PRINCIPLE
Pyrogen test is the measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of a substance being examined. It is designed for products that can be tolerated by the test rabbits in a dose not to exceed 10.0 mL per kg. Weight injected intravenously within a period of to more than 10 min.
SELECTION OF ANIMALS
1. Use healthy, adult rabbits of either sex, weighing not less than 1.5 kg, fed a complete and balanced diet not containing antibiotics and showing no loss of body weight during the week preceding the test.
2. The rabbits must not have been used in a similar test :
a. during the preceding 3 days (as per BP) or 2 days (as per USP and IP)
b. during the preceding 3 weeks (as per BP) or 2 weeks (as per USP and IP) unless the material being examined passed the test Rabbits used in a test for Pyrogens where the mean rise in the rabbits temperature has exceeded 1.2 OC are permanently excluded (as per BP) and (as per USP and IP)
ANIMALS QUARTERS
1. Keep the rabbits individually in a quiet area with an appropriate uniform temperature with a variation of ± 2 OC and uniform humidity and free from disturbance.
2. Carry out the test in a quiet room where there is no risk of disturbance exciting the animals and in which the room temperature is within 3 OC of that of the rabbits living quarters or in which the rabbits have been kept for at least 18 h before the test.
3. Withhold food from the rabbits overnight and until the test is completed; withhold water only during the test.
EQUIPMENT
Thermometer
1. The thermometer or electrical device used indicates the temperature with a precision of 0.1 OC and is inserted in the rectum of the rabbit to a depth of about 5 cm.
2. The depth of inserted is constant for any one rabbit in any one test.
3. When an electrical device is used it should be inserted in the rectum of the rabbit 90 min. before the injection of the solution being examined and left in position throughout the test.
Glassware, Syringes and Needles
All glassware, syringes and needles must be thoroughly washed with water for injections and heated in a hot air oven at 250 OC for 30 min. or at 200 OC for 1 h.
Retaining Boxes
1. The retaining boxes for rabbits in which the temperature is being measured by an electrical device are made in such a way that the animals are retained only by loosely-fitting neck stocks; the rest of the body remains relatively free so that the rabbits may sit in a normal position.
2. The rabbits are not retrained by the use of straps or other similar methods that may harm the animal.
3. The animals must be put into boxes less than 1 h before the test and remain in them throughout the test.
Diluents
1. Treat all diluents and solutions for washing and rinsing of devices or parenteral injections assemblies in a manner that all assure that they are sterile and pyrogen-free.
2. Periodically perform control pyrogen tests on the representative portions of the diluents and solutions for washing or rinsing of the apparatus.
3. Where Sodium Chloride injection is specified as a diligent, use injection containing 0.9 % of Sodium Chloride.
Recording of Temperature
1. Use an accurate temperature-sensing device such as clinical thermometer or thermistor or other suitable probes that have been calibrated t assure an accuracy of ± 0.1 OC and have been tested to determine that a maximum reading is reached in less than 5 min.
2. Insert the thermometer or temperature-sensing probe into the rectum of the test rabbit to a depth of not less than 7.5 cm, and, after a period of time not less than that previously determined as sufficient, record the rabbit’s body temperature, do not use any rabbit having a temperature exceeding 39.8 OC.
3. Not more than 30 min. prior to the injection of the test determine the control temperature of each rabbit. This is the base for the determination of any temperature increase resulting from the injection of test solution.
4. In any one group of test rabbits, use only those rabbits whose control temperatures do not vary by more than 1 OC from each other.
As per IP
Preliminary Test (Sham Test)
1. If animals are used for the first time in a pyrogen test or have not been used during the two previous weeks, condition them one to three days before testing the substance being examined, by injection intravenously into the 10 mL per Kg of body weight of a pyrogen-free saline solution.
2. Record the temperature of the animals, beginning at least 90 min. before injection and continuing for 3 h after injection of the solution being examined. Any animal showing a temperature variation of 0.6 OC or more must not be used in the main test.
Main Test
Carry out the test using a group of three orbits.
Preparation of the Sample
1. Dissolve the substance being examined in, or dilute with, a pyrogen-free saline solution.
2. Warm the liquids being examined to approx. 38 OC before injection.
3. The amount of the sample to be injected varies according to the preparation being examined and is prescribed in the individual monograph.
4. The volume of injection is not less than 0.5 mL/Kg and not more than 10 mL/Kg of body weight.
Procedure
1. Record the temperature of each animal at intervals of not more than 30 min. beginning at least 90 min. before the injection of the solution being examined and continuing for 3 h after the injection.
2. Not more than 40 min. immediately preceding the injection of the test does, record the ‘initial temperature’ of each rabbit, which is the mean of two temperature readings recorded for the rabbit at an interval of 30 min. in the 40 min. period.
3. Rabbits showing a temperature variation greater than 0.2 OC between two successive readings in the determination of ‘initial temperature’ should not be used for the test.
4. Inject slowly the solution being examined into the marginal vein the ear of each rabbit over a period not exceeding 10 min., unless otherwise prescribed in the monograph.
5. Record the temperature of each animal at half-hourly intervals for 3 h after the injection
6. 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.
7. When this difference is negative, the result is counted as a zero response.
INTERPRETATION
1. If the sum of the responses of the group of three rabbits does not exceed 1.4 OC and if the response of any individual rabbit is less than 0.6 OC, the preparation being examined passes the test.
2. If the response of any rabbit exceeds 1.4 OC, continue the test using 5 other rabbits.
3. If not more than 3 of the 8 rabbits show individual responses of 0.6 OC or more, and if the sum of the responses of the group of 8 rabbits does not exceed 3.7 OC, the preparation being examined passes the test.
As per BP
Preliminary Test
1. One to three days before testing the product, inject intravenously into animals, selected as prescribed above but that have not been used during the 2 previous weeks. 10 mL/Kg of body weight of a pyrogen-free 0.9 % w/v solution of Sodium Chloride warmed to about 38.5 OC .
2. Record the temperature of the animals, beginning at least 90 min. before injection and continuing for 3 h after injection of the solution being examined.
3. Any animal showing a temperature variation greater than 0.6 OC, must not be used in the main test.
Main Test
Carry out the test using a group of three rabbits.
Preparation and Injection of the Sample
1. The preparation being examined may be dissolved in, or diluted with, a pyrogen-free 0.9 % w/v solution prescribed in the monograph.
2. Warm the liquid being examined to approx. 38.5 OC, before the injection.
3. Inject the solution slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 min. unless other wise prescribed in the monograph.
4. The amount of the sample to be injected varies according to the preparation being examined and is prescribed in the monograph.
5. The volume of the injections 0.5 to 10 mL/kg of body weight.
Determination of the Initial and Maximum Temperature
1. The ‘initial temperature’ of each rabbit is that mean of two temperature reading recording for that rabbit at an interval of 30 min. in the 40 min. immediately preceding the injection of the material being examined.
2. The ‘maximum temperature’ of each of rabbit is the highest temperature recorded for that rabbit in the 3 h. after the injection.
3. Record the temperature of each animal at intervals of not more than 30 min. beginning at least 90 min. before the injection of the solution being examined and continuing for 3 h after the injection.
4. The difference between the initial temperature and the maximum temperature of each rabbit is taken to be its response.
5. When this difference is negative, the result is counted as a zero response.
6. Rabbits showing a temperature difference greater than 0.2 OC, between any two successive reading taken during the 90 min. before the injection are withdrawn from the test.
7. In any one test, only rabbits having initial temperature that do not differ from one another be more than 1 OC may be used.
8. All rabbits having an initial temperature higher than 39.8 OC pr ;lower than 38.0 OC are excluded from the test.
INTERPRETATION
1. Having carried out the test first on a group of three rabbits, repeat if necessary on further groups of three rabbits to a total of four groups, depending on the results obtained.
2. If the summed response of the first group does not exceed the figure given in second column of the following table the preparation being examined passes the test.
3. If the summed response exceeds the figure of the second column but does not exceed the figure in the third column of the table repeat the test as indicated above.
4. If the summed response is greater than the figure given in the third column of the table the preparation being examined fails the test.
As per USP Procedure
1. Unless otherwise specified in the individual monograph, inject to an ear vein of each of three rabbits 10 mL of the test solution per kg of body weight, completing each injection within 10 min. after start of administration.
2. The test solution is either the product, constituted if necessary as directed in the labeling or the material under test treated as directed in the individual monograph and injected in the dose specified therein.
3. For Pyrogen testing of devices or injection assemblies, use washing or rinsing of the surfaces that come in contact with the parenterally administered material or with the injection site or internal tissues of the patient.
4. Assure that all test solutions are protected from contamination.
5. Perform the injection after warming the test solution to a temperature of 37 ± 2 OC.
6. Record the temperature at 30 min. intervals between 1 and 3 h subsequent to the injection.
Test interpretation and continuation
1. Consider any temperature decreases as zero rise.
2. If no rabbit shows an individual rise in temperature of 0.5 OC or more above its respective control temperature, the product meets the requirements for the absence of the pyrogens.
3. If any rabbits shows individual temperature rise of 0.5 OC or more, continue the test using five other rabbits.
4. If not more than three of the eight rabbits show individual rises in temperature 0.5 OC or more and if the sum of the eight individual maximum temperature rises does not exceed 3.3 OC the material under examination meets the requirements for the absence of Pyrogens.
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