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Pyrogen and Its Determination Using Rabbits

Learn how to determine the pyrogens in parenteral preparations by injecting the sample in rabbits.

Introduction:

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 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 normal temperature greater than 49.8°C.

2.  It was used in positive test during last two weeks or negative test during last two days.

Method for Pyrogen Test:

The test is performed in an air conditioned room. The food and water is withheld to rabbit overnight. Clinical thermometer is inserted in the rectum of each rabbit to a depth of not less than 7.5 cm. Two readings of 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 equipments, injectors and needles used in the test should be pyrogen free. These should be washed with WFI and then heated at 260°C for two hours. The injection is warmed to 38°C before inject to the rabbits. 0.5 to 1.0 ml per kg dose should be injected through the ear vein. Six reading of temperature are recorded at an interval of half an hour.

Test Results:

The response of each rabbit is detected by 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 dose 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 test is done using 5 rabbits, than if 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.

Also see: Pyrogens Testing in Pharmaceuticals