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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study reprot

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
To assess toxicological profile of the test chemical to determine target organ of toxicity, its reversibility and No Observed Adverse Effect Level (NOAEL) in the rat after 28 consecutive days of oral administration.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): 1-hydroxybenzotriazole
- Molecular formula : C6H5N3O
- Molecular weight : 135.126 g/mol
- Substance type:Organic
- Physical state:Solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1-hydroxybenzotriazole
- Molecular formula : C6H5N3O
- Molecular weight : 135.126 g/mol
- Substance type:Organic
- Physical state:Solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:National Institute of Biosciences, Pune
- Females (if applicable) nulliparous and non-pregnant:[yes
- Age at study initiation:6 to 8 weeks
- Weight at study initiation: Male - 173.58 g (= 100 %), Female - 153.64 g (= 100 %)
- Fasting period before study:
- Housing: The rats were housed in polycarbonate cages with paddy as bedding.
After allocation to respective dose groups rats were housed 2/sex/cage.
- Diet (e.g. ad libitum): Rodent feed, ad libitum
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants, which were reasonably expected to be in the dietary materials and water capable of interfering with the conduct of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30% to 70%
- Air changes (per hr): The animal room was independently provided with at least ten air changes per hour of 100% fresh air that has been passed through the HEPA filters
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room

IN-LIFE DATES:
From: 12-12-2017
To: 19-03-2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test item was suspended in corn oil for preparation of suspension(s). The suspension(s) of the test chemical were made at volumes suitable for daily use for 28 days. The suspension(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0 (vehicle), 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 10 ml/kg body weight.
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item formulation analysis for concentration and stability were conducted
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Total: 72
0 mg/kg bw: 6 male, 6 female
250 mg/kg bw: 6 male, 6 female
500 mg/kg bw: 6 male, 6 female
1000 mg/kg bw: 6 male, 6 female

Reversal group
500 mg/kg bw: 6 male, 6 female
1000 mg/kg bw: 6 male, 6 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose were selected basd on Dose Range Finding study:
1) All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 7 days.
2) Male and female animals from control and all the treated dose groups exhibited normal body weight gain at the end of the dosing period of 7 days.
3) Daily clinical observations did not reveal any signs of toxicity in male and female animals from different dose groups during the dosing period of 7 days.
4) Gross pathological examination did not reveal any abnormality attributable to the treatment.
Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment every day for a period of 28 days.

- Rationale for animal assignment (if not random): A total of 72 animals (36 males + 36 females) were selected and randomly distributed into six groups with 6 animals/sex/group for main groups and 6 animals /sex /group for reversal group.
The animals of uniform body weight were selected. The individual body weights of the animals did not exceed ± 20% of group mean body weight. The group means body weights of all the groups were approximately equal..
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily
- Cage side observations checked in table [No.?] were included.: Viability observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on the day of randomization, day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- 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): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:The eyes of all the animals were examined prior to the initiation of the dosing and at scheduled sacrifice. Eye examination was carried out by using a HEINE mini 2000 ophthalmoscope were employed for evaluation after the induction of mydriasis with 1% solution of tropicamide sulfate.
- Dose groups that were examined: All 72 animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of dosing period on day 29 and at termination of recovery period on day 43.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All 72 animals
- Parameters checked in table [No.?] were examined.: Hb (Hemoglobin (g/dL)), RBC (Red Blood Corpuscles (x 106 /µL)), HCT (Hematocrit (%)), MCV (Mean Corpuscular Volume (fL)), MCH (Mean Corpuscular Hemoglobin (pg)), MCHC (Mean Corpuscular Hemoglobin Concentration (g/dL)), Platelets (x 103 /µL), WBC (White Blood Corpuscles (x 103 /µL)), Rt.(Reticulocytes (%)), N (Neutrophils (%)), L (Lymphocytes (%)), E (Eosinophils (%)), M (Monocytes (%)), B (Basophil (%)) and Pt. (Prothrombin time (sec.)) were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of dosing period on day 29 and at termination of recovery period on day 43.
- Animals fasted: Yes
- How many animals:All 72 animals
- Parameters checked in table [No.?] were examined.: Total Protein (g/dL), Blood Urea Nitrogen (mg/dL), Urea Nitrogen (mg/dL) Calculated, ALT : Alanine Aminotransferase (U/L), AST, Aspartate Aminotransferase (U/L), ALP : Alkaline Phosphatase (U/L), GGT : Gamma Glutamyl Transferase (U/L), Glucose (mg/dL), Calcium (mmol/L), Phosphorous (mg/dL), Albumin (g/dL), Total Bilirubin (mg/dL), Creatinine (mmol/L), Total Cholesterol (mg/dL), Triglycerides (mg/dL), Globulin (g/dL) Calculated, Sodium (mmol/L), Potassium (mmol/L), Chloride (mmol/L) and
Bile acid (µmol/L) were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of dosing period and on reversal group rats at termination of recovery period on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined.: Volume , Appearance, Colour, pH , Specific Gravity, Proteins, Glucose, Ketones,Bilirubin, Urobililogen, Occult Blood and Nitrite were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the exposure period of 28 days and towards the end of the recovery period on day 42, sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) assessment of grip strength and motor activity assessment were conducted for all the animals
- Dose groups that were examined: All 72 animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:Arousal level, Sensory Activity, Visual Placing Response, Air righting response, Grip Strength, Motor Activity,

IMMUNOLOGY: Not specified
- Time schedule for examinations:Not specified
- How many animals:Not specified
- Dose groups that were examined:Not specified
- Parameters checked in table [No.?] were examined.Not specified

OTHER: Organ Weights were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, All the rats surviving at the end of the treatment were sacrificed and gross lesions were noted.

HISTOPATHOLOGY: Yes, From each rat, samples or the whole of the tissues listed below were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin.
Procedure for preparation of slides of tissues of various organs from the rats of various dose groups were performed as per the standard operating procedures of Indian Institute of Toxicology, Pune.
Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination.
Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Muscles - Skeletal muscle, Oesophagus, Ovaries, Pancreas, Pharyngeal Lymphnodes, Pituitary, Prostate, Rectum, Sciatic Nerve, Seminal Vesicles with coagulation gland, Skin with Mammary Gland, Spleen, Spinal Cord (Cervical, mid thoracic and lumbar), Sternum with bone marrow, Stomach, Testes, Thymus, Trachea, Thyroid / Parathyroid, Urinary Bladder, Uterus, Vagina.
Statistics:
Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using SYSTAT 13 validated statistical software supplied by Starcom Information Technology Limited, Bangalore developed by Systat Software, Inc. USA. All the parameters characterized by continuous data such as body weight, feed consumption (calculated as gram per animal), organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data does not meet the homogeneity of variance, Student’s t-test were performed to calculate significance.

Significance was calculated at 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male -
Group I (Control, 0 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.1 to 6).
Group II (Control, 0 mg/kg, Reversal): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and during the post-dosing recovery period (animal nos.13 to 18).
Group III (250 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.25 to 30).
Group IV (500 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.37 to 42).
Group V (1000 mg/kg): Polyurea was observed in animals (animal nos. 49, 51 to 54, with onset from day 23) during the dosing period of 28 days.
Group VI (1000 mg/kg, Reversal): Polyurea was observed in animals (animal nos. 61, 63 to 65, with onset from day 23) throughout the dosing period of 28 days and during the post-dosing recovery period (upto day 32).

Female -
Group I (Control, 0 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.7 to 12).
Group II (Control, 0 mg/kg, Reversal): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and during the post-dosing recovery period (animal nos.19 to 24).
Group III (250 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.31 to 36).
Group IV (500 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.43 to 48).
Group V (1000 mg/kg): Polyurea was observed in all animals (animal nos. 55 to 60, with onset from day 22) during the dosing period of 28 days.
Group VI (1000 mg/kg, Reversal): Polyurea was observed in all animals (animal nos.67 to 72, with onset from day 22) throughout the dosing period of 28 days and during the post-dosing recovery period (upto day 32).

Before commencement of treatment:
In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation, rat did not reveal any abnormality from different dose groups and control group.
During treatment:
In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation, rat did not reveal any abnormality from different dose groups and control group.
Detailed clinical observation did not reveal any abnormality in all groups during the dosing period of 28 days and during the post-dosing recovery period.

Mortality:
no mortality observed
Description (incidence):
All animals from control and different dose groups survived throughout the dosing period of 28 days and the post-dosing recovery period of 14 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male -
Reduced body weight gain of 14.94% was observed in male animals from 1000 mg/kg dose group.
All male animals from control, 250 mg/kg, 500 mg/kg and 1000 mg/kg reversal dose groups exhibited normal body weight gain throughout the dosing period of 28 days and the recovery period of 14 days.
Female -
Female animals from control, 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
Reduced body weight gain of 11.36% was observed in female animals from 1000 mg/kg reversal group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Male and Female -
Animals from control and different dose groups exhibited normal feed consumption at the end of the dosing period of 28 days.
Animals from control reversal and high reversal dose groups exhibited normal feed consumption at the end of the recovery period of 14 days.
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 ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Male :
Total RBC and HCT: Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Female :
Hb and HCT: Decreased values were obtained for animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Total RBC: Decreased values were obtained for animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Male :
Globulin: Elevated levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
BUN and Urea Nitrogen: Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Calcium : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Female :
BUN and Urea Nitrogen: Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05),
Alanine Aminotransferase : Decreased levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Alkaline Phosphatase: Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Bile Acid : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urine analysis conducted on male animals during 4th and 6th week of study period (on day 23, 24 and 43), revealed no abnormality attributable to the treatment.
Urine analysis conducted on female animals during 4th week of study period (on day 24 and 26), revealed higher volume of urine from 1000 mg/kg dose group (p<0.05).
Urine analysis conducted on female animals during 6th week of study period (on day 43) revealed no abnormality attributable to the treatment.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory Reactivity Observations:
All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.

Grip Strength:
Grip strength values observed in male and female animals for control and different dose groups were comparable.

Motor Activity:
Motor activity values observed in male and female animals for control and different dose groups were comparable.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from 1000 mg/kg dose group revealed increased relative weights of brain (p<0.05). Increased relative weights of liver and kidneys (p<0.05) were observed in animals from 500 mg/kg and 1000 mg/kg dose groups. Increased relative weights of spleen (p<0.05) was observed in animals from 250 mg/kg and 1000 mg/kg dose groups. Increased relative weights of thymus (p<0.05) was observed in animals from 250 mg/kg dose group.
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group was found to be comparable.
Female -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from 1000 mg/kg dose group revealed increased relative weights of liver (p<0.05). Increased relative weights of kidneys (p<0.05) was observed in animals from 500 mg/kg and 1000 mg/kg dose groups. In addition, decreased relative weights of heart (p<0.05) was observed in animals from 500 mg/kg dose group.
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group revealed increased relative weights of brain, liver, kidneys, spleen and uterus (p<0.05).
Although significant changes in the values of organ weight were observed in male and female animals from 250 mg/kg and 500 mg/kg dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross pathological examination on male and female animals from control and different dose groups did not reveal any abnormality.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed focal to diffuse, minimal to moderate tubular dilatation in kidneys in male (1/6) and female (6/6) animals from 1000 mg/kg dose group, in male (1/6) and female (6/6) animals from 500 mg/kg dose group and in male (0/6) and female (5/6) animals from 250 mg/kg dose group and in female (1/6) from 1000 mg/kg reversal dose group.
Focal to multifocal, minimal mononuclear cells infiltration was observed in kidneys in male (1/6) and female (3/6) animals from 1000 mg/kg dose group and in female animals from 250 mg/kg (2/6) and 500 mg/kg (2/6) dose groups.
The incidence of tubular dilatation and mononuclear cells infiltration were observed in male animals were less as compared to female animals, however, effect observed was found reversible in male animals at the end of 14 day reversal period of the study.
Incidental, physiological and congenital histopathological changes which were covered in the background historical data of the pathology from control and high dose groups includes minimal, focal to multifocal periportal mononuclear cells infiltration in liver; minimal, multifocal tubular eosinophilic secretion in the kidneys; minimal, multifocal brown coloured pigmentation in the spleen; minimal, diffuse dilatation of zona reticularis and/or minimal, multifocal vacuolation in zona fasciculata in the adrenals; minimal, luminal seminal coagulum in urinary bladder; minimal, luminal dilatation in the uterus; presence of persistent Rathke’s pouch in the pituitary; diffuse mucification of epithelium in vagina; presence of ultimobranchial cyst and/or presence of Ectopic thymus in the thyroid; in male and female animals from control and high dose group.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No effect observed
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No effect observed

Target system / organ toxicity

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Any other information on results incl. tables

VIABILITY

 

Dose (mg/kg)

Mortality

Group

Males

Females

Number

Male

Female

Absolute

Relative %

Absolute

Relative %

I

0

0

0/6

0

0/6

0

II

0 (Reversal)

0 (Reversal)

0/6

0

0/6

0

III

250

250

0/6

0

0/6

0

IV

500

500

0/6

0

0/6

0

V

1000

1000

0/6

0

0/6

0

VI

1000 (Reversal)

1000 (Reversal)

0/6

0

0/6

0

GROUP MEAN BODY WEIGHT (g)

Sex - Male

Group

Dose (mg/kg)

 

Week

Number

 

Day 0

Day 1

1

2

3

I

0

Mean

173.60

178.13

221.52

247.95

272.97

±SD

11.14

11.75

14.86

13.60

18.70

II

0 (Reversal)

Mean

172.37

177.30

219.30

242.73

263.93

±SD

11.36

11.89

24.34

22.71

29.12

III

250

Mean

172.62

177.45

215.87

245.08

269.40

±SD

11.08

10.64

13.73

15.22

20.26

IV

500

Mean

173.95

178.78

214.87

239.52

266.13

±SD

10.11

10.50

18.75

17.15

21.25

V

1000

Mean

173.98

178.97

213.95

238.77

250.90

±SD

9.35

9.49

15.17

13.23

15.12

VI

1000 (Reversal)

Mean

174.93

180.52

214.73

239.12

261.53

±SD

6.96

7.63

16.34

15.70

24.82

 

Group

Dose (mg/kg)

 

Week

Number

 

4

5

6

I

0

Mean

292.12

 

±SD

18.94

II

0 (Reversal)

Mean

267.90

287.55

306.47

±SD

23.45

22.01

24.45

III

250

Mean

289.82

 

±SD

16.02

IV

500

Mean

288.53

±SD

17.60

V

1000

Mean

248.47*

±SD

9.77

VI

1000 (Reversal)

Mean

258.12

274.15

304.20

±SD

27.55

25.86

27.69

Sex - Female

Group

Dose (mg/kg)

 

Week

Number

 

Day 0

Day 1

1

2

3

I

0

Mean

153.93

159.88

169.72

186.47

198.95

±SD

13.56

13.08

14.63

14.28

15.82

II

0 (Reversal)

Mean

154.38

157.87

170.48

195.30

207.28

±SD

14.95

16.49

14.06

13.40

17.94

III

250

Mean

154.05

157.45

163.43

175.27

182.67

±SD

16.30

16.67

18.18

19.87

22.00

IV

500

Mean

154.02

157.97

164.27

176.07

183.57

±SD

16.40

15.72

17.56

17.46

19.91

V

1000

Mean

152.82

156.73

166.57

179.75

188.43

±SD

14.81

15.48

19.79

22.07

29.61

VI

1000 (Reversal)

Mean

152.63

157.72

166.75

183.17

189.07

±SD

12.23

12.46

12.41

11.62

14.67

 

Group

Dose (mg/kg)

 

Week

Number

 

4

5

6

I

0

Mean

207.35

 

±SD

20.17

II

0 (Reversal)

Mean

216.73

232.52

238.37

±SD

19.02

19.51

20.78

III

250

Mean

186.47

 

±SD

22.46

IV

500

Mean

188.92

±SD

22.04

V

1000

Mean

186.07

±SD

29.83

VI

1000 (Reversal)

Mean

190.43

200.78

211.28*

±SD

12.36

11.44

7.08

* = Significant at 95% level of confidence (p<0.05)

GROUP MEAN FEED CONSUMPTION (g/animal/day)

Sex - Male

Group

Dose (mg/kg)

 

Day

Number

 

1

8

15

22

28

I

0

Mean

17.63

19.20

19.75

20.62

21.10

II

0 (Reversal)

Mean

17.58

18.42

19.23

20.15

21.12

III

250

Mean

17.15

18.13

19.10

19.77

20.78

IV

500

Mean

17.75

18.57

19.48

20.08

21.17

V

1000

Mean

17.25

18.78

19.48

20.42

21.08

VI

1000 (Reversal)

Mean

17.67

18.87

19.53

20.28

20.95

 

Group

Dose (mg/kg)

 

Day

Number

 

36

42

I

0

Mean

 

II

0 (Reversal)

Mean

21.83

23.00

III

250

Mean

 

IV

500

Mean

V

1000

Mean

VI

1000 (Reversal)

Mean

21.67

23.07

Sex - Female

Group

Dose (mg/kg)

 

Day

Number

 

1

8

15

22

28

I

0

Mean

14.27

15.42

16.45

17.17

17.80

II

0 (Reversal)

Mean

15.32

16.55

17.23

18.20

18.80

III

250

Mean

14.52

15.37

16.07

17.12

17.42

IV

500

Mean

14.08

15.07

15.98

16.73

17.45

V

1000

Mean

15.18

16.60

17.23

17.58

17.92

VI

1000 (Reversal)

Mean

15.82

17.37

18.18

18.60

19.10

 

Group

Dose (mg/kg)

 

Day

Number

 

36

42

I

0

Mean

 

II

0 (Reversal)

Mean

19.63

20.73

III

250

Mean

 

IV

500

Mean

V

1000

Mean

VI

1000 (Reversal)

Mean

19.90

20.77

SUMMARY OF FUNCTIONAL OBSERVATIONAL

Sex   : Male

 

Day   : 28 and 42

 

 

Group Number

I

II

III

IV

V

VI

Dose

0 mg/kg

0 mg/kg Reversal

250 mg/kg

500 mg/kg

1000 mg/kg

1000 mg/kg

Reversal

Number of animals observed

6

6

6

6

6

6

Number of animals within normal limit

6/6

6/6

6/6

6/6

6/6

6/6

Number of animals with

significant deviation

0/6

0/6

0/6

0/6

0/6

0/6

Parameters

 

 

 

 

 

 

Arousal level : Apparently normal

6/6

6/6

6/6

6/6

6/6

6/6

Visual response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Touch response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Auditory response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Tail pinch response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Visual placing response : Early extension of forelimbs to reach for the screen

6/6

6/6

6/6

6/6

6/6

6/6

Air righting response : Lands with all feet on ground

6/6

6/6

6/6

6/6

6/6

6/6

Grip Strength (kg) - Mean ± SD

0.856

± 0.028

0.857

±0.043

0.867

± 0.039

0.825

± 0.043

0.828

± 0.056

0.869

±0.045

Motor Activity -

Interval ‘1’

581.50

±214.63

526.67

±147.13

565.17

±103.93

575.00

±281.40

548.00

±270.57

511.50

±131.34

Mean ± SD

Interval ‘2’

337.17

±143.75

325.50

±135.14

351.33

±61.11

350.33

±179.92

326.83

±58.41

348.00

±46.41

 

Interval ‘3’

205.17

±54.65

239.83

±35.53

255.50

±91.59

214.50

±58.44

221.00

±48.41

255.83

±79.60

Sex   : Female

 

Day   : 28 and 42

 

 

Group Number

I

II

III

IV

V

VI

Dose

0 mg/kg

0 mg/kg Reversal

250 mg/kg

500 mg/kg

1000 mg/kg

1000 mg/kg

Reversal

Number of animals observed

6

6

6

6

6

6

Number of animals within normal limit

6/6

6/6

6/6

6/6

6/6

6/6

Number of animals with

significant deviation

0/6

0/6

0/6

0/6

0/6

0/6

Parameters

 

 

 

 

 

 

Arousal level : Apparently normal

6/6

6/6

6/6

6/6

6/6

6/6

Visual response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Touch response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Auditory response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Tail pinch response : Orienting response

6/6

6/6

6/6

6/6

6/6

6/6

Visual placing response : Early extension of forelimbs to reach for the screen

6/6

6/6

6/6

6/6

6/6

6/6

Air righting response : Lands with all feet on ground

6/6

6/6

6/6

6/6

6/6

6/6

Grip Strength (kg) - Mean ± SD

0.747

±0.032

0.838

±0.040

0.773

±0.044

0.773

±0.051

0.772

±0.021

0.825

±0.039

Motor Activity -

Interval ‘1’

533.50

±92.11

531.50

±137.11

542.67

±122.06

535.17

±120.93

581.33

±81.57

582.00

±87.27

Mean ± SD

Interval ‘2’

385.50

±72.17

373.50

±48.77

340.00

±222.05

345.33

±90.79

343.33

±56.91

326.67

±100.55

 

Interval ‘3’

299.50

±53.94

234.50

±91.11

226.50

±179.55

286.83

±85.16

248.17

±93.12

280.17

±137.76

GROUP MEAN HAEMATOLOGY

Sex : Male

Day : 29 and 43

Group

Dose

(mg/kg)

 

Hb

Total RBC

Rt

HCT

MCV

MCH

MCHC

Number

 

(g/dL)

(6/µL)

(%)

(%)

(fL)

(pg)

(g/dL)

I

0

Mean

14.93

7.69

4.45

46.42

60.48

19.47

32.20

±SD

0.66

0.26

0.85

1.97

4.48

1.46

0.35

II

0 (Reversal)

Mean

15.58

8.56

4.18

46.75

54.62

18.20

33.32

±SD

1.18

0.49

0.52

3.46

2.31

0.98

0.53

III

250

Mean

15.13

7.68

4.65

46.57

60.75

19.75

32.48

±SD

0.52

0.30

0.96

1.15

3.12

1.30

0.61

IV

500

Mean

15.15

7.95

4.68

46.48

58.58

19.08

32.57

±SD

1.11

0.72

0.83

3.54

1.39

0.53

0.20

V

1000

Mean

14.95

7.77

4.92

45.87

59.00

19.22

32.58

±SD

0.81

0.41

1.02

2.35

0.85

0.29

0.33

VI

1000 (Reversal)

Mean

14.52

7.68*

4.28

43.05*

56.23

18.97

33.75

±SD

0.43

0.55

0.69

1.37

2.90

1.08

0.36

 

Group

Dose

(mg/kg)

 

Platelets

Total WBC

Differential %

Pt.

Number

 

(3/ µL)

(3/µL)

N

L

E

M

B

(Sec.)

I

0

Mean

444.50

7.62

18.50

80.00

0.83

0.67

0.00

19.50

±SD

101.64

4.29

3.62

3.46

0.75

0.82

0.00

4.37

II

0 (Reversal)

Mean

387.17

12.13

16.17

82.17

0.83

0.83

0.00

18.67

±SD

72.20

2.89

3.19

3.60

0.75

0.75

0.00

4.46

III

250

Mean

411.83

8.95

18.00

80.17

1.00

0.83

0.00

21.00

±SD

53.25

3.62

3.63

3.76

0.89

0.75

0.00

5.22

IV

500

Mean

479.00

10.40

16.83

81.50

0.83

0.83

0.00

21.00

±SD

92.55

2.45

2.79

2.43

0.75

0.41

0.00

4.05

V

1000

Mean

422.00

9.80

18.50

79.83

1.17

0.50

0.00

20.50

±SD

63.60

4.12

3.83

3.66

0.75

0.84

0.00

4.23

VI

1000 (Reversal)

Mean

382.67

12.50

17.33

81.00

0.67

1.00

0.00

20.33

±SD

79.61

3.64

3.27

3.22

0.52

0.89

0.00

5.54

Sex : Female

Day : 29 and 43

Group

Dose

(mg/kg)

 

Hb

Total RBC

Rt

HCT

MCV

MCH

MCHC

Number

 

(g/dL)

(6/µL)

(%)

(%)

(fL)

(pg)

(g/dL)

I

0

Mean

15.47

7.85

4.38

47.87

61.10

19.72

32.30

±SD

1.07

0.73

0.49

3.52

1.81

0.65

0.54

II

0 (Reversal)

Mean

14.77

8.00

4.25

43.50

54.43

18.48

33.95

±SD

1.25

0.80

0.59

3.95

2.26

0.91

0.55

III

250

Mean

13.58

6.70*

4.68

41.98

62.73

20.30

32.35

±SD

1.97

1.01

0.69

6.13

1.43

0.57

0.69

IV

500

Mean

11.98*

5.83*

4.03

36.33*

62.43

20.58

32.97

±SD

1.09

0.55

0.66

3.05

1.30

0.36

0.55

V

1000

Mean

13.83

6.78

4.47

41.88

61.80

20.40

33.00

±SD

1.00

0.48

0.59

2.84

1.57

0.50

0.64

VI

1000 (Reversal)

Mean

14.50

7.58

4.37

41.88

55.30

19.15

34.62

±SD

0.67

0.44

0.63

1.94

2.37

0.97

0.52

 

Group

Dose

(mg/kg)

 

Platelets

Total WBC

Differential %

Pt.

Number

 

(3/ µL)

(3/µL)

N

L

E

M

B

(Sec.)

I

0

Mean

440.00

9.00

18.00

80.17

1.00

0.83

0.00

20.67

±SD

91.40

1.63

3.58

3.49

0.89

0.75

0.00

4.32

II

0 (Reversal)

Mean

423.83

11.02

16.50

82.17

0.83

0.50

0.00

20.67

±SD

109.01

3.96

2.74

2.86

0.75

0.55

0.00

3.78

III

250

Mean

433.33

8.90

16.67

82.00

0.83

0.50

0.00

20.33

±SD

80.53

1.07

3.72

4.00

0.75

0.55

0.00

5.05

IV

500

Mean

327.00

8.00

17.00

81.50

1.00

0.50

0.00

19.83

±SD

70.50

2.38

3.46

3.39

0.63

0.84

0.00

4.79

V

1000

Mean

437.50

9.67

17.00

81.17

0.83

1.00

0.00

21.33

±SD

98.60

4.29

3.03

2.64

0.75

0.89

0.00

5.09

VI

1000 (Reversal)

Mean

443.00

11.07

18.00

80.67

0.83

0.50

0.00

20.00

±SD

79.47

3.07

3.35

3.44

0.75

0.55

0.00

6.23

Sex : Male

Day : 29 and 43

Group

Number

Dose (mg/kg)

Animal Nos.

Cell Morphology

I

0

1 - 6

Normocytic, Normochromic

II

0 (Reversal)

13 - 18

Normocytic, Normochromic

III

250

25 - 30

Normocytic, Normochromic

IV

500

37 - 42

Normocytic, Normochromic

V

1000

49 - 54

Normocytic, Normochromic

VI

1000 (Reversal)

61 - 66

Normocytic, Normochromic

 Sex : Female

Day : 29 and 43

Group

Number

Dose (mg/kg)

Animal Nos.

Cell Morphology

I

0

7 - 12

Normocytic, Normochromic

II

0 (Reversal)

19 - 24

Normocytic, Normochromic

III

250

31 - 36

Normocytic, Normochromic

IV

500

43 - 48

Normocytic, Normochromic

V

1000

55 - 60

Normocytic, Normochromic

VI

1000 (Reversal)

67 - 72

Normocytic, Normochromic

GROUP MEAN CLINICAL BIOCHEMISTRY

Sex : Male

Day : 29 and 43

Group Number

Dose (mg/kg)

 

TotalProtein (g/dL)

BUN (mg/dL)

Urea

Nitrogen

(mg/dL)

ALT (U/L)

AST (U/L)

ALP (U/L)

Glucose (mg/dL)

I

0

Mean

6.47

17.83

38.88

60.33

94.83

131.67

87.33

±SD

0.19

2.93

6.38

13.69

9.26

48.48

14.12

II

0 (Reversal)

Mean

6.46

15.33

33.43

49.83

111.17

198.33

94.00

±SD

0.26

1.75

3.82

5.42

18.53

34.03

9.01

III

250

Mean

6.90

13.50*

29.43*

54.83

87.50

101.83

84.50

±SD

0.25

2.43

5.30

8.47

7.01

30.39

5.82

IV

500

Mean

6.60

13.67

29.79

54.17

84.00

113.67

83.67

±SD

0.38

1.63

3.56

9.58

10.95

11.81

7.74

V

1000

Mean

6.45

16.00

34.88

57.83

86.17

131.67

85.67

±SD

0.50

3.79

8.27

17.78

15.84

50.23

14.58

VI

1000 (Reversal)

Mean

6.40

19.00

41.42

48.17

98.67

170.33

92.50

±SD

0.20

3.95

8.61

8.59

9.05

55.87

6.83

 

Group Number

Dose (mg/kg)

 

Calcium(mmol/L)

Phospho-rous (mg/dL)

GGT

(U/L)

Total

Bilirubin (mg/dL)

Albumin (g/dL)

Globulin (g/dL)

Creatinine (mg/dL)

I

0

Mean

3.70

8.57

6.00

0.18

1.10

5.37

0.46

±SD

0.06

0.92

1.10

0.02

0.11

0.12

0.06

II

0 (Reversal)

Mean

4.02

8.65

6.17

0.16

1.02

5.43

0.34

±SD

0.17

1.00

1.33

0.02

0.14

0.14

0.05

III

250

Mean

3.60

8.27

6.17

0.14

1.06

5.83*

0.46

±SD

0.13

0.84

0.75

0.01

0.13

0.21

0.05

IV

500

Mean

3.70

7.95

5.83

0.16

1.03

5.58

0.43

±SD

0.06

0.55

0.98

0.04

0.11

0.29

0.03

V

1000

Mean

3.76

8.98

5.67

0.16

1.10

5.33

0.43

±SD

0.12

0.81

1.03

0.02

0.12

0.45

0.08

VI

1000 (Reversal)

Mean

3.81*

9.80

6.33

0.16

0.98

5.43

0.30

±SD

0.09

1.52

0.52

0.01

0.13

0.23

0.06

Sex : Male

Day : 29 and 43 

Group Number

Dose (mg/kg)

 

 

Sodium

(mmol/L)

Potassium

(mmol/L)

Chloride

(mmol/L)

Total Cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Bile Acids (µmol/L)

I

0

Mean

146.60

4.24

109.28

49.17

61.33

20.12

±SD

0.50

0.41

1.90

9.37

24.06

4.84

II

0 (Reversal)

Mean

147.27

4.65

105.35

52.67

62.00

12.85

±SD

1.84

0.51

2.37

7.23

17.57

11.60

III

250

Mean

147.37

4.33

109.17

53.00

76.00

18.14

±SD

0.69

0.55

2.47

6.23

24.84

5.59

IV

500

Mean

147.65

4.27

107.99

46.67

58.83

14.41

±SD

0.92

0.45

1.61

9.20

13.38

5.42

V

1000

Mean

147.59

4.31

108.24

45.83

73.33

17.74

±SD

0.88

0.54

1.89

8.42

33.50

8.26

VI

1000 (Reversal)

Mean

145.48

4.59

106.15

58.33

52.33

19.71

±SD

1.26

0.69

2.28

11.11

5.35

6.88

Sex : Female

Day : 29 and 43

Group Number

Dose (mg/kg)

 

TotalProtein (g/dL)

BUN (mg/dL)

Urea

Nitrogen

(mg/dL)

ALT (U/L)

AST (U/L)

ALP (U/L)

Glucose (mg/dL)

I

0

Mean

7.10

18.50

40.33

58.67

124.00

124.50

90.83

±SD

0.57

2.66

5.81

11.36

29.60

46.47

8.28

II

0 (Reversal)

Mean

6.58

16.17

35.24

35.50

97.00

105.33

97.00

±SD

0.42

2.14

4.66

5.39

11.92

19.50

5.44

III

250

Mean

6.81

15.00

32.70

39.83*

108.83

69.00*

82.33

±SD

0.27

0.89

1.95

5.08

5.95

14.76

19.43

IV

500

Mean

6.92

14.67*

31.97*

40.50*

121.33

84.83

85.00

±SD

0.47

1.97

4.29

6.44

15.07

13.53

14.03

V

1000

Mean

7.56

18.50

40.33

60.50

118.67

105.00

81.67

±SD

0.26

3.51

7.65

15.24

39.12

57.40

5.61

VI

1000 (Reversal)

Mean

6.72

18.17

39.60

37.67

94.33

129.17

103.33

±SD

0.22

2.64

5.75

3.88

9.58

34.06

14.95

 

Group Number

Dose (mg/kg)

 

Calcium(mmol/L)

Phospho-rous (mg/dL)

GGT

(U/L)

Total

Bilirubin (mg/dL)

Albumin (g/dL)

Globulin (g/dL)

Creatinine (mg/dL)

I

0

Mean

3.80

8.23

6.67

0.15

1.17

5.92

0.55

±SD

0.12

0.58

1.37

0.02

0.07

0.53

0.07

II

0 (Reversal)

Mean

3.67

7.03

6.33

0.16

1.03

5.57

0.38

±SD

0.14

0.99

0.82

0.02

0.11

0.37

0.03

III

250

Mean

3.92

7.83

6.00

0.14

1.13

5.68

0.49

±SD

0.12

0.92

0.89

0.02

0.12

0.25

0.05

IV

500

Mean

3.87

7.67

6.67

0.14

1.12

5.80

0.47

±SD

0.17

1.08

1.03

0.02

0.17

0.41

0.06

V

1000

Mean

3.64

7.48

6.50

0.14

1.18

6.38

0.48

±SD

0.13

0.62

1.87

0.02

0.10

0.17

0.14

VI

1000 (Reversal)

Mean

3.72

8.85

6.33

0.15

1.00

5.70

0.35

±SD

0.18

2.28

1.21

0.01

0.10

0.14

0.02

Sex : Female

Day : 29 and 43

Group Number

Dose (mg/kg)

 

 

Sodium

(mmol/L)

Potassium

(mmol/L)

Chloride

(mmol/L)

Total Cholesterol

(mg/dL)

Triglycerides

(mg/dL)

Bile Acids (µmol/L)

I

0

Mean

147.04

4.20

107.88

56.33

77.83

27.91

±SD

1.14

0.31

0.78

5.05

19.63

11.14

II

0 (Reversal)

Mean

146.50

4.06

108.19

57.17

44.67

11.89

±SD

1.26

0.37

1.37

12.78

9.16

2.05

III

250

Mean

144.62

3.92

105.49

66.33

95.83

24.08

±SD

1.61

0.45

1.37

5.75

21.74

2.93

IV

500

Mean

145.73

4.17

107.83

65.50

63.17

18.74

±SD

1.84

0.48

2.31

13.43

25.79

2.92

V

1000

Mean

147.20

4.06

108.93

67.17

113.33

24.12

±SD

2.01

0.60

2.39

7.39

33.63

7.86

VI

1000 (Reversal)

Mean

147.53

4.55

108.51

61.50

51.17

19.62*

±SD

1.33

1.06

1.54

8.26

14.39

6.93

GROUP MEAN ABSOLUTE ORGAN WEIGHTS (g)

Sex : Male

Day : 29 and 43

GroupNumber

Dose

(mg/kg)

 

Body Weight (g)

Brain

Liver

Kidneys

Adrenals

Testes

Prostate + Seminal

Vesicle with

Coagulation gland

I

0

Mean

273.800

1.847

8.275

1.831

0.045

2.872

1.398

±SD

18.670

0.118

0.965

0.188

0.011

0.173

0.301

II

0 (Reversal)

Mean

286.017

1.944

11.316

2.254

0.049

2.939

1.784

±SD

23.665

0.202

3.511

0.777

0.006

0.226

0.404

III

250

Mean

271.600

2.028

9.412

1.747

0.048

2.646

1.481

±SD

15.799

0.055

1.546

0.324

0.006

0.492

0.207

IV

500

Mean

270.383

1.821

9.993

2.138

0.040

2.877

1.273

±SD

17.921

0.151

1.408

0.244

0.008

0.394

0.208

V

1000

Mean

231.450

1.786

8.585

1.795

0.049

2.920

1.359

±SD

8.227

0.090

0.631

0.081

0.005

0.363

0.065

VI

1000 (Reversal)

Mean

285.750

1.967

12.290

1.873

0.049

2.807

1.870

±SD

27.413

0.296

2.552

0.449

0.006

0.223

0.330

 

GroupNumber

Dose

(mg/kg)

 

Heart

Spleen

Thymus

Epididymides

I

0

Mean

0.929

0.734

0.352

0.834

±SD

0.180

0.333

0.062

0.128

II

0 (Reversal)

Mean

1.137

1.017

0.258

1.075

±SD

0.245

0.206

0.092

0.121

III

250

Mean

1.093

1.259

0.477

0.857

±SD

0.091

0.298

0.074

0.047

IV

500

Mean

1.023

1.058

0.320

0.812

±SD

0.206

0.194

0.044

0.111

V

1000

Mean

0.773

1.045

0.390

0.825

±SD

0.051

0.327

0.099

0.085

VI

1000 (Reversal)

Mean

1.037

1.075

0.284

1.016

±SD

0.195

0.132

0.076

0.133

 Sex : Female

Day : 29 and 43

GroupNumber

Dose

(mg/kg)

 

Body Weight (g)

 

Brain

        Liver

 

 

Kidneys

 

 

Adrenals

 

 

Ovaries

 

I

0

Mean

192.800

1.843

6.823

1.377

0.052

0.120

±SD

20.929

0.241

1.286

0.213

0.010

0.030

II

0 (Reversal)

Mean

223.700

1.908

7.828

1.762

0.079

0.115

±SD

19.578

0.187

1.558

0.261

0.015

0.022

III

250

Mean

171.950

1.698

5.448

1.280

0.046

0.074

±SD

25.311

0.030

1.263

0.330

0.008

0.011

IV

500

Mean

174.983

1.748

6.005

1.568

0.052

0.099

±SD

23.341

0.107

0.832

0.254

0.009

0.010

V

1000

Mean

177.633

1.777

7.922

2.019

0.051

0.117

±SD

27.447

0.144

1.709

0.431

0.008

0.028

VI

1000 (Reversal)

Mean

197.333

1.865

8.602

1.861

0.063

0.113

±SD

7.196

0.052

1.062

0.140

0.007

0.017

 

Group No.

Dose

(mg/kg)

 

Heart

Spleen

Thymus

Uterus

I

0

Mean

0.853

0.854

0.318

0.424

±SD

0.130

0.227

0.048

0.138

II

0 (Reversal)

Mean

1.006

0.915

0.346

0.466

±SD

0.086

0.186

0.066

0.099

III

250

Mean

0.655

0.787

0.365

0.376

±SD

0.267

0.357

0.097

0.111

IV

500

Mean

0.632

0.886

0.302

0.409

±SD

0.093

0.222

0.068

0.064

V

1000

Mean

0.748

0.913

0.310

0.343

±SD

0.187

0.242

0.104

0.064

VI

1000 (Reversal)

Mean

0.922

1.022

0.349

0.684

±SD

0.196

0.094

0.093

0.228

GROUP MEAN RELATIVE ORGAN WEIGHTS (%)

Sex : Male

Day : 29 and 43 

GroupNumber

Dose

(mg/kg)

 

Body Weight (g)

Brain

Liver

Kidneys

Adrenals

Testes

Prostate + Seminal

Vesicle with

Coagulation gland

I

0

Mean

273.800

0.677

3.020

0.669

0.017

1.054

0.510

±SD

18.670

0.069

0.249

0.047

0.004

0.105

0.101

II

0 (Reversal)

Mean

286.017

0.679

3.947

0.775

0.017

1.029

0.618

±SD

23.665

0.027

1.209

0.211

0.002

0.054

0.096

III

250

Mean

271.600

0.749

3.459

0.640

0.018

0.979

0.548

±SD

15.799

0.053

0.472

0.090

0.003

0.205

0.095

IV

500

Mean

270.383

0.676

3.704*

0.789*

0.015

1.063

0.469

±SD

17.921

0.069

0.551

0.045

0.004

0.122

0.050

V

1000

Mean

231.450

0.773*

3.707*

0.776*

0.021

1.267

0.588

±SD

8.227

0.052

0.180

0.041

0.002

0.189

0.034

VI

1000 (Reversal)

Mean

285.750

0.689

4.304

0.651

0.017

0.986

0.651

±SD

27.413

0.083

0.861

0.109

0.001

0.085

0.071

 

GroupNumber

Dose

(mg/kg)

 

Heart

Spleen

Thymus

Epididymides

I

0

Mean

0.339

0.270

0.129

0.305

±SD

0.058

0.119

0.020

0.044

II

0 (Reversal)

Mean

0.396

0.353

0.089

0.376

±SD

0.065

0.050

0.029

0.031

III

250

Mean

0.404

0.467*

0.175*

0.317

±SD

0.047

0.125

0.020

0.035

IV

500

Mean

0.380

0.391

0.119

0.300

±SD

0.083

0.066

0.020

0.029

V

1000

Mean

0.334

0.452*

0.168

0.356

±SD

0.024

0.144

0.042

0.031

VI

1000 (Reversal)

Mean

0.361

0.376

0.099

0.355

±SD

0.040

0.025

0.021

0.024

Sex : Female

Day : 29 and 43

GroupNumber

Dose

(mg/kg)

 

Body Weight (g)

 

Brain

        Liver

 

 

Kidneys

 

 

Adrenals

 

 

Ovaries

 

I

0

Mean

192.800

0.954

3.538

0.712

0.027

0.062

±SD

20.929

0.047

0.526

0.048

0.004

0.012

II

0 (Reversal)

Mean

223.700

0.857

3.480

0.785

0.035

0.051

±SD

19.578

0.094

0.496

0.059

0.007

0.010

III

250

Mean

171.950

1.006

3.144

0.739

0.028

0.043

±SD

25.311

0.147

0.373

0.113

0.008

0.006

IV

500

Mean

174.983

1.007

3.437

0.900*

0.030

0.057

±SD

23.341

0.070

0.277

0.131

0.004

0.008

V

1000

Mean

177.633

1.011

4.435*

1.136*

0.029

0.066

±SD

27.447

0.098

0.452

0.165

0.005

0.010

VI

1000 (Reversal)

Mean

197.333

0.945*

4.349*

0.942*

0.032

0.057

±SD

7.196

0.027

0.396

0.042

0.004

0.010

 

Group No.

Dose

(mg/kg)

 

Heart

Spleen

Thymus

Uterus

I

0

Mean

0.443

0.442

0.165

0.218

±SD

0.052

0.100

0.017

0.061

II

0 (Reversal)

Mean

0.451

0.408

0.155

0.208

±SD

0.037

0.073

0.029

0.039

III

250

Mean

0.378

0.446

0.212

0.215

±SD

0.128

0.155

0.050

0.044

IV

500

Mean

0.362*

0.505

0.171

0.235

±SD

0.034

0.111

0.022

0.038

V

1000

Mean

0.417

0.527

0.171

0.197

±SD

0.040

0.168

0.031

0.044

VI

1000 (Reversal)

Mean

0.465

0.519*

0.177

0.345*

±SD

0.088

0.061

0.046

0.109

* = Significant at 95% level of confidence (p<0.05)

Applicant's summary and conclusion

Conclusions:
No Observed Adverse Effect Level (NOAEL) of the test chemical in the Sprague Dawley rat via oral route, over a period of 28 days was found to be 250 mg/kg body weight in male animals and less than 250 mg/kg body weight in female animals.
Executive summary:

In a repeated dose oral toxicity study, Sprague-Dawley male and female rats were treated with the test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for 28 days. All the male and female animals from control and different dose groups up to 1000 mg/kg survived throughout the dosing period of 28 days and the recovery period of 14 days. At the end of the dosing period reduced body weight gain of 14.94% was observed in male animals from 1000 mg/kg dose group. All the male animals from control, 250, 500 and 1000 mg/kg reversal dose groups exhibited normal body weight gain throughout the dosing period of 28 days and the recovery period of 14 days. Female animals from control, 250, 500 and 1000 mg/kg dose groups exhibited normal body weight gain throughout the dosing period of 28 days. Reduced body weight gain of 11.36% was observed in female animals from 1000 mg/kg reversal group. Feed intake of animals from control and different dose groups was found to be comparable throughout the dosing period of 28 days and the recovery period of 14 days. Ophthalmoscopic examination, conducted prior to and at the end of dosing period on animals from control and different dose groups did not reveal any abnormality. No signs of toxicity were observed in male and female animals from 250 and 500 mg/kg dose groups during the dosing period of 28 days. Polyurea was observed in male and female animals from 1000 mg/kg and 1000 mg/kg reversal dose groups during the dosing period of 28 days and the recovery period of 14 days. Similarly, Detailed clinical observations conducted at weekly interval (upto 6thweek) did not reveal any abnormality in all male and female animals from control and different dose groups during the dosing period of 28 days and the recovery period of 14 days. Towards the end of the exposure period in week 4, functional observation battery such as sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), Grip strength and Motor activity values revealed no abnormalities attributable to the treatment. No statistically significant changes in the values of various Haematological parameters were at the end of the dosing period on day 29. At the end of the recovery period on day 43, statistically significant decrease in the values of Total RBC and HCT at 1000 mg/kg in male. Statistically significant decrease in the values of Hb and HCT at 500 mg/kg and Total RBC at 250 and 500 mg/kg were observed in female rat. At the end of the recovery period on day 43, no statistically significant changes in the values of various parameters in female rats. The decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. Clinical biochemistry analysis at the end of the dosing period on day 29, revealed statistically significant increase in the values of Globulin at 250 mg/kg in male rat. In addition statistically significant decrease was observed in the values of BUN and Urea Nitrogen at 250 mg/kg in male, BUN and Urea Nitrogen at 500 mg/kg in female rat, Alanine Aminotransferase at 250 and 500 mg/kg in female and Alkaline Phosphatase at 250 mg/kg in female rat. At the end of the recovery period on day 43 (Reversal groups) statistically significant increase was observed in the values of Bile Acid at 1000 mg/kg in female and statistically significant decrease was observed in the values of Calcium at 1000 mg/kg in male. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. Urine analysis conducted on male animals during 4thand 6thweek of study period (on day 23, 24 and 43), revealed no abnormality attributable to the treatment. Urine analysis conducted on female animals during 4thweek of study period (on day 24 and 26), higher volume of urine was observed in female at 1000 mg/kg dose group. During 6th week of study period (on day 43) revealed no abnormality attributable to the treatment. At termination of dosing on day 29, male animals from 1000 mg/kg dose group revealed increased relative weights of brain. In addition, increased relative weights of liver and kidneys were observed in male at 500 and 1000 mg/kg dose groups when compared with that of controls. Increased relative weights of spleen were observed in male at 250 and 1000 mg/kg as compared to controls. Increased relative weights of thymus were observed in male at 250 mg/kg as compared to controls. No effect on male organ weight sacrificed on day 43 from 1000 mg/kg as comparable to controls. At termination of dosing on day 29, at 1000 mg/kg increased relative weights of liver were observed in female as compared to controls. In addition, increased relative weights of kidneys were observed in female at 500 and 1000 mg/kg as compared to controls. At 500 mg/kg dose decreased relative weights of heart in female rats as compared to controls. Organ weight data of female animals sacrificed on day 43 at 1000 mg/kg increased relative weights of brain, liver, kidneys, spleen and uterus were observed in female as compared to controls. Although significant changes in the values of organ weight of brain, liver, spleen, thymus, heart and uterus were observed in male and female animals at 250 mg/kg and 500 mg/kg dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. Gross pathological examination did not reveal any abnormality attributable to the treatment. Histopathological examination revealed focal to diffuse, minimal to moderate tubular dilatation in kidneys in male (1/6) and female (6/6) animals from 1000 mg/kg dose group, in male (1/6) and female (6/6) animals from 500 mg/kg dose group and in male (0/6) and female (5/6) animals from 250 mg/kg dose group and in female (1/6) from 1000 mg/kg reversal dose group. Focal to multifocal, minimal mononuclear cells infiltration was observed in kidneys in male (1/6) and female (3/6) animals from 1000 mg/kg dose group and in female animals from 250 mg/kg (2/6) and 500 mg/kg (2/6) dose groups. All histopathological the changes observed in male animals were reversible during the recovery period of 14 days. The histopathological changes observed in female animals were evident in reversal group one animal, during the recovery period of 14 days. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemcal in the Sprague Dawley rat via oral route, over a period of 28 days was found to be 250 mg/kg body weight in male animals and less than 250 mg/kg body weight in female animals.