05
Group 3	Mean	2.17	5.32	51.00	97.60	139.34	4.87*	99.34	3.38	0.41
	SD	0.30	0.70	6.60	16.09	1.15	0.42	2.12	1.24	0.11
Group 4	Mean	2.34	5.10	47.20	91.20	139.24	4.40	99.48	1.82	0.45
	SD	0.10	0.20	9.86	11.90	0.36	0.15	2.24	1.01	0.03

! Significant at p ≥ 0.05 level with group 1, * Significant at p ≥ 0.05 level with group 1.

 

CLINICAL BIOCHEMISTRY – MALES - RECOVERY

WEEK -06

		GLU	Urea	CREA	BLI	ALT	AST	ALP	TPO
		mmol /L	mmol /L	µmol /L	µmol /L	U/L	U/L	U/L	g/L
Group 1R	Mean	91.60	45.20	0.74	0.10	60.00	161.80	363.60	7.76
	SD	23.44	5.40	0.05	0.03	4.00	27.87	98.28	0.57
Group 4R	Mean	92.80	41.00	1.32	0.12	75.80	165.40	281.20	8.52
	SD	15.91	7.04	0.86	0.07	36.48	37.27	193.31	1.76

 

		ALB	GLB	CHOL	TRIGL	Na	K	Cl	GGT	A/G
		g/L	g/L	mmol /L	mmol /L	mmol /L	mmol /L	mmol /L	U/L	_
Group 1R	Mean	2.27	5.48	36.02	86.20	137.58	4.52	98.60	1.28	0.42
	SD	0.13	0.66	17.97	25.72	1.83	0.21	1.86	0.34	0.07
Group 4R	Mean	2.31	6.20	44.60	89.60	146.90	4.87	106.22	31.94	0.42
	SD	0.30	2.05	17.08	41.84	9.21	0.40	9.02	36.68	0.15

 

CLINICAL BIOCHEMISTRY – FEMALES

WEEK -04

		GLU	Urea	CREA	BLI	ALT	AST	ALP	TPO
		mmol /L	mmol /L	µmol /L	µmol /L	U/L	U/L	U/L	g/L
Group 1	Mean	89.80	40.40	0.70	0.09	59.80	141.80	246.40	21.68
	SD	14.08	4.77	0.00	0.05	15.88	11.95	60.95	32.04
Group 2	Mean	86.20	30.80	0.78	0.10	61.80	159.40	287.60	7.78
	SD	8.41	7.82	0.04	0.01	12.28	8.96	71.00	0.72
Group 3	Mean	75.00	47.40	0.78	0.09	53.40	137.80	305.60	7.70
	SD	11.22	7.54	0.08	0.02	12.12	28.50	134.46	0.57
Group 4	Mean	60.40#	39.40	0.72	0.12	55.20	151.00	204.00	7.54
	SD	8.17	5.50	0.04	0.05	6.38	15.36	71.40	0.47

# Significant at p ≤ 0.05 level with group 1

		ALB	GLB	CHOL	TRIGL	Na	K	Cl	GGT	A/G
		g/L	g/L	mmol /L	mmol /L	mmol /L	mmol /L	mmol /L	U/L	_
Group 1	Mean	2.37	5.06	58.40	122.60	137.94	4.33	98.62	1.98	0.46
	SD	0.12	0.28	8.56	49.27	0.90	0.43	2.27	1.51	0.03
Group 2	Mean	2.16	5.64	44.40!	49.00!	137.86	4.74	100.54	2.54	0.38
	SD	0.27	0.75	5.73	6.52	0.66	0.19	0.99	0.65	0.08
Group 3	Mean	2.31	4.70	56.60	92.80	139.06	4.71	100.24	14.36	0.43
	SD	0.28	1.91	11.33	28.97	0.64	0.31	1.45	27.42	0.10
Group 4	Mean	2.45	5.08	54.80	80.40	138.32	4.45	99.46	1.34	0.48
	SD	0.15	0.51	6.22	28.14	0.87	0.41	1.74	0.86	0.06

! Significant at p ≤ 0.05 level with group 1, ! Significant at p ≤ 0.05 level with group 1

 

CLINICAL BIOCHEMISTRY – FEMALES - RECOVERY

WEEK -06

		GLU	Urea	CREA	BLI	ALT	AST	ALP	TPO
		mmol /L	mmol /L	µmol /L	µmol /L	U/L	U/L	U/L	g/L
Group 1R	Mean	90.80	45.20	0.80	0.14	49.00	139.20	188.00	7.90
	SD	8.93	7.16	0.00	0.08	4.90	21.11	66.43	0.41
Group 4R	Mean	97.80	47.00	0.82	0.12	31.38	136.00	262.20	7.76
	SD	9.42	8.09	0.08	0.05	25.66	7.97	135.13	0.31

 

		ALB	GLB	CHOL	TRIGL	Na	K	Cl	GGT	A/G
		g/L	g/L	mmol /L	mmol /L	mmol /L	mmol /L	mmol /L	U/L	_
Group 1R	Mean	2.31	6.20	44.60	89.60	146.90	4.87	106.22	31.94	0.42
	SD	0.30	2.05	17.08	41.84	9.21	0.40	9.02	36.68	0.15
Group 4R	Mean	2.51	5.26	53.80	117.60#	139.98#	4.24	100.50	9.58	0.46
	SD	0.08	0.34	8.58	23.28	1.08	0.42	0.74	17.32	0.03

#Significant at p ≥ 0.05 level with group 1, # Significant at p ≥ 0.05 level with group 1

 

URINANALYSIS – MALES

TREATMENT (Week 4)

		pH	SPECIFIC GRAVITY	VOLUME
		(pH)	(SG)	mL
Group 1	Mean	9.00	1.006	11.40
	SD	0.00	0.01	1.95
Group 2	Mean	9.00	1.008	12.40
	SD	0.00	0.00	1.67
Group 3	Mean	8.80	1.010	10.40
	SD	0.45	0.01	1.67
Group 4	Mean	8.80	1.015	11.00
	SD	0.45	0.00	1.41
Group 1R	Mean	8.80	1.013	12.40
	SD	0.45	0.01	1.67
Group 4R	Mean	8.80	1.018	12.40
	SD	0.45	0.01	1.67

 

URINANALYSIS – FEMALES

TREATMENT (Week 4)

		pH	SPECIFIC GRAVITY	VOLUME
		(pH)	(SG)	mL
Group 1	Mean	8.80	1.011	12.40
	SD	0.45	0.01	1.67
Group 2	Mean	9.00	1.011	10.00
	SD	0.00	0.01	1.67
Group 3	Mean	8.80	1.013	10.80
	SD	0.45	0.01	2.28
Group 4	Mean	8.80	1.010	9.60
	SD	0.45	0.01	1.14
Group 1R	Mean	8.80	1.010	10.00
	SD	0.45	0.01	1.67
Group 4R	Mean	8.80	1.013	11.40
	SD	0.45	0.01	1.95

 

ORGAN WEIGHTS (GRAM) – MALES

		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight (G)	Mean	201.6	206.5	205.6	205.8	233.8	233.3
	SD	2.48	2.48	1.33	0.88	1.65	2.59
	N	5	5	5	5	5	5
Brain	Mean	1.7346	1.7108	1.6158	1.7310	1.6298	1.6892
	SD	0.10311	0.08949	0.13100	0.15775	0.15236	0.11628
	N	5	5	5	5	5	5
Heart	Mean	0.7662	0.9258	0.8216	1.0210#	0.9108	0.7960
	SD	0.16332	0.12457	0.07909	0.20868	0.16627	0.12221
	N	5	5	5	5	5	5
Liver	Mean	7.2060	8.3154	8.3516	8.8132	9.3868	8.5660
	SD	3.53997	2.41791	1.32709	1.22586	4.82931	3.92057
	N	5	5	5	5	5	5
Spleen	Mean	1.1056	0.7510	1.0822	1.2202	0.9134	0.8304
	SD	1.11615	0.42077	0.41242	0.35144	0.64380	0.59054
	N	5	5	5	5	5	5
Kidneys	Mean	1.5062	1.7180	1.5344	1.7140	1.6102	1.6560
	SD	0.32228	0.32495	0.14521	0.23357	0.44694	0.40197
	N	5	5	5	5	5	5

Adrenals	Mean	0.0558	0.0556	0.0550	0.0550	0.0534	0.0528
	SD	0.00691	0.00873	0.00919	0.00510	0.01656	0.01322
	N	5	5	5	5	5	5
Testes	Mean	2.1732	2.2650	2.3306	2.5016	2.4852	2.4964
	SD	0.20189	0.36690	0.13797	0.40516	0.56094	0.26159
	N	5	5	5	5	5	5
Epididymes	Mean	0.7882	0.9554	1.0426	1.1044	1.1042	1.0708
	SD	0.21886	0.40228	0.26590	0.41419	0.20895	0.05645
	N	5	5	5	5	5	5
Thymus	Mean	0.3498	0.3568	0.3510	0.4766	0.6034	0.2596
	SD	0.15497	0.12788	0.11518	0.05962	0.48764	0.09413
	N	5	5	5	5	5	5
Pro + Sem. Vesical with Coagulating glands	Mean	0.6402	0.7834	0.6666	1.0854	0.8446	0.9298
	SD	0.19241	0.26254	0.26304	0.66523	0.50951	0.24046
	N	5	5	5	5	5	5

# Significant at p ≥ 0.05 level with group 1

 

ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES

		Group 1	Group 2	Group 3	Group 4		Group 1R		Group 4R
Body Weight (G)	Mean	205.0	206.6	208.2	206.7		234.7		234.1
	SD	1.60	1.94	3.26	4.05		1.72		4.99
	N	5	5	5	5		5		5
Brain	Mean	1.6048	1.5174	1.4656	1.5704		1.6350		1.3958##
	SD	0.06488	0.07779	0.25213	0.10088		0.04643		0.21369
	N	5	5	5	5		5		5
Heart	Mean	0.6312	0.5936	0.6156	0.7820#		0.6228		0.6962
	SD	0.08837	0.05679	0.08325	0.08404		0.10838		0.14596
	N	5	5	5	5		5		5
Liver	Mean	6.0136	5.2958	5.2068	5.7436		5.5752		6.5608
	SD	1.01187	0.70639	0.74267	0.20056		0.70287		1.42551
	N	5	5	5	5		5		5
Spleen	Mean	0.7854	0.5778	0.5220	0.7640		0.4272		0.8990
	SD	0.49005	0.09975	0.12471	0.38037		0.08106		0.65253
	N	5	5	5	5		5		5
Kidneys	Mean	1.1310	1.1960	1.0722	1.1430	1.1392		1.1034
	SD	0.23607	0.23121	0.09131	0.09180	0.10037		0.15315
	N	5	5	5	5	5		5

		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Adrenals	Mean	0.0594	0.0700	0.0622	0.0638	0.0568	0.0586
	SD	0.00635	0.01235	0.01295	0.01439	0.00642	0.01544
	N	5	5	5	5	5	5
Ovaries	Mean	0.1120	0.1112	0.1040	0.0946	0.1098	0.1332
	SD	0.02102	0.00981	0.02233	0.01264	0.03997	0.00896
	N	5	5	5	5	5	5
Uterus	Mean	0.2886	0.3960	0.3238	0.3164	0.3154	0.3800
	SD	0.07681	0.09858	0.15767	0.08780	0.04489	0.11493
	N	5	5	5	5	5	5
Thymus	Mean	0.3396	0.2238	0.2766	0.3476	0.2258	0.3510
	SD	0.16634	0.06073	0.06431	0.03428	0.06864	0.11910
	N	5	5	5	5	5	5

# Significant at p ≥ 0.05 level with group 1, ##Significant at p≤0.05 level with group 1

 

ORGAN WEIGHTS RATIO (%) – MALES

		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight (G)	Mean	201.6	206.5	205.6	205.8	233.8	233.3
	SD	2.48	2.48	1.33	0.88	1.65	2.59
	N	5	5	5	5	5	5
Brain	Mean	0.8606	0.8286	0.7857	0.8410	0.6969	0.7241
	SD	0.05237	0.04426	0.06274	0.07721	0.0618	0.0525
	N	5	5	5	5	5	5
Heart	Mean	0.3798	0.4484	0.3994	0.4962	0.3896	0.3412
	SD	0.07968	0.06098	0.03717	0.10243	0.0709	0.0531
	N	5	5	5	5	5	5
Liver	Mean	3.5679	4.0309	4.0602	4.2812	4.0087	3.6717
	SD	1.74103	1.19784	0.63616	0.59293	2.0455	1.6904
	N	5	5	5	5	5	5
Spleen	Mean	0.5464	0.3635	0.5258	0.5925	0.3896	0.3555
	SD	0.55140	0.20211	0.19879	0.16868	0.2729	0.2533
	N	5	5	5	5	5	5

Kidneys	Mean	0.7469	0.8327	0.7461	0.8327	0.6884	0.7101
	SD	0.15903	0.16386	0.06912	0.11382	0.1893	0.1754
	N	5	5	5	5	5	5
Adrenals	Mean	0.0277	0.0269	0.0268	0.0267	0.0228	0.0226
	SD	0.00337	0.00419	0.00455	0.00240	0.0071	0.0056
	N	5	5	5	5	5	5
Testes	Mean	1.0777	1.0981	1.1336	1.2152	1.0626	1.0699
	SD	0.09411	0.18840	0.07181	0.19648	0.2373	0.1117
	N	5	5	5	5	5	5
Epididymes	Mean	0.3907	0.4641	0.5071	0.5364	0.4724	0.4590
	SD	0.10679	0.20087	0.12960	0.20100	0.0894	0.0267
	N	5	5	5	5	5	5
Thymus	Mean	0.1735	0.1729	0.1709	0.2316	0.2580	0.1113
	SD	0.07688	0.06313	0.05658	0.02946	0.2081	0.0404
	N	5	5	5	5	5	5
Pro + Sem. Vesical with Coagulating glands	Mean	0.3170	0.3784	0.3239	0.5275	0.3621	0.3981
	SD	0.09233	0.12309	0.12610	0.32423	0.2208	0.1008
	N	5	5	5	5	5	5

 

ORGAN WEIGHTS RATIO (%) – FEMALES

		Group 1	Group 2	Group 3	Group 4	Group 1R	Group 4R
Body Weight (G)	Mean	205.0	206.6	208.2	206.7	234.7	234.1
	SD	1.60	1.94	3.26	4.05	1.72	4.99
	N	5	5	5	5	5	5
Brain	Mean	0.7826	0.7344	0.7043	0.7603	0.6968	0.5953
	SD	0.02848	0.03509	0.12292	0.05660	0.0196	0.0827
	N	5	5	5	5	5	5
Heart	Mean	0.3078	0.2873	0.2954	0.3785	0.2653	0.2971
	SD	0.04322	0.02761	0.03646	0.04176	0.0451	0.0599
	N	5	5	5	5	5	5
Liver	Mean	2.9320	2.5637	2.5001	2.7790	2.3750	2.7960
	SD	0.48266	0.34348	0.34371	0.09393	0.2893	0.5609
	N	5	5	5	5	5	5
Spleen	Mean	0.3828	0.2799	0.2504	0.3721	0.1821	0.3811
	SD	0.23753	0.04973	0.05710	0.19166	0.0346	0.2708
	N	5	5	5	5	5	5
Kidneys	Mean	0.5512	0.5788	0.5152	0.5535	0.4856	0.4707
	SD	0.11164	0.11155	0.04645	0.05117	0.0441	0.0578
	N	5	5	5	5	5	5

Adrenals	Mean	0.0290	0.0339	0.0298	0.0308	0.0242	0.0250
	SD	0.00321	0.00617	0.00607	0.00650	0.0026	0.0064
	N	5	5	5	5	5	5
ovaries	Mean	0.0546	0.0538	0.0499	0.0458	0.0468	0.0569
	SD	0.01026	0.00468	0.00997	0.00672	0.0171	0.0043
	N	5	5	5	5	5	5
Uterus	Mean	0.1408	0.1917	0.1555	0.1527	0.1343	0.1625
	SD	0.03780	0.04768	0.07629	0.04020	0.0184	0.0496
	N	5	5	5	5	5	5
Thymus	Mean	0.1660	0.1084	0.1326	0.1685	0.0963	0.1493
	SD	0.08221	0.02991	0.02901	0.01953	0.0296	0.0479
	N	5	5	5	5	5	5

 

GROSS PATHOLOGY

Male

Gross observation	Group
	control (G1)		Low dose (G2)	Intermediate dose (G3)	High dose (G4)
External
NAD	5/5		5/5	5/5	5/5
Internal
NAD		5/5	5/5	5/5	5/5

 

Female

Gross observation	Group
	control (G1)	Low dose (G2)	Intermediate dose (G3)	High dose (G4)
External
NAD	5/5	5/5	5/5	5/5
Internal
NAD	5/5	5/5	5/5	5/5

Key: M – Male; F – Female; No. of animals showing observation/Total no. of animals

 

HISTOPATHOLOGY

ORGAN	OBSERVATIONS	Control (G1)		High dose (G4)
		M	F	M	F
Liver	Sinusoidal Haemorrhage	2/5	0/5	1/5	1/5
	Foci of centrilobular/ Periportalnecrosis//Inflammation	3/5	1/5	3/5	2/5
	No Abnormality Detected (NAD)	1/5	4/5	2/5	2/5
Lungs	Thickening of Alveolar wall/Alveolar Inflammation	4/5	3/5	2/2	2/2
	No Abnormality Detected (NAD)	1/5	2/5	3/5	3/5
Spleen	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Heart	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Aorta	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Kidney	Tubular Haemorrhage	1/5	0/5	0/5	0/5
	Tubular /interstitial inflammation	1/5	1/5	1/5	1/5
	No Abnormality Detected (NAD)	3/5	4/5	4/5	4/5
Adrenals	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Brain	Foci of Cerebral Necrosis	1/5	1/5	1/5	0/5
	No Abnormality Detected (NAD)	4/5	4/5	4/5	5/5
Pituitary	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Thyroid and Para thyroid	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Trachea	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Oesophagus	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Jejunum	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Duodenum	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5

Ileum	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Colon	Sub mucosal lymphoid tissue hyperplasia	2/5	0/5	3/5	0/5
	No Abnormality Detected (NAD)	3/5	5/5	2/5	5/5
Rectum	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Caecum	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Lymph nodes	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Pancreas	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Thymus	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Urinary bladder	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Testes	No Abnormality Detected (NAD)	5/5	-	5/5	-
Epididymis	No Abnormality Detected (NAD)	5/5	-	5/5	-
Prost+Seminal Vesicles+coagula Ting glands	No Abnormality Detected (NAD)	5/5	-	5/5	-
Ovary	No Abnormality Detected (NAD)	-	5/5	-	5/5
Uterus	No Abnormality Detected (NAD)	-	5/5	-	5/5
Cervix+Vagina	No Abnormality Detected (NAD)	-	5/5	-	5/5
Stomach	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Skeletal muscle	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Sciatic nerve	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Eye	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Spinal cord	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5
Sternum	No Abnormality Detected (NAD)	5/5	5/5	5/5	5/5

Key: M – Male; F – Female; No. of animals showing observation/Total no. of animals.

Applicant's summary and conclusion

Conclusions:

Based on all the available data, it was concluded that after repeated exposures of the test chemical via oral route, it caused adverse effects on hepatobiliary system. Therefore, the NOAEL and LOAEL for the test chemical was 60 and 120 mg/kg bw/day, respectively after 28 days of exposure.

Executive summary:

A study according to the OECD Test Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents), performed using male and female Wistar Rats. The test chemical was administered via oral: gavage route. 5 animals per sex dose were used in the study. 6 groups were used in the study. The test chemical was administered in a dose range of 0, 30, 60 and 120 mg/kg bw/day as a control group, low dose, mid dose and high dose groups, respectively. Two recovery groups (0 mg/kg bw/day-Control Recovery and 1000 mg/kg bw/day-High Dose Recovery Group) were used in the study to observe the reversibility of the effects that would be observed in the main groups, if caused due to the test chemical. Distilled water was used as a vehicle. The test chemical was administered to the animals, once daily. The animals were dosed for 28 days, consecutively. The doses were selected on the basis of a Dose Range Finding (DRF) Study. In this study, the doses for the DRF study were 0, 50, 100 and 200 mg/kg bw/day. During the treatment 03 Males and 04 Females showed Mortality in High dose (200 mg/kg Body weight). There was no mortality in Control, Low Dose and Mid Dose. Piloerection and dullness were observed in High dose group. Hence, based on the Dose Range Finding Study, the doses of 30, 60 and 120 mg/kg body weight were confirmed for main study. As per the requirement according to OECD test guideline 407, animals was observed twice daily for morbidity and mortality while for Clinical Signs, Once daily during acclimatization phase and twice daily on first 3 days of treatment; once daily thereafter. During Recovery period, animals were observed once daily. Detailed clinical observations were done, once before the first exposure and weekly after. Body weights were examined once during Acclimatization and before randomization and Weekly Once during Treatment Period and Recovery Period. Also, feed consumption was observed Once weekly during treatment and recovery period. Opthalmoscopic examinations was performed once during acclimatization, during week 4 in all animals of groups 1 and 4. Blood samples for hematology and clinical biochemistry was collected from all animals under Isoflurane anesthesia system. The animals were placed individually in metabolic cages and fasted overnight before blood and urine sampling but allowed access to water ad libitum. Urine samples were collected in the morning before the blood collection. Blood samples were collected in the morning to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit heparinized glass capillary tube. Blood samples were centrifuged and plasma was separated for clinical biochemistry analysis. Examinations for functional observational battery was performed by end of week 4 for main groups and by the end of week 6 for recovery groups. Necropsy and histopathology was done on day 29 and day 43 for main group and recovery group animals, respectively. It was observed that, No clinical signs were observed in all the dose groups examined including the recovery groups. No mortality was observed in all the dose groups examined including the recovery groups. No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicological relevant changes in the body weight between treated and control group were observed. No significance in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control group. No effects were observed after ophthalmological examinations in both the control groups and treatment groups. No treatment related effects were observed in the both male and female animals with respect to hematological parameters viz RBC, WBC Hb, MCV, MCHC, PCV, PT and APTT when compared with control group. However, in the treatment group, Platelets count was increased in Low Dose (G2), Intermediate Dose (G3) and high dose (G4) group male rats when compared with control group was observed and in high dose recovery group females Monocytes count was decreased comparable with control. This significance may not be attributed to treatment since all the hematological values fall within the normal biological range and no biological significance was noted. There was no dose related effect on few biochemical parameters. There was a significant decrease in both Asparate aminotransferase (AST) and Alanine aminotransferase (ALT) levels in males. Significant changes were observed indicating the test chemical's effect on liver. In males, Urea levels, Bilirubin levels decreased, while Potassium levels increased in low dose group. In Mid Dose Group, Bilirubin levels decreased, while alkaline phosphatase levels and Potassium levels increased, when compared with the control group. In females, Glucose and Sodium levels increased in High dose group, while cholesterol and triglyceride levels decreased, when compared with control group. No significant changes were observed in any of the treated groups and recovery group. No test chemical related effects on the grip strength, motor activity, sensory activity was noted in both treated and recovery group animals. Both in males and females when organ weights of Heart increased High dose group (G4) compared with control group. In Recovery group Females, organ weights of Brain decreased High dose group (G4), when compared with control group. During necropsy, macroscopic lesions were observed on liver and lungs where hemorrhagic spots were observed in both the organs in high dose group. Apart from these observations, no other treatment related macroscopic finding in any of the animals in all groups including Systemic and Reproductive organs. In microscopic observations, sinusoidal hemorrhages were observed in liver in control males, but such effect was observed in both the sexes in high dose group. Also, Foci of necrosis/inflammation of hepatocytes were observed in centri lobular of liver in three male and two female animals from G4 group. Alveolar wall thickening or alveolar inflammation noticed in the lungs of two male and two female animals from G4 group. Thus, based on all the available data, it was concluded that after repeated exposures of the test chemical via oral route, it caused adverse effects on hepatobiliary system. Therefore, the NOAEL and LOAEL for the test chemical was 60 and 120 mg/kg bw/day, respectively after 28 days of exposure.