1.1 To lay down a standard procedure to be followed for conducting Pyrogen Tests in Laboratory Rabbits by the method prescribed in United State Pharmacopoeia (USP).
2.0 SCOPE
2.1 This Standard Operating Procedure (SOP) shall be applicable for conducting Pyrogen Tests in Laboratory Rabbits by the USP method in the Animal House Facility.
3.0 RESPONSIBILITY
3.1 Head- Animal House Facility / Study Director
3.2 All Personnel involved in the study.
4.0 DEFINITIONS
4.1 Nil
5.0 PROCEDURE
5.1 Pre-Experimental Activities
5.1.1 IAEC approval for conducting Pyrogen Test of test item 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 for 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 following SOP…………..
5.1.8 Numbering and identification of animals shall be carried out by following the currently running version of SOP……….
5.1.9 If the test item needs to be formulated (prepared) / diluted in any vehicle then dose calculation and formulation of the test item shall be carried out as per the sponsor’s instruction / recommended procedure.
5.2 Experimental Activities
5.2.1 The test involves the measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance under examination. It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10 ml per kg injected intravenously within a period of not more than 10 minutes.
5.3 Selection of Animals
5.3.1 Use healthy, adult rabbits of either sex, preferably of the same variety, weighing not less than 1.5 kg, fed on a complete and balanced diet.
5.3.2 House the animals individually in an area of uniform temperature between 20° C and 23° C, preferably with uniform humidity, and free from disturbances likely to excite them. The temperature varies not more than ±3 from the selected temperature. Before using a rabbit for the first time in a Pyrogen test, condition it not more than seven days before use by a sham test that includes all of the steps as directed for Procedure except injection.
5.3.3 Do not use a rabbit for pyrogen testing more frequently than once every 48 hours, nor prior to 2 weeks following a maximum rise of its temperature of 0.6 or more while being subjected to the pyrogen test, or following its having been given a test specimen that was adjudged pyrogenic.
5.4 Preparation of Test Solution
5.4.1 All glassware’s used in testing must be thoroughly washed with water for injections and depyrogenated by heating in a hot air oven at 250° for not less than 30 minutes or at 200° for 1 hour for depyrogenation (SOP……….).
5.4.2 Treat all diluents and solutions for washing and rinsing of devices in a manner that will assure that they are sterile and pyrogen-free.
5.4.3 Dissolve the substance under examination in, or dilute with, pyrogen-free saline solution or other solution prescribed in the monograph. Warm the liquid under examination to approximately 38 degrees Celsius before injection.
5.5 Test Procedure
5.5.1 Description of Test Procedure
5.5.1.1 The retaining boxes /restrainer for rabbits in which the temperature is being measured by an electrical device should be made in such a way that the animals are retained only by loosely-fitting neck stocks and the rest of the body remains relatively free so that the rabbits may sit in a normal position.
5.5.1.2 The animals must be put in the boxes/restrainers 1 hour before the test and remain in them throughout the test. Ensure that the room temperature where the test is carried out is between 20° C and 23° C.
5.5.1.3 Perform the test in a separate area designated solely for pyrogen testing and under environmental conditions similar to those under which the animals are housed and free from disturbances likely to excite them.
5.5.1.4 Withhold all food from the rabbits used during the period of the test.
5.5.1.5 Access to water is allowed at all times but restricted during the test.
5.5.1.6 Record the temperature of each animal at intervals of not more than 30 minutes, beginning at least 90 minutes before the injection of the solution under examination and continuing for 3 hours after the injection.
5.5.1.7 Not more than 40 minutes immediately preceding the injection of the test dose, record the "initial temperature" of each rabbit, which is the mean of two temperatures recorded for that rabbit at an interval of 30 minutes in the 40-minute period.
5.5.2 Animal acceptance criteria
5.5.2.1 Rabbits showing a temperature variation greater than 0.2° C between two successive readings in the determination of "initial temperature" should not be used for the test.
5.5.2.2 In any one group of test animals, use only those animals whose "initial temperatures" do not vary by more than 1° C from each other, and do not use any rabbit having a temperature higher than 39.8° and lower than 38° C.
5.5.3 Administration of Test Item
5.5.3.1 Inject the solution under examination slowly into the marginal vein of the ear of each rabbit by following the currently running version of SOP………, over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph.
5.5.3.2 The amount of sample to be injected varies according to the preparation under examination and is prescribed in the individual monograph.
5.5.3.3 The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight.
5.5.4 Observations
5.5.4.1 Record the temperature of each animal by using a precise digital tela-thermometer as per SOP……….., at half-hourly intervals for 3 hours after the injection. The difference between the "initial temperature" and the "maximum temperature" which is the highest temperature recorded for a rabbit is taken to be its response. When this difference is negative, the result is counted as a zero response. Rectal temperature raw data shall be recorded in format number F……………...
5.5.4.2 Animals shall be observed for morbidity or mortality as per SOP………. and observation shall be recorded in associated format to SOP.
5.5.4.3 All observations shall be systematically recorded, with individual records being maintained for each animal.
5.5.4.4 Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress shall be humanely killed. When animals are killed for humane reasons or found dead, the time of death shall be recorded as precisely as possible.
5.5.5 Data Recording and Result Interpretation
5.5.6 Use an accurate temperature sensing device such as a thermometer, thermistor probes, or similar probes that have been calibrated to assure an accuracy of ±0.10C and have been tested to determine that a maximum reading is reached in less than 5 minutes. Insert the temperature-sensing probes into the rectum of the test rabbit to a depth of not less than 7.5 cm.
5.5.7 If no rabbit shows an individual rise in temperature of 0.6 °C or more above its respective control temperature, and if the sum of the 3 temperature rises does not exceed 1.4 °C, the tested material meets the requirements for the absence of pyrogens.
5.5.8 If 1 or 2 rabbits show a temperature rise of 0.6 °C or more, or if the sum of the temperature rises exceeds 1.4 °C, continue the test using 5 other rabbits.
5.5.9 If not more than 3 of the 8 rabbits show individual rises in temperature of 0.6 °C or more, and if the sum of the 8 temperature rises does not exceed 3.7 °C, the tested material meets the requirements for the absence of pyrogens.
5.6 Data and Reporting
5.6.1 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, and the number of animals that died during the test or were killed for humane reasons.
5.7 Test report
5.7.1 Draft 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 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 (i.e. animals showing clinical signs including mortality, nature, severity and duration of effects)
Individual weights of animals at the day of dosing
Discussion and interpretation of results:
Discussion and interpretation of results shall be mentioned clearly
Conclusions:
The study shall be concluded as appropriate
5.8 Archive
5.8.1 After the approval of the draft study report from the sponsor, the study file and all related materials / raw data shall be achieved.
|
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 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 (i.e. animals showing
clinical signs including mortality, nature, severity and duration of effects) Individual weights of animals on
the day of dosing |
|
Discussion
and interpretation of results |
: |
Discussion and interpretation
of results shall be mentioned clearly |
|
Conclusions |
: |
The study shall be concluded as
appropriate |
5.10 Checklist of SOPs required for conducting Pyrogen Test in Experimental Animals (By USP 38 Method)
|
SOPs
required |
SOP
No. |
Title
of SOP |
|
Pre-experimental
activities |
SOP/----------- |
|
|
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 items by oral, parenteral, and topical routes in laboratory animals |
|
|
SOP/-------- |
Standard
Operating Procedure for Digital Tele-thermometer |
|
|
SOP/-------- |
Standard
Operating Procedure for the handling of animal morbidity and mortality in animal house facility |
5.11 Archive
5.11.1 After the approval of the draft study report from the sponsor, the study file and all related materials / raw data shall be achieved.
5.11.2 Flow chart for conducting Pyrogen Test in Experimental Animals (By IP / USP Method)
6.1 Nil
7.0 ABBREVIATIONS
7.1 SOP: Standard Operating Procedure
7.2 QAD: Quality Assurance Department
7.3 DRS: Data Recording sheets
7.4 USP: United States Pharmacopoeia
8.0 REFERENCES
8.1 USP 38
9.0 REVISION HISTORY Nil
END OF DOCUMENTS
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