REPORT Title SUB-ACUTE TOXICITY STUDY (28 DAYS) ON POLY … · 2018-11-14 · teena biolabs pvt. ltd study no. : tfrcrdpct02022015 (rat). name of the test item : poly herbal formulation

TEENA BIOLABS PVT. LTD Study No. : TFRCRDPCT02022015 (RAT).

Name of the Test item : POLY HERBAL FORMULATION

Page 1 of 87

Registration No. 177/PO/cb/99/CPCSEA

Ministry of Environment and Forest, Government of India.

REPORT

Title

SUB-ACUTE TOXICITY STUDY (28 DAYS)

ON

POLY HERBAL FORMULATION

IN

IN WISTAR RATS.

Sponsor

M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA OUSHADHI,

Chollaveedu (V), Racherla (M), Prakasam (Dist.)

A.P., INDIA.

PIN Code- 523372

Performing Laboratory

M/s.TEENA BIOLABS PVT. LTD.,

Pharmacology Division

Plot No.41, S.V. Co-operative Indl. Estate,

Bachupally (V), Quthbullapur (M),

R.R. (Dist).A.P.,

INDIA

Study No: TFRCRDPCT02022015 Date: 11th

Mar 2015.

Total Number of pages: 87

105 & 106, Sree Rama Towers, Kukatpally, Hyderabad – 500072, Telangana State., INDIA.

Tel.: +91 040 23068027 Tel.Fax : + 91 040 23065808,

e-mail : [email protected]

PHARMACOLOGY DIVISION

mailto:[email protected]



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2.0 STUDY INFORMATION

Study Number : TFRCRDPCT02022015

Study Title : POLY HERBAL FORMULATION

Sponsor : M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI,

Chollaveedu (V), Prakasam (Dist.)

A.P., INDIA.

Testing Facility : M/s. TEENA BIOLABS PVT. LTD.,




R.R. (Dist).Telangana State,

INDIA.

Date of Submission of final report : Date: 11th

Mar. 2015

Study Director : Dr. C.SRIDEVI.



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3.0 CONTENTS

S.NO TITLE PAGE No.

1.0 PROJECT TITLE …………. 1

2.0 STUDY INFORMATION …………. 2

3.0 CONTENTS …………. 3 – 4

4.0 STATEMENT OF COMPLIANCE …………. 5

5.0 ABSTRACT …………. 6

6.0 INTRODUCTION …………. 7

7.0 SUMMARY …………. 8– 9

8.0 ABBREVIATIONS …………. 10

9.0 STUDY PERSONNEL …………. 11

10.0 GENERAL INFORMATION …………. 12 – 13

11.0 MATERIALS AND METHODS …………. 14 – 16

12.0 OBSERVATIONS …………. 17 – 22

13.0 STUDY RESULTS …………. 23 – 26

14.0 CONCLUSION …………. 27

15.0 TABLE INDEX …………. 28

I PRETERMINAL DEATHS …………. 29

II FOOD INTAKE …………. 30 – 33

III GROUP MEAN FOOD INTAKE – GRAPH …………. 34 – 35

IV BODY WEIGHTS …………. 36 – 39

V GROUP MEAN BODY WEIGHTS – GRAPH …………. 40 – 41

VI HOME CAGE OBSERVATIONS …………. 42 – 43

VII HAND HELD OBSERVATIONS …………. 44 – 46

VII OPEN FIELD ACTIVITY …………. 47 – 48

IX STIMULUS RESPONSE …………. 49

X NERVOUS AND MUSCLE MEASUREMENTS …………. 50

XI HAEMATOLOGY …………. 51 – 54



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XII BIOCHEMISTRY …………. 55 – 58

XIII ORGAN WEIGHTS …………. 59 – 60

XIV SUMMARY OF GROSS NECROPSY FINDINGS …………. 61 – 64

XV HISTOPATHOLOGY OBSERVATIONS …………. 65 – 67

16.0 PATHOLOGY REPORT …………. 68

17.0 APPENDICES INDEX …………. 69

I DOSAGE CALCULATION AND SCHEDULE …………. 70 – 71

II STANDARD COMPOSITION OF FEED …………. 72 – 74

II MICROBIAL LIMIT TEST …………. 75

18.0 PHOTOGRAPHS INDEX …………. 76

I MICROSCOPIC OBSERVATION OF HEART (CONTROL GROUP) …………. 77

II MICROSCOPIC OBSERVATION OF HEART (HIGH GROUP). …………. 78

III MICROSCOPIC OBSERVATION OF LUNGS( CONTROL GROUP) …………. 79

IV MICROSCOPIC OBSERVATION OF LUNGS (HIGH GROUP) …………. 80

V MICROSCOPIC OBSERVATION OF SPLEEN (CONTROL GROUP) …………. 81

VI MICROSCOPIC OBSERVATION OF SPLEEN (HIGH GROUP) …………. 82

VII MICROSCOPIC OBSERVATION OF KIDNEY (CONTROL

GROUP)

…………. 83

VII MICROSCOPIC OBSERVATION OF KIDNEY (HIGH GROUP) …………. 84

IX MICROSCOPIC OBSERVATION OF LIVER (CONTROL GROUP) …………. 85

X MICROSCOPIC OBSERVATION OF LIVER (HIGH GROUP) …………. 86

19.0 REFERENCES …………. 87



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4.0 STATEMENT OF COMPLIANCE

Test Item: POLY HERBAL FORMULATION

The objective of this sub-acute toxicity for 28 days study in rats was to assess the toxicological profile

of Poly herbal formulation when administered to rats orally for 28 days. Results of this study should indicate

potential target organs and may be useful for establishing a dosage regimen in sub-acute studies. The study was

carried out in full compliance with the guidelines laid down in “Schedule Y of the Drugs and Cosmetics Act,

1940 and Experiments on Animals (CPCSEA, M/s. Teena Biolabs Pvt. Ltd. Registration No.

177/PO/cb/99/CPCSEA).

Dr. C.SRIDEVI

_______________ ______________________ _________________

Study Director Signature Date



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5.0 ABSTRACT

REPEATED DOSE SUB ACUTE TOXICITY STUDY (28 DAYS) ON

POLY HERBAL FORMULATION

IN WISTAR RATS BY ORAL ROUTE

The present investigation in Wistar rats is to evaluate the pre clinical safety of Poly herbal

formulation. The study design involves four groups consisting 12 animals per group (6 males & 6

females). The vehicle control group received D.M water where as low dose, middle dose and high dose

group animals have received test item at various dose levels. The test item has been administered orally in

a constant volume of.1.0 ml per animals for 28 days.

Conclusion:

The results of repeated dose 28 days toxicity study indicated no toxicity and mortality in control

and treated group animals. There were no significant changes recorded in physical, clinical, behavioral,

hematological, biochemical and histopathological parameters under experimental conditions up to high

dose levels in this experiment with Wistar rats.



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6.0 INTRODUCTION

The herbal formulation is for the therapecutic treatment of polycarcinoma.The preparation was made

after phyto chemical screening of plants extracts.



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7.0 SUMMARY

7.1. Title : Repeated dose 28 days toxicity study of Poly herbal

formulation in rats by oral route.

7.2. Objective : To assess the safety profile of Poly herbal formulation in

rats.

7.3. Sponsor : M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI

7.4. Test item details

7.4.1. Test item name : Poly herbal formulation

7.4.2. Therapeutic activity : Anti-Cancer activity

7.4.3. Appearance : Brownish colored powder.

7.4.4. Dosage

regimen(Clinical)

: 1.0 ml

7.4.5. Duration of

experiment

: 28 days.

7.4.6. Route : Oral.

7.5. Test System : Wistar rats.

7.6. Test groups :

S. No Study

Type Group details Dose

Duration of

exposure

Study

period

1 Repeated

dose 28

days

toxicity

study

Vehicle control 0.0 mg/kg

28 days 28 days 2 Low Dose ( 1.0 x TD)

1350.0 mg/kg

3 Middle Dose (2.0 x TD) 2700.0 mg/kg

4 High Dose ( 3.0 x TD) 4050.0 mg/kg

7.7. Methodology

The animals were acclimatized to the laboratory conditions and exposed to the test item

through oral route for 28 days. The animals were observed twice daily for 28 days for any

symptoms of toxicity after exposure to the test item. All the animals were sacrificed on 29th

day.



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7.8.

Observations

A daily physical and clinical examination of all the groups and neurological activity -

functional observation battery was monitored once weekly in all the animals till the end of the

experiment. The haematology, biochemistry, gross necropsy, histopathology of all major

organs was undertaken in all the animals which were euthanized at the end of the experiment.

7.9. Statistics

Data was compiled and mean and standard deviation calculated then analyzed for significant

differences between vehicle control and treatment groups using GraphPad Prism software 6.0.

7.10. Results

There were no pre-terminal deaths observed in vehicle control, low dose, middle dose and high

dose group animals in repeated dose for 28 days toxicity study.

There was progressive increase in food intake; body weight of all treated groups when

compared with vehicle control animals.

Hematological parameters in all treated groups of animals at three dose levels were found to be

in normal range when compared with vehicle control animals.

Biochemical parameters in all groups of animals at three dose levels were found to be in

normal range when compared with vehicle control group.

No test item induced pathological changes was observed in the systemic organs studied



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8.0 ABBREVIATIONS

ALP = Alkaline phosphates

ALT = Alanine aminotransferase

AST =

Aspartate aminotransferase

B = Basophiles

BUN = Blood Urea Nitrogen

CPCSEA = Committee for the Purpose of Control and

Supervision on Experiments on animals

Dl = Decilitre

E = Eosinophils

Fl = Femto litre

Gm = Gram

Hb = Haemoglobin

HCT = Haematocrit

HD = High dose

IU = International unit

Kg = Kilogram

L = Lymphocytes

LD = Low dose

M = Monocytes

MCV = Mean Corpuscular Volume

MD = Middle dose

Mg = Milligram

Ml = Millilitre

N = Neutrophils

µl = Micro litre

PLT = Platelet count

RBC = Red blood cell

SD = Standard deviation

TD = Therapeutic dose

TI = Test item

VC = Vehicle control

WBC = White blood cell



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9.0 STUDY PERSONNEL, FACILITY AND ACCREDITATION

I Test Facility Management : K.Anjaiah.

II Study director : Dr.Sridevi

III Study Personnel : V.A Nageswara Rao

: B.Raju

: D.Sandhya Rani

: M.Sunitha

1 Clinical chemist : Md.Ghouse

2 Histo-technician : Sanoj kumar yadav.

3 Pathologist : Dr. K.Pradip

4 Biostatistician : V.A Nageswara Rao

IV Test Facility : M/s. TEENA BIOLABS PVT. LTD.

V Sponsor : M/s. CIREX PHARMACEUTICALS LIMITED

VI Accreditation : ISO 9001:2008 Certified

: NABL-ISO/IEC 17025:2005

VII Approvals : CPCSEA Approved

: DCGI Approved

: DCA Approved



Page 12 of 87

10.0 GENERAL INFORMATION

Study number : TFRCPCT01022015

Sponsor

: M/s. SRI SRI SRI VEMAYASWAMYAYURVEDA

OUSHADHI,

Chollaveedu (V), Prakasam (Dist.)

A.P., INDIA.

Test facility

: M/s. TEENA BIOLABS PVT. LTD.




R.R. (Dist).

A.P., INDIA.

Study centre : Animal house (Biological Testing Lab)

IAEC approval date : 02.02.2015

Receipt of test item : 04.02.2015

Receipt of animals : 04.02.2015

Study initiation : 08.02.2015

First day of drug exposure : 08.02.2015

Necropsy : 01.03.2015

Raw data completion : 05.03.2015

Submission of final report : 11.03.2015



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10.1 Data retention

All raw data, documentation, records, specimens, slides, study plans, draft reports and final

reports generated as a result of this study were kept confidential and archived by the

archiving incharge at archives room of the main building of M/s. Teena Biolabs Pvt. Ltd.

The retention duration of these records and the excess test material provided by the sponsor

kept under appropriate storage conditions will be for five years after the release of the final

report.



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11.0 MATERIALS AND METHODS

11.1. TEST ITEM

11.1.1. Test item:

Poly herbal formulation supplied by the sponsor was used.

11.1.2. Test item preparation:

The test item for administration is in standard volume of 1.0ml per animal for 28

days.

11.2. TEST SYSTEM

11.2.1. Age & weight of animals:

Wistar rats 6-8 weeks of age, at the commencement of treatment, the weight

variation of the animals will not exceed ± 20 % of the mean body weight in each

sex and group.

11.2.2. Source of animals:

M/s. Teena Biolabs Pvt. Ltd.

11.2.3. Rationale for the selection of Wistar rats as test system:

Wistar rats are one of the recommended species as test system for the conduct of

toxicity studies. They have been widely used throughout the industry for the

evaluation of product safety. Extensive data available for comparative purpose.

11.2.4. Route of administration and reason for choice:

Oral route has been chosen because it is the proposed route for administration to

humans. The dosage can be accurately administered. The oral route is one of the

proposed routes for toxicity testing.

11.2.5. Randomization and numbering of animals:

All the animals were acclimatized to the laboratory conditions for a minimum

period of five days, after acclimatization all the animals were randomized and

assigned to four groups of 12 animals consisting 6 males and 6 females. The

females were nulliparous and non pregnant.



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Group Number of animals Animal No.

Male Female

Vehicle control 12 (6M+6F) R 101 – 106 R 151 – 156

Low dose 12 (6M+6F) R 201 – 206 R 251 – 256

Middle dose 12 (6M+6F) R 301 – 306 R 351 – 356

High dose 12 (6M+6F) R 401 – 406 R 451 – 456

11.2.6. Animal husbandry:

The animals were housed in polypropylene cages as group wise and sex wise in

experimental room. Animal rooms were maintained under standard animal

husbandry conditions. The temperature of the animal room was maintained at

22±30C and relative humidity 30 to 70%. The photoperiodicity was 12 hours light

and 12 hours dark.

11.2.7. Feed and water:

The animals were fed on pelleted feed containing standard composition of all

macro and micronutrients (Appendix–III), purified water collected through aqua

guard was provided to animals ad libitum.

11.2.8. Identification of animals:

Animals were identified by body marking. Each cage was identified with

individual cage tags by study number, animal numbers, dose, group, route, species,

sex and experiment starting date.

11.2.9. Route of administration:

The test item was administered by oral route.



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11.3. STUDY DESIGN

The repeated dose 28 days toxicity study consists total of 48 rats (24 males + 24 females)

were divided into four groups. The test item Poly herbal formulation was administered

Orally at a volume of 1.0ml per animal for 28 days with various concentrations 1350.0 mg/

kg, 2700.0 mg/kg and 4050.0 mg/kg. All the animals were sacrificed on 29th

day after first

dosing of the test item. All the animals were observed twice daily for morbidity and

mortality. Home cage observations, hand held observations, open field activity, stimulus

response and nervous and muscle measurements were conducted at regular intervals. All the

animals were subjected to haematology, biochemistry and followed by necropsy and

histopathological examination of all vital organs (viz., Thymus, brain, heart, lungs, spleen,

adrenal glands, kidney and liver. Pancreas, testes or ovary and uterus).

11.4. RANDOMIZATION

Randomization ensured the allocation of treatments to animals/groups was independent of

their characteristics and were similar in all the groups. It was also taken care while

randomization, base variables were homogenized and were allotted to different groups.

The need for randomization applies not only to the allocation of the animals to the different

(control as well as treatment) groups but also to anything that can materially affect the

recorded response.

11.5. STATISTICAL ANALYSIS

The data on food intake, body weight, hematology, biochemistry and various organ

weights were subjected for analysis. Data was compiled and mean and standard deviation

calculated then analyzed for significant differences between vehicle control and treatment

groups using GraphPad Prism software 6.0.



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12.0 OBSERVATIONS

12.1. PRE-TERMINAL DEATHS:

The animals were observed twice daily after exposure to the test item for 28 days to record the

symptoms of toxicity and mortality, if any. In case of mortality, autopsy was conducted and

vital organs were collected for histopathological examination.

12.2. PHYSIOLOGICAL/ CLINICAL ACTIVITY:

12.2.1. Food intake

The food intake profile was recorded once in four days during the experimental period.

12.2.2. Body weights

The body weight of each animal was recorded once a week during the experimental period.

12.3. FUNCTIONAL OBSERVATION BATTERY:

12.3.1. Home-cage observations:

i. Body posture:

The body posture in all groups of animals during the experimental phase was

observed.

ii. Respiration character:

Respiration rate was observed in all groups of animals during the experimental

phase.

iii. Tremors:

The presence of tremors, if any was recorded.

iv. Convulsions:

Convulsions, if any were recorded immediately after the exposure to the test

item and daily during the post exposure.

v. Vocalization:

The vocalizations, if any were recorded.

vi. Palpebral closure:

The appearances of eye and eyelid closure, if any were observed.

12.3.2. Hand held observations:

The following physiological activities were monitored once weekly.



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i. Response to handling:

The behavior observed while removing from the cage.

ii. Mucous membrane:

The eye mucous membrane observed during the experimental period.

iii. Skin:

The condition of skin observed during the experimental period.

iv. Fur:

The physical appearance of fur coat, if any was observed.

v. Lacrimation:

The lacrimation, if any was observed during the experimental period in all the

groups.

vi. Salivation:

The salivation, if any was observed during the experimental period in all the

groups.

vii. Piloerection:

Piloerection, if any were observed in all the groups.

12.3.3. Open field activity:

The following physical observations were recorded periodically.

i. Number of rearings:

The numbers of rearings, if any were observed in 3 minutes time during the

experimental period.

ii. Gait:

The gaits, if any were observed in all groups of animals.

iii. Tail elevation:

The tail position of the animal whether it is lifted while walking or

occasionally lifted was observed.

iv. Head position:

The head position was observed without tilt.

v. Occurrence of stereotype:

The stereotype (Bizarre ehavior), if any were observed in all

vi. Faeces colour:

The faeces colour during the experimental period in all the groups of animals

was observed.



Page 19 of 87

vii. Urine color:

The color of urine observed in all groups.

12.3.4. Stimulus response:

The following activities were monitored once weekly.

i. Pinna reflex:

The Pinna touch response was noted by touching with a blunt object inside

Pinna and the response was recorded.

ii. Righting reflex:

The righting reflex, if any were observed in all the groups

12.3.5. Nervous and muscle measurements:

The following nervous and muscle measurements were monitored once weekly.

i. Abdominal tone:

The muscle tones, if any were observed in all groups.

ii. Limb tone:

The limb tones, if any were observed in all groups.

iii. Grip strength:

The grip strength was observed in all the groups.

iv. Locomotor activity:

The locomotor activities, if any were observed in all groups of animals.

12.4. CLINICAL SIGNS:

General clinical observations were conducted once daily during the study period.

12.5. CLINICAL LABORATORY INVESTIGATIONS

12.5.1. Haematology:

The whole blood samples drawn from retro orbital plexus were collected in K2

EDTA vacutainers and were subjected for haematological evaluation in

haematology cell counter, SYSMEX-KX 21, to study the following parameters.

1) Total white blood cell count (WBC)

2) Total Red blood cell count (RBC)

3) Haemoglobin (Hb)

4) Haematocrit (PCV)

5) Mean corpuscular volume (MCV)

6) Mean hemoglobin (MCH)

7) Mean hemoglobin concentration (MCHC)

8) Platelets (PLT)



Page 20 of 87

12.5.2. Biochemistry:

The blood samples drawn from the retro orbital plexus collected in plain

vacutainers and centrifuged at 3000 rpm for about 10 minutes to separate the serum

for biochemistry analysis using ERBA biochemical analyzer. Following principles

are used.

12.5.2.1. Albumin:

Albumin binds with bromocresol green (BCG) at pH

4.2 causing a shift

in absorbance of the yellow BCG dye. The blue-green colour formed is

proportional to concentration of albumin present, when measured

photo metrically between 580 – 630 nm.

12.5.2.2. ALP:

Para-nitro phenyl phosphate reacts with water in the presence of ALP

and magnesium para nitro phenol and phosphate.

12.5.2.3. Bilirubin:

Bilirubin reacts with diazotized sulphanilic acid in acidic medium to

form pink coloured azobilirubin with absorbance directly proportional

to bilirubin concentration. Direct bilirubin, being water-soluble

directly reacts in acidic medium. However indirect or unconjugated

bilirubin is solubilised using a surfactant and then it reacts similar to

direct bilirubin.

12.5.2.4. Creatinine:

Creatinine reacts with alkaline picrate to produce an orange-yellow

colour (the Jaffe’s reaction). Specificity of the assay has been

improved by the introduction of an initial rate method. The absorbance

of the orange-yellow colour formed is directly proportional to

creatinine concentration and is measured photometrically at 500 – 520

nm.



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12.5.2.5. Glucose:

Glucose Oxidase is an enzyme; it converts glucose to gluconic acid

and hydrogen peroxide in the presence of water and oxygen. Hydrogen

peroxide reacts with 4 – aminoantipyrine and 4 – ehavior benzoic

acid in the presence of enzyme peroxidase, it is converted into

quinoneimine dye (pink colour) and water. Pink colour is

proportional to the glucose concentration and can be measured photo

metrically between 500 to 570 nm.

12.5.2.6. AST (SGOT):

L–aspartate reacts with 2–oxoglutarate in the presence of AST to form

oxaloacetate and L– ehavior . Pyruvate reacts with NMDH in the

presence of MDH to form malate and NMD. Sample pyruvate reacts

with NMDH in the presence of LDH forms L-lactate and NMD.

12.5.2.7. ALT (SGPT):

L–alanine reacts with 2–oxoglutarate in the presence of ALT to form

pyruvate and L–glutamate. Pyruvate reacts with NMDH in the

presence of LDH to form L-lactate and NMD.

12.5.2.8. Total protein:

The peptide bonds of the proteins react with copper II ions in alkaline

solution to form blue- violet complex. Tartrate is used as stabilizer.

The colour developed is proportional to protein concentration and is

measured at 576 nm.

12.5.2.9. Cholesterol:

Cholesterol ester is converted to cholesterol and fatty acid in the

presence of cholesterol esterase (CE) and the cholesterol reacts with

oxygen in the presence of cholesterol Oxidase (CHOD) to form

cholest-4-en-3-one and hydrogen peroxide (H2O2) and then H2O2 reacts

with 4-aminoantipyrne (4AAP) and phenol in the presence of

peroxidase POD to form water and quinoneimine. The absorbance of

Quinoneimine present in the specimen is directly proportional to

cholesterol concentration.



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12.5.3 NECROPSY & HISTOPATHOLOGY

12.5.3.1. Gross observations:

After the experimental period, the rats were euthanized by carbon dioxide and

subjected to gross necropsy and findings recorded. External features suggesting any

abnormality were also recorded. After opening the chest and abdominal cavities, an in

situ examination of organs was done. The individual organs were again examined for

gross morphological changes (if any) after removal.

12.5.3.2. Organ weights:

After detailed gross necropsy examination, Liver , spleen, kidney, lungs, heart,

brain, adrenal, thymus, testes and uterus with ovary the following organs were

collected from each animal and the weights were recorded using electronic

weighing machine (Shimadzu AX-200).

Ttestes,Ute

and uterus with ovary.

12.5.3.3. Histopathology

The brain, spleen, kidney, heart, lungs, liver, thymus, pancreas, intestines, testes,

ovary and uterus were collected from the control and treated animals and preserved

in 10% neutral buffered formalin: The organs were sliced adequately after fixation,

they were sampled and processed by conventional methods, paraffin blocks were

made and 6 m paraffin sections obtained which were stained with hematoxylin and

eosin and examined under a research microscope. Histological changes in tissues

were recorded and compared with corresponding controls. Histopathological findings

were given in Table-XVI.



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13.0 STUDY RESULTS

13.1. PRE-TERMINAL DEATHS:

There were no pre-terminal deaths recorded in animals receiving the vehicle, low, middle and

high dose group animals in repeated dose 28 days toxicity study (Table-I).

13.2. PHYSIOLOGICAL ACTIVITY:

13.2.1. Food intake:

There was progressive increase in food intake of all treated animals when compared with

vehicle control animals.

Detail data displayed in Table II A to II D. Group mean was displayed in graphical form as

Table III.

13.2.2. Body weights:

There was progressive increase in body weights of all treated animals when compared with

vehicle control animals.

Detail data displayed in Table IV A to IV D. Group mean was displayed in graphical form as

Table V.

13.3. FUNCTIONAL OBSERVATION BATTERY:

13.3.1. Home-cage observations:

i. Body posture:

There were no significant abnormalities in body posture in all groups of animals

during the experimental phase.

ii. Respiration character:

There were no significant abnormalities in respiration character and rate in all

groups of animals during the experimental phase.

iii. Tremors:

There were no tremors in the animals before and after exposure to the test item.

iv. Convulsions:

There were no convulsions recorded immediately after the administration of

test item and during post exposure in any group of animals.

v. Vocalization:

There was no spontaneous vocalization observed.

vi. Palpebral closure:

There was no abnormal closure of the animal eyes with in the cage.



Page 24 of 87

13.3.2. Hand held observations:

The following physiological activities were monitored once weekly.

i. Response to handling:

There was no abnormal ehavior observed while removing from the cage.

ii. Mucous membrane:

There was no abnormal eye mucous membrane observed during the

experimental period. iii. Skin:

There was no abnormal condition of skin observed during the study period.

iv. Fur:

The clean groomed fur was observed in all the animals exposed to test item.

v. Lacrimation:

No excessive lacrimation was observed during the experimental period in all

the groups.

vi. Salivation:

No excessive salivation was observed during the experimental period in all the

groups.

vii. Piloerection:

No piloerection was observed in all the groups.

13.3.3. Open field activity:

The following physical observations were recorded periodically.

i. Number of rearings:

There was no abnormal number of rearings observed in 3 minutes time during

the experimental period.

ii. Gait:

There was no abnormal gait in all groups of animals.

iii. Tail elevation:

All the animals in all groups lifted tail while walking during the observation

period.

iv. Head position:

Head position was normal without tilt.

v. Occurrence of stereotype:

There was no abnormal stereotype (Bizarre behavior) observed in all groups.



Page 25 of 87

vi. Faces color:

There were no abnormalities in the faeces colour during the experimental period

in all the groups of animals.

vii. Urine colour:

There was no abnormal urine colour observed in all groups.

13.3.4. Stimulus response:

The following activities were monitored once weekly.

i. Pinna reflex:

There was no abnormal pinna touch response in all groups of animals.

ii. Righting reflex:

There was no abnormal righting reflex observed in all the groups

13.3.5. Nervous and muscle measurements:

The following nervous and muscle measurements were monitored once weekly.

i. Abdominal tone:

There was no abnormal muscle tone observed in all groups.

ii. Limb tone:

There was no abnormal limb tone observed in all groups.

iii. Grip strength:

There was no abnormal change in grip strength in all groups.

iv. Loco motor activity:

The locomotor activity has found to be normal in all groups of animals.

13.4. CLINICAL OBSERVATIONS:

We did not find any clinical symptoms of toxicity in all experimental group during entire

experimental period. The observations include gross evaluation of skin and fur, eyes and

mucous membranes, respiratory, autonomic and central nervous systems and behavior pattern.

13.5. CLINICAL LABORATORY INVESTIGATIONS:

13.5.1. Haematology:

The hematological parameters in all the treated groups of rats were found to be in

normal when compare with vehicle control group animals. (Table XI).



Page 26 of 87

13.5.2. Biochemistry:

The biochemical parameters tested were not altered to rats in various doses. All the

parameters were found to be normal when compare to vehicle control group

animals (Table-XII).

13.6. NECROPSY AND HISTOPATHOLOGY:

13.6.1. Gross observations:

No significant gross changes in the organs of animals in any of the groups (VC, LD,

and MD & HD) were present (Table-XIV.

13.6.2. Organ weights:

There were no significant changes in the animals organ weights of liver, kidneys,

lungs, heart, spleen, brain, testes, adrenal, thymus, tested and uterus with ovary and

the weights of all organs were observed to be in normal range in treated groups of

animals and comparable to vehicle control group animals (Table-XIII).

13.6.3. Histopathology:

Based on the summary of histopathological observation the drug may not have

produced any major reactive and toxic changes in the systemic organs (Brain,

spleen, heart, lungs, kidney, adrenal glands, thymus and intestines) including

reproductive organs such as testes and uterus. Histopathological observations of

all the vital organs are given in table XV.



Page 27 of 87

14.0. CONCLUSION

All the animals exposed to the test item at three dose levels were active, there were no pre-terminal

deaths recorded in vehicle control, low dose, middle dose & high dose. There were no significant test

item “Poly herbal formulation” associated changes in physical, clinical and behavioral profile.

Food intake was normal in all groups receiving vehicle and test item at various doses throughout the

study period. But mild increase in the food consumption in female of treated group when compared

with control animals.

There was a progressive increase in body weight in both male and female animals of all treated

groups animals when compared with vehicle control animals.

Hematological parameters in all treated groups of animals at three dose levels were found to be in

normal range when compared with control animals.

Biochemical parameters in all treated groups of animals at three dose levels were found to be in

normal range when compared with vehicle control group.

There were no gross changes in the vital organs collected after euthanization. There were no

significant changes in the weights of the organs in all groups of animals exposed to the test item at

various dose levels.

Based on summary of histopathological observation, we could not observe any toxic reaction in

major systemic organ such as brain, liver, lungs, heart, spleen, pancreas, kidney, testes, uterus, ovary,

thymus, adrenal and intestine.

Based on the summary of gross pathological observation the drug may not produced any major

reactive and toxic changes in systemic organs.

The study established that Poly herbal formulation has not produced any significant changes in

clinical, behavioral, physical, physiological, biochemical, hematological parameters at three different

dose levels on administration test item at recommended clinical dosage schedule under experimental

conditions in the animals studied.



Page 28 of 87

15.0 TABLE INDEX

S.NO. TITLE PAGE NO.

I Pre-Terminal Death(s) ……………………….. 29

II Food Intake ……………………….. 30 – 33

III Group Mean Food Intake – graph ……………….……… 34 – 35

IV Body Weights ………………………. 36 – 39

V Group Mean Body Weights – graph ………………………. 40 – 41

VI Home Cage Observations ………………………... 42 – 43

VII Hand held Observations ………………………... 44 – 46

VIII Open field Activity ………………………… 47 – 48

IX Stimulus Response ………………………… 49

X Nervous and Muscle Measurements ……………………….. 50

XI Haematology …………………………. 51 – 54

XII Biochemistry ………………………… 55 – 58

XIII Organ Weights ………………………… 59 – 60

XIV Summary of Gross Necropsy Findings ………………………….. 61 – 64

XV Histopathological Observations …………………………. 65 – 67



Page 29 of 87

TABLE – I

PRETERMINAL DEATH(S) TABLE

Days of

observation

VC LD MD HD

M F M F M F M F

1 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

2 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

3 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

4 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

5 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

7 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

8 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

9 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

10 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

11 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

12 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

13 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

14 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

15 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

16 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

17 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

18 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

19 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

20 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

21 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

22 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

23 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

24 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

25 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

26 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

27 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6

28 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6



Page 30 of 87

TABLE –II-A

FOOD INTAKE PER DAY

Test item: POLY HERBAL FORMULATION

Dose: 0.0 mg/kg (VC)

MALES

Animal ID No. gm/day (Average/Animal)

4 8 12 16 20 24 28

101

8.33 8.58 9.16 9.83 10.75 11.25 12.16 102

103

Mean 8.33 8.58 9.16 9.83 10.75 11.25 12.16


4 8 12 16 20 24 28

104

8.41 8.66 9.00 9.66 10.50 11.33 12.08 105

106

Mean 8.41 8.66 9.00 9.66 10.50 11.33 12.08

FEMALES


4 8 31 16 20 24 28

151

8.16 8.41 9.00 9.33 9.66 10.41 12.00 152

153

Mean 8.16 8.41 9.00 9.33 9.66 10.41 12.00


4 8 31 16 20 24 28

154

8.33 8.66 8.83 9.50 9.91 11.00 12.02 155

156

Mean 8.33 8.66 8.83 9.50 9.91 11.00 12.02



Page 31 of 87

TABLE –II-B

FOOD INTAKE


Dose: 1350.0 mg/kg (LD)

MALES


4 8 12 16 20 24 28

201

8.66 9.00 9.66 10.66 11.25 12.00 12.75 202

203

Mean 8.66 9.00 9.66 10.66 11.25 12.00 12.75


4 8 12 16 20 24 28

204

8.75 8.91 9.58 10.41 10.91 11.75 12.58 205

206

Mean 8.75 8.91 9.58 10.41 10.91 11.75 12.58

FEMALES


4 8 31 16 20 24 28

251

8.50 8.58 9.16 9.83 10.25 11.50 12.50 252

253

Mean 8.50 8.58 9.16 9.83 10.25 11.50 12.50


4 8 31 16 20 24 28

254

8.41 8.83 9.58 10.16 10.66 11.66 12.61 255

256

Mean 8.41 8.83 9.58 10.16 10.66 11.66 12.61



Page 32 of 87

TABLE –II-C

FOOD INTAKE


Dose: 2700.0 mg/kg (MD)

MALES


4 8 12 16 20 24 28

301

9.00 9.08 10.33 11.00 11.66 12.50 13.33 302

303

Mean 9.00 9.08 10.33 11.00 11.66 12.50 13.33


4 8 12 16 20 24 28

304

9.16 9.83 10.50 11.25 11.75 12.41 13.50 305

306

Mean 9.16 9.83 10.50 11.25 11.75 12.41 13.50

FEMALES


4 8 31 16 20 24 28

351

8.75 9.16 10.25 10.50 11.25 11.91 13.00 352

353

Mean 8.75 9.16 10.25 10.50 11.25 11.91 13.00


4 8 31 16 20 24 28

354

9.00 9.66 10.41 10.75 11.66 12.50 13.50 355

356

Mean 9.00 9.66 10.41 10.75 11.66 12.50 13.50



Page 33 of 87

TABLE –II-D

FOOD INTAKE


Dose: 4050.0 mg/kg (HD)

MALES


4 8 12 16 20 24 28

401

9.66 10.75 11.33 12.25 12.83 13.50 14.25 402

403

Mean 9.66 10.75 11.33 12.25 12.83 13.50 14.25


4 8 12 16 20 24 28

404

10.00 10.66 11.50 12.00 13.00 13.66 14.33 405

406

Mean 10.00 10.66 11.50 12.00 13.00 13.66 14.33

FEMALES


4 8 31 16 20 24 28

451

9.58 10.66 10.75 11.00 11.83 12.58 13.75 452

453

Mean 9.58 10.66 10.75 11.00 11.83 12.58 13.75


4 8 31 16 20 24 28

454

9.83 10.41 10.91 11.50 12.33 13.00 14.00 455

456

Mean 9.83 10.41 10.91 11.50 12.33 13.00 14.00



Page 34 of 87

III. MEAN FOOD CONSUMPTION OF ALL GROUPS - MALE



Page 35 of 87

III. MEAN FOOD CONSUMPTION OF ALL GROUPS - FEMALE



Page 36 of 87

TABLE –IV – A

BODY WEIGHT



MALES

Animal ID No. gm/day

0 7 14 21 28

101 161 179 194 210 230

102 163 180 197 212 231

103 167 183 198 215 235

Mean 164 181 196 212 232

S.D. 3.06 2.08 2.08 2.52 2.65


0 7 14 21 28

104 160 177 193 208 228

105 164 180 194 211 229

106 168 185 198 216 238

Mean 164 181 195 212 232

S.D. 4.00 4.04 2.65 4.04 5.51

FEMALES


0 7 14 21 28

151 162 176 188 204 220

152 165 178 191 210 228

153 169 182 196 214 230

Mean 165 179 192 209 226

S.D. 3.51 3.06 4.04 5.03 5.29


0 7 14 21 28

154 160 172 185 203 220

155 162 176 189 203 223

156 168 181 195 213 231

Mean 163 176 190 206 225

S.D. 4.16 4.51 5.03 5.77 5.69



Page 37 of 87

TABLE –IV – B

BODY WEIGHT



MALES


0 7 14 21 28

201 161 180 196 210 231

202 162 179 198 214 233

203 165 183 200 217 236

Mean 163 181 198 214 233

S.D. 2.08 2.08 2.00 3.51 2.52


0 7 14 21 28

204 162 178 193 207 224

205 165 183 199 212 232

206 169 186 203 217 237

Mean 165 182 198 212 231

S.D. 3.51 4.04 5.03 5.00 6.56

FEMALES


0 7 14 21 28

251 161 174 186 204 221

252 164 179 193 211 227

253 170 184 197 215 230

Mean 165 179 192 210 226

S.D. 4.58 5.00 5.57 5.57 4.58


0 7 14 21 28

254 163 175 190 207 225

255 167 179 195 214 233

256 169 182 198 215 232

Mean 166 179 194 212 230

S.D. 3.06 3.51 4.04 4.36 4.36



Page 38 of 87

TABLE –IV – C

BODY WEIGHT



MALES


0 7 14 21 28

301 160 175 189 209 227

302 165 181 196 215 232

303 170 186 204 222 240

Mean 165 181 196 215 233

S.D. 5.00 5.51 7.51 6.51 6.56


0 7 14 21 28

304 161 175 190 206 224

305 164 181 195 212 231

306 168 186 201 219 239

Mean 164 181 195 212 231

S.D. 3.51 5.51 5.51 6.51 7.51

FEMALES


0 7 14 21 28

351 161 173 187 203 221

352 163 179 192 209 227

353 167 181 195 214 231

Mean 164 178 191 209 226

S.D. 3.06 4.16 4.04 5.51 5.03


0 7 14 21 28

354 160 175 186 203 220

355 162 178 190 208 224

356 168 183 200 218 233

Mean 163 179 192 210 226

S.D. 4.16 4.04 7.21 7.64 6.66



Page 39 of 87

TABLE –IV – D

BODY WEIGHT



MALES


0 7 14 21 28

401 162 177 191 207 223

402 168 180 195 213 229

403 170 185 202 220 239

Mean 167 181 196 213 230

S.D. 4.16 4.04 5.57 6.51 8.08


0 7 14 21 28

404 160 174 187 202 219

405 167 180 196 214 232

406 169 187 205 222 240

Mean 165 180 196 213 230

S.D. 4.73 6.51 9.00 10.07 10.60

FEMALES


0 7 14 21 28

451 162 178 195 211 227

452 164 179 193 209 223

453 171 185 201 219 235

Mean 166 181 196 213 228

S.D. 4.73 3.79 4.16 5.29 6.11


0 7 14 21 28

454 160 173 188 205 219

455 166 178 195 211 227

456 169 183 199 215 230

Mean 165 178 194 210 225

S.D. 4.58 5.00 5.57 5.03 5.69



Page 40 of 87

V. GROUP MEAN BODY WEIGHTS MALE



Page 41 of 87

V. GROUP MEAN BODY WEIGHTS FEMALE



Page 42 of 87

TABLE – VI

HOME CAGE OBSERVATIONS


Groups Days Body posture (%) Respiration character (%) Tremors

Normal Abnormal Normal Abnormal None

VC

1st Day 100(12) Nil 100(12) Nil 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12)

LD

1st Day 100(12) Nil 100(12) Nil 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12)

MD

1st Day 100(12) Nil 100(12) Nil 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12)

HD

1st Day 100(12) Nil 100(12) Nil 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12)

Male & Female (12) No. of animals 100 – Normal,



Page 43 of 87

TABLE – VI

HOME CAGE OBSERVATIONS

Test item: Poly herbal formulation

Groups Days Convulsions

Vocalization Palpebral closure

None Open Eyelids slightly

lowered

VC

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

LD

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

MD

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

HD

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

Male & Female (12) No. of animals 100 - Normal



Page 44 of 87

TABLE – VII

HAND HELD OBSERVATIONS


Groups

Days

Behaviour while removing from cage

Groups Days Very easy Easy/ normal Runs around

cage Vocalization Very difficult

VC

1st Day 100(12) Nil Nil Nil Nil

8th

Day 100(12) Nil Nil Nil Nil

15th


22nd


28th


LD


8th


15th


22nd


28th


MD


8th


15th


22nd


28th


HD


8th


15th


22nd


28th





Page 45 of 87

TABLE – VII



Groups Days

Eye Mucous membrane Skin Fur

Normal Congested Normal Cleanly

groomed Slightly soiled

VC

1st Day 100(12) Nil 100(12) 100(12) Nil

8th

Day 100(12) Nil 100(12) 100(12) Nil

15th

Day 100(12) Nil 100(12) 100(12) Nil

22nd

Day 100(12) Nil 100(12) 100(12) Nil

28th

Day 100(12) Nil 100(12) 100(12) Nil

LD

1st Day 100(12) Nil 100(12) 100(12) Nil

8th

Day 100(12) Nil 100(12) 100(12) Nil

15th

Day 100(12) Nil 100(12) 100(12) Nil

22nd

Day 100(12) Nil 100(12) 100(12) Nil

28th

Day 100(12) Nil 100(12) 100(12) Nil

MD

1st Day 100(12) Nil 100(12) 100(12) Nil

8th

Day 100(12) Nil 100(12) 100(12) Nil

15th

Day 100(12) Nil 100(12) 100(12) Nil

22nd

Day 100(12) Nil 100(12) 100(12) Nil

28th

Day 100(12) Nil 100(12) 100(12) Nil

HD

1st Day 100(12) Nil 100(12) 100(12) Nil

8th

Day 100(12) Nil 100(12) 100(12) Nil

15th

Day 100(12) Nil 100(12) 100(12) Nil

22nd

Day 100(12) Nil 100(12) 100(12) Nil

28th

Day 100(12) Nil 100(12) 100(12) Nil

Male & Female (12) No. of animals 100 – Normal



Page 46 of 87

TABLE – VII



Groups Days Lacrimation Salivation Piloerection

None None None

VC

1st Day 100(12) 100(12) 100(12)

8th

Day 100(12) 100(12) 100(12)

15th

Day 100(12) 100(12) 101(1)

22nd

Day 100(12) 100(12) 100(12)

28th

Day 100(12) 100(12) 100(12)

LD

1st Day 100(12) 100(12) 100(12)

8th

Day 100(12) 101(1) 100(12)

15th

Day 100(12) 100(12) 100(12)

22nd

Day 100(12) 100(12) 100(12)

28th

Day 100(12) 100(12) 100(12)

MD

1st Day 100(12) 100(12) 100(12)

8th

Day 100(12) 100(12) 101(1)

15th

Day 100(12) 100(12) 100(12)

22nd

Day 100(12) 100(12) 100(12)

28th

Day 100(12) 100(12) 100(12)

HD

1st Day 100(12) 100 (12) 100(12)

8th

Day 100(12) 100(12) 100(12)

15th

Day 100(12) 101(1) 100(12)

22nd

Day 100(12) 100(12) 100(12)

28th

Day 100(12) 100(12) 100(12)

Male & Female (12) No. of animals 100 – Normal,101- Piloerection ,101- Salivation



Page 47 of 87

TABLE – VIII

OPEN FIELD ACTIVITY


Groups Days

No. of rearings Gait Tail elevation

Normal Normal Lifted while

walking Occasionally lifted

VC

1st Day 100(12) 100(12) 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil

LD

1st Day 100(12) 100(12) 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil

MD

1st Day 100(12) 100(12) 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil

HD

1st Day 100(12) 100(12) 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil




Page 48 of 87

TABLE – VIII

OPEN FIELD ACTIVITY


Groups Days

Head position Occurrence of

stereotype Faeces colour Urine colour

Normal None Normal Pale Dark Normal Pale/dark

yellow

VC

1st Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

LD

1st Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

MD

1st Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

HD

1st Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

8th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

15th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

22nd

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil

28th

Day 100(12) 100(12) 100(12) Nil Nil 100(12) Nil




Page 49 of 87

TABLE – IX

STIMULUS RESPONSE


Groups Days

Pinna touch response Righting reflex

Auricle

Twitched/normal

Auricle does not

twitch

Normal Abnormal

VC

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

LD

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

MD

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil

HD

1st Day 100(12) Nil 100(12) Nil

8th

Day 100(12) Nil 100(12) Nil

15th

Day 100(12) Nil 100(12) Nil

22nd

Day 100(12) Nil 100(12) Nil

28th

Day 100(12) Nil 100(12) Nil




Page 50 of 87

TABLE – X

NERVOUS AND MUSCLE MEASUREMENTS


Groups Days

Abdominal tone Limb tone Grip

strength

Locomotor

Activity

Normal Abnormal Normal Abnormal Normal Normal

VC

1st Day 100(12) Nil 100(12) Nil 100(12) 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

LD

1st Day 100(12) Nil 100(12) Nil 100(12) 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

MD

1st Day 100(12) Nil 100(12) Nil 100(12) 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

HD

1st Day 100(12) Nil 100(12) Nil 100(12) 100(12)

8th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

15th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

22nd

Day 100(12) Nil 100(12) Nil 100(12) 100(12)

28th

Day 100(12) Nil 100(12) Nil 100(12) 100(12)




Page 51 of 87

TABLE – XI– A

HAEMATOLOGY



MALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

101 10.54 7.89 15.56 46.81 52.98 17.94 31.23 649

102 11.19 8.62 13.85 43.57 55.13 18.85 30.98 693

103 12.85 7.38 14.32 46.60 56.45 17.39 31.06 708

104 12.69 9.24 15.16 49.32 53.76 18.55 30.65 727

105 11.91 8.51 14.20 43.80 58.91 18.12 30.81 664

106 13.13 8.36 13.46 45.78 56.22 17.24 32.17 734

Mean 12.05 8.33 14.43 45.98 55.58 18.02 31.15 695.83

SD 1.03 0.64 0.79 2.14 2.12 0.63 0.54 34.03

FEMALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

151 7.28 7.59 13.28 42.58 56.39 16.57 30.98 647

152 8.25 7.81 13.75 43.68 56.24 16.36 30.77 693

153 8.13 7.95 14.02 42.69 54.86 17.21 31.34 648

154 7.49 8.26 14.89 43.12 55.13 16.98 30.98 714

155 8.99 8.54 13.92 41.99 56.48 17.35 32.57 708

156 8.56 7.96 14.60 42.10 57.68 17.68 31.99 724

Mean 8.12 8.02 14.08 42.69 56.13 17.03 31.44 689.00

SD 0.64 0.34 0.58 0.63 1.02 0.49 0.64 33.68



Page 52 of 87

TABLE – XI– B

HAEMATOLOGY



MALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

201 12.68 8.31 14.35 42.89 54.69 17.50 32.57 754

202 10.51 8.59 13.62 46.58 56.84 17.89 31.98 698

203 12.69 9.12 14.20 43.60 55.23 18.75 30.80 743

204 13.54 8.56 14.28 48.12 53.51 17.24 32.56 675

205 10.69 7.99 15.16 45.68 55.96 18.40 32.50 699

206 11.85 8.23 15.01 43.81 57.54 17.97 31.88 719

Mean 11.99 8.47 14.44 45.11 55.63 17.96 32.05 714.67

SD 1.21 0.39 0.57 2.02 1.47 0.56 0.68 29.88

FEMALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

251 8.35 7.35 14.35 43.68 56.98 17.35 31.38 615

252 8.62 8.22 13.31 42.66 57.61 16.51 31.63 658

253 8.31 7.69 14.02 43.81 55.93 17.99 30.97 712

254 7.98 9.15 14.53 43.58 57.24 16.85 31.62 668

255 8.12 8.42 13.88 42.83 56.81 17.68 30.70 689

256 8.35 7.14 13.93 43.27 55.60 16.89 32.37 716

Mean 8.29 8.00 14.00 43.31 56.70 17.21 31.45 676.33

SD 0.22 0.68 0.39 0.47 0.78 0.56 0.53 37.88



Page 53 of 87

TABLE – XI– C

HAEMATOLOGY



MALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

301 12.35 8.45 14.24 43.36 57.59 17.96 31.21 714

302 11.91 8.69 13.99 48.02 56.94 18.27 31.98 698

303 12.46 9.12 15.32 47.66 52.16 18.02 30.16 781

304 13.04 9.52 14.85 44.87 55.57 18.97 31.00 735

305 11.58 8.27 15.24 42.51 58.87 17.25 32.18 675

306 12.01 9.41 13.43 47.12 54.89 18.57 32.19 691

Mean 12.23 8.91 14.51 45.59 56.00 18.17 31.45 715.67

SD 0.51 0.52 0.75 2.35 2.36 0.59 0.81 38.00

FEMALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

351 8.69 7.39 14.61 43.71 57.24 17.68 30.69 698

352 7.68 8.47 13.86 43.60 56.12 17.98 31.97 646

353 8.23 8.19 14.97 42.98 57.35 16.96 32.41 687

354 8.67 7.98 14.89 44.36 58.10 17.38 30.78 718

355 8.64 8.75 13.02 43.97 56.17 16.21 32.38 634

356 8.01 7.49 13.35 44.21 57.68 17.40 31.47 669

Mean 8.32 8.05 14.12 43.81 57.11 17.27 31.62 675.33

SD 0.42 0.54 0.83 0.50 0.81 0.62 0.70 31.87



Page 54 of 87

TABLE – XI– D

HAEMATOLOGY



MALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

401 12.68 8.99 15.69 47.55 58.65 18.46 31.23 698

402 11.25 8.45 13.86 43.98 56.32 17.39 32.61 646

403 12.86 9.31 15.80 46.67 53.29 17.42 32.67 742

404 13.54 9.26 14.38 42.89 57.13 18.96 30.90 696

405 11.58 8.96 13.54 48.41 59.71 17.86 33.14 664

406 12.89 9.12 14.56 43.86 54.50 18.23 32.18 645

Mean 12.47 9.02 14.64 45.56 56.60 18.05 32.12 681.83

SD 0.87 0.31 0.93 2.27 2.43 0.62 0.88 37.53

FEMALES

Animal ID No.

Total

WBC

103/µl

Total

RBC

106/µl

(HB%)

gm/dl

PCV

(%)

MCV

(fl)

MCH

(pg)

MCHC

(%)

Platelets

103/µl

Limits 6.2 - 17.9 7.12 - 9.44 13.2 - 16.7 40.8 - 53.1 50.2 - 63.6 16.1 - 20.1 30.6 - 33.2 543 - 876

451 8.69 7.15 14.03 44.38 56.89 16.24 32.45 745

452 8.47 8.92 13.47 44.13 56.35 17.86 31.90 812

453 8.13 7.26 14.40 42.90 57.38 17.69 31.47 789

454 7.54 8.14 14.23 43.86 56.40 16.35 30.83 768

455 8.99 7.42 13.97 43.59 58.69 16.98 32.58 735

456 8.65 8.75 14.63 43.68 56.88 17.29 30.81 814

Mean 8.41 7.94 14.12 43.76 57.10 17.07 31.67 777.17

SD 0.51 0.78 0.40 0.51 0.87 0.67 0.70 33.49



Page 55 of 87

TABLE – XII– A

BIOCHEMISTRY



MALES

Animal ID

No. S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

101 83.21 14.87 0.43 0.12 42.68 165.34 143.69 6.24 3.99 55.58

102 85.59 16.74 0.39 0.16 48.23 162.67 146.22 6.98 4.02 53.86

103 81.96 14.61 0.51 0.25 49.37 162.96 142.41 7.14 4.32 51.96

104 86.35 17.14 0.64 0.31 43.39 164.32 143.82 6.86 4.23 57.99

105 82.61 13.80 0.49 0.13 46.72 166.47 142.72 7.32 4.56 55.38

106 86.15 12.72 0.71 0.21 45.87 166.38 148.34 6.79 4.69 58.73

Mean 84.31 14.98 0.53 0.20 46.04 164.69 144.53 6.89 4.30 55.58

SD 1.94 1.70 0.12 0.07 2.63 1.65 2.30 0.37 0.28 2.52

FEMALES

Animal ID

No. S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

151 87.94 16.31 0.43 0.13 46.38 162.59 148.89 6.21 3.95 65.37

152 84.37 17.92 0.53 0.15 47.29 168.34 141.84 5.86 4.21 69.15

153 82.64 15.82 0.41 0.24 42.12 163.94 146.24 6.35 3.76 62.62

154 85.31 14.35 0.58 0.29 45.72 161.25 149.08 6.93 4.11 64.75

155 89.60 16.69 0.49 0.18 46.71 166.84 147.86 6.98 4.38 66.73

156 83.81 17.31 0.51 0.25 43.01 163.69 145.29 7.11 4.26 62.81

Mean 85.61 16.40 0.49 0.21 45.21 164.44 146.53 6.57 4.11 65.24

SD 2.65 1.25 0.06 0.06 2.13 2.66 2.74 0.50 0.23 2.47



Page 56 of 87

TABLE – XII– B

BIOCHEMISTRY



MALES

Animal ID

No.

S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

201 83.75 14.89 0.46 0.26 42.69 166.37 142.87 6.15 3.69 56.75

202 85.94 17.67 0.38 0.23 45.53 162.68 144.90 6.03 3.82 59.46

203 80.36 15.75 0.73 0.17 43.86 168.39 148.46 7.25 4.21 55.70

204 87.65 13.72 0.65 0.21 47.71 162.36 145.98 6.97 4.59 58.81

205 82.69 14.55 0.55 0.29 43.92 167.65 143.64 6.89 4.20 53.68

206 86.31 16.38 0.49 0.18 42.16 165.49 146.37 6.25 4.37 56.42

Mean 84.45 15.49 0.54 0.22 44.31 165.49 145.37 6.59 4.15 56.80

SD 2.69 1.41 0.13 0.05 2.03 2.51 2.02 0.51 0.34 2.11

FEMALES

Animal ID

No.

S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

251 85.67 14.92 0.54 0.29 45.38 166.96 142.87 5.96 4.06 67.57

252 83.89 15.82 0.58 0.21 42.06 161.34 145.74 6.39 3.85 64.70

253 86.75 16.74 0.48 0.13 44.86 168.41 148.28 6.95 4.12 67.82

254 84.32 16.95 0.59 0.18 40.84 166.36 144.34 6.05 4.41 63.71

255 82.87 17.24 0.51 0.25 47.11 162.89 148.71 7.15 4.25 62.50

256 87.79 16.25 0.53 0.19 45.89 169.09 143.31 6.91 4.39 64.87

Mean 85.22 16.32 0.54 0.21 44.36 165.84 145.54 6.57 4.18 65.20

SD 1.86 0.85 0.04 0.06 2.40 3.09 2.50 0.50 0.21 2.11



Page 57 of 87

TABLE – XII– C

BIOCHEMISTRY



MALES

Animal ID

No.

S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

301 84.34 14.56 0.57 0.28 47.68 168.47 143.91 6.45 3.35 58.35

302 81.69 16.78 0.53 0.19 46.81 169.25 147.62 7.26 3.89 53.72

303 80.74 15.69 0.47 0.21 48.93 162.38 143.94 6.94 3.76 53.29

304 85.28 13.32 0.61 0.24 43.97 163.45 146.37 7.90 3.91 55.94

305 88.37 17.51 0.68 0.23 47.37 165.69 148.75 6.91 4.26 52.86

306 85.86 13.49 0.77 0.29 43.41 167.92 144.97 6.71 4.85 57.34

Mean 84.38 15.23 0.61 0.24 46.36 166.19 145.93 7.03 4.00 55.25

SD 2.81 1.73 0.11 0.04 2.19 2.82 2.00 0.50 0.51 2.30

FEMALES

Animal ID

No. S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

351 85.75 17.38 0.52 0.24 45.63 169.86 140.75 6.39 3.92 64.80

352 83.62 15.30 0.49 0.18 48.71 166.50 145.39 7.03 3.68 67.28

353 86.74 14.56 0.51 0.29 46.79 163.82 143.72 7.29 4.28 66.42

354 82.58 16.82 0.39 0.13 43.84 165.75 147.38 6.57 3.84 61.98

355 86.37 16.75 0.56 0.15 47.25 168.49 144.03 6.91 4.21 63.21

356 89.70 17.61 0.45 0.21 41.86 163.84 148.13 6.05 4.35 66.78

Mean 85.79 16.40 0.49 0.20 45.68 166.38 144.90 6.71 4.05 65.08

SD 2.51 1.21 0.06 0.06 2.48 2.45 2.69 0.46 0.27 2.13



Page 58 of 87

TABLE – XII– D

BIOCHEMISTRY



MALES

Animal ID

No.

S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

401 89.25 13.37 0.57 0.13 43.84 166.38 147.59 6.26 3.89 78.90

402 86.47 18.82 0.71 0.19 47.97 162.81 146.62 7.28 4.06 75.36

403 83.78 15.71 0.69 0.26 44.65 167.44 147.98 6.95 4.26 74.25

404 80.62 16.80 0.43 0.30 48.27 164.87 143.85 7.15 3.85 71.69

405 85.99 14.99 0.67 0.29 46.12 162.32 146.99 6.32 4.09 72.68

406 87.41 17.84 0.52 0.22 42.13 163.50 143.94 7.33 4.17 72.80

Mean 85.59 16.26 0.60 0.23 45.50 164.55 146.16 6.88 4.05 74.28

SD 3.02 1.98 0.11 0.06 2.41 2.04 1.82 0.48 0.16 2.61

FEMALES

Animal ID

No.

S.Glucose

mg/dl

BUN

mg/dl

Serum

Creatinine

mg/dl

S.Total

Bilirubin

mg/dl

SGPT

(ALT)

IU/L

SGOT

(AST)

IU/L

S.Alkaline

Phosphatase

IU/L

S.Total

Protein

mg/dl

S.Albumin

mg/dl

S.Total

Cholesterol

mg/dl

Limits 45 - 100 09 - 25 0.37 - 0.8 0.09 - 0.35 23 - 55 92 - 194 91 - 235 5.8 - 7.4 3.6 - 4.7 47 - 80

451 87.69 16.28 0.48 0.17 43.75 163.41 148.23 6.75 3.76 69.56

452 84.71 14.69 0.51 0.24 41.73 166.36 141.76 5.83 4.12 63.81

453 86.97 15.37 0.56 0.14 48.64 169.86 147.32 6.64 4.29 67.78

454 85.16 17.82 0.53 0.27 43.91 166.40 144.88 6.09 4.06 65.92

455 89.37 16.84 0.57 0.20 48.35 163.48 149.73 6.29 3.91 62.56

456 83.72 17.29 0.46 0.29 43.26 161.89 145.91 6.91 4.40 68.23

Mean 86.27 16.38 0.52 0.22 44.94 165.23 146.31 6.42 4.09 66.31

SD 2.11 1.18 0.04 0.06 2.86 2.88 2.80 0.42 0.24 2.72



Page 59 of 87

TABLE – XIII – A

ORGAN WEIGHTS (in gm)



MALES

Animal ID

No. Brain Heart Liver Spleen Thymus Kidneys Adrenals Testis Lungs

101 1.56 0.83 6.92 0.62 0.31 1.51 0.02 2.53 1.43

102 1.54 0.72 7.66 0.83 0.41 1.63 0.03 2.80 1.41

103 1.73 0.85 7.51 0.74 0.52 1.46 0.03 2.54 1.60

104 1.63 0.84 7.43 0.53 0.43 1.43 0.07 2.71 1.87

105 1.51 0.73 8.32 0.76 0.43 1.51 0.04 2.38 1.63

106 1.58 0.93 8.92 0.83 0.35 1.47 0.06 2.43 1.83

Mean 1.59 0.82 7.79 0.72 0.41 1.50 0.04 2.57 1.63

SD 0.08 0.08 0.71 0.12 0.07 0.07 0.02 0.16 0.19

FEMALES

Animal ID

No. Brain Heart Liver Spleen Thymus Kidneys Adrenals Uterus Lungs

151 1.63 0.67 6.10 0.62 0.34 1.30 0.06 0.72 1.45

152 1.48 0.63 6.01 0.48 0.27 1.34 0.03 0.42 1.20

153 1.43 0.67 6.67 0.53 0.43 1.40 0.06 0.85 1.49

154 1.47 0.75 7.30 0.45 0.29 1.52 0.07 0.85 1.74

155 1.59 0.74 6.82 0.63 0.44 1.16 0.07 0.47 1.32

156 1.43 0.68 5.74 0.42 0.32 1.21 0.06 0.47 1.13

Mean 1.51 0.69 6.44 0.52 0.35 1.32 0.06 0.63 1.39

SD 0.09 0.05 0.59 0.09 0.07 0.13 0.02 0.20 0.22



Page 60 of 87

TABLE – XIII – B

ORGAN WEIGHTS (in gm)



MALES

Animal ID

No. Brain Heart Liver Spleen Thymus Kidneys Adrenals Testis Lungs

401 1.43 0.83 8.34 0.85 0.34 1.23 0.05 2.70 1.53

402 1.63 0.73 8.68 0.74 0.32 1.39 0.06 1.72 1.72

403 1.88 0.93 9.94 0.47 0.34 1.54 0.05 2.40 1.63

404 1.68 0.95 9.34 0.49 0.53 1.46 0.07 2.18 2.21

405 1.60 0.86 8.26 0.63 0.37 1.72 0.06 2.32 1.87

406 1.67 0.97 9.34 0.51 0.58 1.85 0.04 2.60 1.82

Mean 1.65 0.88 8.98 0.62 0.41 1.53 0.06 2.32 1.80

SD 0.15 0.09 0.66 0.15 0.11 0.22 0.01 0.35 0.24

FEMALES

Animal ID

No. Brain Heart Liver Spleen Thymus Kidneys Adrenals Uterus Lungs

451 1.52 0.76 6.84 0.47 0.31 1.23 0.06 0.43 1.35

452 1.38 0.55 5.26 0.42 0.20 1.09 0.08 0.41 1.43

453 1.42 0.72 5.89 0.35 0.20 1.25 0.05 0.38 1.27

454 1.48 0.63 6.15 0.56 0.25 1.37 0.07 0.42 1.32

455 1.45 0.60 6.63 0.52 0.38 1.19 0.08 0.39 1.36

456 1.68 0.74 6.20 0.65 0.35 1.22 0.07 0.44 1.39

Mean 1.49 0.67 6.16 0.49 0.28 1.23 0.07 0.41 1.35

SD 0.11 0.08 0.56 0.11 0.08 0.09 0.01 0.02 0.06



Page 61 of 87

TABLE – X IV A

SUMMARY OF GROSS NECROPSY FINDINGS

Species: Rat Dose/Group: 0.0 mg/ kg b.wt (VC)

Organ

Lesions

Sex Total

Number of

animals M F

External No abnormality detected 6/6 6/6 12/12

Kidney Swollen/enlarged 0/6 1/6 1/12

Lung Emphysema 0/6 1/6

1/12

M- Male; F – Female



Page 62 of 87

TABLE – X IV B


Species: Rat Dose/Group: 1350.0 mg/kg b.wt (LD)

Organ

Lesions

Sex Total

Number of

animals M F

External

No abnormality detected

6/6

6/6

12/12

Lung

Hemorrhage

2/6

0/6

2/12

M-Male; F – Female



Page 63 of 87

TABLE – X IV C


Species: Rat Dose/Group: 2700.0 mg/ kg b.wt (MD)

Organ

Lesions

Sex Total

Number of

animals M F

External No abnormality detected 6/6 6/6 12/12

Kidney Swollen 0/6 0/6 0/12

Lung Emphysema 1/6 0/6 1/6




Page 64 of 87

TABLE – X IV D


Species: Rat Dose/Group: 4050.0 mg/ kg b.wt (HD)

Organ

Lesions

Sex Total

Number of

animals M F

External

No abnormality detected

6/6

6/6

12/12

Lung Emphysema 0/6 0/6 0/12




Page 65 of 87

TABLE – XV

HISTOPATHOLOGICAL OBSERVATIONS

Organ wise lesions Vehicle

Control

High dose

Kidney

1.Congestion and hemorrhage

2.Tubular degeneration

3.Tubulonephritis/ inflammation in tubular region

M F

0/6 0/6

0 /6 0/6

0/ 6 0/6

M F

0/6 1/6

0/6 1/6

0/6 0/ 6

Liver

1.Congestion & hemorrhage

2. Degeneration

3.Necrosis/ Inflammation and infiltration of inflammatory cells

4. Extra meduallry hematopoiesis

0/ 6 0/6

0/ 6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 1/6

0/6 0/6

0/6 0/6

Lungs

1. Congestion and hemorrhage

2.Edema/emphysema

2.Degeneration and necrosis

3.Inflammation with infiltration of inflammatory cells (bronchi

& alveoli)

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

Heart

1. Congestion and hemorrhage in myocardium

2. Degeneration myocardium

3. Inflammatory Foci &Infiltration of inflammatory cells

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6



Page 66 of 87

Spleen

1.Congestion & hemorrhage

2. .Lymphoid follicular depletion

3.Lymphoid follicular hyperplasia

4.Fibrosis/follicular atrophy

0/6 0/6

0/ 6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

Brain

1.Meneigeal congestion & hemorrhage

2. Vacoular degeneration

3.Epithelial cell hyperplasia

4.Necrosis with infiltration of inflammatory cells

0/6 0/6

0/ 6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6

Pancreas


2. Cystic Degeneration

3.Degeneration/ necrosis of non glandular region

4.Inflammation with infiltration of inflammatory cells

0/6 0/6

0/6 0/6

0/ 6 0/6

0/ 6 0/6

0/6 0/6

0/6 0/6

0/6 6/6

0/6 0/6

Adrenal


2. Cystic Degeneration

3.Necrosis of non glandular region

4.Inflammation with infiltration of inflammatory cells

0/6 0/6

0/6 0/6

0/ 6 0/6

0/ 6 0/6

0/6 0/6

0/6 2/6

0/6 0/6

0/6 0/6

Testes

1.Seminiferous tubular degeneration

2. Infiltration of multinucleated giant cells

3.Oligospermia

0/6 -

0/6 -

0/6 -

0/6 -

0/6 -

0/6 -



Page 67 of 87

Uterus/ Ovary

1.Follicular degeneration

2. Inflammation/infiltration of inflammatory cells

- 0/6

- 0/6

- 0/6

- 0/6

Intestine

1.Degeneration

2.Submucosal lymphocytic infiltration/inflammation

1/ 6 0/6

0/6 2/6

1/6 0/6

0/6 0/6

Thymus

1. Congestion/ hemorrhage

2. Degeneration/Inflammation

3. Depletion of cells

0/6 0/6

0/6 0/6

6/6 0/6

0/6 0/6

0/6 0/6

0/6 0/6



Page 68 of 87

16.0 PATHOLOGY REPORT

Based on summary of histopathological observation, we could not observe major toxic reaction

(degenerative, necrotic, inflammatory and hyper plastic changes) in spleen, pancreas, testes, ovary,

stomach, intestine and lymph node.

No adverse toxic reaction was observed in Brain, heart, spleen, pancreas, adrenal, thymus, testes, ovary

and intestine of all treated group animals when compared with vehicle control animals.

The histopathological photographs of Heart, Lungs, Spleen, Kidney and Liver of the vehicle control

animals & all treated animals (Photographs 1-10) are presented.

Based on the histopathological observation the drug may not be produced major reactive and toxic

Changes in the systemic organs up to the high dose used in this experiment.



Page 69 of 87

17.0. APPENDICES INDEX

PAGE NO.

I Dose Calculation & Dosage Schedule 72 – 73

II Standard Composition of Feed 74 – 76

III Microbial Limit Test 75



Page 70 of 87

I. DOSE CALCULATION

Recommended Human Dose : 15000 mg per day.

Calculated Rat Dose:

Conversion factor from man to rat (f*) = 0.018 for 200 gm of body weight.

15000 mg x f* 0.018 =270.0 mg / 200 gm

1 X recommended human dose converted into Rat = 1350.0 mg/kg body weight

Dose Selection

Low dose : 1 X recommended human dose

Middle Dose : 2 X recommended human dose

High Dose : 3 X recommended human dose

.

VOLUME – TEST ITEM PREPARATION

A constant volume of 1.0ml per animal for 28 days was administered to all groups of animals.

Concentration of LD: 1350.0 mg / kg body weight.

Concentration of MD: 2700.0 mg / kg body weight.

Concentration of HD: 4050.0 mg / kg body weight.

Formulation of Test Item:

The test item was formulated with D.M water.



Page 71 of 87

DOSAGE SCHEDULE AND GROUP DETAILS OF RATS EXPOSED TO

“POLY HERBAL FORMULATION”

Group

no. Group Details

No. of

animals/sex

Dosage Schedule Duration

of

Study mg/kg Exposure (days)

I Vehicle control (VC) 12 (6M+6F) 0.0 28 days 28 days

II Low dose (LD) 12 (6M+6F) 1350.0 28 days 28 days

III Middle dose (MD) 12 (6M+6F) 12.50 28 days 28 days

IV High dose (HD) 12 (6M+6F) 4050.0 28 days 28 days



Page 72 of 87

II. STANDARD COMPOSITION OF FEED

Diet composition for rats

a Wheat flour 22.5%

b Roasted Bengal gram 60.0%

c Skim milk powder 5.0%

d Casein 4.0%

e Refined ground nut oil 4.0%

f Salt mixture 4.0%

g Vitamin mixture 0.5%



Page 73 of 87

Vitamin mixture composition

1 (dl) – a Tocopherol Acetate (E)

50% dry powder 12.0 g

2 Menadione (K) 0.15 g

3 Thiamine (B1) 1.2 g

4 Riboflavin (B2) 0.5 g

5 Pyridoxine (B6) 0.6 g

6 Niacin 1.0 g

7 Pantothenic acid (calcium salt) 1.2 g

8 Cynacobalamine (B12) 0.5 mg

9 Folic acid 0.1 g

10 Para Amino Benzoic Acid (PABA) 10.0 g

11 Biotin 40.0 mg

12 Inositol 10.0 g

13 Choline chloride 100.0 g

Total vitamins put together 176.76 g

Starch 323.34 g

500.00 g

i.e., 500 g of vitamin mixture is used for every 100 kg of the diet prepared.



Page 74 of 87

Salt mixture composition

Mineral Per 100 kg diet (g)

1 Dicalcium phosphate 1250.00

2 Calcium carbonate (CaCO3) 555.00

3 Sodium chloride (NaCl) 300.00

4 Magnesium sulphate (MgSO47H2O) 229.20

5 Ferrous sulphate (FeSO47H2O) 50.00

6 Magnesium sulphate (MgSO4H2O) 16.04

7 Potassium iodide (KI) 1.00

8 Zinc sulphate (ZnSO47H2O) 2.192

9 Copper sulphate (CuSO45H2O) 1.908

10 Cobalt chloride (CoCl26H2O) 0.012

All minerals together 2405.35 g

Starch 1594.65 g

4000.00 g



Page 75 of 87

III. MICROBIAL LIMIT TEST

S.no. Name of the test Result Specification

01

Total microbial count

a) Bacterial count

b) Fungal count

32 cfu/ml

Nil cfu/ml

NMT 500 cfu/ml

NMT 100 cfu/ml

02

Test for pathogens

a) E.coli

b) Salmonella

c) S.aureus

d) Pseudomonas

Absent

Absent

Absent

Absent

Should be absent

Should be absent

Should be absent

Should be absent



Page 76 of 87

18.0. PHOTOGRAPHS INDEX

S.NO. TITLE PAGE NO.

I Microscopic Observation of Heart (Control group)…………………………. 77

II Microscopic Observation of Heart (High group)...…….................................... 78

III Microscopic Observation of Lungs (Control group)………………………… 79

IV Microscopic Observation of Lungs (High group)…………………………… 80

V Microscopic Observation of Spleen (Control group)……………………….. 81

VI Microscopic Observation of Spleen (High group)………………………….. 82

VII Microscopic Observation of Kidney (Control group)…………………......... 83

VIII Microscopic Observation of Kidney (High group)………………………….. 84

IX Microscopic Observation of Liver (Control group)…………………………. 85

X Microscopic Observation of Liver (High group)……………………………. 86



Page 77 of 87

I. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF HEART

Control group



Page 78 of 87

II. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF HEART

High group



Page 79 of 87

III. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF LUNGS

Control group



Page 80 of 87

IV. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF LUNGS

High group



Page 81 of 87

V. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF SPLEEN

Control group



Page 82 of 87

VI. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF SPLEEN

High group



Page 83 of 87

VII. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF KIDNEY

Control group



Page 84 of 87

VIII. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF KIDNEY

High group



Page 85 of 87

IX. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF LIVER

Control group



Page 86 of 87

X. PHOTOGRAPH

MICROSCOPIC OBSERVATION OF LIVER

High group



Page 87 of 87

19.0 REFERENCES

D.R. Lawrence and A.L. Bacharach (1964). Evaluation of drug activities. Pharmacometrics, Vol. 1,

Academic Press, London.

S.C Gad and C.S weil: Statistics and Experimental Design for Toxicologists. The Telford press,

Caldwell, New Jersey, 43-45 (1986).

Michael J.Derelanko and Mannfred A. Hollinger : Hand Book of Toxiclogy, Second Edition.

CPCSEA Guidelines for Laboratory Animal Facility, Government of India.

Drugs and Cosmetic Act-1945 : Schedule – Y.

Good Laboratory Practice (GLP):World Health Organisation, Second Edition.

REPORT Title SUB-ACUTE TOXICITY STUDY (28 DAYS) ON POLY … · 2018-11-14 · teena biolabs pvt. ltd study no. : tfrcrdpct02022015 (rat). name of the test item : poly herbal formulation

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