1.0 OBJECTIVE
1.1 To lay down a standard procedure to be followed for conducting a Bioassay of Test Items for Human Chorionic Gonadotrophin (HCG) activity in experimental rats by the following method prescribed in Indian Pharmacopoeia (I.P.)-2014 and British Pharmacopoeia (B.P.)-2013.
2.0 SCOPE
2.1 This Standard Operating Procedure (SOP) shall be applicable for conducting a Bioassay of Test Items for Human Chorionic Gonadotrophin (HCG) activity in experimental rats by Indian Pharmacopoeia (I.P.)-2014 and British Pharmacopoeia (B.P.)-2013 method.
3.0 RESPONSIBILITY
3.1 Head- In-vivo Facility
3.2 Study Director
3.3 Lab Technician- Animal House Facility
4.0 DEFINITIONS
4.1 Nil
5.0 PROCEDURE
5.1 Pre-Experimental Activities
5.1.1 IAEC approval for conducting bioassay shall be taken as per the currently running version of SOP……….. study shall be initiated after getting approval from the IAEC committee for the conduct of the experiment.
5.1.2 Detailed Study Plan for the study shall be prepared by following recommended method prescribed.
5.1.3 Study personnel shall be given study-specific training if any (if required) as per the currently running version of SOP……………..
5.1.4 After getting approval of the study plan from the sponsor, the required number of data recording sheets (DRS) formats shall be issued from QAD.
5.1.5 Animal requisition shall be given to animal house facility and animals shall be issued as per the currently running version of SOP…………...
5.1.6 After issue animals shall be acclimatized to standard laboratory conditions for a period of at least 5 days as per the currently running version of SOP…………...
5.1.7 After completion of the acclimatization period animals shall be randomized and grouped by the following SOP……………...
5.1.8 Numbering and identification of animals shall be carried by following the currently running version of SOP…………….
5.2 Experimental Activities
5.2.1 HCG Activity as per Indian Pharmacopoeia (I.P.)-2014
5.2.1.1 Selection of Animals
5.2.1.1.1 It causes an increase in the weight of the seminal vesicles or of the prostate glands of immature male rats when administered as directed in the Assay.
5.2.1.1.2 Use immature male rats of the same strain, approximately 21 days old and of approximately equal weight within the range of 25 to 35 g.
5.2.1.1.3 The animals are given ad libitum water and food, commonly used for laboratory animals.
5.2.1.1.4 Assign the rats at random to four equal groups of at least eight animals. If sets of four littermates are available, allot one littermate from each set to each group and mark according to litter.
5.2.1.2 Selection of Dose
5.2.1.2.1 Choose two doses of the standard preparation and two of the test solution such that the smaller dose is sufficient to produce a positive response in some of the rats and the larger dose does not produce a maximum response in all of the rats. Use doses in geometric progression. As an initial approximation, doses of 7.5 and 15 Units may be tried although the dose will depend on the sensitivity of the animals used that may vary widely.
5.2.1.3 Preparation of Test Solution
5.2.1.3.1 Dissolve a sufficient quantity of the injection under examination corresponding to the daily doses to be used in sufficient albumin-phosphate buffer pH 7.2 so that the daily dose is about 0.2 mL.
5.2.1.4 Preparation of Albumin-phosphate buffer of pH 7.2
5.2.1.4.1 Dissolve 10.75 g of disodium hydrogen phosphate, 7.6 g of sodium chloride and 10 g of bovine albumin insufficient water to produce 1000 ml. Before use, adjust the pH to 7.2 with 2 M sodium hydroxide or a 10 percent w/v solution of phosphoric acid as required so that the daily dose is about 0.5 mL.
5.2.1.4.2 Add a suitable antimicrobial preservative such as 0.4% w/v of phenol or 0.002% w/v of thiomersal.
5.2.1.4.3 Store the solutions at a temperature of 2° to 8°.
5.2.1.5 Administration of Test Item
5.2.1.5.1 Inject subcutaneously (by following the currently running version of SOP…………) into each rat the daily dose allocated to its group on 4 consecutive days at the same time each day.
5.2.1.6 Observations
5.2.1.6.1 Clinical signs of toxicity after dosing shall be recorded as per the currently running version of SOP…………… and data shall be recorded in associated format to the SOP. All animals shall be observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for the time as specified in the individual monograph, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead.
5.2.1.6.2 Animals shall be observed daily during the observation period for mortality as per SOP…………… and observation shall be recorded in associated format to SOP.
5.2.1.6.3 Individual weights of animals shall be determined shortly before the test substance is administered and daily thereafter. Bodyweight shall be recorded as per the currently running version of SOP………………. and data shall be recorded in associated format to SOP. At the end of the test surviving animals shall be weighed and then humanely killed.
5.2.1.6.4 All test animals shall be subjected to gross necropsy on the fifth day, about 24 hours after the last injection as per the currently running version of SOP……………. and all gross pathological changes shall be recorded in associated format to SOP. Animals who died during the test or are removed from the study for animal welfare reasons shall also be subjected to the same procedures.
5.2.1.6.5 Seminal vesicles or prostate gland shall be removed and freed from extraneous fluid and tissue and weighed immediately.
5.2.1.7 Data Recording and Result interpretation
5.2.1.7.1 Calculate the result of the assay by standard statistical methods, using the weight of the vesicles or the prostate gland as the response.
5.2.1.7.2 The estimated potency is not less than 80% and not more than 125% of the stated potency. The fiducial limits of error are not less than 64% and not more than 156% of the stated potency.
5.2.2 HCG Activity as per British Pharmacopoeia (B.P.)-2013
5.2.2.1 Selection of Animals
5.2.2.1.1 The potency of chorionic gonadotrophin is estimated by comparing under given conditions its effect of increasing the mass of the seminal vesicles (or the prostate gland) of immature rats with the same effect of the International Standard of chorionic gonadotrophin or of a reference preparation calibrated in International Units.
5.2.2.1.2 The International Unit is the activity contained in a stated amount of the International Standard, which consists of a mixture of a freeze-dried extract of chorionic gonadotrophin from the urine of pregnant women with lactose. The equivalence in International Units of the International Standard is stated by the World Health Organization.
5.2.2.1.3 Use immature male rats of the same strain, 19 to 28 days old, differing in age by not more than 3 days and having body masses such that the difference between the heaviest and the lightest rat is not more than 10 g.
5.2.2.1.4 The animals are given ad libitum water and food, commonly used for laboratory animals.
5.2.2.1.5 Assign the rats at random to 6 equal groups of at least 5 animals. If sets of 6 littermates are available, assign one littermate from each set to each group and mark according to litter.
5.2.2.2 Selection of Dose
5.2.2.2.1 Choose 3 doses of the reference preparation and 3 doses of the preparation to be examined such that the smallest dose is sufficient to produce a positive response in some of the rats and the largest dose does not produce a maximal response in all the rats. Use doses in geometric progression and as an initial approximation total doses of 4 IU, 8 IU, and 16 IU may be tried although the dose will depend on the sensitivity of the animals used, which may vary widely.
5.2.2.3 Preparation of Test Solution
5.2.2.3.1 Dissolve separately the total quantities of the preparation to be examined and of the reference preparation corresponding to the daily doses to be used in sufficient phosphate-albumin buffered saline pH 7.2 R such that the daily dose is administered in a volume of about 0.5 mL.
5.2.2.4 Preparation of Albumin-phosphate buffer of pH 7.2
5.2.2.4.1 Dissolve 10.75 g of disodium hydrogen phosphate, 7.6 g of sodium chloride, and 10 g of bovine albumin insufficient water to produce 1000 ml. Before use, adjust the pH to 7.2 with 2 M sodium hydroxide or a 10 percent w/v solution of phosphoric acid as required so that the daily dose is about 0.5 mL.
5.2.2.4.2 Add a suitable antimicrobial preservative such as 4 g/L of phenol or 0.02 g/L of thiomersal.
5.2.2.4.3 Store the solutions at 5 ± 3 °C.
5.2.2.5 Administration of Test Item
5.2.2.5.1 Inject subcutaneously (by following the currently running version of SOP.............) into each rat the daily dose allocated to its group on 4 consecutive days at the same time each day.
5.2.2.6 Observations
5.2.2.6.1 Clinical signs of toxicity after dosing shall be recorded as per the currently running version of SOP……………. and data shall be recorded in associated format to the SOP. All animals shall be observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for the time as specified in the individual monograph, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead.
5.2.2.6.2 Animals shall be observed daily during the observation period for mortality as per SOP…………… and observation shall be recorded in associated format to SOP.
5.2.2.6.3 Individual weights of animals shall be determined shortly before the test substance is administered and daily thereafter. Bodyweight shall be recorded as per the currently running version of SOP……………… and data shall be recorded in associated format to SOP. At the end of the test surviving animals shall be weighed and then humanely killed.
5.2.2.6.4 All test animals shall be subjected to gross necropsy on the fifth day, about 24 hours after the last injection as per the currently running version of SOP…………….. and all gross pathological changes shall be recorded in associated format to SOP. Animals who died during the test or are removed from the study for animal welfare reasons shall also be subjected to the same procedures.
5.2.2.6.5 Seminal vesicles or prostate gland shall be removed and freed from extraneous fluid and tissue and weighed immediately.
5.2.2.7 Data Recording and Result interpretation
5.2.2.7.1 Calculate the result of the assay by standard statistical methods, using the weight of the vesicles or the prostate gland as the response. (The precision of the assay may be improved by a suitable correction of the organ mass with reference to the body mass of the animal from which it was taken; an analysis of covariance may be used).
5.2.2.7.2 The estimated potency is not less than 80 percent and not more than 125 percent of the stated potency. The confidence limits (P = 0.95) of the estimated potency are not less than 64 percent and not more than 156 percent of the stated potency.
5.3 Data and Reporting
5.3.1 Individual animal data shall be provided. Additionally, all data shall be summarized in tabular form, showing for each test group the number of animals used, the number of animals found dead during the test or killed for humane reasons, necropsy findings, Organ weight (Seminal vesicles or prostate gland weights), Bodyweight and change in body weight.
5.3.2 Statistical analysis, if required shall be carried out by using the licensed version of Graphpad instant 3.01 or by Graphpad 5.04 by following its user manual.
5.4 Test report
5.4.1 Test report shall be prepared and shall include the following information, as appropriate:
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Test substance
|
:
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Certificate of analysis of Test
Item
Vehicle (if appropriate):
justification for the choice of vehicle.
|
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Test animals
|
:
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Species/strain of animals used
Number, age, and sex of animals
(including, where appropriate, a rationale for use of males instead of
females)
Source, housing conditions, diet
etc.
|
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Test conditions
|
:
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Details of the test substance
formulation, including details of the physical form of the material
administered
Details of the administration of
the test substance including dosing volumes and time of dosing
Details of food and water
quality (including diet type/source, water)
The rationale for the selection
of the starting dose
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Results
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:
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Tabulation
of response data and dose level for each animal
Tabulation of body weight/body
weight changes
Individual weights of animals at
the day of dosing, daily thereafter, and at time of death or sacrifice.
Date and time of death if prior
to scheduled sacrifice
Necropsy findings and organ
weight change as compared to standard.
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Discussion
and interpretation of results
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:
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Discussion and interpretation
of results shall be mentioned clearly
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Conclusions
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:
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The study shall be concluded as
appropriate
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5.5 Archive
5.5.1 After the approval of the draft study report from the sponsor, the study file and all related materials / raw data shall be achieved.
5.6 Flow chart for conducting Bioassay of Test Item for HCG Activity
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5.7 Checklist of SOPs required for conducting Bioassay of Test Item for HCG Activity
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SOPs
required
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SOP No.
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Title
of SOP
|
|
Pre-experimental
activities
|
SOP…………
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Standard Operating Procedure for
Institutional Animal Ethics Committee meeting and obtaining protocol approval
for experimentation on animals
|
|
SOP…………
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Standard Operating Procedure for personnel
training for animal care and use
|
|
SOP…………
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Standard Operating Procedure for the supply of
laboratory animals to studies
|
|
SOP…………
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Standard Operating Procedure for acclimatization
of laboratory animals
|
|
SOP…………
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Standard Operating Procedure
for randomization of experimental
animals
|
|
SOP…………
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Standard Operating Procedure for
animal numbering and identification procedure for laboratory animals
|
|
experimental
activities
|
SOP…………
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Standard Operating Procedure for weighing of
laboratory animals
|
|
SOP………….
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Standard Operating Procedure for the administration of test item by oral, the parenteral, and topical route in laboratory animals
|
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SOP………….
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Standard Operating Procedure for observation of
clinical signs in experimental animals
|
|
SOP………….
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Standard Operating Procedure for the handling of animal
morbidity and mortality in Animal House Facility
|
|
SOP………….
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Standard Operating Procedure for
gross necropsy and collection, weighing & fixation of organs/tissues in
laboratory animals
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6.0 ENCLOSURES
6.1 Formats
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Sr. No.
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Format Title
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Format No.
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No. of pages
|
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1
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Animal Bio-waste Record
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F……………………..
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01
|
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2
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Label For Transport of Bio-Medical Waste
Containers/Bags
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F…………………….
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01
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6.2 Annexures
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Sr. No.
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Annexure Title
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Annexure No.
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No. of Pages
|
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1.
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Categories of Bio-Medical Waste
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A……………………..
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01
|
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2.
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Color Coding & Type of Container For
Disposal Of Bio-Medical Waste
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A……………………..
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01
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7.0 ABBREVIATIONS
7.1 SOP : Standard Operating Procedure
7.2 QAD : Department of Quality Assurance
7.3 AHF : Animal House Facility
7.4 MBD : Department of Microbiology
7.5 SP : Study plan
8.0 REFERENCES
8.1 NIH Guide for Grants and Contracts. 7/17/2002, Notice: OD-02-062.
8.2 CCAC Guidelines on Euthanasia.
8.3 AVMA Panel on Euthanasia. 2000 Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc 2001, 218:669-696.
9.0 REVISION HISTORY
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Sr. No.
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Change Control No.
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Reason for change
|
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1.
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CC…………………
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Change of Format of SOP
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END OF DOCUMENT
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