Guidelines for toxicity / safety profile
evaluation
Of
Bhasma/raskalpas
Standard Operating Procedure (SOP) for Administration of
Bhasma/Rasakalpas with Honey as Vehicle
Principle: To validate the classical method of administration of the test
compound Bhasma/Rasakalpas in all test centers.
Materials:
Test compounds : Bhasma/Rasakalpas
Vehicle control :Honey : 2 parts
De-ionized water :3 parts
Syrings
Needles
(200-500-l)
:1 ml tuberculin syringe/insulin syringe
:Aspirator needles (16/18nos) 6’ balled or Eppendorf pipette
Centrifuge tubes: Plastic graduated 10 ml tubes
Balance
: Metler balance(0-1000mg)
Mixer
: Vortex Mixer
Butter paper
: Small pieces
Dosage schedule:
The required dose for mice/rat will be calculated by using the standard dose
calculation procedure from recommended clinical dose.
Conversion formula:
If human dose is 125mg,BD
Total clinical dose (a) x conversion factor (b) 0.018 = (c) per 200 gm of rat
125 mg x 2(a) x 0.018 (b) = 4.5 mg (c) /200gms of rat
4.5x1000/200 = 22.5mg/kg
Ref: Paget and Branes (1964) Evaluation of Drug Activities :Pharmacometric eds.Laurence and
Bocharach,Vol.1.Academic Press,New York.
Experimental Doses Calculated as per the standard procedures are:
S.No
Groups
Dose /kg, weight
Dose /200
gms. weight
Volume of
administration
1
2
3
4
Vehicle Control
Therapeutic Dose
Average Dose
High Dose
-22.5mg
112.5mg
225mg
-4.5mg
22.5mg
45mg
0.5 ml
0.5 ml
0.5 ml
0.5 ml
If Human Dose is 125mg,BD
Total quantity of drug:
67.5 mg for TD,
337 mg for AD
675 mg for HD
Preparation of Compound:
1.The required test compound will be weighted on the metler balance as per
standard procedures on a butter paper.
2.Weighed test compound will be transferred into 10-15 ml centrifuge tube.
3.Appropriate volume to be administered will be made as per the calculated dose.
4.The diluted honey will be prepared fresh by taking 3 ml honey and diluting with
4.5 ml de ionized water.
(67.5 mg of the Bhasma in 7.5 ml of diluted Honey is equivalent to 4.5 mg in 0.5
ml/200gms body wt.of rat)
5.Vortex the mixture.
6.The concentration of test compound to be administered will be 0.5 ml to each
rat and 0.25 ml to each mouse on tongue.
7.Take the tuberculin syringe (1ml) and use 16-18 gauge bent balled needle or
eppendorf tips and pipettes.
8.Administration of test compound.
9.The test compound should be applied on tongue and not in Oro-pharaynx.
Guidelines for 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
ix.
Haematology - Twice (15th day/30th day)
x.
Clinical chemistry – Twice (15th day/30 day)
xi.
Histopathology
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.
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
Bio-chemical (Blood glucose, SGOT, SGPT, Serum Creatinine, Total protein)
and Hematological investigation (RBC, Hb, Prothrombine time WBC, TC, MCV,
MCH, MCHC)
Histopathology of vital organs:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Liver
Kidney
Lungs
Spleen
Ovaries/testis
Heart
Stomach
Intestine
Sciatic nerve
Brain
Chronic Toxicity Evaluation
Species
No. of Animals
Male
Age
(wks)
Female
Weight
(gms)
Duration of
experiment
TC Exp.
Term.
Exp.
90 days
100%
Mice
40
40
4
15-18
(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.
ii)
Acclimatization of animals
Period – 7 days (Recording of body weight and food intake twice in a
week)
iii)
Urine qualitative test (Ames multiple sticks)
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 - (30/60/90 days) (Hb, RBC, WBC, Prothrombin
time,Platelet count, Differential count,MCV,MCH,MCHC)
j. Clinical chemistry – (30/60/90 days) (Blood glucose, total protein, serum
creatinine, SGOT, SGPT).
k. Histopathology – Liver, heart, brain, lung, kidney, spleen, sciatic nerve,
testes/ ovaries,stomach,intestine.
l. Blood serum metal estimation, Tissue metal esimation
General comments
o Metal free pellet diet and water
o One rat and two mice in each cage
o Test compound, vehicle control, its dosage regimen will be supplied by
sponsor (ISM)
o Scoring of cage side activity, neurological activity according to OECD
guidelines.
Route of Administration:
Oral
Duration:
90 days