Pyrogen Test (Rabbit test or Sham Test)

Ø  1912 : pyrogen test was done by Rabbit Test method.

Ø  USP XII –(Rabbit test ) approved method (1942 to 1980 )

Pyrogen :

Ø  A pyrogen is a foreign substance that causes a fever (temperature elevation) in an animal’s body. Typically, pyrogenic substances include endotoxin and other bacterial by products. Vaccines and other injectable drugs must be confirmed to be pyrogen free according to regulatory requirements of 21CFR, USP, and EP.

Pyrogen Testing :

1.       Rabbit test method (Sham test).

2.       LAL test  ( Bacterial Endotoxin Test).

Rabbit Test Method:

 3 rabbits is selected

 Endo toxin sample is inoculated (10mL/kg per body weight)

 3 days rabbit temperature is observed

 Rabbit temperature is with in 37^oC.(sample does not have endotoxins or toxic)

 Rabbit temperature out off 37^oC.(sample have endotoxins or toxic)

 37^oC =healthy human temperature.

 If <1.4⁰C increase the temperature  acceptable. (product is Pyrogen free).

 If >1.4⁰C increase the temperature  not acceptable. (product is Pyrogenic).

Pyrogen Test Procedure:

Ensure the test material all are pyrogen free.

 Check the pressure or absence of pyrogen in all aqueous parentenals.

 Rabbits are used to perform the test their body temperature increases when pyrogen is

    introduced by parentenals route.

Procedure:

 Three healthy rabbits are selected.

 Each weighing at least 1.5 kg

Note:

. Do not use any rabbit having a temperature higher than 39.8⁰C  and lower than 38⁰C.

. Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes.

Method:

 Test performed in air condition room.

 The food and water is withheld to rabbit overnight.

 Clinical thermometer is in 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.

 Record the “initial temperature” of each rabbit.

 Record the “initial temperature” of each rabbit.

  The equipments, injections and needles used in the test should be pyrogen free.

 The injection is warmed to 38⁰C 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 initial of half an hour.

Pyrogen Test Results:

 Each rabbit is detected by difference of initial temperature and the highest temperature

     recorded.

 Three rabbits are calculated.

 If the sum of responses does not greater than 1.4⁰C and any rabbit shows the response less than

    0.6⁰C, the product passes the test.

 If sum of responses is greater than  1.4⁰C or any of rabbit shows the response 0.6 or greater,

     continue the test using 5 rabbits.

 If test is done using 5 rabbits then if sum responses of all 5 rabbits is greater than 3.7⁰C and the

individual responses of not more than three rabbits is greater than 0.6⁰C the product passes

the test.