This document outlines protocols for toxicity studies of herbal medicines. It discusses types of toxicity studies including acute, sub-acute, chronic and subchronic studies. It describes commonly used laboratory animals for such studies like mice, rats, rabbits and protocols for acute, subacute and chronic toxicity studies. The document provides an example of a toxicity study report format and discusses guidelines for assessing toxicity of herbal medicines from WHO.

1. Department of
RASASASHTRA & BHAISHAJYA KALPANA
WELCOME YOU ALL
108-Feb-17PROTOCOL FOR TOXICITY STUDIES

2. PROTOCOL FOR TOXICITY
STUDY
Presenter:
Dr.SARANYA SASI
2nd Year PG Scholar
Guide:
Dr.GOVINDA SHARMA K
Asso:Professor
Department Of RASASASHTRA & BHAISHAJYA KALPANA
Sri Dharmasthala Manjunatheswara College of Ayurveda & Hospital
Hassan 08-Feb-17 2PROTOCOL FOR TOXICITY STUDIES

3. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 3
• 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

4. •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
08-Feb-17PROTOCOL FOR TOXICITY STUDIES 4

5. TYPES
Systemic toxicity
studies
Reproductive
toxicity studies
Local toxicity
studies
Hypersensitivity
studies
Genotoxicity
studies
Carcinogenicity
studies
1. Male fertility
2. Female reproduction & Developmental studies
08-Feb-17PROTOCOL FOR TOXICITY STUDIES 5

6. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 6
COMMONLY USED LABORATORY ANIMALS
FOR RESEARCH PURPOSE *
Animals Purpose
Laboratory Mouse
(Mus musculusdomesticus)
Cancer research
Diabetes
Arthrosclerosis
Epilepsy
Autoimmune disease
Laboratory Rat (Rattusnorvegicus)
Biochemistry
Nutrition
Toxicology
Oncology
Pharmacology
Endrocrinology
Neurophysiology
Mastomys (Pramyscoucha)
Routine testing of plague suspect
material
Protozological and helminthological
Research
RODENTS

7. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 7
Gerbil (Merioneshurrianae)
Behavioral
Cancer
Epilepsy
Neurological Experiments
Hamster (Mesocricetusauratus) Cancer
Guinea pig
(Caviaporcellus)
Delayed hypersensivity
Biochemistry
Toxicology
Physiology
Pharmacology
Oncology
Vit C Metabolism
Rabbit (Oryctolaguscuniculus)
Immunology
Hypertension
Infectious disease
Virology
Embryology
Toxicology
Teratology
atherosclerosis

8. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 8
NON-RODENTS
Animals Purpose
Monkeys
Virology
Toxicolgy
Reproductive physiology
Contraceptive biology
Endocrinology
Immunology
Dogs (Canisfamiliaris)
Chronic arterial disease
Congestive heart failure
DM
Ulcerative 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 Shilpa
Department of Veterinary Pathology Veterinary college Hassan

9. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 9

10. 08-Feb-17 10PROTOCOL FOR TOXICITY STUDIES
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 dose
Route of administration : Oral
Duration : 10 days

11. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 11
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

12. 08-Feb-17 12PROTOCOL FOR TOXICITY STUDIES
Species Animals No. Age
(wks)
Weight
(gms)
Duration of experiment
Male Female TC
Exp.
Imm.
Exp.
Post
Exp.
Mice
(Swiss)
24
6x4*
24
6x4*
4-6 18-20 15 days
oral
50%
15th
day
50%
30th
day
Rat
Wistar
24
6x4*
24
6x4*
4-6 60-90 15 days
oral
50%
15th
day
50%
30th
day
TC-Test compound exposure
Imm Exp – Immediate (48 hours) after last exposure – instant effect
Post exp – Post-exposure (15 days) after last exposure – reversibility
of toxicity if any

13. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 13
Pre-experimentation phase
I. Acclimatization of animals
•Period – 7 days (Recording of body weight and food intake twice in
a week)
•Urine qualitative test
•Fecal consistency

14. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 14
Experimentation phase
I)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)
I)Food consumption (Daily)
II)Fecal consistency
III)Cage side activity
IV)Neurological examination
V)Urine qualitative test

15. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 15
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

16. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 16
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.

17. 08-Feb-17 17PROTOCOL FOR TOXICITY STUDIES
Species No. of Animals Age
(wks)
Weight
(gms)
Duration of
experiment
Male Female TC Exp. Term.
Exp.
Mice
(Swiss)
40
10 x 4*
40
10 x 4*
4 15-18 90 days 100%
Rat
Wistar
40
10 x 4*
40
10 x 4*
4 60-80 90 days 100%
TC-Test compound exposure
Term. Exp – Termination of experiment immediately after last
exposure (48 hours) euthanization of animals for collection of vital
organs.

18. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 18
Pre-experimentation phase
I. 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)

19. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 19
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

20. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 20
•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

21.  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
08-Feb-17 21PROTOCOL FOR TOXICITY STUDIES

22. 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:
08-Feb-17PROTOCOL FOR TOXICITY STUDIES 22

23. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 23
ASSESSMENT OF HERBAL MEDICINES
WHO - 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/female
No : 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

24. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 24
Frequency of administration : one or more doses – 24 hr period
Observation : 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 STUDY
Species : 2(1 rodent & 1 non-rodent)
Sex : male/female
No : rodent – 10 male & 10 female(each group)
non-rodent – 3 male & 3 female
ROA : the oral route of clinical administration

25. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 25
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 month
Repeated
Btwn 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

26. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 26
Observations and examinations:
1. General signs, body weight and food and water intake
2. Hematological examination
3. Renal and hepatic function tests
4. Other function tests
5. Animal found dead during the examination should be
autopsied as soon as possible

27. Journal of Pharmacology and pharmacotherapeutics
April-June 2011 Vol 2 Issue 2
08-Feb-17PROTOCOL FOR TOXICITY STUDIES 27

28. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 28
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.

29. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 29
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 dose
dependent. At high dose level the drug has the proclivity to depress WBC formation
especially lymphocyte formation and cause hypertriglyceridemia
2. 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.

30. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 30
1. Pharmaceutico – analytical study of NARADIYA LAKSHMI VILASA RASA &
its toxicological evaluation (2016)
Dr.Patel Manjit Dhirubhai
Dept. Of RASASHASTRAAlva'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

31. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 31
•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

32. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 32
• 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

33. 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.
08-Feb-17PROTOCOL FOR TOXICITY STUDIES 33

34. 08-Feb-17PROTOCOL FOR TOXICITY STUDIES 34