2 Mice

Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Mice Reported by Nexus Clinical Research (India) Ltd.
REPORT
SUB-ACUTE TOXICITY STUDY ON
PYRITINOL DIHYDROCHLORIDE MONOHYDRATE IN MICE
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.
Rajasthan, India.
This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd.
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SP-289 (A), RIICO Industrial Area, Chopanki, Dist. Alwar-301019
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 document 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)
Page This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd.
Signature
Clinical Quality Associate
SUB-ACUTE TOXICITY STUDY ON

PYRITINOL DIHYDROCHLORIDE MONOHYDRATE TABLETS IN MICE
Date of Conduct of Study: 06/09/2011 Date of completion of study: 05/10/2011 Date of Reporting: 14/10/2011 Animal Model: Swiss albino mice 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 mice. 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 renal toxicity at 1000 mg/kg and at 750 mg/kg and 1000 mg/kg showed pancreas toxicity. The SGOT and SGPT enzyme level was increased that is marker of liver toxicity and renal toxicity confirmed by BUN and creatinine level. These liver, renal and pancreas toxicities were confirmed by histopathological changes observed at higher dose treated both male and female mice after 30 days treatment of test drug. Liver to body weight ratio were increased in male and female mice at 1000 mg/kg. There was no significant changes were observed in other organs i.e. heart, 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 are 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 (Mice) Page No.
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ABBREVIATIONS Abbreviations
CMM LMM IMM HMM CFM LFM IFM HFM SEM Hb RBC WBC ESR TG BUN SGOT SGPT Na K Cl PCV
Full form
CONTROL DOSE MALE MICE LOW DOSE MALE MICE INTERMEDIATE DOSE MALE MICE HIGH DOSE MALE MICE CONTROL DOSE FEMALE MICE LOW DOSE FEMALE MICE INTERMEDIATE DOSE FEMALE MICE HIGH DOSE FEMALE MICE 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)
Molecular Weight: 441.39 g/mol CAS Number: OBJECTIVES 1. 2. To assess the Sub-acute toxicity of Pyritinol Dihydrochloride Monohydrate in mice. 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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10049-83-9
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 mice 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: Swiss Albino Mice 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
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cage. Standard hygiene procedures were implemented.
PROCEDURE: Mice were weighed and marked. The sub-acute oral toxicity of Pyritinol Dihydrochloride Monohydrate Tablets was evaluated in mice using the schedule Y and Organization of Economic Co-operation and Development (OECD) guideline for testing of chemicals. Healthy adult albino mice 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, body weight 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 HCL DOSAGE Groups No. of animals used Daily dosage of Pyritinol Dihydrochloride Monohydrate for oral use MICE 500 mg/kg 750 mg/kg 1000 mg/kg 1 ml/100gm Sterile water
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 lower 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 animals. 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 exercised 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, body weight change, 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 mice when compared with control. B. MORTALITY IN MICE No mortality was recorded in treated mice 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 mice 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 mice at time interval
Mean Food consumption (gm) 6 5 4 3 2 1 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
CMM
LMM IMM HMM
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 mice at time interval
6
Food consumption (gm)
5 4 3 2 1 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days

CFM LFM IFM HFM
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). Page This Report is generated on 14/10/11 by Nexus Clinical Research (India) Ltd. for Kusum Healthcare Pvt. Ltd.
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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 mice when compared with their control groups. Mean Water consumption (ml) measured during Sub-acute toxicity study in Male mice at time interval
Mean Water consumption (ml 8
6 4 2
0 0 1 3 6 9 12 15 18 21 24 27 30
CMM LMM IMM
HMM
Time interval in Days
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).
Water consumption (ml)
Water consumption (ml) measured during Subacute toxicity study in Female mice at time interval
6 5 4 3 2 1 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
CFM
LFM IFM HFM
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Mice Reported by Nexus Clinical Research (India) Ltd. 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).
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 mice when compared with control group. Mean Body weight (gm) measured during Subacute toxicity study in Male mice at time interval
35 Body weight (gm) 30 25 20 15 10 5 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days
CMM LMM IMM
HMM
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).
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Mean Body weight (gm) measured during Subacute toxicity study in Female mice at time 40 interval
35 Body weight (gm) 30 25
CFM LFM IFM HFM
20
15 10 5 0 0 1 3 6 9 12 15 18 21 24 27 30
Time interval in Days
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 mice during the course of the treatment at all dose groups. G. ORGAN - BODY WEIGHT RATIO IN MICE There was significant increased in liver to body weight ratio when compared with control group in intermediate and higher dose treated male and female mice. There was no significant changes in both male and female mice 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. There was significant changes observed in liver to body weight ratio at higher dose in both male and female mice when compares to control group.
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Mean relative organ body weight ratio
Mean relative organ body weight ratio in Male mice 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 Organ Brain Gonads
CMM LMM IMM HMM
Relative Mean relative organ body weight ratio in Female mice at 30 th day in Sub-acute toxicity study
Relative Mean relative organ body weight ratio 0.045 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 -0.005
CFM LFM IFM HFM
Liver
Lungs Heart Spleen Kidney Brain Ovary

Organ
The results were expressed as Mean SEM (n=6). The data was analyzed using One-way Analysis of
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> 0.05), statistically significant at (p<0.05), (p<0.01), (p<0.001).
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Variance (ANOVA) followed by Dunnetts t-test. No statistical difference between control and test groups (p
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 mice 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 mice 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 mice showed normal hepatic cells (HC) with central vein (CV) and sinusoidal dilation (SS).
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Figure: Effect on histopathology of liver of female mice after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
2. Kidney Male: Male mice treated with high dose of the drug showed visible congestion and dilation of tubular epithelium in several areas. Glomeruli showed distention of Bowman's space and necrotic features in high dose treated mice.
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Mice Reported by Nexus Clinical Research (India) Ltd. Figure: Effect on histopathology of Kidney of male mice after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose
Female: The histopathology of kidney of control and intermediate treated group of female mice showed normal renal corpuscles (RC), tubules (T) and Bowmans space (BS), but at higher dose show moderate congestion and dilation of tubular epithelium.
Figure: Effect on histopathology of Kidney of female mice 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 mice showed normal red pulp (RP) and white pulp (WP).
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Figure: Effect on histopathology of spleen of male mice 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 mice showed normal red pulp (RP) and white pulp (WP).
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Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Mice Reported by Nexus Clinical Research (India) Ltd. Figure: Effect on histopathology of spleen of female mice 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 mice, 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 mice 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 mice 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 mice 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 Mice Reported by Nexus Clinical Research (India) Ltd. Figure: Effect on histopathology of Heart of male mice 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 mice showed normal
muscle fiber (MF), connective tissue (CT) and nucleus of myocytes (NM).
Figure: Effect on histopathology of Heart of female mice 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 mice 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 mice 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 mice showed no alteration of structures such as, bronchioles (B), bronchiolar lumen and alveolar air space (AAS).
Figure: Effect on histopathology of Lungs of female mice after 30 days treatment with (A) Control dose, (B) 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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7.0 Pancreases: The most prominent histopathological changes in male and female mice 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 Mice:
Figure: Effect on histopathology of pancreas s of male mice after 30 days treatment with (A) Control dose, (B) Lower dose, (C) Intermediate dose, (D) Higher dose.
Female Mice:
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Figure: Effect on histopathology of pancreas s of female mice 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 mice, when compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
140 120
BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE 140

120
Glucose (mg/dl)
80 60 40 20 0 CMM LMM IMM HMM
Glucose (mg/dl)
Glucose (mg/dl)
100
100 80 60 40 20 0 CFM LFM IFM HFM
Glucose (mg/dl)
Parameters
Parameters
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).
BUN:
There was no significant changes were obseved in BUN levels of all treated male and female mice, when compared with their control groups.
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BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE

25 20 BUN (mg/dl) 15 10 5 0 CMM LMM IMM Parameters HMM BUN (mg/dl) BUN (mg/dl) 25 20 15
BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE
10
5 0 CFM LFM IFM Parameters HFM
BUN (mg/dl)
Creatinine:
Creatinine levels had shown significant increase at high dose treated male and female mice group as compared with their control groups. There were no significant changes were observed in other treated groups of male and female mice. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
2
Creatinine (mg/dl)
1.5 1 0.5 0 CMM LMM IMM Parameters HMM
Creatinine (mg/dl)
1.5 1 0.5 0
Creatinine (mg/dl)
Creatinine (mg/dl)
CFM
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LFM IFM Parameters
HFM
Sub-Acute Toxicology Report of Pyritinol Dihydrochloride in Mice 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).
Sodium:
Sodium levels had shown significant increase in high dose treated group and significant decrease in low dose treated group male mice and but there were no changes in female mice in all treated group. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
200

200
Na (mEq/L)
Na (mEq/L)
150 100 50 0 CMM LMM IMM HMM
150 100 50 0 CFM LFM IFM HFM
Na (mEq/L)
Na (mEq/L)
Parameters
Parameters
Potassium:
There was significant increased in potassium levels of higher dose treated male and female mice, but there were no changes in other groups when compared with their control groups.
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7 6 5 4 3 2 1 0 CMM LMM IMM HMM 7 6
K (mEq/L)
K (mEq/L)
5 4 3 2 1 0 CFM LFM IFM Parameters HFM

K (mEq/L)
K (mEq/L)
Parameters
Chloride:
No significant changes were obserbed in chlorine level in male and female mice at higher intermediate and lower dose level dose when compared with control group. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
100 100

Cl (mEq/L)
95 90 85 80 75 CFM LFM IFM HFM
Cl (mEq/L)
95 90 85 80 75 CMM LMM IMM HMM
Cl (mEq/L)
Cl (mEq/L)
Parameters
Parameters
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Triglyceride:
There were no significant changes were observed in triglyceride levels of both male and female treated mice at all dose level when compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
300 250 200 150 100 50 0 CMM LMM IMM HMM TG (mg/dl) TG (mg/dl)
BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE 270

260 250 240 230 220 210 200 CFM LFM IFM Parameters HFM
TG (mg/dl)
TG (mg/dl)
Parameters
Cholesterol:
There were no significant changes were observed in cholesterol levels of both male and female treated mice at all dose level when compared with their control groups.
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Cholesterol (mg/dl)
260 250 240 230 220 210 200 190 CMM LMM IMM HMM 215
Cholesterol (mg/dl)
214 213 212 211 210 209 208 CFM LFM IFM HFM
Cholesterol (mg/dl)
Cholesterol (mg/dl)
Parameters
Parameters
SGOT:
SGOT levels had shown significant increase in higher treatment groups of male and female mice and no change was observed in intermediate and low dose group in male and female mice groups as compared with their control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
80 70 60 50 40 30 20 10 0 CMM LMM IMM HMM 80 SGOT (IU/L) 60 40 20 0 CFM LFM IFM HFM
SGOT (IU/L)
SGOT (IU/L)
SGOT (IU/L)
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Parameters
Parameters
SGPT:
SGPT levels had shown significant increase in higher dose treatment groups of both male and female mice and no change was observed in intermediate and low dose group in male and female mice as compared to respective control groups. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
90 80 70 60 50 40 30 20 10 0
CMM LMM IMM Parameters HMM

80 70 60 50 40 30 20 10 0
CFM LFM IFM HFM
SGPT (IU/L)
SGPT (IU/L)
SGPT (IU/L)
SGPT (IU/L)
Parameters
Alkaline phosphatase:
There were no significant changes observed in Alkaline phosphatase levels in female group in all doses when compared with control group. Alkaline phosphatase levels were significantly increased in higher dose treated male mice group when compared with control group.
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100

90 Alk.PO4 (IU/L) 85 80 75 70 Alk.PO4 (IU/L)
Alk.PO4 (IU/L)
95 90 85 80 75 70 CMM LMM IMM HMM
Alk.PO4 (IU/L)
65 CFM LFM IFM Parameters HFM
Parameters
Bilirubin:
No significant changes were obserbed in bilirubin level in male and female mice at intermediate and lower dose level dose but significant increased at higher dose in both male and female mice when compared with control group.

1.15
Bilirubin (mg/dl)
1.12
Bilirubin (mg/dl)
1.1 1.05 1
1.1 1.08 1.06 1.04 1.02 1 0.98 CFM LFM IFM HFM
Bilirubin (mg/dl)
Bilirubin (mg/dl)
0.95
0.9 CMM LMM IMM HMM
Parameters
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Parameters
Proteins:
No significant changes were obserbed in protein level in male and female mice at higher, intermediate and lower dose level when compared with control group. BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE
6.3 6.25 6.2 6.15 6.1 6.05 CMM LMM IMM HMM 6.5 6.4 6.3 6.2 6.1 6 5.9 5.8 5.7 5.6 CFM LFM IFM HFM

Proteins (mg/dl)
Proteins (mg/dl)
Proteins (mg/dl)
Proteins (mg/dl)
Parameters
Parameters
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35
HAEMATOLOGICAL PARAMETERS Hemoglobin:

No significant change was observed in haemoglobin levels in all the treatment groups of both male and female mice when compared with control group. HAEMATOLOGICAL PARAMETERS IN MALE MICE
20
HAEMATOLOGICAL PARAMETERS IN FEMALE MICE 20

15
Hb (gm/dl)
10 5 0 CMM LMM IMM HMM
Hb (gm/dl)
Hb (gm/dl)
15
10 5 0 CFM LFM IFM HFM
Hb (gm/dl)
Parameters
Parameters
RBC:
No significant change was observed in RBC levels in both male and female mice in all the treatment groups when compared with their control groups.
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36
HAEMATOLOGICAL PARAMETERS IN MALE MICE

7.3 7.28 7.26 7.24 7.22 7.2 7.18 7.16 7.14 CMM LMM IMM HMM 8 7 6 5 4 3 2 1 0
HAEMATOLOGICAL PARAMETERS IN FEMALE MICE

RBC (million Cells/mcL)
CFM
LFM
IFM
HFM
Parameters
Parameters
WBC:
Total WBC levels were significantly increased at higher dose male mice and significant decreased in higher and intermediate dose treated female mice as compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE MICE
Total WBC (Cells/mcL)
6000 5000

5000 4000 3000
4000
3000 2000 1000 0
2000
1000 0
CMM LMM IMM HMM

Parameters
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CFM LFM IFM HFM Parameters
Polycyte:
There was significant increased in polycytes were recorded in higher dose group in male mice when compared with their control group. No significant changes were observed in Polycyte levels in female mice at all dose level when compared with their control group. HAEMATOLOGICAL PARAMETERS IN MALE MICE
80 70 60 50 40 30 20 10 0 CMM LMM IMM HMM 70

Polycytes (%)
60
50 40 30 20 10 0 CFM LFM IFM HFM
Polycytes (%)
Polycytes (%)
Polycytes (%)
Parameters
Parameters
Lymphocyte:
No significant changes were observed in lymphocyte levels in male mice when compared to their control group. Significant decrease in lymphocyte level was observed at higher dose treated female mice as copmpared to their control group.
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49 60 50
Lympho. (%)
48
Lympho. (%)
47 46 45 44 43
40 30 20 10 0 CFM LFM IFM HFM
Lympho. (%)
Lympho. (%)
42
CMM LMM IMM Parameters HMM
Parameters
Eosinophil:
Eosinophils level had shown moderate increase in all the treatment groups of both male and female mice, as compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE MICE
1.4 1.2 1 0.8 0.6 0.4 0.2 0 CMM LMM IMM Parameters HMM 1 0.8
Eosino.(%)
Eosino.(%)
Eosino.(%)
0.6 0.4 0.2 0 CFM LFM IFM Parameters HFM

Eosino.(%)
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39
Monocyte:
Significant increased in monocyte was observed in intermediate and higher dose treated male and female mice groups when compared with their control groups. HAEMATOLOGICAL PARAMETERS IN MALE MICE
4 3.5 3 2.5 2 1.5 1 0.5 0 CMM LMM IMM HMM
HAEMATOLOGICAL PARAMETERS IN FEMALE MICE 2.5

2
Mono. (%)
Mono. (%)
Mono. (%)
1.5 1 0.5 0 CFM LFM IFM HFM
Mono. (%)
Parameters
Parameters
ESR:
No significant changes were observed in ESR levels in male and female mice when compared to their control group.
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40

11.6 11.5 ESR (mm/hr) 11.4 11.3 11.2 11.1 11 10.9 CMM LMM IMM Parameters HMM
ESR (mm/hr)

16 14 12 10 8 6 4 2 0
CFM LFM IFM Parameters HFM ESR (mm/hr)
ESR (mm/hr)
PCV:
There were no significant changes obnserved in PCV count in all treated groups of female mice when compared with their control groups.

40 39 38 37 36 35 34 33 32 CMM LMM IMM Parameters HMM 40 39 38 37 36 35 34 33
PCV (%)
PCV (%)
PCV (%)
PCV (%)
CFM
LFM
IFM
HFM
Parameters
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41
Platelets:
No significant changes were observed in platelets count in male and female mice when compared to their control group. HAEMATOLOGICAL PARAMETERS IN MALE MICE
423900 423800 423700 423600 423500 423400 423300 423200 423100 CMM LMM IMM HMM Parameters

376600
Platelets (Cells/mcL)
376500 376400 376300 376200 376100 376000 CFM LFM IFM HFM
Parameters
URINE ANALYSIS
Before sacrifice, urine samples were collected by keeping the mice 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, RBC, Cast, Crystals were absent in both male and female test drug treated and control drug treated mice.
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42
INFERENCES
Mortality was not recorded in both male and female treated mice. No significant changes in food intake were observed in low dose, intermediate and high dose treated in both male and female mice groups throughout the treatment period when compared with their control groups. No Significant change in water intake was observed in low, intermediate and higher dose treatment groups in both male and female mice when compared with their control groups. No significant changes in the body weight were observed in low, intermediate and high treatment groups of male and female mice when compared with control group. No significant variations in the organ-body weight for mice were observed except in liver of both male and female mice. These changes were largely confined to the high dose group. Histopathological study of liver of animals both males & females treated with high dose of the drug revealed severe congestion and dilation of sinusoids especially in the periportal area. Treatment with high doses of the drug, no effect on white pulp area of spleen, in both male and female mice. The kidney of high dose treated male & female mice revealed moderate congestion and dilation of tubular epithelium. The histopathology of lung of control and treated group of male mice showed no alteration of structures such as, bronchioles (B), bronchiolar lumen and alveolar air space (AAS) in both male & female mice. The gonads showed normal stages of gametogenesis in all treated animals. The pancreas at intermediate and higher dose showed marked interstitial oedema and large numbers of variable size vacuoles in the cytoplasm of acinar cells. There were no significant changes were obseved in glucose level, BUN level, Triglyceride level, cholesterol level, Chlorine level and Protein level of all treated male and female mice, when compared with their control groups. Creatinine levels had shown significant increase at high dose treated male and female mice group as compared with their control groups. There were no significant changes were observed in other Sodium levels had shown significant increase in high dose treated group and significant decrease
in low dose treated group male mice. There was significant increased in potassium levels of
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43
treated groups of male and female mice.
higher dose treated male and female mice, but there were no changes in other groups when compared there control groups. SGOT and SGPT levels significantly increased in high dose treated male and female mice. Alkaline phosphatase levels were significantly increased in higher dose treated male mice group. Bilirubin levels were significantly increased in higher dose group in both male and female mice. There were no significant effects observed in hemoglobin level, RBC counts, ESR, PCV and platelet counts in all treated male and female mice. Total WBC levels were significantly increased at higher dose male mice and significant decreased in higher and intermediate dose treated female mice as compared with their control groups. There was significant increased in polycytes were recorded in higher dose group in male mice but no changes in female mice at all dose levels. There were significant decrease in lymphocyte level was observed at higher dose treated female mice. Eosinophils had shown moderate increase in all the treatment groups of male mice and female mice. Significant increased in monocyte was observed in intermediate and higher dose treated male and female mice 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 mice. 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 mice. Treatment with Pyritinol Dihydrochloride Monohydrate induces significant changes in the liver-body weight ratio of animals (male and female mice) treated with high dose levels. Histopathological study reveals significant changes in the organs of animals from the high dose group. Liver and kidney were found to be the target organ which was most affected after Pyritinol Dihydrochloride Monohydrate administration at higher doses. On treatment with the high dose of the drug, liver sections showed
acinar cells show pancreatic cell destruction.

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there was marked interstitial oedema and large numbers of variable size vacuoles in the cytoplasm of
44
severe congestion and swollen hepatocytes in periportal areas. In pancreas histopathological study
The kidney of mice treated with high dose level reveals mild congestion, dilation of tubular epithelium and mild distention of Bowman's space. SGPT, SGOT levels were increased in high dose treated male and female mice, 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 animals. 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 mice 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 mice.
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
Page
2. Schedule Y of Drugs and Cosmetics act, 1940.
45
Toxicity Study in Rodents 2006.
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
46
APPENDIX
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47
Mean Body weight

Mean Body weight (gm) measured during Sub-acute toxicity study in Male mice 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 25.3 25.3 25.2 27.3 28.6 27.9 28.5 29.3 26.5 27.6 31.3 29.3 LMM 24.6 24.7 24.5 26.3 25.5 25.3 26.1 25.4 27.2 28.1 28.3 27.8 IMM 26.5 25.3 28.3 25.3 24.2 24.6 25.9 23.8 23.6 20.3 25.6 25.5 HMM 24.6 26.5 21.5 22.1 26.5 24.5 26.3 25.5 25.3 26.1 25.4 27.2
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 CMM
1.1 1.5 1.9
LMM
1.7 1.3 2.1 1.7 1.6 1.3 2
IMM
1.9 1.6 2.2 1.9 1.3 1.3 1.9 1.5 1.4 2 1.5 1.3
HMM
2.2 1.3
1.7
1.6 1.7 1.5 1.8 1.4 1.6 1.5 1.2 1.9
1.2
1.4 1.5 1.3
1.5
1.5 1.9
1.8
1.1 1.75
1.3
1.4
1,6
1.2
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48
Mean Body weight (gm) measured during Sub-acute toxicity study in Female mice 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 26 25.4 25.8 25.3 28.1 29.3 29.7 30.4 30.8 32.5 31.9 32.6 LFM 25.3 25.8 24.7 24.1 30.6 30.1 28.3 29.1 30.5 28.9 29.4 28.9 IFM 26.3 25.5 24.3 23.7 27.6 28.1 28.7 28.57 28.3 30 28.7 31.1 HFM 30.9 31.4 30.9 26.3 29.4 29.1 28.3 27.9 26.1 30.4 31.8 28.9
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 1.3 1.2 1.4 1.9 2.1 2.4 2.9 1.8 1.5 2 2.3 1.9 LFM 1.2 1.3 1.4 1.9 2 2.3 2.1 1.9 2.4 2.1 1.9 2 IFM 1.4 1.2 1.6 2.1 2.3 1.9 1.5 2 2.2 1.9 1.7 2.1 HFM 1.3 1.6 2.1 1.9 2.5 1.4 1.7 2.4 1.9 1.8 1.6 2
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49
Mean Food Consumption

Mean Food consumption (gm) measured during sub-acute toxicity study in Male mice 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 4.5 4.9 4.6 4.8 4.5 4.5 4.3 4.3 4.2 4.8 4.3 4.6 LMM 5.3 3.9 4.5 4.6 4.2 4.1 4.2 4.5 3.9 4.9 4.6 4.9 IMM 4.5 4.6 4.9 4.6 4.8 4.9 4.6 4.9 4.6 4.8 4.9 4.6 HMM 5.5 4.3 4.6 4.9 4.6 4.8 4.9 4.6 4.8 4.9 4.5 4.3
Food consumption (gm) measured during sub-acute toxicity study in Female mice 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 4.4 3.7 4.4 4.2 3.7 3.8 3.7 3.6 4.4 4.6 4.4 4.3 LFM 4.8 4.3 4.1 4.5 4.1 3.6 3.8 3.6 3.9 3.8 3.9 4.2 IFM 5.2 5.4 4.9 4.3 4.8 4.8 4.8 4.5 4.5 4.6 5.1 4.5 HFM 5.3 4.9 4.8 4.5 4.9 4.9 4.6 5 4.7 4.8 4.6 5.2
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50
Mean Water Consumption

Mean Water consumption (ml) measured during Sub-acute toxicity study in Male mice 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 6.2 6.1 6.3 5.5 5.7 5.5 5.6 5.2 5.4 5.3 5.6 6 LMM 6.2 6.1 5.9 4.9 5.9 4.9 5.6 5.3 5.2 6.1 6.3 6.5 IMM 5.3 6.3 6.2 5.2 6.1 6.3 5.5 5.7 5.5 5.6 5.6 6.2 HMM 5.9 6.3 6.2 5.2 6.1 6.3 5.5 5.7 5.5 5.6 5.2 5.4
Mean Water consumption (ml) measured during Sub-acute toxicity study in Female mice 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 4.5 4.8 4.6 5 4.9 5.3 5.1 5.3 5.1 5 5.3 5.6 LFM 4.8 4.9 4.7 4.6 4.3 5.1 5.2 4.7 4.3 4.7 4.1 4.3 IFM 4.9 4.5 4.9 4.4 4.2 4.6 4.4 3.9 4.1 4.2 3.9 4.1 HFM 5.2 5.5 4.2 4 4.5 4.9 4.2 4.1 4.2 4.3 4.2 3.9
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Mean relative organ body weight ratio in Male mice 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 0.0345 0.0286 0.0066 0.0013 0.0036 0.0042 0.0057 LMM 0.037 0.0273 0.0067 0.0021 0.0043 0.0056 0.0068 IMM 0.0435 0.0281 0.0058 0.0023 0.0041 0.0058 0.0057 HMM 0.0465 0.0285 0.0058 0.0016 0.0034 0.0057 0.0055
SEM
Sr. No. 1 2 3 4 5 6 7
Organ Liver Lungs Heart Spleen Kidney Brain Gonads
CMM 0.0018 0.0014 0.0002 0.0003 0.0012 0.0009 0.0013
LMM 0.0013 0.001 0.0003 0.0003 0.0019 0.0012 0.0014
IMM 0.0014 0.0011 0.0007 0.0004 0.0009 0.0009 0.0013
HMM 0.0009 0.0015 0.0012 0.0005 0.0007 0.0011 0.0012
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Mean relative organ body weight ratio in Female mice 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 0.0322 0.0138 0.0059 0.0015 0.0038 0.0047 0.0048 LFM 0.0321 0.0139 0.0061 0.0013 0.0045 0.0054 0.0049 IFM 0.0364 0.0141 0.0053 0.0014 0.0044 0.0061 0.0053 HFM 0.0385 0.0152 0.0054 0.0015 0.0038 0.0059 0.0055
SEM
Sr. No. 1 2 3 4 5 6 7 Organ Liver Lungs Heart Spleen Kidney Brain Ovary CFM
0.0003 0.0011 0.0004 0.0005 0.0009 0.0011 0.0012
LFM
0.0002 0.0014 0.0005 0.0012 0.0011 0.0013 0.0014
IFM
0.0007 0.0013 0.0007 0.0017 0.0007 0.0012 0.0011
HFM
0.0005 0.0015 0.0018 0.0015 0.0012 0.0015 0.0013
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53
Blood Biochemical Parameters in Male Mice Blood Biochemical Parameters in Male Mice
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) 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 110.22 17.22 1.06 238 47.21 45.24 137 4.29 89.11 82.22 1.03 6.22 234.33 CMM 18.11 3.35 0.5 23.4 5.3 4.2 1.4 0.3 5.6 2.1 0.01 0.02 12.25 LMM 108.26 18.4 1.15 238 48.17 47.55 114.55 4.21 88.24 81.36 1.03 6.21 225.38 LMM 13.33 3.6 0.3 30.52 4.4 2.4 1.6 0.2 5.2 1.5 0.02 0.023 10.37 IMM 111.5 17.9 1.03 235 50.11 45.38 134.28 4.35 89.21 81.92 1.02 6.22 235.11 IMM 14.55 4.53 0.23 24.51 4.6 2.8 1.9 0.19 4.6 2.6 0.012 0.065 13.5 HMM 113 18.3 1.64 236 70.16 80.16 155.5 6.1 90.45 93.62 1.12 6.24 239.43 HMM 16.56 3.66 0.25 23.21 3.5 3.71 2.4 0.22 5.9 1.36 0.015 0.023 12.2
SEM
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 13
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Blood Biochemical Parameters in Female Mice Blood Biochemical Parameters in Female Mice
Sr. No. Parameters CFM 106.13 Glucose (mg/dl) 1 16.22 BUN (mg/dl) 2 1.1 Creatinine (mg/dl) 3 244 TG (mg/dl) 4 48.76 SGOT (IU/L) 5 47.25 SGPT (IU/L) 6 141 Na (mEq/L) 7 4.25 K (mEq/L) 8 91.45 Cl (mEq/L) 9 80.25 Alk.PO4 (IU/L) 10 1.03 Bilirubin (mg/dl) 11 6.22 Proteins (mg/dl) 12 Cholesterol (mg/dl) 212.41 13 LFM 103.22 17.4 1.09 248 47.19 46.43 142.35 4.21 92.33 82.36 1.04 6.18 211.66 LFM 14.94 5.42 0.023 13.45 3.98 3.27 9.14 0.038 3.21 4.19 0.0053 0.25 0.22 IFM 108.5 17.3 1.08 245 48.17 43.55 145.39 4.15 89.13 83.96 1.04 6.22 213 IFM 15.44 2.45 0.012 11.55 4.93 2.92 11.39 0.022 4.22 2.33 0.0085 0.18 0.95 HFM 105.6 16.25 1.73 240 70.51 70.64 146.55 5.8 90.51 83.62 1.1 6.2 211.65 HFM 16.54 3.85 0.014 13.68 5.44 3.11 11.44 0.025 2.24 2.42 0.0075 0.16 1.65
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 14.44 6.58 0.032 18.17 4.73 2.28 10.23 0.052 2.29 5.33 0.0031 0.028 0.18
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Hematological Parameters in Male Mice Hematological parameters in Male Mice

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 14.22 7.27 4564 51.11 46.18 2 0.71 11.4 37.6 423539 LMM 15.65 7.22 4755 51.26 47 2.1 0.91 11.2 36.25 423652 IMM 13.5 7.24 4630 50.64 46.12 3.2 1.16 11.23 36.06 423602 HMM 14.7 7.25 5529 65.5 47.12 3.42 1.15 11.45 37 423685
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 2.1 0.02 36.2 1.2 1.6 0.001 0.0020 0.05 1.24 135 LMM 2.3 0.03 45.3 1.6 1.5 0.002 0.0015 0.06 1.23 145 IMM 3.5 0.01 40.2 2.1 1.2 0.0012 0.0016 0.04 1.25 144 HMM 1.6 0.009 52.9 1.9 1.06 0.0015 0.0021 0.035 1.14 145
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Hematological Parameters in Female Mice Hematological parameters in Female Mice

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 12.7 5.66 4548 50.9 46.1 1.4 0.6 11.12 36.89 376326 LFM 13.5 5.35 4337 50.46 47.16 1.19 0.85 11.85 37.25 376341 IFM 13.6 5.44 3992 50.54 45.73 1.89 0.83 12.28 37.33 376314 HFM 12.9 5.59 3365 51.93 37.28 1.9 0.89 12.55 37.68 376372
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 2.1 0.02 36.2 1.2 1.6 0.001 0.002 0 0.05 1.24 135 LFM 2.3 0.03 45.3 1.6 1.5 0.002 0.0015 0.06 1.23 145 IFM 3.5 0.01 40.2 2.1 1.2 0.0012 0.0016 0.04 1.25 144 HFM 1.6 0.009 52.9 1.9 1.06 0.0015 0.0021 0.035 1.14 145
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URINALYSIS
SIGNIFICANT/ NON SIGNIFICANT URINALYSIS SIGNIFICANT (SHOWED PROTEIN IN URINE) Group Parameters pH ALBUMIN SUGAR ACT. BILE OB PUS RBC CMM 5.60 Ab Ab Ab Ab Ab Ab Ab LMM 6.00 Ab Ab Ab Ab Ab Ab Ab IMM 5.75 Ab Ab Ab Ab Ab Ab Ab HMM 5.70 Ab Ab Ab Ab Ab Ab Ab CFM 5.75 Ab Ab Ab Ab Ab Ab Ab DOSE INCREASE/
FORM DECREASE ALL INCREASED OUTPUT LFM 5.75 Ab Ab Ab Ab Ab Ab Ab IFM 5.65 Ab Ab Ab Ab Ab Ab Ab HFM 5.75 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 Mice Reported by Nexus Clinical Research (India) Ltd.

(CPCSEA Registration No. 843/AC/04)

Clinical Research Organization

SP-289 (A), RIICO Industrial Area, Chopanki, Dist. Alwar-301019

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)

cage. Standard hygiene procedures were implemented.

Low Dose Intermediate Dose High Dose Control

Food consumption (gm)

5 4 3 2 1 0 0 1 3 6 9 12 15 18 21 24 27 30 Time interval in Days

Time interval in Days

Water consumption (ml)

Time interval in Days

Mean relative organ body weight ratio

CFM LFM IFM HFM

Lungs Heart Spleen Kidney Brain Ovary

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

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

CLINICAL BIOCHEMISTRY& HEMATOLOGY Glucose:

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE 140

80 60 40 20 0 CMM LMM IMM HMM

100 80 60 40 20 0 CFM LFM IFM HFM

BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

1.5 1 0.5 0 CMM LMM IMM Parameters HMM

LFM IFM Parameters

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

150 100 50 0 CMM LMM IMM HMM

150 100 50 0 CFM LFM IFM HFM

BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

5 4 3 2 1 0 CFM LFM IFM Parameters HFM

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

95 90 85 80 75 CMM LMM IMM HMM

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE 270

BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

95 90 85 80 75 70 CMM LMM IMM HMM

65 CFM LFM IFM Parameters HFM

BLOOD BIOCHEMICAL PARAMETERS IN MALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

BLOOD BIOCHEMICAL PARAMETERS IN FEMALE MICE

HAEMATOLOGICAL PARAMETERS Hemoglobin:

HAEMATOLOGICAL PARAMETERS IN FEMALE MICE 20

10 5 0 CMM LMM IMM HMM

10 5 0 CFM LFM IFM HFM

HAEMATOLOGICAL PARAMETERS IN MALE MICE

HAEMATOLOGICAL PARAMETERS IN FEMALE MICE

RBC (million Cells/mcL)

RBC (million Cells/mcL)

RBC (million Cells/mcL)

HAEMATOLOGICAL PARAMETERS IN FEMALE MICE