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To test sterile preparations for the presence of pyrogens.

1. Three rabbits of similar variety of any sex, weighing not less than 1.5 kilogram; housed individually in an area of uniform temperature and humidity.
2. Glassware, syringe and needles washed with water for injection and sterilized in hot air oven at 250*c for 30 minutes.
3. Retaining boxes to rest the animal well fitted with loosely fitting neck locks. Rest of the bodies of the animals must be freely rested.
4. Clinical thermometer with an accuracy of 0.1 *c. It should show the reading in less than 5 minutes.

Preliminary Test (Sham Test):

? It is done to select the animal groups for the main test.
? Animals used for the first time or those not used during the two previous weeks should be conditioned 1-3 days before testing by injecting IV- 10 ml/kg of pyrogen free saline solution warmed to 38.5 *c.
? Temperatures are recorded 90 minutes before injection and upto 3 hours after injection at regular intervals.
? Any animal showing variation of 0.6 *c or more than 0.6 *c shouldn't be used for main test.

Main Test:

? A batch of three rabbits that passed in the sham test is taken.
? The test sample may be dissolved or diluted with pyrogen free saline solution or solutions prepared in official monographs; warm the liquid to 38.5 *c before injection.

? Temperatures are noted from 90 minutes before injection to 3 hours after injection and at intervals of not more than 30 minutes.
? Initial temperature is noted for each rabbit. This temperature is the mean of two successive readings taken at an interval of 30 minutes in the 40 minutes preceding the injection.
? Rabbits which show a difference of 0.2 *c or more are not taken into the test procedure.
? Only those rabbits must be used in the test whose initial temperatures do not vary by more than one degree from each other. So there should be uniformity in the initial temperature among the animals tested.
? Further, neither those rabbits whose initial temperature is more than 39.8 *c nor those whose initial temperature is less than 38 *c may be used in the test. So, necessarily the initial temperature of the test rabbits must be in the range of 38 *c and 39.8 *c.
? Now, the test solution is injected into the marginal vein of ear within a period of 4 minutes, amount injected depending upon the preparation. The amount is prescribed in the individual monographs and the volume limits are 0.5- 10 ml/ kg.
? Temperatures are recorded for every 30 minutes for 3 hours; total 10 readings are taken. The maximum temperature is recorded as response; the difference between the initial temperature and maximum temperature is calculated, if the result is negative it is said to be zero response.

Interpretation of results:

1. If sum of responses from three rabbits is less than 0.6 *c then the sample is said to have passed.
2. Sum of responses is more than 1.4 *c or response of anyone is 0.6 *c or more than 0.6 *c then continue the test, using five other rabbits.
3. If not more than 3 out of 8 rabbits show individual response of 0.6 *c or more than 0.6 *c and sum of responses of eight rabbits is not more than 3.7 *c then the sample is said to have passed the test for apyrogenesity.

Reference: Indian Pharmacopoeia 1996, Test for pyrogens (2.6).

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kranthi kumar's picture
Author: kranthi kumar


Santosh kumar. JH's picture

Excellent write up easily understandable at a single sight. Thank You!
kranthi kumar's picture

I feel glade upon your comment.
Prof. J. Vijaya Ratna's picture

Kranthi You have neatly explained the rabbits test for pyrogens. Can you put some images to supplement your write up? Shall I tell you a funny experience we had in our final year? When our batch of final years students were doing the pyrogens test under the excellent guidance of our Sir, Prof. Satyanarayana; There was no clinical thermometer in the lab and so our SIR went to some place and borrowed a thermometer. It is to be placed in the rectum of the rabbit to record the temperature. He started the work, told us how to record the temperature and went into his room to look into some work. We started finding the temperature, one after another. We were about 12 students in the batch. As each one was recording, and putting the thermometer in its rectum, the rabbit must have felt some irritation; it resisted, and the thermometer broke when it was in the rabbit. With great difficulty we removed it out so that the rabbit was fine. But then, the experiment got stuck, there was no thermometer, we broke someone else's thermometer. and at that time, SIR came out. He found what happened, was very angry and asked us " Who broke this thermometer?" We were quiet, no reply! He questioned us for nearly 30 minutes but we kept mum. He warned us that he would put the charge on all of us but we still kept mum. He then laughed and let us off and we repeated the experiment in the next class. SIR was careful about not leaving us with the rabbit. One should be very careful when working with animals and many more times careful when working with human beings. Vijaya Ratna
kranthi kumar's picture

Dear Madam, Thank you for sharing your experience as a student. Its funny. Yes, one should be very much careful while handling with the animals. Surely i will try to find a picture related to it.
P.V.ABHIGNA's picture

Dear Kranthi, Nicely written in very simple mannar.


kranthi kumar's picture

Dear Abhigna, Thank you for the comment. I am glad that my blog can be understood by anyone.

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