1.0 OBJECTIVE
1.1 To lay down a standard procedure to be followed conducting Bioassay of Menotrophin for Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) activity in experimental rats by the following method prescribed in British Pharmacopoeia (B.P.)-2013.
2.0 SCOPE
2.1 This Standard Operating Procedure (SOP) shall be applicable for conducting Bioassay of Test Menotrophin for Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) activity in experimental rats.
3.0 RESPONSIBILITY
3.1 Head- Animal House 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………… The 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 current 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 current running version of SOP……………….
5.2 Experimental Activities
5.2.1 Follicle Stimulating Hormone (FSH) Activity
5.2.1.1 Selection of Animals
5.2.1.1.1 FSH activity causes enlargement of the ovaries of immature female rats when administered as directed in the Assay.
5.2.1.1.2 Select female rats of the same strain, 19 to 28 days old and differing in age by not more than 3 days, and having weights such that the difference between the heaviest rat and the lightest rat is not more than 10 g.
5.2.1.1.3 Assign the rats at random to six equal groups of at least five animals. If sets of six littermates are available, allot one littermate from each set to each group and mark according to litter.
5.2.1.1.4 House the animals individually in an area of uniform temperature (± 2°), preferably with uniform humidity, and free from disturbances likely to excite them.
5.2.1.1.5 The animals are given ad libitum water and food, commonly used for laboratory animals.
5.2.1.1.6 Assign the rats at random to 6 equal groups of at least 5 animals.
5.2.1.2 Selection of Dose
5.2.1.2.1 Choose three doses of the Standard Preparation and three doses of the preparation being examined such that the smallest dose produces a positive response in some of the rats and the largest dose does not produce a maximum response in all of the rats. Use doses in geometric progression. As an initial approximation, total doses of 1.5, 3.0 and 6.0 IU may be tried although the dose will depend on the sensitivity of the animals used, which 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 containing not less than 70 IU of chorionic gonadotrophin per mL 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°.
5.2.1.4.4 The buffer solution shall contain in the daily dose not less than 14 IU of chorionic gonadotrophin to ensure complete luteinization.
5.2.1.5 Administration of Test Item
5.2.1.5.1 Inject subcutaneously into each rat the daily dose allocated to its group by following the currently running version of SOP……………..
5.2.1.5.2 Repeat the procedure after 24 and 48 hours.
5.2.1.6 Observations
5.2.1.6.1 Clinical signs of toxicity after dosing shall be recorded as per the current running version of SOP/AHF/025 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 fourth, 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 Ovaries shall be removed and freed from extraneous fluid and tissue and weighed immediately. The combined weight of both ovaries from each rat shall be recorded.
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 ovaries as the response. (The precision of the assay may be improved by a suitable correction of the organ weight with reference to the weight of the animal from which it was taken; an analysis of covariance may be used).
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 Luteinizing Hormone (LH) Activity
5.2.2.1 Selection of Animals
5.2.2.1.1 LH activity causes increases in the weight of the seminal vesicles and prostate gland of immature male rats when administered as directed in the Assay.
5.2.2.1.2 Select male rats of the same strain, 19 to 28 days old and differing in age by not more than 3 days, and having weights such that the difference between the heaviest rat and the lightest rat is not more than 10 g.
5.2.2.1.3 Assign the rats at random to six equal groups of at least five animals. If sets of six littermates are available, allot one littermate from each set to each group and mark according to litter.
5.2.2.1.4 House the animals individually in an area of uniform temperature (± 2°), preferably with uniform humidity, and free from disturbances likely to excite them.
5.2.2.1.5 The animals are given ad libitum water and food, commonly used for laboratory animals.
5.2.2.1.6 Assign the rats at random to 6 equal groups of at least 5 animals.
5.2.2.2 Selection of Dose
5.2.2.2.1 Choose three doses of the Standard Preparation and three doses of the preparation being examined such that the smallest dose produces a positive response in some of the rats and the largest dose does not produce a maximum response in all of the rats. Use doses in geometric progression. As an initial approximation, total doses of 7 IU, 14 IU and 28 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 being examined and of the Standard Preparation 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.
5.2.2.3.2 Add a suitable antimicrobial preservative such as 0.4% w/v of phenol or 0.002% w/v of thiomersal.
5.2.2.3.3 Store the solutions at a temperature of 2° to 8°.
5.2.2.4 Administration of Test Item
5.2.2.4.1 Inject subcutaneously into each rat the daily dose (by following the currently running version of SOP………………) allocated to its group on 4 consecutive days at the same time each day.
5.2.2.5 Observations
5.2.2.5.1 Clinical signs of toxicity after dosing shall be recorded as per the current 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.
5.2.2.5.2 Animals shall be observed daily during the observation period for mortality as per SOP……………….. and observation shall be recorded in associated format to SOP.
5.2.2.5.3 Individual weights of animals shall be determined shortly before the test substance is administered and daily thereafter. Bodyweight shall be recorded as per the current 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.
5.2.2.5.4 All test animals shall be subjected to gross necropsy on the fifth day, about 24 hours after the last injection as per the current 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.
5.2.2.5.5 Seminal vesicles or prostate gland shall be removed and freed from extraneous fluid and tissue and weighed immediately.
5.2.2.6 Data Recording and Result interpretation
5.2.2.6.1 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 weight with reference to the weight of the animal from which it was taken; an analysis of covariance may be used).
5.2.2.6.2 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.
5.3 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 for LH and Combined ovaries weight for FSH activity), 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 5.01 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:
Test substance | : | Certificate of analysis of Test Item Vehicle (if appropriate): justification for the choice of vehicle. |
Test animals | : | 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. |
Test conditions | : | 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 |
Results | : | 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. |
Discussion and interpretation of results | : | Discussion and interpretation of results shall be mentioned clearly |
Conclusions | : | The study shall be concluded as appropriate |
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 FSH and LH Activity
5.7 Checklist of SOPs required for conducting Bioassay of Test Item for FSH and LH Activity
SOPs required | SOP No. | Title of SOP |
Pre-experimental activities | SOP………… | Standard Operating Procedure for Institutional Animal Ethics Committee meeting and obtaining protocol approval for experimentation on animals |
SOP………… | Standard Operating Procedure for personnel training for animal care and use |
SOP………… | Standard Operating Procedure for the supply of laboratory animals to studies |
SOP………… | Standard Operating Procedure for acclimatization of laboratory animals |
SOP………… | Standard Operating Procedure for randomization of experimental animals |
SOP………… | Standard Operating Procedure for animal numbering and identification procedure for laboratory animals |
experimental activities | SOP………… | Standard Operating Procedure for weighing of laboratory animals |
SOP…………. | Standard Operating Procedure for the administration of test item by oral, the parenteral, and topical route in laboratory animals |
SOP…………. | Standard Operating Procedure for observation of clinical signs in experimental animals |
SOP…………. | Standard Operating Procedure for the handling of animal morbidity and mortality in Animal House Facility |
SOP…………. | Standard Operating Procedure for gross necropsy and collection, weighing & fixation of organs/tissues in laboratory animals |
6.0 ENCLOSURES
6.1 Formats
Sr. No. | Format Title | Format No. | No. of pages |
1 | Animal Biowaste Record | F…………………….. | 01 |
2 | Label For Transport of Bio-Medical Waste Containers/Bags | F……………………. | 01 |
6.2 Annexures
Sr. No. | Annexure Title | Annexure No. | No. of Pages |
1. | Categories of Bio-Medical Waste | A…………………….. | 01 |
2. | Color Coding & Type of Container For Disposal Of Bio-Medical Waste | A…………………….. | 01 |
7.0 ABBREVIATIONS
7.1 SOP : Standard Operating Procedure
7.2 AHF : Animal House Facility
7.3 DRS : Data Recording sheets
7.4 FSH : Follicle Stimulating Hormone
7.5 LH : Luteinizing Hormone
7.6 HCG : Human Chorionic Gonadotrophin
8.0 REFERENCES
8.1 British Pharmacopoeia (B.P.)-2013.
9.0 REVISION HISTORY
Sr. No. | Change Control No. | Reason for change |
1. | CC………………… | Change of Format of SOP |
END OF DOCUMENTS
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