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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:
key study
Study period:
Feb 2020 to Jul 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Cinnamyl alcohol
EC Number:
203-212-3
EC Name:
Cinnamyl alcohol
Cas Number:
104-54-1
Molecular formula:
C9H10O
IUPAC Name:
3-phenylpropan-1-ol
Test material form:
solid
Details on test material:
Name: Cinnamyl Alcohol
CAS No. 104-54-1
IUPAC Name: 3-Phenyl-2-propen-1-ol
Molecular Formula: C9-H10-O
Molecular Weight: 134.177 g/mol
SMILES: OC\C=C\c1ccccc1

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rats are the standard laboratory rodent species used as a reliable test model to assess the Toxicity of the test item and recommended by the guidelines The strain Wistar was chosen on account of available historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sipra Labs Limited, Plot No.13, IDA, Balanagar, Hyderabad, India.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 Weeks
- Weight at study initiation:
Male: 128.1g -136.9 g
Female: 125.7 g -134.4 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: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C – 23.4°C.
- Humidity (%): 51 – 61 %.
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle per day

IN-LIFE DATES: From: To: No Data Available

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was weighed in pre-weighed glass beakers on an appropriate weighing balance. The test chemical was transferred to mortar and triturated with pestle. 10 mL of the vehicle was added to mortar and again triturated. This was transferred to a measuring cylinder. Sufficient quantity of vehicle was added to make up the required volume of formulation. This was transferred back to beaker. Homogeneity of the test chemical 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: 0 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml for control, low dose, mid dose and high dose groups, respectively.
- Amount of vehicle (if gavage): 10 ml/kg
- 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. Test item 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.

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=0.9982).

Accuracy: Accuracy was performed at three levels ie., 50, 100, 150%. The mean recovery values of samples for 50% level is 102.35% , for 100% level is 100.18% and for 150% level is 97.14%.

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.

The dose concentration verification was carried out for test item fortified in Distilled water pertaining to low dose (25mg/mL), mid dose (50mg/mL) and high dose (100mg/mL). The recovery of test item in the prepared dose formulation was within the guideline specification.
The test item was found to be homogeneously dispersed in Distilled water, which was evinced from the recovery of test item from different layers (Top, middle and bottom layers of Distilled water).
Duration of treatment / exposure:
28 days consecutively.
Frequency of treatment:
Once Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (G1)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Group (Recovery) (G1R)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Low Dose Group (G2)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Intermediate Dose Group (G3)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (G4)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Dose Group (Recovery) (G4R)
No. of animals per sex per dose:
5 animals per sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the study were selected based on the dose range finding 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.
- Fasting period before blood sampling for clinical biochemistry: Yes, the animals were fasted before the blood sampling.
- Rationale for selecting satellite groups: Recovery groups for vehicle control and high dose were included to know the reversibility of treatment related effects.
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): No Data Available
- Other: No Data Available
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on first 3 days of treatment; once daily thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first exposure and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Once weekly
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: All dose groups were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 for main groups and Week 6 for recovery group
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals were fasted.

The following hematology parameters were determined:
Erythrocyte count (RBC)
Hemoglobin (Hb)
Hematocrit (PCV)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration
(MCHC)
Platelet (thrombocyte) count (PLT)
Total leukocyte count (WBC)
Differential leukocyte count (DC)
Prothrombin time (PT)
Activated Partial Thromboplastin time (APTT)
Reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 for main groups and Week 6 for recovery group
- Animals fasted: Yes

The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Cholesterol, total
Triglycerides
Aspartate aminotransferase (AST)
Alanine aminotransferase (ALT)
Alkaline phosphatase (ALP)
γ-glutamyl transferase
Bilirubin
Sodium (Na)
Potassium (K)
Chloride (Cl)
Protein, total
Albumin
Globulin
A G ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 for main groups and Week 6 for recovery group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
The following urinalysis parameters were determined: Volume, specific gravity, colour, clarity, pH, urobilinogen, bilirubin, ketone bodies, proteins and glucose. Urine analysis was done using Test Strips with urine analyzer.
Microscopic examination of urine was done for epithelial cells, pus cells, RBC, cast and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before sacrifice
- Dose groups that were examined: All the dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity /
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, All animals in main study (Group 1 to Group 4) was sacrificed at the end of week 4 and animals in recovery group (Group 1R and Group 4R) were sacrificed at the end of week 6 of experimental period by Carbon dioxide asphyxiation. All animals were weighed and sent to necropsy.

Samples of the following tissues and organs were collected from Allocation A & B Animals at necropsy and fixed in 10% Neutral Buffered Formalin solution (NBF) except testes and eyes, which were fixed in Modified Davidson’s fixative for 24 hours and then transferred to NBF. Adrenal glands, Aorta, Bone marrow (Sternum), Brain (3 levels), Cecum Cervix, Colon, Duodenum, Epididymides, Eye, Heart, Ileum, with Peyer's patches Jejunum, Kidneys, Liver, Lungs (inflated with NBF at necropsy), Lymph nodes (mesenteric, axillary), Oesophagus, Ovaries, Pancreas, Rectum, Prostate + Seminal vesical with coagulating glands, Pituitary, Sciatic nerve, Skeletal muscle, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder (inflated with NBF at necropsy), Uterus, Vagina, All Gross lesions

The following organs were weighed and recorded on the scheduled dates of necropsy. Wet weight was taken after dissection to avoid drying.
Brain Spleen Thymus Epididymides
Heart Kidneys Testes Ovaries
Liver Adrenals Uterus
Prostate + Seminal vesical with coagulating glands


HISTOPATHOLOGY: Yes, 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.
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 of toxicity were observed in the study.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in body weight or body weight gain were observed.
For tabular data, please refer to Table 1-2 below.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant changes in food intake were observed. For tabular data, please refer to pp. 27-30 in the attached report).
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No eye abnormalities were observed in the study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with cinnamyl alcohol had no effect on both male and female animals with respect to haematological parameters viz RBC, Hb, MCV, MCHC, PCV, PLT, PT and APTT when compared with control group. However, in males, WBC count was decreased in the high dose (G4) group, when compared with control group, was observed and in females, WBC values were comparable with control. This significance may not be attributed to treatment since all the haematological values fall within the normal biological range and no biological significance was noted.
For tabular data, please refer to Table 3 and 4 below.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters revealed no significant changes in the test item treated and recovery males and female animals with an exception of Bilirubin, ALT, Total proteins, ALB, GLB and Cholesterol in males, glucose, creatinine, Bilirubin, triglycerides in females of treatment groups. There was no dose-related effect on any of the biochemical parameters. In males, when compared with control group, bilirubin levels increased in low dose group (G2), ALT levels decreased in high dose (G4), total protein levels decreased in low dose (G2) and intermediate dose (G3) compared to control, albumin levels increased in low (G2), intermediate and high dose, Globulin levels decreased in low dose (G2), intermediate (G3) and high dose (G4), cholesterol levels increased in low dose (G2) and high dose (G4). In females, when compared with control group, creatinine levels decreased in intermediate dose group (G3), bilirubin levels decreased in high dose (G4), triglyceride levels increased in intermediate dose (G3). All the recovery group male and female animals showed no effect on biochemical parameters. This change in the biochemical parameters is considered to have arisen by chance or caused by slightly higher/lower control values and not to represent a change of any biological significance.
For tabular data, please refer to Table 5 and 6 below.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No significant changes in urinalysis were observed in the study.
For tabular data, please refer to pp. 65-67 in the attached report.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant changes in the functional observatory battery were observed. For tabular data, please refer to pp. 49-54 in the attached report.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant changes in absolute or relative organ weight were observed.
For tabular data, please refer to Table 7-10 below.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross findings were observed in the study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Observed microscopic lesions in G1 and G4 occured at comparable incidences and were considered to be common background findings in Wistar rats.
For tabular data, please refer to Table 11 below.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1. BODY WEIGHTS (G) – SUMMARY MALES

























































































































 



Group 1



Group 2



Group 3



Group 4



 


 


 


 


Day 1



Mean



130.6



132.2



133.3



132.0



SD



1.63



2.00



2.48



2.85



Min



128.4



130.7



130.9



128.3



Max



131.7



135.4



136.8



136.3



N



5



5



5



5



 


 


 


 


Day 8



Mean



146.9



148.5



148.5



148.3



SD



2.20



2.75



1.50



3.33



Min



144.3



147.0



146.7



143.3



Max



148.8



153.4



150.8



152.4



N



5



5



5



5



 


 


 


 


Day 15



Mean



161.7



163.8



163.9



163.9



SD



1.38



3.51



1.29



2.96



Min



159.9



161.4



162.1



159.2



Max



163.3



169.9



165.4



167.2



N



5



5



5



5



 


Table 1.BODY WEIGHTS (G) – SUMMARY (CONTD.) MALES





















































































 



Group 1



Group 2



Group 3



Group 4



 


 


 


 


Day 22



Mean



176.8



178.2



179.2



179.1



SD



1.48



3.29



1.98



3.12



Min



175.1



175.9



176.7



174.0



Max



178.8



184.0



181.5



182.4



N



5



5



5



5



 


 


 


 


Day 28



Mean



190.2



190.5



192.6



192.5



SD



1.59



2.53



2.72



3.41



Min



187.8



188.3



189.5



188.6



Max



192.1



194.6



196.0



196.1



N



5



5



5



5



 


Table 2. BODY WEIGHTS (G) – SUMMARY FEMALES

























































































































 



Group 1



Group 2



Group 3



Group 4



 


 


 


 


Day 1



Mean



130.1



129.7



129.9



129.8



SD



2.91



2.93



2.83



2.84



Min



125.7



126.5



126.6



127.2



Max



133.4



133.7



133.7



134.4



N



5



5



5



5



 


 


 


 


Day 8



Mean



145.0



145.8



146.6



145.6



SD



3.30



3.76



3.09



3.62



Min



140.1



141.2



143.4



141.1



Max



149.3



151.1



151.1



150.2



N



5



5



5



5



 


 


 


 


Day 15



Mean



160.2



161.4



162.7



161.4



SD



3.08



3.97



3.68



4.11



Min



156.4



156.3



158.6



156.8



Max



164.8



167.3



167.0



166.7



N



5



5



5



5



 


Table 2. BODY WEIGHTS (G) – SUMMARY (CONTD.) FEMALES





















































































 



Group 1



Group 2



Group 3



Group 4



 


 


 


 


Day 22



Mean



175.0



175.8



177.1



175.9



SD



2.69



4.10



3.98



4.27



Min



171.1



170.0



173.2



171.1



Max



178.6



181.5



182.6



180.4



N



5



5



5



5



 


 


 


 


Day 28



Mean



185.9



185.8



188.0



187.1



SD



3.03



3.70



3.50



3.84



Min



182.7



180.6



184.8



182.6



Max



190.6



190.6



193.2



191.3



N



5



5



5



5



 


Table 3. HEMATOLOGY SUMMARY – MALES




























































































































































































































































































































































































Week -04



 



RBC



Hb



PCV



MCV



MCH



MCHC



Reticulocyte



x106Cells/µL



g/dL



%



fL



pg



g/dL



%



Group 1



Mean



8.12



14.78



45.44



56.02



32.54



32.54



1.84



SD



0.299



0.335



1.504



2.150



0.989



0.989



0.039



Group 2



Mean



8.02



15.10



47.86



59.80



18.84



31.54



1.86



SD



0.647



0.787



1.858



2.781



0.541



0.654



0.049



Group 3



Mean



8.41



14.50



48.92



58.22



17.26



29.78



1.87



SD



0.441



2.190



3.044



3.072



2.543



5.094



0.049



Group 4



Mean



8.11



15.24



47.60



58.74



18.78



32.04



1.88



SD



0.360



0.336



2.021



1.050



0.502



0.808



0.059



 



PLT



WBC



DC (%)



PT



APT T



X103cel ls/µl



X103c


ells/ µl



 


Neut



 


Lymph



 


Mono



 


Eos



 


Baso



 


Sec



 


Sec



Group 1



Mean



882.80



21.54



10.60



84.00



3.60



1.80



0.00



17.20



43.00



SD



94.896



2.644



2.191



2.345



0.548



0.447



0.000



1.095



3.464



Group 2



Mean



824.80



20.54



11.20



84.60



2.80



1.40



0.00



17.20



41.80



SD



106.251



4.239



2.490



3.647



0.837



0.548



0.000



1.095



1.095



Group 3



Mean



901.60



16.06



13.40



81.60



3.20



1.80



0.00



17.20



41.40



SD



155.191



6.491



4.336



6.025



1.643



0.447



0.000



1.095



1.342



Group 4



Mean



678.50



13.78*



15.80



79.60



3.20



1.40



0.00



17.20



42.80



SD



327.366



2.266



5.167



3.782



0.837



0.894



0.000



1.095



1.304



*Significant at p ≤ 0.05 level with group 1



Week -06



 



 



RBC



Hb



PCV



MCV



MCH



MCHC



Reticulocyte


 

x106Cells/µL



g/dL



%



fL



pg



g/dL



%


 

Group 1R



Mean



8.63



15.54



49.04



56.88



31.64



31.64



1.85


 

SD



0.226



0.498



1.616



2.241



0.428



0.428



0.073


 

Group 4R



Mean



8.90



16.16



51.36



57.70



18.16



31.48



1.87


 

SD



0.389



0.680



2.502



0.579



0.321



0.497



0.082


 

 



PLT



WBC



DC (%)



PT



AP TT


 

x103cells


/µL



x103cells


/ µL



Neut



Lymph



Mono



Eos



Baso



Sec



Sec


 

 


Group 1R



Mean



 


784.00



 


15.06



 


15.20



 


79.00



 


3.60



 


2.20



 


0.00



16.4


0



42.2


0


 

SD



 


87.227



 


4.194



 


2.490



 


3.391



 


0.894



 


0.447



 


0.000



1.14


0



2.77


5


 

 


Group 4R



Mean



 


805.40



 


17.12



 


14.20



 


80.40



 


3.40



 


2.00



 


0.00



16.8


0



42.4


0


 

SD



 


147.025



 


3.432



 


1.643



 


1.140



 


0.548



 


0.000



 


0.000



1.09


5



2.79


3


 

 


Table 4. HEMATOLOGY SUMMARY – FEMALES



























































































































































































































































































































































































Week -04



 



RBC



Hb



PCV



MCV



MCH



MCHC



Reticulocyte



x106Cells/µL



g/dL



%



fL



pg



g/dL



%



Group 1



Mean



7.67



15.40



46.22



60.30



20.10



35.34



1.84



SD



0.520



0.778



2.600



1.471



0.632



4.286



0.055



Group 2



Mean



7.41



14.38



43.20



58.36



19.44



33.34



1.85



SD



0.640



1.013



3.652



0.817



0.550



0.723



0.056



Group 3



Mean



7.45



14.74



45.04



60.54



19.80



30.72



1.95



SD



0.382



0.351



0.921



2.454



0.570



4.803



0.099



Group 4



Mean



7.24



14.50



43.30



59.90



20.06



33.48



7.24



SD



0.578



0.831



2.788



1.134



0.594



0.729



0.578



 



PLT



WBC



DC (%)



PT



AP TT



X103cel ls/µl



X103cel ls/ µl



Neut



Lymp h



Mono



Eos



Baso



Sec



Sec



 


Group 1



Mean



 


784.20



 


12.44



 


15.80



 


83.00



 


3.20



 


1.40



 


0.00



 


17.20



42.6


0



SD



 


89.926



 


3.197



 


8.871



 


5.431



 


1.095



 


0.548



 


0.000



 


1.095



3.71


5



 


Group 2



Mean



 


857.40



 


19.94



 


13.40



 


82.40



 


2.80



 


1.40



 


0.00



 


17.60



41.8


0



SD



 


65.416



 


6.303



 


1.817



 


2.881



 


0.837



 


0.548



 


0.000



 


0.894



1.09


5



 


Group 3



Mean



 


883.60



 


15.04



 


12.40



 


83.20



 


2.80



 


1.60



 


0.00



 


16.80



41.8


0



SD



138.14


8



 


5.772



 


2.510



 


3.271



 


0.447



 


0.548



 


0.000



 


1.095



1.09


5



 


Group 4



Mean



 


863.60



 


12.46



 


12.60



 


83.40



 


2.40



 


1.60



 


0.00



 


17.20



42.4


0



 



SD



 


87.048



 


3.446



 


2.881



 


4.336



 


1.140



 


0.548



 


0.000



 


1.095



2.51


0



 



Week -06



 



 



RBC



Hb



PCV



MCV



MCH



MCHC



Reticulocytes



 



x106Cells/µL



g/dL



%



fL



pg



g/dL



%



 



 


Group 1R



Mean



8.20



15.48



47.76



58.32



18.92



32.44



1.87



 



SD



0.425



0.585



2.089



2.203



1.083



0.820



0.036



 



 


Group 4R



Mean



8.26



15.64



48.92



59.28



18.96



32.00



1.87



 



SD



0.455



0.472



2.321



1.791



0.680



0.620



0.036



 



 



PLT



WBC



DC (%)



PT



AP TT



 



x103cells/ µL



x103cell s/ µL



Neut



Lymph



Mono



Eos



Baso



Sec



Sec



 



 


Group 1R



Mean



 


783.40



 


15.44



 


12.40



 


82.80



 


3.20



 


1.60



 


0.00



16.4


0



41.


80



 



SD



 


63.885



 


3.523



 


3.050



 


3.564



 


0.447



0.54


8



 


0.000



1.14


0



3.4


21



 



 


Group 4R



Mean



 


867.60



 


13.56



 


11.20



 


84.80



 


2.60



 


1.40



 


0.00



16.2


0



40.


80



 



SD



 


104.333



 


2.651



 


1.924



 


2.683



 


0.548



0.54


8



 


0.000



0.83


7



3.1


14



 



 


Table 5. CLINICAL BIOCHEMISTRY SUMMARY – MALES
























































































































































































































































 



 


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



78.80



47.60



0.70



0.07



59.20



147.80



427.00



7.52



SD



24.160



10.550



0.000



0.012



15.627



15.450



82.592



0.342



 


Group 2



Mean



77.20



46.00



0.78



0.09!



55.00



162.00



308.00



6.96!



SD



8.044



12.708



0.084



0.011



7.141



26.739



155.816



0.097



 


Group 3



Mean



90.40



36.00



0.74



0.07



46.20



155.60



312.60



6.76#



SD



10.502



14.782



0.055



0.010



5.718



17.387



150.324



0.152



 


Group 4



Mean



72.80



56.60



0.74



0.08



37.40*



162.20



321.00



7.14



SD



14.822



7.057



0.055



0.008



3.507



16.858



162.691



0.404



 



 



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



2.61



4.92



41.80



95.20



139.28



5.27



99.14



1.38



0.53



SD



0.222



0.466



3.633



27.289



1.018



0.880



1.141



0.779



0.086



Group 2



Mean



3.01!



3.84!



57.20!



70.20



140.58



4.99



100.66



2.32



0.78



SD



0.244



0.313



8.786



6.943



0.722



0.358



1.301



1.639



0.088



Group 3



Mean



3.12#



3.64#



49.40



73.80



143.90



4.89



102.60



1.58



0.86



SD



0.175



0.207



10.644



36.602



8.514



0.977



3.574



0.383



0.092



Group 4



Mean



3.04*



4.10*



57.00*



96.20



140.80



5.14



100.84



2.36



0.74



SD



0.213



0.308



7.681



49.877



1.693



0.328



1.950



0.760



0.065



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



 


Table 5. CLINICAL BIOCHEMISTRY SUMMARY – MALES (CONTD.)



































































































































































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



106.40



42.60



0.70



0.09



42.20



147.00



433.20



6.64


 

SD



18.229



4.615



0.000



0.018



10.010



16.000



115.818



0.251


 

Group 4R



Mean



94.80



47.20



0.74



0.08



42.60



146.20



224.00



6.90


 

SD



9.524



3.114



0.055



0.015



8.081



19.992



78.026



0.187


 

 



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



3.02



3.64



44.80



122.80



140.82



5.36



101.04



1.90



0.83



SD



0.104



0.270



13.368



43.797



0.680



0.129



2.257



1.020



0.080



Group 4R



Mean



2.96



3.94



57.80



109.80



140.90



5.05



103.02



1.58



0.75



SD



0.148



0.207



10.640



37.930



0.721



0.516



1.514



0.988



0.066



 


 


Table 6. CLINICAL BIOCHEMISTRY SUMMARY – 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



88.20



50.20



0.84



0.12



41.00



145.60



190.40



7.00



SD



9.121



3.962



0.055



0.035



5.385



16.920



70.769



0.224



 


Group 2



Mean



78.40



43.00



0.80



0.11



44.80



153.60



221.80



7.36



SD



6.804



9.539



0.000



0.023



2.280



10.431



98.609



0.428



 


Group 3



Mean



75.40



53.00



0.74#



0.09



53.80



152.00



284.40



7.24



SD



18.447



11.726



0.055



0.027



13.936



18.069



97.999



0.089



 


Group 4



Mean



114.60!



59.00



0.82



0.07*



33.60



165.60



182.00



7.02



SD



7.701



5.431



0.045



0.007



8.849



10.574



47.911



0.319



 



 



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



3.18



3.82



48.58



63.40



139.10



4.58



100.48



2.12



0.83



SD



0.114



0.277



24.039



15.258



0.308



0.160



0.986



0.915



0.086



Group 2



Mean



3.01



4.36



62.80



82.20



151.72



4.83



102.18



1.64



0.70



SD



0.214



0.550



13.535



20.389



26.888



0.065



0.680



0.835



0.127



Group 3



Mean



3.09



4.16



62.60



121.60#



139.52



5.25



101.14



1.96



0.74



SD



0.203



0.288



14.363



54.998



1.165



0.946



1.665



0.971



0.099



Group 4



Mean



3.03



3.98



60.40



70.60



140.62



4.70



102.32



1.30



0.76



SD



0.112



0.396



7.021



32.708



1.411



0.136



1.399



1.356



0.095



!#* Significant at p ≤0.05 level with group 1



 


Table 6. CLINICAL BIOCHEMISTRY SUMMARY – FEMALES (CONTD.)



































































































































































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



84.00



47.40



0.82



0.11



43.40



156.20



232.00



7.18


 

SD



6.519



7.701



0.045



0.023



2.302



13.882



101.993



0.295


 

 


Group 4R



Mean



109.40



51.60



0.86



0.09



35.00



154.80



180.00



7.40


 

SD



15.043



9.072



0.055



0.008



5.244



12.194



30.927



0.367


 

 



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



3.21



3.98



61.00



68.00



140.00



4.90



102.38



2.58



0.81



SD



0.067



0.268



4.528



28.169



0.791



0.174



1.057



0.460



0.050



Group 4R



Mean



3.47



3.94



62.00



73.20



140.08



4.83



102.52



1.74



0.88



SD



0.185



0.371



7.583



23.091



1.092



0.319



0.726



1.680



0.102



 


Table 7. ORGAN WEIGHTS (GRAM) – SUMMARY MALES























































































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


Body Weight (G)



Mean



188.0



187.8



190.0



190.0



225.5



226.6



SD



1.98



1.75



2.94



2.94



2.64



2.25



N



5



5



5



5



5



5



 


Brain



Mean



1.5430



1.7382



1.7330



1.4304



1.7530



1.6732



SD



0.04971



0.06478



0.15706



0.47356



0.22155



0.05758



N



5



5



5



5



5



5



 


 


Heart



Mean



0.8344



0.9568



0.9258



0.8464



1.1662



0.9258



SD



0.17248



0.15895



0.20795



0.08252



0.14443



0.18871



N



5



5



5



5



5



5



 


 


Liver



Mean



8.1652



8.7424



8.7168



8.0322



11.8724



8.4078



SD



1.15197



1.56685



1.48162



0.57004



3.58843



1.77379



N



5



5



5



5



5



5



 


 


Spleen



Mean



1.0728



1.2652



0.9974



0.8522



1.0582



2.0620



SD



0.08228



0.30047



0.30545



0.12619



0.08389



2.57129



N



5



5



5



5



5



5



 


Kidneys



Mean



1.5832



1.5464



1.6710



1.4906



2.0448



1.6392



SD



0.18549



0.17077



0.38220



0.10094



0.26631



0.30563



N



5



5



5



5



5



5



 


Table 7. ORGAN WEIGHTS (GRAM) – SUMMARY MALES (CONTD.)


 



























































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


 


Adrenals



Mean



0.0566



0.0776



0.0672



0.0544



0.0482



0.0668



SD



0.02176



0.02492



0.02007



0.00777



0.00712



0.00705



N



5



5



5



5



5



5



 


Testes



Mean



2.0332



0.7208



2.4070



2.0160



2.6044



2.5612



SD



1.05259



0.93357



0.36166



0.89828



0.12315



0.29673



N



5



5



5



5



5



5



 


Epididymes



Mean



0.3388



0.3450



0.3526



0.4338



0.6208



0.3088



SD



0.09377



0.04554



0.08898



0.09132



0.43014



0.11852



N



5



5



5



5



5



5



 


Thymus



Mean



0.9638



1.2862



1.2254



1.0340



1.3868



1.0706



SD



0.36533



1.02182



0.61066



0.15234



0.39887



0.46435



N



5



5



5



5



5



5



Pro + Sem. Vesical with Coagulating glands



Mean



1.1048



1.0556



1.2926



0.9480



1.2198



1.3300



SD



0.44539



0.54158



0.37314



0.49306



0.63471



0.40374



N



5



5



5



5



5



5



 


Table 8. ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES























































































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


Body Weight (G)



Mean



183.6



183.6



185.9



184.7



222.7



227.2



SD



3.08



3.53



3.75



3.86



2.60



2.61



N



5



5



5



5



5



5



 


Brain



Mean



1.6490



1.6490



1.6518



1.7262



1.6706



1.7658



SD



0.28800



0.10695



0.09125



0.04262



0.12188



0.08867



N



5



5



5



5



5



5



 


 


Heart



Mean



0.7794



0.7518



0.8738



0.7846



0.7848



0.7976



SD



0.08175



0.09130



0.47131



0.03816



0.14482



0.09502



N



5



5



5



5



5



5



 


 


Liver



Mean



7.4798



6.9852



6.7412



6.4838



6.4204



6.8684



SD



0.63619



0.49866



0.81772



0.57539



0.28058



1.25711



N



5



5



5



5



5



5



 


 


Spleen



Mean



0.9762



1.0416



0.9322



0.8784



0.8114



0.7872



SD



0.18277



0.19668



0.07474



0.22041



0.07522



0.15867



N



5



5



5



5



5



5



 


Kidneys



Mean



1.4362



1.3130



1.3230



1.2294



1.3412



1.4292



SD



0.09410



0.12204



0.18534



0.07887



0.16524



0.17327



N



5



5



5



5



5



5



 


Table 8. ORGAN WEIGHTS (GRAM) – SUMMARY FEMALES (CONTD.)































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


 


Adrenals



Mean



0.0626



0.0768



0.0712



0.0692



0.0906



0.0778



SD



0.01337



0.03611



0.01686



0.01585



0.05127



0.01457



N



5



5



5



5



5



5



 


 


Ovaries



Mean



0.7036



0.6836



0.2088



0.1798



0.2632



0.3420



SD



1.03313



0.91919



0.07944



0.06956



0.13995



0.44364



N



5



5



5



5



5



5



 


 


Uterus



Mean



0.4514



0.5074



0.2090



0.3390



0.3394



0.3248



SD



0.11485



0.53257



0.08184



0.08909



0.27956



0.16199



N



5



5



5



5



5



5



 


 


Thymus



Mean



0.4582



0.5594



0.4322



0.3574



0.3838



0.3694



SD



0.21840



0.35996



0.20254



0.12132



0.31577



0.06308



N



5



5



5



5



5



5



 


Table 9. ORGAN WEIGHTS RATIO (%) – SUMMARY MALES



























































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


Body Weight (G)



Mean



188.0



187.8



190.0



190.4



225.5



226.6



SD



1.98



1.75



2.94



3.41



2.64



2.25



N



5



5



5



5



5



5



 


Brain



Mean



0.8207



0.9258



0.9122



0.7496



0.7780



0.7385



SD



0.02740



0.03976



0.08319



0.24591



0.10199



0.03021



N



5



5



5



5



5



5



 


 


Heart



Mean



0.4444



0.5090



0.4868



0.4443



0.5171



0.4086



SD



0.09613



0.08011



0.10691



0.03796



0.06335



0.08436



N



5



5



5



5



5



5



 


 


Liver



Mean



4.3427



4.6518



4.5857



4.2171



5.2691



3.7113



SD



0.63238



0.80538



0.76966



0.24423



1.61179



0.78835



N



5



5



5



5



5



5



 


 


Spleen



Mean



0.5704



0.6728



0.5240



0.4478



0.4693



0.9089



SD



0.04130



0.15404



0.15559



0.06641



0.03585



1.13188



N



5



5



5



5



5



5



 


Table 9. ORGAN WEIGHTS RATIO (%) – SUMMARY MALES (CONTD.)























































































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


Kidneys



Mean



0.8426



0.8230



0.8791



0.7832



0.9068



0.7236



SD



0.10575



0.08447



0.20039



0.05407



0.11811



0.13762



N



5



5



5



5



5



5



 


 


Adrenals



Mean



0.0301



0.0414



0.0353



0.0286



0.0214



0.0295



SD



0.01158



0.01342



0.01020



0.00442



0.00318



0.00330



N



5



5



5



5



5



5



 


Testes



Mean



1.0838



0.3852



1.2662



1.0603



1.1549



1.1303



SD



0.56357



0.50072



0.18394



0.47313



0.04812



0.13338



N



5



5



5



5



5



5



 


Epididymes



Mean



0.1806



0.1836



0.1854



0.2273



0.2213



0.1365



SD



0.05174



0.02314



0.04612



0.04421



0.22054



0.05340



N



5



5



5



5



5



5



 


Thymus



Mean



0.5140



0.6813



0.6473



0.5436



0.6159



0.4719



SD



0.19857



0.53443



0.32900



0.08214



0.18016



0.20480



N



5



5



5



5



5



5



Pro + Sem. Vesical with Coagulating glands



Mean



0.5875



0.5608



0.6813



0.5007



0.5421



0.5857



SD



0.23452



0.28481



0.20256



0.26528



0.28509



0.17395



N



5



5



5



5



5



5



 


Table 10. ORGAN WEIGHTS RATIO (%) – SUMMARY FEMALES























































































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


Body Weight (G)



Mean



183.6



183.6



185.9



184.7



222.7



227.2



SD



3.08



3.53



3.75



3.86



2.60



2.61



N



5



5



5



5



5



5



 


Brain



Mean



0.8979



0.8990



0.8884



0.9350



0.7504



0.7774



SD



0.15129



0.06615



0.04569



0.03864



0.05824



0.04080



N



5



5



5



5



5



5



 


 


Heart



Mean



0.4247



0.4102



0.4716



0.4250



0.3521



0.3513



SD



0.04570



0.05469



0.25812



0.02442



0.06263



0.04372



N



5



5



5



5



5



5



 


 


Liver



Mean



4.0724



3.8064



3.6250



3.5092



2.8833



3.0245



SD



0.29584



0.27864



0.41819



0.29140



0.13552



0.55605



N



5



5



5



5



5



5



 


 


Spleen



Mean



0.5313



0.5674



0.5017



0.4761



0.3647



0.3464



SD



0.09671



0.10625



0.04320



0.12289



0.03784



0.06855



N



5



5



5



5



5



5



 


Kidneys



Mean



0.2043



0.2253



0.2303



0.6657



0.6028



0.6296



SD



0.02841



0.03481



0.03571



0.04328



0.08087



0.08059



N



5



5



5



5



5



5



 


Table 10. ORGAN WEIGHTS RATIO (%) – SUMMARY FEMALES (CONTD.)































































































































 



Group 1



Group 2



Group 3



Group 4



Group 1R



Group 4R



 


 


Adrenals



Mean



0.3576



0.3653



0.3559



0.0375



0.0409



0.0342



SD



0.02979



0.02776



0.03506



0.00876



0.02375



0.00605



N



5



5



5



5



5



5



 


 


ovaries



Mean



0.0882



0.0913



0.1025



0.0974



0.1185



0.1510



SD



0.01530



0.02638



0.02438



0.03789



0.06409



0.19642



N



5



5



5



5



5



5



 


Uterus



Mean



0.3496



0.3715



0.4139



0.1841



0.1523



0.1432



SD



0.06959



0.14679



0.18006



0.05147



0.12629



0.07161



N



5



5



5



5



5



5



 


 


Thymus



Mean



0.9552



0.9515



1.0181



0.1928



0.1734



0.1625



SD



0.09352



0.08186



0.05783



0.06338



0.14280



0.02663



N



5



5



5



5



5



5



 


Table 11. SUMMARY OF INCIDENCE OF MICROSCOPIC FINDINGS















































































































































































































































































































































































































































































































































































 



 


 


Organs/Microscopic findings



G1



G4



Male


0 mg/kg bw (Vehicle control)



Female 1000 mg/kg bw


(High dose)



Male


0 mg/kg bw (Vehicle control)



Female 1000 mg/kg bw


(High dose)



Liver



 



 



 



 



Vacoular degeneration



0/5



1/5



0/5



0/5



Foci of inflammation/ Necrosis in Perfortal/ Centrilobular


region



 


4/5



 


1/5



 


2/5



 


1/5



NAD



1/5



4/5



3/5



4/5



Spleen



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Lungs



 



 



 



 



Alveolar Hemorrhage



1/5



0/5



0/5



1/5



Alveolar


Inflammation/Peribrancheolar Lymphoid Hyperplesia



 


3/5



 


2/5



 


3/5



 


3/5



NAD



1/5



3/5



2/5



1/5



Heart



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Aorta



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Kidney



 



 



 



 



Tubular/Interptitial inflammation/Infilteration of


inflammatory cells



 


2/5



 


1/5



 


2/5



 


3/5



NAD



3/5



4/5



3/5



2/5



Adrenals



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Brain



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Pituitary



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Thyroid



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Trachea



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Oesophagus



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Duodenum



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Jejunum



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Ileum



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Colon



 



 



 



 



Sub mucosal Lymphoid tissue


Hyperplasia



1/5



1/5



0/5



1/5



NAD



4/5



4/5



5/5



4/5



Rectum



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Caecum



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Lymph Node



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Pancreas



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Thymus



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Urinary Bladder



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Testes



 



 



 



 



NAD



5/5



-



5/5



-



Prostate



 



 



 



 



Submucosal Inflammation



1/5



-



0/5



-



NAD



4/5



-



5/5



-



Seminal Vesicles with Coagulating Glands



 



 



 



 



NAD



5/5



-



5/5



-



Epididymes



 



 



 



 



NAD



5/5



-



5/5



-



Ovary



 



 



 



 



NAD



-



5/5



-



5/5



Uterus



 



 



 



 



NAD



-



5/5



-



5/5



Cervix + Vagina



 



 



 



 



NAD



-



5/5



-



5/5



Stomach



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Skeletal Muscle



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Eye



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Spinal Cord



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Bone Marrow (Sternum)



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Sciatic Nerve



 



 



 



 



NAD



5/5



5/5



5/5



5/5



Key- NAD: No Abnormality Detected

Applicant's summary and conclusion

Conclusions:
NOAEL was determined to be 1000 mg/kg bw/day in both sexes.
Executive summary:

A 28 days repeated dose toxicity study was performed in accordance with the OECD TG 407 under GLP conditions using male and female Wistar rats. In this study, the animals of age 6 -7 weeks were used. 5 animals per sex per dose were used in the study. A total of 6 dose groups were selected for the study viz. 0 mg/kg bw/day (control group), 250 mg/kg bw (Low Dose Group), 500 mg/kg bw (Intermediate Dose Group) and 1000 mg/kg bw (High Dose Group). Further, two recovery groups were also included in the study in order to check the reversibility of the effects that are observed due to the administration of the test chemical, namely, Control Group Recovery (0 mg/kg bw/day) and High Dose Recovery (1000 mg/kg bw/day). 5 animals per sex per dose were used in the study. The animals were treated daily once via oral gavage route. The vehicle used for the study was distilled water. The parameters that were checked in the study were clincal signs, clinical observations (behavioral parameters), body weight and feed consumption, hematological and clinical biochemistry parameters, urinalysis, absolute and relative organ weights, gross pathology and histopathology. The animals of main groups were dosed consecutively for 28 days and on day 29, they were sacrificed. The animals were kept in metabolic cages a day prior to sacrifice in order to collect urine for urinalysis. Before sacrifice, blood was collected from all the animals from all the dose groups, using isofluorane anesthesia for hematological and biochemistry parameters. The same procedure was followed for recovery group animals before sacrifice on day 43. The in-life observations showed that No clinical signs were observed in any of the vehicle control(G1), low(G2), intermediate (G3), high dose(G4), vehicle control recovery (G1R) and recovery high dose (G4R) group animals. No mortality was observed in vehicle control (G1), low (G2), intermediate (G3), high dose (G4), recovery control (G1R) and recovery high dose (G4R) group animals. No adverse effect on body weight was observed for treated groups when compared with control group. No significant and toxicologically relevant changes in the body weight between treated and control group were observed. No significant changes related to the test chemical in the feed consumption was observed in any of the treated and recovery groups when compared with vehicle control (G1) group. No adverse effects on opthalmological findings were observed during the study. In hematological parameters, males in high dose group (G4) were showed decrease in WBC count when compared with control group. However, no changes in the females were observed. Treatment with the test chemical had no effects on both male and female animals with respect to hematological parameters viz RBC, Hb, MCV, MCHC, PCV, PLT, PT and APTT. In males, when compared with control group, bilirubin levels increased in low dose group (G2), ALT levels decreased in high dose, total protein levels decreased in low dose and intermediate dose compared to control, albumin levels increased in low, intermediate and high dose, Globulin levels decreased in low dose, intermediate and high dose, cholesterol levels increased in low dose and high dose. In females, when compared with control group, creatinine levels decreased in intermediate dose group (G3), bilirubin levels decreased in high dose (G4), triglyceride levels increased in intermediate dose (G3). All the recovery group male and female animals showed no effect on biochemical parameters. This change in the biochemical parameters is considered to have arisen by chance or caused by slightly higher/lower control values and not to represent a change of any biological significance. No significant changes in any of the treated groups and recovery groups were observed in urinalysis findings. No significant and test chemical related findings were observed during functional observation battery. There were no test chemical related changes in the in absolute and relative organ weights in both sexes in main group and recovery group animals. No test chemical related effects were observed during the necropsy of the animals in any organs in main groups or also in the recovery group. No test chemical related changes in the histopathology was observed in the animals of all dose groups in main groups or also in the recovery group. Therefore, based on all the available data, it was observed that the test chemical did not induce any systemic and local toxicity in the tested animals in main groups and recovery groups, respectively. Therefore, on the basis of all the observations and results, it was concluded that the NOAEL for the test chemical was 1000 mg/kg bw/day.