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Department of RASASASHTRA & BHAISHAJYA KALPANA

WELCOME YOU ALL

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Presenter:Dr.SARANYA SASI 2nd Year PG Scholar

Guide:Dr.GOVINDA SHARMA KAsso:Professor

Department Of RASASASHTRA & BHAISHAJYA KALPANASri Dharmasthala Manjunatheswara College of Ayurveda & Hospital

Hassan 08-Feb-17

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• INTRODUCTION•TYPES•COMMONLY USED LABORATORY ANIMALS• ACUTE TOXICTY STUDIES• SUB ACUTE TOXICITY STUDIES• CHRONIC STUDIES • SUBCHRONIC STUDIES• TEST REPORT • ASSESSMENT OF HERBAL MEDICINE •ARTICLE• CCRAS RESEARCH REPORT • DISCUSSION •CONCLUSION • REFERENCE

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•Toxicology is classically defined as the study of poisons.

•It is concerned mainly with the study of adverse effects of xenobiotics.

•Casarett 1996 defined it as a science that defines the limits of safety of chemical agents for human and animal population.

INTRODUCTION

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TYPES

Systemic toxicity studies

Reproductive toxicity studies

Local toxicity studies

Hypersensitivity studies

Genotoxicity studies

Carcinogenicity studies

1. Male fertility 2. Female reproduction & Developmental studies

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COMMONLY USED LABORATORY ANIMALS FOR RESEARCH PURPOSE *

Animals Purpose

Laboratory Mouse(Mus musculusdomesticus)

Cancer researchDiabetesArthrosclerosisEpilepsyAutoimmune disease

Laboratory Rat (Rattusnorvegicus)

Biochemistry NutritionToxicologyOncologyPharmacologyEndrocrinologyNeurophysiology

Mastomys (Pramyscoucha)Routine testing of plague suspect material Protozological and helminthological Research

RODENTS

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Gerbil (Merioneshurrianae)Behavioral CancerEpilepsyNeurological Experiments

Hamster (Mesocricetusauratus) Cancer

Guinea pig (Caviaporcellus)

Delayed hypersensivityBiochemistryToxicologyPhysiology Pharmacology Oncology Vit C Metabolism

Rabbit (Oryctolaguscuniculus)

Immunology HypertensionInfectious disease Virology Embryology Toxicology Teratology atherosclerosis

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NON-RODENTSAnimals Purpose

Monkeys

VirologyToxicolgy Reproductive physiology Contraceptive biology Endocrinology Immunology

Dogs (Canisfamiliaris)

Chronic arterial diseaseCongestive heart failure DMUlcerative colitis Lymphocytic leukemia Glaucoma

Cats (Feliscatus)Neurophysiology Reflexes Synoptic transmission Perception of light and sound

*Girish B.C, C.Ravikumar, C.R Santosh, V.T ShilpaDepartment of Veterinary Pathology Veterinary college Hassan

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ACUTE TOXICITY STUDIES

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Sl.No. Test Groups No. of Animals

1. Control group 10(5M + 5F)

2. Therapeutic Dose(TD)

10(5M + 5F)

3. Average Dose(TD x 5)

10(5M + 5F)

4. Highest Dose(TD x 10)

10(5M + 5F)

Test dose : Single doseRoute of administration : Oral Duration : 10 days

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a) Mortality : To be observed on 24, 48 & 72 hours

b) Clinical signs: Monitoring of convulsions, lethargy, sleep, coma, salivation, diarrhoea

•Cage side examination (daily)

•Skin colour, fur, eyes & mucous membrane (daily)

•Spontaneous and voluntary motor activity on every ½ hour, 1 hour, 2 hours, 4 hours and 24 hours after drug administration.

•0,1,7th day spontaneous motor activity

•Necropsy - In case of animal dies

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SUBACUTE TOXICITY

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Species Animals No. Age (wks)

Weight (gms)

Duration of experimentMale Female TC

Exp.Imm. Exp.

Post Exp.

Mice (Swiss)

246x4*

246x4*

4-6 18-20 15 days oral

50% 15th day

50% 30th day

Rat Wistar

246x4*

246x4*

4-6 60-90 15 days oral

50% 15th day

50% 30th day

TC-Test compound exposureImm Exp – Immediate (48 hours) after last exposure – instant effectPost exp – Post-exposure (15 days) after last exposure – reversibility of toxicity if any

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Pre-experimentation phaseI. Acclimatization of animals•Period – 7 days (Recording of body weight and food intake twice in a week)

•Urine qualitative test

•Fecal consistency

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Experimentation phaseI)Test compound exposure multiple dose (once daily for 15 days) and dosage schedule (as recommended by sponsor)

II)Mortality 6/12/24 hours

III)Body weight (Twice in a week)

IV)Food consumption (Daily)

V)Fecal consistency

VI)Cage side activity

VII)Neurological examination

VIII)Urine qualitative test

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IX) Haematology - Twice (15th day/30th day) (Hb, RBC, WBC, Platelet count, differential count)

X) Clinical chemistry – Twice (15th day/30 day) (Blood glucose, total protein, serum Creatinine, SGOT, SGPT).

XI) Histopathology -Liver, Kidney, Lungs, Spleen, Ovaries, Testis, Stomach, Intestine

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Observations:a. Mortality: To be observed on 24, 48 and 72 hrs.

b. Clinical Signs:

• A careful cage side examination will be made daily.

changes in:

- Skin, fur, eyes and mucous membrane.

- Convulsions, lethargy, sleep, coma, salivation, diarrhoea and date of death.

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CHRONIC TOXICITY

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Species No. of Animals Age (wks)

Weight (gms)

Duration of experiment

Male Female TC Exp. Term. Exp.

Mice (Swiss)

4010 x 4*

4010 x 4*

4 15-18 90 days 100%

Rat Wistar

4010 x 4*

4010 x 4*

4 60-80 90 days 100%

TC-Test compound exposureTerm. Exp – Termination of experiment immediately after last exposure (48 hours) euthanization of animals for collection of vital organs.

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Pre-experimentation phaseI. Acclimatization of animals•Period – 7 days (Recording of body weight and food intake twice in a week)

•Urine qualitative test (Ames multiple sticks)

•Fecal consistency (Filter paper technique)

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Experimentation phase

I)Test compound exposure - multiple dose (once daily for 90 days) and dosage schedule (as recommended by sponsor)

II)Mortality 6/12/24 hours

III)Body weight (Twice in a week)

IV)Food consumption (Twice/weekly)

V)Fecal consistency

VI)Cage side activity

VII)Neurological examination

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•Urine qualitative test (30/60/90 days)

•Haematology - Twice (30/60/90 days) (Hb, RBC, WBC, Platelet count, differential count)

•Clinical chemistry – Twice (30/60/90 days) (Blood glucose, total protein, serum creatinine, SGOT, SGPT).

•Histopathology – Liver, heart, brain, lung, kidney, spleen, sciatic nerve, testes/ ovaries

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SUBCHRONIC TOXICITY 13 week study +/- 4 wk recovery (3 doses and

control) Species 2 species – rodents, dogs In-life observations (+/- ophthamology) Clinical pathology Necropsy Histopathology Used to set doses for carcinogenicity studies

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TEST REPORT

Test substance: •Physical nature•Purity•Physicochemical properties •Identification data •Source of substance•Batch number

Vehicle (if appropriate): •Justification for choice of vehicle (if other than water)

Test animals: •Species/strain used and justification for choice made; •Number, age, and sex of animals at start of test; •Source, housing conditions, diet, etc.; •Individual weights of animals at the start of the test.

The test report should include the following information:

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ASSESSMENT OF HERBAL MEDICINESWHO - published guidelines for the assessment and evaluation of toxicities of herbal medicines These guidelines can be utilized in testing of Ayurvedic medicines These guidelines are intended to indicate the standard methods of non-clinical toxicological studies related to assessing the safety of herbal medicines. ACUTE TOXICITY Species : 2(1 rodent & 1 non-rodent)Sex : male/femaleNo : rodent – 5 animal per sex at least (each group) non-rodent – 2 animal at least ROA : ordinarily, the oral route of clinical administration Parenteral route Dose : a sufficient dose levels should be used in rodents to determine the approximate lethal dose sufficient dose level should be used for the observation of overt toxic signs

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Frequency of administration : one or more doses – 24 hr periodObservation : toxic sign

• severity• onset• progression and reversibility of signs should observed and • recorded in relation to dose and time

(animal should be observed for at least 7 to 14 days – general rule)• animals dying during the observation period, as well as rodent surviving • to the end of observation period should be autopsied.• histopathological examination should conducted - if necessary

LONG TERM TOXICITY STUDYSpecies : 2(1 rodent & 1 non-rodent)Sex : male/femaleNo : rodent – 10 male & 10 female(each group) non-rodent – 3 male & 3 femaleROA : the oral route of clinical administration

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Administration period: depend on expected period of clinical use.

Expected period of clinical use Administration period for toxicity study Single/repeated for Less than 1 week 2 weeks – 1 monthRepeatedBtwn 1 week to 4 wk

4 wk – 3 months

Repeated Btwn 1 – 6 months

3 – 6 months

Long term repeated More than 6 months

9- 12 months

Dose levels –• groups receiving at least 3 different dose levels should be used. • one dose level should not cause toxic changes and one dose level that

produces overt toxic effects should be included

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Observations and examinations:1. General signs, body weight and food and water intake2. Hematological examination3. Renal and hepatic function tests4. Other function tests5. Animal found dead during the examination should be

autopsied as soon as possible

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Journal of Pharmacology and pharmacotherapeutics April-June 2011 Vol 2 Issue 2

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Conclusion Naga bhasma has got no acute toxic effect as on GIT, liver, testis, kidney, and seminal vesicle in the dose upto 416mg/100 gm body weight. Whereas for a period of 60 days study shows, no toxic effect has been observed in therapeutic dose and intermediate dose.

But in higher dose significant toxicity has been attributable to naga bhasma. However, further giving haritaki churna and swarna bhasma to the highest dose animals seems lesser amount of toxicity. So the concept of bhasma vikara shanti upaya described by different autors in different RS literatures proves significance.

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RESEARCH - CCRAS

1. Chronic Toxicity study of Mahalaxmivilasa Rasa with Gold in rats. conclusion :The test drug has no serious toxicity potential. Though some changes were observed in spleen and lungs on histopathological examination they were not consistent and

dosedependent. At high dose level the drug has the proclivity to depress WBC formation especially lymphocyte formation and cause hypertriglyceridemia2. Acute Toxicity study of Mahasudarshan Ghan Vati in mice Coclusion:Effect of test drug was studied after a single administration up to eight dose levels with 3000mg/kg as the maximum dose in rats. The animals were observed for 72 hrs periodically for general behavioral changes to screen its effect on CNS and mortality was observed up to 7 days. No mortality and behavioral changes was observed during the study.

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1. Pharmaceutico – analytical study of NARADIYA LAKSHMI VILASA RASA & its toxicological evaluation (2016) Dr.Patel Manjit Dhirubhai Dept. Of RASASHASTRA Alva's Ayurvedic Medical College Moodbidiri Karnataka

RTESULT By observing the haematological, bio chemical and food concersion parameters, Naradiya lakshmi vilasa rasa is safe at therapeutic dose, 5 times therapeutic dose and upto the 10 times of therapeutic dose. Its careful administration is not likely to cause any serious toxic outcome at the therapeutic dose level

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•Acute toxicity is involved in estimation of LD50 (the dose which has proved to be lethal (causing death) to 50% of the tested group of animals). •Determination of acute oral toxicity is usually an initial screening step in the assessment and evaluation of the toxic characteristics of all compounds•The methods so far utilized for the determination of median lethal dose (LD50) and the new changes which could be made. •Sub acute and chronic toxicity studies are designed to characterize the toxic effects of drugs upon repeated daily administration for periods of time ranging from 2 weeks to 1 year and to determine no-toxic-effect dosage levels for short to long-term repeated dosing.

DISCUSSION

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• The presence of heavy metals by chemical analysis - above the permissible limits in some drugs - may be in different forms by which the elements are bound. • Since these elements could be chelated in the formulation either hydrolytic or nonhydrolytic form of the metal will be safe to use. • The results of the researches by CCRAS also confirms the nontoxic nature of the drug as the final product in bhasma/rasakalpas are different from the raw materials since they would be transformed to therapeutic compounds by different processes like detoxification, titration, heating etc.• Hence it is suggested that any claim made on safety of ayurvedic herbo mineral/ metallic preparations should be made only after the toxicity clinical studies

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REFERENCE

•CRC hand book of toxicology• Safety/Toxicity study report of some ayurvedic drugs (CCRAS Department of AYUSH India)•www.Oecd-library.org.cited •www.http://toxipedia.org.

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