1.0 TEST SYSTEM DETAILS:
Species : Rattus norvegicus (Rat)
Strain : Wistar or Sprague Dawley
Age : 8-10 weeks
Body Wight : 120-200 g
Sex : Male/Female
No. of animals : Model-1 6 /Group; Model-2 6 /Group
Total Animals : Model-1 (12-Female); Model-2 (60- Male/Female)
2.0 ALLOCATION OF GROUPS:
Model -1: OECD-423
|
Groups |
Treatment |
Dose;
ROA |
No. of
Animals |
|
G1 |
Normal Control |
Normal saline |
6 |
|
G2 |
Test Group-I |
2000 mg/kg; p.o. |
6 |
*The doses and ROA (Routes of administration) will be decided based on physical state test formulation mainly oral dose.
Model -2: OECD-407
|
Groups |
Treatment |
Dose;
ROA |
No. of
Animals |
|
GI |
Normal Control |
Normal saline |
12 |
|
GII |
Lower Dose |
X mg/kg; p.o. |
12 |
|
GIII |
Median Dose |
XX mg/kg; p.o. |
12 |
|
GIV |
Higher Dose |
XXX mg/kg; p.o. |
12 |
|
GV |
Satellite Group |
XXX mg/kg; p.o. |
12 |
The doses and ROA (Routes of administration) will be decided based on physical state test formulation mainly oral dose. X, XX & XXX shows the concentration of Lower, Median & Higher respectively.
3.0 METHOD:
Model-1
· As per available reference of Natural Probiotic formulation the starting dose will be 2000 mg/kg b.wt and observed for clinical signs of toxicity and mortality.
· After 72 hrs further doses will be administered in more 3 animals (as per annex-2d)
· All the animals will be observed individually after dosing at least once during first 30 minutes, periodically during first 24 hrs, with special attention given during the first 4 hrs, and daily thereafter, for a total of 14 days.
· These observations include changes in skin and fur, eyes and mucous membrane, and also respiratory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Attention will be directed towards tremor, convulsion, salivation, diarrhea, lethargy, sleep and coma.
Model-2
· The experiment will be conducted on four different groups with control, high, median & lower doses in 6 male and 6 female Wistar rats in each group. Dosing will be dose till 28 days.
· Observe for clinical signs, hematological, biochemical changes and mortality.
· If clinical signs, hematological, biochemical changes will not occur significantly
· Select 3 subsequent doses and administer in separate group of animals & observe for clinical signs, hematological and biochemical changes.
4.0 END POINT PARAMETER(S):
· Clinical Signs : All visible clinical signs will be observed at least twice a day for a total of 14 days in model-1 & 28 days in model-2. The health condition of the animals should be recorded.
· Mortality: All the animals will be checked twice daily for morbidity and mortality.
· Body Weight and food consumption: All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly.
· Hematology The blood sample will be collected on 14th day (Model-1) & 29th day (Model-2)of experimentation and following hematological examination will be done-Hemoglobin concentration, erythrocyte count, total and differential leukocyte count and blood clotting time.
· Clinical biochemistry: The blood sample will be collected from overnight fasting animals and following biochemical parameters will be examined- Sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein, albumin, bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase.
· Urine analysis: The urine sample will be done at the end of the experiment. The urine will be collected aseptically directly from bladder of each rat via catheter or intraperitonially and examined for appearance, volume, specific gravity, pH, protein, glucose and blood/blood cells.
· Pathology (Necropsy): All test animals will be subjected to gross necropsy at termination of the study to examine gross pathological changes.
· Histopathology : The histopathological study will be done on the preserved organs and tissues of all animals in the control as well as treated groups.
5.0 REFERENCE(S):
5.1 OECD, Test No. 423: Acute Oral toxicity - Acute Toxic Class Method, OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. 2002.
5.2 OECD, 2008. Guidelines for testing of chemicals: repeated dose 28-day oral toxicity in rodents. Test No 407, adopted in 1981, and revised method adopted 3rd October 2008. OECD, Paris.
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