1 Rat

Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Rats Reported by Nexus Clinical Research (India) Ltd.
REPORT
SUB-ACUTE TOXICITY STUDY ON
PYRITINOL DIHYDROCHLORIDE MONOHYDRATE IN RATS Date: 14/10/2011
Study Site
Appasaheb Birnale College of Pharmacy, South Shivaji Nagar, Miraj Road, Sangli, Maharashtra- 416 416
(CPCSEA Registration No. 843/AC/04)
Clinical Research Organization

Nexus Clinical Research (India) Ltd.
Anuj, Plot No. 45, 1st floor, Near DY Patil Stadium Mumbai - Pune Highway, Sec.13, Nerul (East) New Mumbai 400706
Sponsor of Study
Kusum Healthcare Pvt. Ltd. SP-289 (A), RIICO Industrial Area, Chopanki, Dist. Alwar-301019
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Rajasthan, India.
This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd.
GLP & GAE COMPLIANCE STATEMENT

The investigation reported in this document was performed by the Nexus Clinical Research (India) Ltd., New Mumbai 400 706, India at Appasaheb Birnale College of Pharmacy, South Shivaji Nagar, Miraj Road, Sangli, Maharashtra (CPCSEA Registration No. 843/AC/04). The study, carried out as reported in this document, confirms to the Quality Assurance guidelines followed at the study center. The animal study was performed in compliance with the guideline of Good Animal ethics practice followed at Animal Testing Unit and complied with the ethical norms laid down in guideline issued by CPCSEA, India. This report fully and accurately reflects the procedures used and data generated in the study. The work was performed in compliance with the guideline of Good Animal Ethics Practice and the study has been subjected to procedure and documentation inspection by the Quality Manager. This information contains confidential and trade secretes information. It should not be photo-copied or microfilmed; nor should be released in any form to an outside party. It is made available solely for the use by the sponsor to support the registration of the product described herein. Information contained herein should not be reviewed, abstracted, quoted or used to support the registration of any other product without the express written agreement of company/ sponsor of the study and Nexus Clinical Research (India) Ltd. This is to certify that the objectives laid down in the protocol were achieved and as nothing occurred to adversely affect the quality or integrity of the study, the data generated is considered to be valid. The report has been approved for issue. This report constitutes a true record of actions taken and results obtained in the study. Name Report reviewed and authorized by Dr. Amit Bhatt, President & CEO Nexus Clinical Research (India) Ltd. New Mumbai (India) Amol Mohite Nexus Clinical Research (India) Ltd. New Mumbai (India) Signature
Clinical Quality Associate
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SUB-ACUTE TOXICITY STUDY ON

PYRITINOL DIHYDROCHLORIDE MONOHYDRATE TABLETS IN RATS
Date of Conduct of Study: 06/09/2011 Date of completion of study: 05/10/2011 Date of Reporting: 14/10/2011 Animal Model: Wistar rats Period of Treatment : 30 days Sample: Pyritinol Dihydrochloride Monohydrate Tablets Description: Yellow color film coated, biconvex, round tablets with plain surface
(Batch No.: R&D/1a/C1/11); Mfg. Date: 07-2011)

Route of Administration : Oral Number of dose Groups: 04
Low Dose Intermediate Dose High dose Control
500 mg/kg 750 mg/kg 1000 mg/kg 1 ml/100gm Sterile water
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Number of animals: 6 males and 6 females in each group
Summary Results
Pyritinol Dihydrochloride Monohydrate Tablets manufactured by Kusum Healthcare Pvt. Ltd. presented for oral route of administration was subjected to sub-acute toxicity studies in rats. Three doses of the drug viz., low dose, intermediate dose and high dose had been employed in the study based on sub-acute toxicity study. Pyritinol Dihydrochloride Monohydrate tablets were tested in this study causes hepatotoxicity and pancreas toxicity at 750 mg/kg and 1000 mg/kg. The SGOT and SGPT enzyme level was increased that is marker of liver toxicity. These liver and pancreas toxicities were confirmed by histopathological changes observed at higher dose treated both male and female rat after 30 days treatment of test drug. Liver to body weight ratio were increased in male and female rat at 1000 mg/kg. There was no significant changes were observed in other organs i.e. heart, Kidney, lungs, brain and gonads in all doses treatment group with Pyritinol Dihydrochloride Monohydrate. Hematological analysis revealed no abnormalities attributable to the treatment. Species-dependent variations in the toxic response are evident in the study. Sex-dependent variations were not very evident in the toxic response of the animals.
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CONTENTS
Sr. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 11. 12. 13. 14. 18. 19. Introduction Objectives Materials & Method Results Inferences General Conclusions Project Team Place of Conduct of Study & Report issued by APPENDIX Mean Body Weights Mean Food Intake Mean Water Intake Mean Relative Organ Body Weight Ratio Blood Biochemical Parameters Hematological Parameters Urine Analysis Mortality Record (Rats) Page No.
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ABBREVIATIONS Abbreviations
CMR LMR IMR HMR CFR LFR IFR HFR SEM Hb RBC WBC ESR TG BUN SGOT SGPT Na K Cl PCV
Full form
CONTROL DOSE MALE RAT LOW DOSE MALE RAT INTERMEDIATE DOSE MALE RAT HIGH DOSE MALE RAT CONTROL DOSE FEMALE RAT LOW DOSE FEMALE RAT INTERMEDIATE DOSE FEMALE RAT HIGH DOSE FEMALE RAT STANDARD ERROR MEAN HAEMOGLOBIN RED BLOOD CORPUSCLES WHITE BLOOD CORPUSCLES ERYTHROCYTE SEDIMENTATION RATE TRIGLYCERIDES BLOOD UREA NITROGEN SERUM GLUTAMIC-OXALOACETIC TRANSAMINASE SERUM GLUTAMIC-PYRUVIC TRANSAMINASE SODIUM POTASSIUM CHLORINE PACKED CELL VOLUME
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SUB-ACUTE TOXICITY STUDY

PYRITINOL DIHYDROCHLORIDE MONOHYDRATE TABLETS INTRODUCTION Pyritinol Dihydrochloride Monohydrate is given to increase cerebral brain activity in patients with impaired cerebral function. It helps to improve memory, concentration, co-ordination, physical weakness and psychological weaknesses. It works by promoting the brain's glucose reuptake. Pyritinol Dihydrochloride Monohydrate is often prescribed for various cerebrovascular disorders. PRODUCT DETAILS Nonproprietary Name: Pyritinol Dihydrochloride Monohydrate Chemical Name: 5,5;-[dithiobis(methylene)]bis[4-(hydroxymethyl)-2-methylpyri-din-3ol]dihydrochloride monohydrate Structural Formula:
Empirical Formula: C16H20N2O4S2.2 (HCl) 10049-83-9
Molecular Weight: 441.39 g/mol CAS Number: OBJECTIVES 1. 2. To assess the Sub-acute toxicity of Pyritinol Dihydrochloride Monohydrate in rats. To evaluate the toxic effect of Pyritinol Dihydrochloride Monohydrate for oral use at different dose levels i.e. low, intermediate and high dose.
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3.
To evaluate the toxicity (if any) by studying various hematological and tissue parameters of the treated animals.
4.
To assess the safety of Pyritinol Dihydrochloride Monohydrate for use in the rat models.
MATERIALS & METHOD: SAMPLE: Pyritinol Dihydrochloride Monohydrate for oral use PRESENTATION: Tablets Finished formulation for oral use (Batch No.: R&D/1a/C1/11); Mfg. Date: 07-2011) MANUFACTURER Kusum Healthcare Pvt. Ltd. ROUTE OF ADMINISTRATION: Oral ANIMALS USED: Wistar rats were used as experimental models. Both species were supplied as fresh animals (untreated normal animals with filial lineage records) by Krishna Institutes of Medical Sciences Karad, Maharashtra. ANIMAL GROUPING: The selected animals were separated on the basis of sex and were assigned to the requisite experimental groups. Each experimental group consisted of twelve animals, six from each sex. The groups were assigned to the animals by randomization technique using a random distribution table. ANIMAL MAINTENANCE: The animals were housed in polyurethane cages in groups of six. They were exposed to room temperature (29 2C) with 12 hours light and dark cycle. The relative humidity was generally maintained at 40-70%. Humidity indices lower than 40% and higher than 70% were avoided for prolonged periods. All the animals were acclimatized for 5 days to the animal house conditions prior to the start of experimental protocol and fed with standard feed (pellets) supplied by Pranav Agro Industry Ltd., Sangli. Water was supplied to every cage ad libitum, in glass bottles. The cages were placed randomly on the racks and their positions changed daily to avoid any bias or any influence due to the specific location of the cage. Standard hygiene procedures were implemented.
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PROCEDURE: Rats were weighed and marked. The sub-acute oral toxicity of Pyritinol Dihydrochloride Monohydrate Tablets was evaluated in rat using the schedule Y and Organization of Economic Co-operation and Development (OECD) guideline for testing of chemicals. Healthy adult wistar rat received repeated doses of Pyritinol Dihydrochloride Monohydrate at different dose levels for different groups. Finally, these animals were then maintained for further 30 days drug administration with daily observations like sign of toxicity or mortality, organ-body weight ratio and food- water consumption. DOSE AND ADMINISTRATION: The necessary dosages of Pyritinol Dihydrochloride Monohydrate for oral use were made DAILY in sterile water just before use. Also, sterile water was used in animals of control group as sham treatment method. Sterile disposable syringes with needles were used for oral administration of the drug. Oral administration of the drug was done on each day, after physical restriction of the animal. The treatment was done daily in the afternoon including holidays. The drug was administered in three doses (see dosage chart) with corresponding controls. The animals were divided into four groups with control and each experimental group was administered with the drug at doses given below; PYRITINOL DIHYDROCHLORIDE MONOHYDRATE DOSAGE Groups No. of animals used Daily dosage of Pyritinol Dihydrochloride Monohydrate for oral use Rats 500 mg/kg 750 mg/kg 1000 mg/kg 1 ml/100 gm sterile water
Page This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd.
Low Dose Intermediate Dose High Dose Control
Males 6 6 6 6
Females 6 6 6 6
NOTE: The maximum adult human dose reported for Pyritinol Dihydrochloride Monohydrate is 600 mg/day. In the present study the low dose was higher to the adult human dose while the intermediate dose and high doses was lover to LD50 value. The period of administration was standardized for all dose groups to avoid mortality. A control group was also maintained comprising of 6 male and 6 female rats. Sterile water was administrated to the animals of control group. The administration was done once daily in the afternoon, for a period of 30 days (including holidays) PERIOD OF TREATMENT: 30 (thirty) days including holidays. PARAMETERS OBSERVED: The general behavioral symptoms and any local reactions were observed during the treatment period. Mortality, if any, in all the groups, during the course of the experiment was recorded. Food intake was measured daily and the mean food intake for each interval of one day was computed separately for each group. Known amounts of the food and water were supplied to each group and their daily food intake and water intake were recorded. The animals which died (if any) during the course of the experiment were subjected to autopsy. Those which survived were sacrificed after 24 hours of the last dosage by overdose of anesthetic ether and subjected to autopsy. But, none of the animal died during the study before sacrifice urine samples were collected by keeping the animals in metabolic cages. Urine samples were subjected to routine qualitative urinalysis. Before autopsy blood samples were collected by cardiac puncture under ether anesthesia. Blood samples were analyzed for routine hematological parameters and serum was analyzed for clinical biochemical parameters. The body weights were recorded as soon as the animals were anesthetized and then dissected. After a gross pathological examination, liver, adrenal, Lungs, gonads, spleen, heart and kidney were excised during autopsy. These organs were quickly blotted, weighed on a digital balance and samples of the tissue were processed for histopathological studies.
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The organ weights were used to calculate the organ/body weight ratio. The tissues were fixed in neutral formalin and processed for routine Haematoxylin-Eosin staining. Any other organ showing signs of gross pathology or areas with tissue lesions were also similarly processed. STATISTICAL ANALYSIS Body weights, absolute and relative organ weights, and clinical pathology data were analyzed statistically. Quantitative results were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnetts t-test (If the ANOVA indicated statistical significance among experimental groups then the Dunnetts t-test was used to delineate which groups (if any) differed from the control). The term significant was used throughout the text of the report to indicate statistical significance at p<0.05. RESULTS: A. GENERAL SYMPTOMS There were no significant change in behavior and clinical sign was noticed in the treated male and female rat when compared with control. B. MORTALITY IN RATS No mortality was recorded in treated rats at all dose levels. C. FOOD INTAKE Food intake was measured daily and the mean food intake for each interval of three day was computed separately for each group. The results of the treated animals were compared with those of the control animals. No significant changes in food intake were observed in low dose, intermediate and high dose treated in both male and female rat groups throughout the treatment period when compared with their control groups.
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Mean Food consumption (gm) measured during subacute toxicity study in Male rats at time interval
Mean Food consumption (gm)
25 20 15 10
CMR LMR IMR
5 0
0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
HMR
The results were expressed as Mean. The data was analyzed using One-way Analysis of Variance (ANOVA) followed by Dunnetts t- test. No statistical difference between control and test groups (p > 0.05), statistically significant at (p<0.05), (p<0.01), (p<0.001).
Food consumption (gm) measured during sub-acute toxicity study in female rats at time interval
30
Food consumption (gm)
25 20 15 10
CFR LFR IFR HFR
5
0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
The results were expressed as Mean. The data was analyzed using One-way Analysis of Variance (ANOVA)
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significant at (p<0.05), (p<0.01), (p<0.001).
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followed by Dunnetts t- tes.t No statistical difference between control and test groups (p > 0.05), statistically
D. WATER INTAKE Water intake was measured daily and the mean water intake for each interval of three day was computed separately for each group. The results of the treated animals were compared with those of the control animals. No Significant change in water intake was observed in low, intermediate and higher dose treatment groups in both male and female rat when compared with their control groups. Mean Water consumption (ml) measured during Subacute toxicity study in Male rats at time interval
Mean Water consumption (ml)
45 40 35 30 25 20 15 10 5 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
CMR LMR IMR
HMR
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Water consumption (ml) measured during Subacute toxicity study in Female rat at time interval
Water consumption (ml) 50 40 30 20 10
CFR LFR IFR HFR
0
0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
E. BODY WEIGHT Body weight was measured daily and the mean body weight for each interval of three day was computed separately for each group before analysis. The results of the treated animals were compared with those of the control animals. No significant changes in the body weight were observed in low, intermediate and high treatment groups of male and female rat when compared with control group.
Mean Body weight (gm) measured during Subacute toxicity study in Male rats at time interval
280 Body weight (gm) 260 240 220 200 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
CMR LMR IMR HMR
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Rats Reported by Nexus Clinical Research (India) Ltd. The results were expressed as Mean SEM (n=6). The data was analyzed using One-way Analysis of Variance (ANOVA) followed by Dunnetts t- test. No statistical difference between control and test groups (p > 0.05), statistically significant at (p<0.05), (p<0.01), (p<0.001).
Mean Body weight (gm) measured during Subacute toxicity study in Female rats at time interval
300 Mean Body weight (gm) 250 200 150 100 50 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
CFR
LFR
IFR HFR
The results were expressed as Mean SEM (n=6). The data was analyzed using One-way Analysis of Variance (ANOVA) followed by Dunnetts t- test. No statistical difference between control and test groups (p > 0.05), statistically significant at (p<0.05), (p<0.01), (p<0.001).
F. AUTOPSY Autopsy was carried out on animals, which died (if any) during the course of the treatment, and after completion of observational period (30 days). No mortality was recorded in treated rats during the course of the treatment at all dose groups. G. ORGAN - BODY WEIGHT RATIO IN RAT There was significant increased in liver to body weight ratio when compared with control group in intermediate and higher dose treated male and female rats. There was no significant changes in both male and female rat lungs, Heart, Spleen, Kidney, Brain and Gonads to body weight ratio when compared with control group at low dose, intermediate and higher dose treated group.
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Mean relative organ body weight ratio
Mean relative organ body weight ratio in Male rats at 30 th day in Sub-acute toxicity study
0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 Liver Lungs Heart Spleen Kidney Brain Gonads Organ
CMR LMR IMR HMR
Relative Mean relative organ body weight ratio
Relative Mean relative organ body weight ratio in Female rats at 30 th day in Sub-acute toxicity study
0.05 0.04
0.03
0.02 0.01 0 Liver Lungs Heart Spleen Kidney Brain Ovary Organ
CFR LFR IFR
HFR
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H. HISTOPATHOLOGY: In the microscopic evaluation of the histological sections the following observations were noted. Any sex specific changes, if seen, are mentioned separately. 1. Liver Male: The histopathology of liver of control and lower dose and intermediate treated group of male rat showed normal hepatic cells (HC) with central vein (CV) and sinusoidal dilation (SS) but Mild congestion, swollen hepatocytes and dilatation of sinusoids were visible in animals of high dose groups.
Figure: Effect on histopathology of liver of male rats after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
Female: The histopathology of liver of control treated group of female rat showed normal hepatic cells (HC) with central vein (CV) and sinusoidal dilation (SS). But mild congestion, swollen hepatocytes and dilatation of sinusoids were visible in animals of high dose groups.
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Figure: Effect on histopathology of liver of female rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
2. Kidney Male: Male rat treated with high dose of the drug showed normal tubular epithelium, normal glomeruli and normal bowman's capsules in all dose groups treated rats.
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Rats Reported by Nexus Clinical Research (India) Ltd. Figure: Effect on histopathology of Kidney of male rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
Female: The histopathology of kidney of control, low dose, intermediate dose and higher dose treated groups of female rat showed normal renal corpuscles (RC), tubules (T) and Bowmans space (BS).
Figure: Effect on histopathology of Kidney of female rats after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
3. Spleen Male: The histopathology of spleen of control and treated group of male rat showed normal red pulp (RP) and white pulp (WP).
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Figure: Effect on histopathology of spleen of male rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
Female: The histopathology of spleen of control and treated group of female rat showed normal red pulp (RP) and white pulp (WP).
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Rats Reported by Nexus Clinical Research (India) Ltd. Figure: Effect on histopathology of spleen of female rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
4. Gonads Male: The photomicrographs of testes of control male rats, showed the normal histoarchitechure such as sertoli cells of normal size with normal stage of spermatogenesis with normal somniferous tubules spermatogenesis.
Figure: Effect on histopathology of Testis of male rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
Female: The photomicrographs of control female mice ovary and test drug treated mice, showed normal developing follicles with oocytes and corpus luteum.
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Figure: Effect on histopathology of ovary of female rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose 5.
Heart
Male: The histopathology of heart of control and treated group of male rat showed normal muscle
fiber (MF), connective tissue (CT) and nucleus of myocytes (NM).
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Rats Reported by Nexus Clinical Research (India) Ltd. Figure: Effect on histopathology of Heart of male rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose. Female: The histopathology of heart of control and treated group of female rat showed normal muscle
fiber (MF), connective tissue (CT) and nucleus of myocytes (NM).
Figure: Effect on histopathology of Heart of female rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose.
6. Lungs: Male: The histopathology of lung of control and treated group of male rat showed no alteration of structures such as, bronchioles (B), bronchiolar lumen and alveolar air space (AAS).
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Figure: Effect on histopathology of Lungs of male rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose.
Female: The histopathology of lung of control and treated group of female rat showed no alteration of structures such as, bronchioles (B), bronchiolar lumen and alveolar air space (AAS).
Lower dose, (C) Intermediate dose, (D) Higher dose. This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd.
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Figure: Effect on histopathology of Lungs of female rat after 30 days treatment with (A) Control dose, (B)
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7.0 Pancreas: The most prominent histopathological changes in male and female rat in pancreases at intermediate and higher doses were presence of marked interstitial oedema and large numbers of variable size vacuoles in the cytoplasm of acinar cells. These histopathological changes are fully characteristic of this form of pancreatitis. Male Rats:
Figure: Effect on histopathology of pancreas s of male rat after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose.
Female Rats:
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Figure: Effect on histopathology of pancreas of female rats after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose.
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CLINICAL BIOCHEMISTRY& HEMATOLOGY Glucose:

There was no significant change were obseved in glucose levels of all treated male and female rats, when compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
90 Glucose (mg/dl) 86 84 82 80 78 CMR LMR IMR Parameters HMR Glucose (mg/dl) 88
Glucose (mg/dl)
BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

90 88 86 84 82 80 78 76 CFR LFR IFR Parameters HFR
Glucose (mg/dl)
BUN:
There was no significant changes were obseved in BUN levels of all treated male and female rats, when compared with their control groups.
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BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS

30 25 BUN (mg/dl)
BUN (mg/dl)

15 10 5 0 CMR LMR IMR Parameters HMR
BUN (mg/dl)
20
BUN (mg/dl)
Creatinine:
There was no significant changes were observed in creatinine level of all treated groups of male and female rats. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
1.04 Creatinine (mg/dl) 1.035 1.025 1.02 1.015 1.01 1.005 CMR LMR IMR Parameters HMR Creatinine (mg/dl) 1.03
Creatinine (mg/dl)

1.4 1.2 1 0.8 0.6 0.4 0.2 0 CFR LFR IFR Parameters HFR
Creatinine (mg/dl)
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Sodium:
Sodium levels had shown significant increase in high dose treated male rats groups but there were no changes in female rat in all treated group when compared with control rats. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
200 Na (mEq/L) 150 100 50 0 CMR LMR IMR Parameters HMR Na (mEq/L)
Na (mEq/L)
BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 200

150 100 50 0 CFR LFR IFR Parameters HFR
Na (mEq/L)
Potassium:
There was significant increased in potassium levels of higher dose treated male and female rats, but there were no changes in other groups when compared there control groups.
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7 6 5 4 3 2 1 0 CMR LMR IMR Parameters HMR

5 K (mEq/L) 4 3 2 1
K (mEq/L)
K (mEq/L)
K (mEq/L)
0
CFR LFR IFR Parameters HFR
Chloride:
No significant changes were obserbed in chlorine level in male and female rats at higher, intermediate and lower dose level dose when compared with control group. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
90 88 86 84 82 80 78 76 74 CMR LMR IMR Parameters HMR

Cl (mEq/L) 90 85 80 75 CFR LFR IFR Parameters HFR
Cl (mEq/L)
Cl (mEq/L)
Cl (mEq/L)
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Triglyceride:
There were no significant changes were observed in triglyceride levels of both male and female treated rats at all dose level when compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
140 120 100 80 60 40 20 0 CMR LMR IMR Parameters HMR
TG (mg/dl)

TG (mg/dl)
TG (mg/dl)
TG (mg/dl)
Cholesterol:
There were no significant changes were observed in cholesterol levels of both male and female treated rats at all dose level when compared with their control groups.
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Cholesterol (mg/dl)
105 Cholesterol (mg/dl) 100 95 90 85 80 75 CMR LMR IMR Parameters HMR
Cholesterol (mg/dl)

100 95 90
Cholesterol (mg/dl)
85
80 75 CFR LFR IFR Parameters HFR
SGOT:
SGOT levels had shown significant increase in higher and intremiadate treatment groups of male and female rats and no change was at low dose group in male and female rats groups as compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
90 80 70 60 50 40 30 20 10 0 CMR LMR IMR Parameters HMR

100 SGOT (IU/L) 80 60 40 20 0
SGOT (IU/L)
SGOT (IU/L)
SGOT (IU/L)
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CFR
LFR IFR Parameters
HFR
SGPT:
SGPT levels had shown significant increase in higher and intremiadate treatment groups of male and female rats and no change was at low dose group in male and female rats groups compared to respective control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
90 80 70 60 50 40 30 20 10 0 CMR LMR IMR Parameters HMR

100 SGPT (IU/L) 80 60 40 20
SGPT (IU/L)
SGPT (IU/L)
SGPT (IU/L)
0
Alkaline phosphatase:
There were no significant changes observed in Alkaline phosphatase levels in male amd female rats at all doses group when compared with control group.
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86 84 82 80 78 76 74 72 70 68 66 CMR LMR IMR Parameters HMR
100 Alk.PO4 (IU/L) 80 60 40 20 0
Alk.PO4 (IU/L)
Alk.PO4 (IU/L)
Alk.PO4 (IU/L)
CFR
LFR IFR Parameters
HFR
Bilirubin:
No significant changes were obserbed in bilirubin level in male and female rats at higher, intermediate and lower dose level when compared with their respective control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
0.88 Bilirubin (mg/dl) 0.876 0.874 0.872 Bilirubin (mg/dl)
BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 0.886

0.884 0.882 0.88 0.878 0.876 0.874 0.872 0.87 CFR LFR IFR Parameters HFR
0.878
0.87
0.868 0.866 CMR LMR IMR Parameters HMR
Bilirubin (mg/dl)
Bilirubin (mg/dl)
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Proteins:
No significant changes were obserbed in protein level in male and female rats at higher, intermediate and lower dose level dose when compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS
5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 CMR LMR IMR Parameters HMR 5.75 5.7 5.65 5.6 5.55 5.5 5.45 5.4 5.35 5.3
Proteins (mg/dl)
Proteins (mg/dl)
Proteins (mg/dl)
Proteins (mg/dl)
CFR
LFR IFR Parameters
HFR
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HAEMATOLOGICAL PARAMETERS Hemoglobin:

No significant change was observed in haemoglobin levels in all the treatment groups of both male and female rats when compared with control group. HAEMATOLOGICAL PARAMETERS IN MALE RATS
18 17 Hb (gm/dl) 15 14 13 12 CMR LMR IMR Parameters HMR Hb (gm/dl) 16
Hb (gm/dl)
HAEMATOLOGICAL PARAMETERS IN FEMALE RATS

20 15 10 5
Hb (gm/dl)
0
RBC:
No significant change was observed in RBC levels in both male and female rats in all the treatment groups when compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE RATS
RBC (million Cells/mcL) RBC (million Cells/mcL) 6.35 6.3 6.25 6.2 6.15 6.1 CMR LMR IMR Parameters HMR
RBC (million Cells/mcL)
HAEMATOLOGICAL PARAMETERS IN FEMALE 5.7 RATS

5.65 5.6
5.55
5.5 5.45 CFR LFR IFR Parameters HFR
RBC (million Cells/mcL)
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WBC:
No significant change was observed in WBC levels in both male and female rats in all the treatment groups when compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE RATS
4800
Total WBC (Cells/mcL)
HAEMATOLOGICAL PARAMETERS IN FEMALE RATS 4100

Total WBC (Cells/mcL) 4000 3900 3800 3700
4700 4600
4500
4400 4300 4200 CMR LMR IMR Parameters HMR
3600
Polycyte:
No significant changes were observed in Polycyte counts in both male and female rats at all dose levels when compared with their control groups.
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37
HAEMATOLOGICAL PARAMETERS IN MALE RATS

70 60 50 40 30 20 10 0 CMR LMR IMR Parameters HMR 60 58 56 54 52 50 48 46 44
Polycytes (%)
Polycytes (%)
Polycytes (%)
Polycytes (%)
CFR
LFR IFR Parameters
HFR
Lymphocyte:
There was significant no significant changes were observed in lymphocyte levels in male and female rats at higher, Intermediate and low dose levels when compared to their control group. HAEMATOLOGICAL PARAMETERS IN MALE RATS
50 Lympho. (%) Lympho. (%) 40 30 20 10 0 CMR LMR IMR Parameters HMR
Lympho. (%)

60 50 40 30 20 10 0 CFR LFR IFR Parameters HFR
Lympho. (%)
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38
Eosinophil:
Eosinophils level had shown significant increase in high and intermediate treatment groups of both male and female rats, as compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE RATS
1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 CMR LMR IMR Parameters HMR 2 Eosino.(%) 1.5 1 0.5 0 CFR LFR IFR Parameters HFR
Eosino. (%)
Eosino.(%)
Eosino. (%)
Monocyte:
Significant increased in monocyte was observed in intermediate and higher dose treated male and female rats groups when compared with their control groups.
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39

4 3.5 3 2.5 2 1.5 1 0.5 0 CMR LMR IMR Parameters HMR

5 Mono. (%) 4 3 2 1 0 CFR LFR IFR Parameters HFR
Mono. (%)
Mono. (%)
Mono. (%)
ESR:
No significant changes were observed in ESR levels in male and female rat when compared to their control group. HAEMATOLOGICAL PARAMETERS IN MALE RATS
25 ESR (mm/hr) 20 15 10 5 0 CMR LMR IMR Parameters HMR
ESR (mm/hr)

ESR (mm/hr)
15 10 5
ESR (mm/hr)
0
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40
PCV:
There were no significant changes observed in PCV count in all treated groups of female and male rats when compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE RATS
60 50

40
PCV (%)
PCV (%)
40 30
PCV (%)
30 20 10 0
PCV (%)
20 10 0 CMR LMR IMR Parameters HMR
CFR
LFR IFR Parameters
HFR
Platelets:
No significant changes were observed in platelets count in male and female rats when compared to their control groups.
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41

377500 Platelets (Cells/mcL) 377000 376500 376000 375500 375000
Platelets (Cells/mcL)

413500 413000 412500 412000 411500 411000 410500
CMR LMR IMR Parameters
HMR
CFR
LFR IFR HFR Parameters
URINE ANALYSIS
Before sacrifice, urine samples were collected by keeping the rat in metabolic cages. Urine samples were subjected to routine qualitative urinalysis. There were no significant difference were noticed in urinary pH, Color. Albumin, sugar, bile, pus cells, RBCs, Cast, Crystals were absent in both male and female test drug treated and control drug treated rats.
INFERENCES
There were no significant change in behavior and clinical sign & symptoms were noticed and no mortality was recorded in both male and female treated rats. No significant changes in food, water intakes and body weight were observed in low dose, intermediate and high dose treated in both male and female rat groups throughout the treatment period. No significant variations in the organ-body weight for rats were observed except in liver of both male and female rats. These changes were largely confined to the high dose group.
42
There were significant changes in histopathology of liver and pancreas was observed at intermediate and higher dose treated male and female rats. There were no significant changes were obseved in glucose level, BUN level, Creatinine level, Triglyceride level, Bilrubin, Cholesterol level, Chlorine level, Alkaline phosphatase level and Protein level of all treated male and female rats, when compared with their control groups. Sodium levels had shown significant increase in high dose treated male rats groups but there were no changes in female rats in all treated group. There was significant increased in potassium levels of higher dose treated male and female rats, but there were no changes in other groups. SGOT and SGPT levels significantly increased in high dose and intermediate treated male and female rats. There were no significant effects observed in hemoglobin level, RBC, WBC and Platelets counts, Polycyte, lymphocyte, ESR and PCV in all treated male and female rats. Eosinophils level had shown significant increase in high and intermediate treatment groups of both male and female rats, as compared with their control groups. Significant increased in monocyte was observed in intermediate and higher dose treated male and female rats groups when compared with their control groups. GENERAL CONCLUSIONS Pyritinol Dihydrochloride Monohydrate Tablets manufactured by Kusum Healthcare Pvt. Ltd., presented for oral administration were subjected to sub-acute toxicity studies in rats. Three doses of the drug viz., low dose, intermediate dose and high dose have been employed in the study. The lower dose used in the study is significantly higher than the expected therapeutic dose in human. Mortality was not observed in all groups of treated rats. Treatment with Pyritinol Dihydrochloride Monohydrate induces significant changes in the liver-body weight ratio of male and female rats treated with high dose levels. Histopathological study reveals
Dihydrochloride Monohydrate administration. On treatment with the high dose of the drug, liver
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and pancreas were found to be the target organ which was most affected after Pyritinol
43
significant changes in the organs of animals from the high dose and intermediate dose groups. Liver
sections showed severe congestion and swollen hepatocytes in periportal areas. In pancreas histopathological study there was marked interstitial oedema and large numbers of variable size vacuoles in the cytoplasm of acinar cells show pancreatic cell destruction. SGPT, SGOT levels were increased in high dose treated male and female rats, the changes seen in the treated animals shown correlation with the histopathological observations. The mode of toxicity of the drug cannot be fully explained but liver and pancreas was founded to be the major target organ leading to toxic response. Species-dependent variations in the toxic response are not evident in the study. Sex-dependent variations are not very evident in the toxic response of the rats. The maximum adult human dose reported for Pyritinol Dihydrochloride Monohydrate is 600 mg/day. No mortality and clinical signs of compound-related toxicity were noted even at 500 mg/kg (50 times higher than maximum human dose), the no observed adverse effect level (NOAEL) of Pyritinol Dihydrochloride Monohydrate HCl in rat is 500 mg/kg (50 times higher than maximum human dose) when administered orally for 30 consecutive days. Hence it is concluded that Pyritinol Dihydrochloride Monohydrate Tablets manufactured by Kusum Healthcare Pvt. Ltd., is safe at 500 mg/kg in rats. Record Retention All study data, including (but not limited to) animal data, clinical pathology data, necropsy data, pathology data, professional reports, study protocol (including amendments), final study report, and any communications concerning the conduct of the study will be retained in a period of 5 years following completion of the final report. These materials will be retained on site at which the work was performed. Following the 5 year period (or before at Sponsors request), the Sponsor will be contacted to determine the disposition of these materials. References: 1. OECD Guideline for the Testing of Chemicals: Guidelines (407) Repeated Dose 28-Day Oral Toxicity Study in Rodents 2006. 2. Schedule Y of Drugs and Cosmetics act, 1940.
This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd. Page
44
PROJECT TEAM Dr. Amit Bhatt (PhD, PDM) Dr. Nilofar S. Naikwade M.Pharm, Ph.D Mr. Sandip B patil M.Pharm Mr. Sachin Sajane Project Manager Project Assistant Attendant Study Director
PLACE OF CONDUCT OF STUDY Appasaheb Birnale College of Pharmacy, South Shivaji Nagar, Miraj Road, Sangli, Maharashtra- 416 416 (CPCSEA Registration no. 843/AC/04) REPORT PREPARED BY Pushpendra Kumar Sharma M. Pharm (Pharmacology) Medico-Regulatory Associate Nexus Clinical Research (India) Ltd. New Mumbai (India) QUALITY ASSURED BY Amol Mohite Clinical Quality Associate Nexus Clinical Research (India) Ltd. New Mumbai (India) REPORT REVIEWED AND AUTHORIZED BY Dr. Amit Bhatt (President & CEO) Nexus Clinical Research (India) Ltd. New Mumbai (India)
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Date: 14/10/2011
45
APPENDIX
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46
Mean Body weight

Mean Body weight (gm) measured during Sub-acute toxicity study in Male rats at time interval Body weight (gm)
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CMM 238 241 245.3 245 246.2 248.56 254.32 260 256.56 258.45 265 263.12 LMM 230 239 245 245 247.65 248 249.32 256.89 260.12 257 259 269.23 IMM 235.4 240.56 242.56 241.32 245.65 250 252.87 256.78 258.56 257.45 266.78 273.21 HMM 235.2 248.56 243.12 241 245.23 241.56 248.56 254.32 260 256 258 265
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CMM
4.5 4.3 5.6
LMM
7.3 7.8 7.5 7.2 7.0 7.6 5.6
IMM
5.6 5.1 4.5 4.3 4.9 4.2 6.3 5.8 8.2 2.3 5.9 4.7
HMM
3.6 6.5
4.5
4.3 4.8 6.3 6.5 4.6 4.8 4.6 8.3 7.2
6.3
5.6 4.6 3.6
5.9
4.6 4.8
4.5
6.3 2.3
4.7
5.8
4.5
3.5
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Mean Body weight (gm) measured during Sub-acute toxicity study in Female rats at time interval Body weight (gm)
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CFM 200 224.53 226.32 232 236.45 245.12 248.45 246.32 252.25 249.56 258.26 259.8 LFM 210 226.45 231 237.16 240 241.58 246.23 248.23 245.78 249.56 259.45 258.6 IFM 205.3 224.89 232.12 226.56 245.19 241.59 247.48 249.89 250.2 251 250 260.1 HFM 212.5 234.12 236.52 240.18 235 251 256.5 254.8 250.5 256.4 260 261.2
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CFM 5.3 5.3 6.5 5.4 5.8 5.7 4.6 4.8 7.3 6.5 7.8 8.2 LFM 5.6 4.5 6.2 2.5 5.6 4.5 6.5 6.8 6.4 5.9 5.7 4.6 IFM 4.2 3.2 1.2 2.5 3.9 4.5 2.6 2.5 2.8 3.1 3.6 3.4 HFM 4.5 2.5 6.5 6.8 4.8 6.2 6.5 2.5 2.8 2.4 3.1 3.6
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48
Mean Food Consumption

Mean Food consumption (gm) measured during sub-acute toxicity study in Male rats at time interval
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CMM 16.3 18.5 14.3 16.5 17.5 17.12 16.73 15.45 18.98 18.46 15.56 18.45 LMM 16.5 17.23 17.56 15.6 17.56 18.54 17.98 15.46 16.98 17.45 18.59 17.6 IMM 19.45 18.23 17.56 16.25 18.12 17.58 18.45 19.45 20.15 21.1 21.3 22.56 HMM 21.56 18.49 19.56 22.12 16.23 15.23 18.6 20.5 21.3 19.5 20.5 21.2
Food consumption (gm) measured during sub-acute toxicity study in Female rats at time interval
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CFM 15.9 17.45 16.89 19.45 20.15 20.78 18.56 17.45 18.98 19.77 20.56 22.15 LFM 17.5 17.68 18.04 19.33 20.36 21.45 18.25 17.89 15.69 18.59 21.58 23.65 IFM 18.5 16.83 19.53 20 21.45 22.14 19.56 18.45 21.45 23.56 22.56 24.37 HFM 18.45 19.46 20.31 22.35 18.6 19.21 19.5 20.5 22.1 19.6 22.5 21.3
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Mean Water Consumption

Mean Water consumption (ml) measured during Sub-acute toxicity study in Male rats at time interval
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CMM 38.6 41.26 39.56 35.26 39.65 37.56 38.25 39.61 37.45 36.89 36.12 38.59 LMM 37.9 36.48 36.45 39.48 38.7 35.48 36.45 35.48 38.49 36.49 35.12 39.38 IMM 35.2 35.89 33.26 35.62 35.48 32.15 36.45 36.95 35.48 32.46 35.48 36.45 HMM 36.45 36.54 36.54 38.45 39.54 32.23 36.49 35.98 35.58 32.45 35.98 36.45
Mean Water consumption (ml) measured during Sub-acute toxicity study in Female rats at time interval
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 Days 0 1 3 6 9 12 15 18 21 24 27 30 CFM 42.3 41 37.45 35.23 36.45 35.45 38.48 36.95 38.6 35.2 38.5 36.7 LFM 38.9 37 38.45 36 41.2 36.45 38 39 41.23 35.45 32.98 35.45 IFM 37.5 36.56 35.89 35.68 38.59 36.45 37.89 35 38.25 38.55 36 37.56 HFM 38.4 39.45 41.23 35.62 32.45 36.45 37.89 34.78 38.63 37.45 36.7 40
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Mean relative organ body weight ratio in Male rats at 30th day in Sub-acute toxicity study Mean relative organ body weight ratio
Sr. No. 1 2 3 4 5 6 7 Organ Liver Lungs Heart Spleen Kidney Brain Gonads CMM 0.029 0.02 0.0074 0.0029 0.0032 0.0068 0.0035 LMM 0.03 0.021 0.0076 0.003 0.003 0.0059 0.0036 IMM 0.037 0.019 0.0075 0.0027 0.0038 0.0067 0.004 HMM 0.058 0.021 0.0078 0.0032 0.004 0.0065 0.0037
SEM
Sr. No. 1 2 3 4 5 6 7
Organ Liver Lungs Heart Spleen Kidney Brain Gonads
CMM LMM IMM HMM 0.00048 0.0005 0.0006 0.0005 0.0003 0.0003 0.0004 0.00028 0.0003 0.00035 0.00035 0.00035 0.0003 0.00028 0.0003 0.0003 0.0003 0.0004 0.00045 0.00035 0.00044 0.0005 0.00066 0.00058 0.0002 0.0002 0.0003 0.0002
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Mean relative organ body weight ratio in Female rats at 30th day in Sub-acute toxicity study

Sr. No. 1 2 3 4 5 6 7 Organ Liver Lungs Heart Spleen Kidney Brain Ovary CFM 0.028 0.014 0.008 0.003 0.0035 0.0056 0.0038 LFM 0.029 0.014 0.0078 0.0034 0.0037 0.005 0.0037 IFM 0.04 0.015 0.0081 0.0029 0.004 0.0058 0.0041 HFM 0.046 0.014 0.0084 0.0035 0.0041 0.0056 0.0038
SEM
Sr. No. 1 2 3 4 5 6 7 Organ Liver Lungs Heart Spleen Kidney Brain Ovary CFM 0.0005 0.0002 0.0003 0.0002 0.0003 0.0004 0.0001 LFM 0.00047 0.0003 0.00035 0.00027 0.0003 0.0005 0.0002 IFM 0.00045 0.00027 0.00042 0.00022 0.0004 0.0006 0.00026 HFM 0.00047 0.0003 0.00044 0.0003 0.00041 0.0005 0.00025
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Blood Biochemical Parameters in Male Rats Blood Biochemical Parameters in Male Rats
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 Parameters Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) TG (mg/dl) SGOT (IU/L) SGPT (IU/L) Na (mEq/L) K (mEq/L) Cl (mEq/L) Alk.PO4 (IU/L) Bilirubin (mg/dl) Proteins (mg/dl) Cholesterol (mg/dl) CMM 85 24 1.026 110 44.25 52.23 132 4.1 85.23 80.32 0.875 5.6 93.48 LMM 84.23 25.23 1.03 115 44.45 53.26 135 4.1 84.56 78.36 0.876 5.6 95.65 IMM 85.05 24.56 1.028 116 56.65 62.45 136 4.23 85.23 78.25 0.874 5.6 93.45 HMM 84.55 25.45 1.025 112 78.56 81.45 175 6.25 84.25 79.5 0.874 5.5 94.56
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 Parameters Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) TG (mg/dl) SGOT (IU/L) SGPT (IU/L) Na (mEq/L) K (mEq/L) Cl (mEq/L) Alk.PO4 (IU/L) Bilirubin (mg/dl) Proteins (mg/dl) Cholesterol (mg/dl) CMM 2.5 1.5 0.005 16.56 4.5 5.6 14.56 0.2 3.2 3.6 0.003 0.15 5.9 LMM 2.5 1.9 0.005 15.48 4.6 4.3 15.65 0.3 3.6 4.5 0.002 0.14 5.4 IMM 2.6 1.5 0.005 14.56 4.9 3.9 18.65 0.3 3.9 4.8 0.002 0.11 5.6 HMM 3.2 0.96 0.008 15.36 5.2 3.8 14.23 0.32 4.2 4.9 0.003 0.15 8.3
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Blood Biochemical Parameters in Female Rats Blood Biochemical Parameters in Female Rats
Sr. No. Parameters CFM 84.65 Glucose (mg/dl) 1 24.14 BUN (mg/dl) 2 1.02 Creatinine (mg/dl) 3 110.21 TG (mg/dl) 4 45.2 SGOT (IU/L) 5 47.15 SGPT (IU/L) 6 135 Na (mEq/L) 7 4.3 K (mEq/L) 8 89.56 Cl (mEq/L) 9 78.58 Alk.PO4 (IU/L) 10 0.88 Bilirubin (mg/dl) 11 5.57 Proteins (mg/dl) 12 Cholesterol (mg/dl) 92.25 13 LFM 84.12 23.4 1.05 116 47 47.5 134 4.2 88.45 78.35 0.88 5.55 93.45 IFM 84.14 25.14 1.25 118 62.4 58.45 136 4.3 86.45 77.45 0.88 5.6 93.45 HFM 84.14 24.48 1.2 115 82.45 80.12 135.21 4.2 88.59 77.48 0.88 5.6 91.82
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 Parameters Glucose (mg/dl) BUN (mg/dl) Creatinine (mg/dl) TG (mg/dl) SGOT (IU/L) SGPT (IU/L) Na (mEq/L) K (mEq/L) Cl (mEq/L) Alk.PO4 (IU/L) Bilirubin (mg/dl) Proteins (mg/dl) Cholesterol (mg/dl) CFM 2.3 1.12 0.005 15.23 3.2 4.6 15.45 0.3 2.5 6.3 0.002 0.12 6.5 LFM 2.5 0.9 0.006 14.26 3.6 4.8 14.23 0.3 3.6 5.3 0.003 0.11 6.5 IFM 3.2 0.87 0.004 13.25 3.5 4.3 16.54 0.5 3.5 8.5 0.002 0.06 5.8 HFM 3.6 1.3 0.005 12.54 4.8 3.9 15.62 0.25 3.4 6.5 0.005 0.09 4.9
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Hematological Parameters in Male Rats Hematological parameters in Male Rats

Sr. No. 1 2 3 4 5 6 7 8 9 10 Parameters Hb (gm/dl) RBC (million Cells/mcL) Total WBC (Cells/mcL) Polycytes (%) Lympho. (%) Mono. (%) Eosino.(%) ESR (mm/hr) PCV (%) Platelets (Cells/mcL) CMM 15.56 6.25 4521 55.26 42.15 2.1 0.95 17.59 42.12 376423 LMM 15.7 6.3 4532 52.15 42.15 2.2 1 16.59 41.25 376890 IMM 15.89 6.3 4536 50.13 41.12 3.2 1.5 17.48 40.65 376595 HMM 15.6 6.25 4531 52.14 42.65 3.8 1.55 17.56 43.65 376381
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 Parameters Hb (gm/dl) RBC (million Cells/mcL) Total WBC (Cells/mcL) Polycytes (%) Lympho. (%) Mono. (%) Eosino.(%) ESR (mm/hr) PCV (%) Platelets (Cells/mcL) CMM 1.3 0.05 120 2.5 4.6 0 0.005 1.5 4.5 480 LMM 1.4 0.005 150 2.6 4.2 0 0.005 1.4 4.6 458 IMM 1.2 0.005 135 2.9 4.3 0.005 0.006 1.2 4.5 468 HMM 1.2 0.0052 126 2.8 4.5 0 0.003 1.1 4.3 459
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Hematological Parameters in Female Rats Hematological parameters in Female Rats

Sr. No. 1 2 3 4 5 6 7 8 9 10 Parameters Hb (gm/dl) RBC (million Cells/mcL) Total WBC (Cells/mcL) Polycytes (%) Lympho. (%) Mono. (%) Eosino.(%) ESR (mm/hr) PCV (%) Platelets (Cells/mcL) CFM 13.56 5.6 3946 53.12 43.45 2.1 0.98 14.56 38.45 412550 LFM 13.45 5.62 3925 53.23 45 2 1.0 15.45 38.59 412563 IFM 14.23 5.63 3958 52.45 44.89 3.2 1.6 15.65 39 412590 HFM 12.45 5.61 3912 54.23 44.58 5.3 1.5 16.89 39.58 412556
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 Parameters Hb (gm/dl) RBC (million Cells/mcL) Total WBC (Cells/mcL) Polycytes (%) Lympho. (%) Mono. (%) Eosino.(%) ESR (mm/hr) PCV (%) Platelets (Cells/mcL) CFM 1.2 0.05 100 2.3 4.5 0.02 0.03 1.2 6.2 500 LFM 1.3 0.06 102 2.5 4.2 0 0.05 1.3 5.6 652 IFM 0.9 0.01 105 3.4 3.2 0.06 0.01 0.6 4.9 785 HFM 1.5 0.023 110 3.6 3.6 0.05 0.05 0.9 3.9 786
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URINALYSIS
SIGNIFICANT/ NON SIGNIFICANT URINALYSIS Group Parameters pH ALBUMIN SUGAR ACT. BILE OB PUS RBC CMM 5.40 Ab Ab Ab Ab Ab Ab Ab LMM 6.00 Ab Ab Ab Ab Ab Ab Ab IMM 5.23 Ab Ab Ab Ab Ab Ab Ab HMM 5.75 Ab Ab Ab Ab Ab Ab Ab DOSE INCREASE/
FORM DECREASE ALL CFM 5.45 Ab Ab Ab Ab Ab Ab Ab OUTPUT LFM 5.45 Ab Ab Ab Ab Ab Ab Ab IFM 5.50 Ab Ab Ab Ab Ab Ab Ab HFM 5.45 Ab Ab Ab Ab Ab Ab Ab
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DAYS Groups CMR LMR IMR HMR CFR LFR IFR HFR 0 Nil Nil Nil Nil Nil Nil Nil Nil 3 Nil Nil Nil Nil Nil Nil Nil Nil 6 Nil Nil Nil Nil Nil Nil Nil Nil 9 Nil Nil Nil Nil Nil Nil Nil Nil 12 Nil Nil Nil Nil Nil Nil Nil Nil 15 Nil Nil Nil Nil Nil Nil Nil Nil 18 Nil Nil Nil Nil Nil Nil Nil Nil 21 Nil Nil Nil Nil Nil Nil Nil Nil 24 Nil Nil Nil Nil Nil Nil Nil Nil 27 Nil Nil Nil Nil Nil Nil Nil Nil 30 Nil Nil Nil Nil Nil Nil Nil Nil
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Rats Reported by Nexus Clinical Research (India) Ltd.

(CPCSEA Registration No. 843/AC/04)

Clinical Research Organization

GLP & GAE COMPLIANCE STATEMENT

Clinical Quality Associate

SUB-ACUTE TOXICITY STUDY ON

(Batch No.: R&D/1a/C1/11); Mfg. Date: 07-2011)

Low Dose Intermediate Dose High dose Control

500 mg/kg 750 mg/kg 1000 mg/kg 1 ml/100gm Sterile water

Number of animals: 6 males and 6 females in each group

SUB-ACUTE TOXICITY STUDY

Empirical Formula: C16H20N2O4S2.2 (HCl) 10049-83-9

Low Dose Intermediate Dose High Dose Control

Food consumption (gm)

significant at (p<0.05), (p<0.01), (p<0.001).

CMR LMR IMR

Mean relative organ body weight ratio

Relative Mean relative organ body weight ratio

CFR LFR IFR

fiber (MF), connective tissue (CT) and nucleus of myocytes (NM).

fiber (MF), connective tissue (CT) and nucleus of myocytes (NM).

CLINICAL BIOCHEMISTRY& HEMATOLOGY Glucose:

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

15 10 5 0 CMR LMR IMR Parameters HMR

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 200

BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 6

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 95

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 105

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

LFR IFR Parameters

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

BLOOD BIOCHEMICAL PARAMETERS IN MALE RATS

100 Alk.PO4 (IU/L) 80 60 40 20 0

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

LFR IFR Parameters

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS 0.886

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE RATS

LFR IFR Parameters

HAEMATOLOGICAL PARAMETERS Hemoglobin:

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS

HAEMATOLOGICAL PARAMETERS IN FEMALE 5.7 RATS

RBC (million Cells/mcL)

Total WBC (Cells/mcL)

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS 4100

Total WBC (Cells/mcL)

HAEMATOLOGICAL PARAMETERS IN MALE RATS

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS

LFR IFR Parameters

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS

HAEMATOLOGICAL PARAMETERS IN MALE RATS

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS 6

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS 20

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS 50

20 10 0 CMR LMR IMR Parameters HMR

LFR IFR Parameters

HAEMATOLOGICAL PARAMETERS IN MALE RATS

HAEMATOLOGICAL PARAMETERS IN FEMALE RATS