Guidelines for toxicity / safety profile evaluation
Of
Ayurveda & Siddha plant drugs
In most cities of India, and even some overseas countries, Ayurvedic herbal medicines
(AHMs) are becoming increasingly popular as an effective and relatively alternative to
allopathic drugs. In order to make global acceptance the quality and its safety are under
scrutiny though traditional practitioners prescribed them since long time.
In the recent past several western research groups have high lightened these
pitfalls reporting the prevalence and concentration of heavy metals in Ayurvedic HMPs.
In Ayurvedic system of medicine the formulations prepared with minerals are called as
herbo-mineral preparations or Bhasmas in traditional language. The report of JAMA has
mentioned presence of heavy metals in herbal medicine products but did not hint
anything about Bhasmas. However, we also feel that it is mandatory to test raw materials
used in Ayurvedic formulation preparation for the presence of various toxic materials
including heavy metal content.
'Bhasmas' a mineral preparation in Ayurveda, is prepared under special physicochemical processes that, according to the ancient Indian belief, 'detoxify', toxic heavy
metals in it. Strictly speaking, these constituents are thus not contaminants but ingredients
deliberately included for a specific curative purpose. India being a signatory to WTO, to
promote its products in the international market, it is imperative to study their safety for
human consumption.
The pre-clinical toxicology unit of NIN in association with CCRAS has designed
protocol for Bhasmas which includes acute and long term testing as per international
guidelines (WHO/OECD).
1.
2.
Acute toxicity will be carried out in mice and rats with a single exposure
of 10 times of the recommended therapeutic dose of test compound the
study duration will be 25 days.
Long terms toxicity testing duration is for 90-100 days followed with
investigation for 2 months where 14 tissues will be examined for
histopathological changes if any, in approximately 200 animals. In
addition levels of various metal ions will be monitored using
ICPMS/AAS.
In any condition, the above procedures cannot be avoided since they are as per the
regulatory requirements and follow international guidelines of WHO and OECD.
This protocol may be adopted by acknowledging to CCRAS and NIN
CENTRAL COUNCIL FOR RESEARCH IN AYURVEDA &
SIDDHA
(An autonomous Organisation under Ministry of Health & Family Welfare, Govt. of
India)
Jawahar Lal Nehru Bhartiya Chikitsa Avum Homoeopathy Anusandhan Bhawan
No.61-65, Institutional Area, Opp.’D’ Block, Janak Puri, New Delhi-110 058
Acute Toxicity Test evaluation:
Sl.No.
1.
2.
3.
4.
Test Groups
VC
(Control)
Therapeutic Dose
(TD)
Average Dose
(TD x 5)
Highest Dose
(TX x 10)
No. of Animals
10
(5M + 5F)
10
(5M + 5F)
10
(5M + 5F)
10
(5M + 5F)
Test dose
:
Single dose
Route of administration
:
Oral
Duration
:
10 days
:
To be observed on 24, 48 & 72 hours
Observations:
a)
Mortality
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
Annexure I
Safety/Toxicity Evaluation of Selective Plant Based Product
for Ayurvedic Usage
Sub-Acute Toxicity evaluation:
Species
Mice
(Swiss)
Rat
Wistar
Animals No.
Male
Female
24
6x4*
24
6x4*
24
6x4*
24
6x4*
Age
(wks)
Weight
(gms)
4-6
18-20
4-6
60-90
Duration of experiment
TC Exp.
Imm.
Post
Exp.
Exp.
15 days
50% 15th 50%
oral
day
30th day
15 days
50% 15th 50%
oral
day
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
Four groups (1) TH (Recommended Therapeutic dose)
(2) 5 times of TH
(3) 10 times of TH
(4) Vehicle control
Pre-experimentation phase
I.
Acclimatization of animals
i)
ii)
iii)
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)
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)
iv.
Food consumption (Daily)
v.
Local consistency
vi.
Cage side activity
vii.
Neurological examination
viii. Urine qualitative test
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
ix.
General comments
 Metal free pellet diet and water
 One rat and two mice in each cage
 Test compound, vehicle control, its dosage regimen will be supplied by sponsor
(ISM)
 Scoring of cage side activity, neurological activity according to OECD
guidelines.
Route of Administration:
Oral
Duration:
30 days
Observations:
a.
Mortality:
To be observed on 24, 48 and 72 hrs.
b.
Clinical Signs:
A careful cage side examination will be made daily.
Daily include changes in:
Skin, fur, eyes and mucous membrane
Convulsions, lethargy, sleep, coma, salivation, diarrhoea and date of death
Chronic Toxicity Evaluation
Species
No. of Animals
Male
Mice
(Swiss)
Rat
Wistar
40
10 x 4*
40
10 x 4*
Age
(wks)
Weight
(gms)
4
15-18
Duration of
experiment
TC Exp.
Term.
Exp.
90 days
100%
4
60-80
90 days
Female
40
10 x 4*
40
10 x 4*
100%
TC-Test compound exposure
Term. Exp – Termination of experiment immediately after last exposure (48 hours)
euthanization of animals for collection of vital organs.
* Four groups (1) TH (Recommended Therapeutic dose)
(2) 5 times of TH
(3) 10 times of TH
(4) Vehicle control
Pre-experimentation phase
I.
Acclimatization of animals
i)
Period – 7 days (Recording of body weight and food intake twice in a
week)
ii)
Urine qualitative test (Ames multiple sticks)
iii)
Fecal consistency (Filter paper technique)
Experimentation phase
a. Test compound exposure - multiple dose (once daily for 90 days) and
dosage schedule (as recommended by sponsor)
b. Mortality 6/12/24 hours
c. Body weight (Twice in a week)
d. Food consumption (Twice/weekly)
e. Fecal consistency
f. Cage side activity
g. Neurological examination
h. Urine qualitative test (30/60/90 days)
i. Haematology - Twice (30/60/90 days) (Hb, RBC, WBC, Platelet count,
differential count)
j. Clinical chemistry – Twice (30/60/90 days) (Blood glucose, total protein,
serum creatinine, SGOT, SGPT).
k. Histopathology – Liver, heart, brain, lung, kidney, spleen, sciatic nerve,
testes/ ovaries.
General comments




Metal free pellet diet and water
One rat and two mice in each cage
Test compound, vehicle control, its dosage regimen will be supplied by sponsor
(ISM)
Scoring of cage side activity, neurological activity according to OECD
guidelines.
Route of administration:
Normally, the expected clinical route of administration should be used.
Administration Period:
The period of administration of the test substance to animals will depend on the
expected period of clinical use. The period of administration of the toxicity study may
vary from country to country, according to its individual regulations.
The following table reflects commonly used ranges of administration periods:
Expected period of Clinical use
Single administration or repeated
administration for less than one week
Repeated administration, between one
week to four weeks
Repeated administration, between one to
six months
Long term repeated administration for
more than six months
Administration period for the
toxicity study
2 weeks to 1 month
4 weeks to 3 months
3 to 6 months
9 to 12 months
As a rule, the test substance should be administered seven days a week.
Administration periods for the toxicity study must be recorded in each result.
Dose levels:
Groups receiving at least three different dose levels should be used.
One dose level should not cause toxic changes (no effect dose) and one dose level
that produces over toxic effects should be included. Within this range, the addition of at
least one more dose may enhance the possibility of observing a dose response
relationship for toxic manifestations. All studies should include a vehicle control group of
test animals.
General observations and examinations:
Observations and examinations should be performed on the following items (from
1 to 6):
1.
General signs, body weight and food and water intake:
For all experimental animals, the general signs should be observed daily and body
weight and
food intake should be measured periodically. If useful, water intake should
also be determined. The frequency of measurements should normally be as follows:
2.
 Body weight: before the start of drug administration, at least once a week
for the first three months of administration and at least once every four
weeks thereafter.
 Food intake: before the start of drug administration, at least once a week
for the first three months of administration and at least once every four
weeks thereafter. If the test substance is administered mixed in the food,
the intake should be measured once a week.
Haematological examination:
For rodents, blood samples should be taken before autopsy. For non-rodents,
blood samples should be taken before the start of the drug administration, at least once
during the administration
period (for studies of longer than one month), and before
autopsy.
For both haematological and blood chemistry examinations, it is desirable to
include as many
parameters as possible.
3.
Renal and hepatic function tests:
Since the liver and kidneys are the usual organs of metabolism and excretion,
potentially toxic
agents easily affect them; their functions should be monitored in
long term toxicity studies.
For rodents, a fixed number of animals from each group should be selected and
urinalysis should
be performed before the start of drug administration, and at least
once during the administration
period.
4.
Other function tests:
If appropriate, ECG and visual, auditory tests should be performed. For rodents,
opthalmological
examination should be performed on a fixed number of animals
from each group at least once
during the administration period; for non-rodents,
examination should be performed on all
animals before the start of drug
administration and at least once during the period of
administration.
5.
Animals found dead during the examination should be autopsied as soon as
possible. A macroscopic examination should be made of organs and tissues. In
addition, where possible, organ weight measurements and histopathological
examinations should be performed in an attempt to identify the cause of death and
the nature (severity or degree) of the toxic changes present.
6.
In order to maximize the amount of useful information that can be obtained
during the administration period, all moribund animals should be sacrificed rather
than allowed to die. Prior to sacrifice, clinical observations should be recorded
and blood samples collected for haematological and blood chemical analysis. At
autopsy, a macroscopic examination of organs and tissues and measurement of
organ weights should be recorded. A full histopathological examination should be
performed in an attempt to characterize the nature (severity of degree) of all toxic
changes.
All survivors should be autopsied at the end of the administration period or of the
recovery period after taking blood samples for haematological (including blood
chemistry) examinations, organs and tissues should be examined macroscopically
and organ weights measures. Histopathological examinations of the organs and
tissues of animals receiving lower dosage should also be performed, if changes
are found on gross or macroscopic examination of their organs and tissues of
these animals, or if the highest dose group reveal significant changes. On the
other hand, histopathological examination of all rodents will further improve the
chances of detecting toxicity.
Recovery from toxicity:
In order to investigate the recovery from toxic changes, animals that are allowed
to live for varying lengths of time after cessation of the period of administration of the
test substance, should be examined.
Animal species:
Some regulatory agencies require that at least two species be used, one of them to
be selected
from rodents and the other from non-rodents.
Sex:
In at least one of the species, males and females should be used.
Number of animals:
In the case of rodents, each group should consist of at least five animals per sex.
In the case of non-rodents, each group should consist of at least two animals per sex.
Route of administration:
Ordinarily, the oral route is sufficient as this is the normal route of clinical
administration.
However, some regulatory agencies suggest in addition a
parenteral route of administration.
In cases where it is proposed to administer the herbal preparation to a human
subject by the parenteral route, it may be sufficient to use this route alone for animal
testing.
Dose levels:
A sufficient number of dose levels should be used in rodents to determine the
approximate lethal
dose. In non-rodents, sufficient dose levels should be used for the
observation of over toxic
signs.
Frequency of administration:
The test substance should be administered in one or more doses during a 24 hour
period.
Observation:
Toxic signs and the severity onset, progression and reversibility of the signs
should be observed and record relation to dose and time. As a general rule, the
animals should be observed for at least seven to days.
Animals dying during the observation period, as well as rodents surviving to the
end of the
observation period should be autopsied.
If necessary, a histopathological examination should be conducted on any organ or tissue
showing macroscopic changes at autopsy