The test involves 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.
The retaining boxes for rabbits in which the temperature is being measured by the electrical device should be made in such a way that the animals are retained only by loosely-fitting neckstocks and the rest of the body remains relatively free so that the rabbits may sit in a normal position. The animals must be put in the boxes 1 hour before the test and remain in them throughout the test. Ensure that the room temperature where the test is carried out is within 3° of that of the rabbits living quarters or in which the rabbits have been kept for 18 hours before the test. Withhold food from the animals overnight and until the test is completed; withhold water during the test.
Related: Bacterial Endotoxin Test Methods
Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined. Any animal showing a temperature variation of 0.6° or more must not be used in the main test.
Inject the solution under examination slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. The amount of sample to be injected varies according to the preparation under examination and is prescribed in the individual monograph. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Record the temperature of each animal 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.
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Test Animals
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 and not showing loss of body weight during the week preceding the test. House the animals individually in an area of uniform temperature (± 2°), preferably with uniform humidity, and free from disturbances likely to excite them. Do not use animals for pyrogen tests more frequently than once every 48 hours. After a pyrogen test in the course of which a rabbit's temperature has risen by 0.6° or more, or after a rabbit has been given a test substance that was adjudged pyrogenic, at least 2 weeks must be allowed to elapse before the animal is used again.Materials
All glassware, syringes and needles must be thoroughly washed with water for injections and heated in a hot air oven at 250° for 30 minutes or at 200° for 1 hour. Treat all diluents and solutions for washing and rinsing of devices in a manner that will assure that they are sterile and pyrogen-free.The retaining boxes for rabbits in which the temperature is being measured by the electrical device should be made in such a way that the animals are retained only by loosely-fitting neckstocks and the rest of the body remains relatively free so that the rabbits may sit in a normal position. The animals must be put in the boxes 1 hour before the test and remain in them throughout the test. Ensure that the room temperature where the test is carried out is within 3° of that of the rabbits living quarters or in which the rabbits have been kept for 18 hours before the test. Withhold food from the animals overnight and until the test is completed; withhold water during the test.
Recording of Temperature
Use an accurate temperature-sensing device such as a clinical thermometer or thermistor or other suitable probes that have been calibrated to assure an accuracy of 0.1° and have been tested to determine that a maximum reading is reached in less than 5 minutes. Insert the thermometer or temperature-sensing probe into the rectum of the test rabbit to a depth of about 5 cm. The depth of insertion is constant for anyone rabbit in any one test. If an electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before the injection of the solution being examined and left in position throughout the test. After a period of time not less than that previously determined as sufficient, record the rabbit's body temperature.Related: Bacterial Endotoxin Test Methods
Preliminary Test (Sham Test)
If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1 to 3 days before testing the substance under examination by injecting intravenously into them 10 ml per kg of body weight of a pyrogen-free saline solution warmed to about 38.5°.Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined. Any animal showing a temperature variation of 0.6° or more must not be used in the main test.
Main Test
Carry out the test using a group of three rabbits. Preparation of the sample. 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 38.5° before injection.Procedure
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. 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. Rabbits showing a temperature variation greater than 0.2° between two successive readings in the determination of "initial temperature" should not be used for the test. In anyone group of test animals, use only those animals whose "initial temperatures" do not vary by more than I° from each other, and do not use any rabbit having a temperature higher than 39.8° and lower than 38°.Inject the solution under examination slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. The amount of sample to be injected varies according to the preparation under examination and is prescribed in the individual monograph. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Record the temperature of each animal 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.
Interpretation of results
If the sum of the responses of the group of three rabbits does not exceed 1.4° and if the response of any individual rabbit is less than 0.6°, the preparation under examination passes the test. If the response of any rabbit is 0.6° or more, or if the sum of the response of the three rabbits exceeds 1.4°, continue the test using five other rabbits. If not more than three of the eight rabbits show individual responses of 0.6° or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7°, the preparation under examination passes the test.Related: BET: Bacterial Endotoxin (LAL) Test
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