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Guidelines for toxicity safety profile evaluation by pptfiles

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									      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 physico-
chemical 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.      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.
       2.      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.     Test Groups          No. of Animals
                       1.            VC                    10
                                 (Control)             (5M + 5F)
                       2.     Therapeutic Dose             10
                                    (TD)               (5M + 5F)
                       3.      Average Dose                10
                                  (TD x 5)             (5M + 5F)
                       4.      Highest Dose                10
                                 (TX x 10)             (5M + 5F)

Test dose                      :      Single dose

Route of administration        :      Oral

Duration                       :      10 days

Observations:

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
                                                                                Annexure I

     Safety/Toxicity Evaluation of Selective Plant Based Product
                        for Ayurvedic Usage
Sub-Acute Toxicity evaluation:

Species           Animals No.           Age      Weight        Duration of experiment
                 Male   Female         (wks)     (gms)      TC Exp.     Imm.       Post
                                                                        Exp.       Exp.
Mice              24         24         4-6     18-20       15 days   50% 15th 50%
(Swiss)          6x4*       6x4*                            oral      day        30th day
Rat               24         24         4-6     60-90       15 days   50% 15th 50%
Wistar           6x4*       6x4*                            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)        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
         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
               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

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          Age     Weight          Duration of
                                               (wks)    (gms)           experiment
                       Male      Female                             TC Exp.     Term.
                                                                                 Exp.
     Mice            40         40         4           15-18       90 days     100%
     (Swiss)         10 x 4*    10 x 4*
     Rat             40         40         4           60-80       90 days       100%
     Wistar          10 x 4*    10 x 4*

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            Administration period for the
                                                                   toxicity study
            Single administration or repeated           2 weeks to 1 month
            administration for less than one week
            Repeated administration, between one        4 weeks to 3 months
            week to four weeks
            Repeated administration, between one to     3 to 6 months
            six months
            Long term repeated administration for       9 to 12 months
            more than six 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:
           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.
2.     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

								
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