Pyrogen Test and Its Determination Using Rabbits : Pharmaguideline

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Pyrogen Test and Its Determination Using Rabbits

Learn how to determine the pyrogen in parenteral preparations by injecting the sample in rabbits for pyrogen testing.


Pyrogen test is performed to check the presence or absence of pyrogens in all aqueous parenterals. Rabbits are used to perform the test because their body temperature increases when pyrogen is introduced by the parenteral route.

For this test, three healthy rabbits are selected each weighing at least 1.5 kg. No rabbit should be selected if:
1.  It has a normal temperature greater than 49.8°C.
Pyrogen in Parenterals2.  It was used in a positive test during last two weeks or negative test during last two days.

Method for Pyrogen Test:
The pyrogen testing is performed in an air-conditioned room. The food and water is withheld to rabbit overnight. A clinical thermometer is inserted in the rectum of each rabbit to a depth of not less than 7.5 cm. Two readings of the temperature of rabbit in normal conditions should be taken at the interval of half an hour before start the test and mean of the both should be calculated to determine the initial temperature.

The equipment, injectors and needles used in the test should be pyrogen-free. These should be washed with water for injection and then heated at 260°C for two hours. The injection is warmed to 38°C before injecting to the rabbits. 0.5 to 1.0 ml per kg dose should be injected through the ear vein. Six reading of temperature is recorded at an interval of half an hour.

Pyrogen Test Results:

The response of each rabbit is detected by the difference of initial temperature and the highest temperature recorded. The response of all three rabbits gives the sum of responses and can be concluded as:
i)  If the sum of responses does not greater than 1.4°C and any of rabbit shows the response less than 0.6° C, the product passes the test.
ii)  I sum of responses is greater than 1.4 °C or any of rabbit shows the response 0.6 or greater, continue the test using 5 rabbits.
iii)  If the test is done using 5 rabbits, then if the sum of responses of all 5 rabbits is greater than 3.7°C and the individual response of not more than three rabbits is greater than 0.6°C, the product passes the test.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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