One way of detecting pyrogens in a pharmaceutical preparation is to inject the material into animals. The USP Pyrogen Test uses a standardized protocol using 8 adult rabbits under controlled conditions.


Mechanism: cytokine release



(1) Select 8 healthy adult rabbits free of infection and raised under controlled conditions.

(2) The baseline temperature for the rabbits is recorded and the rabbits weighed. The temperatures for all 8 rabbits should agree within 1 degree of each other and not be above normal.

(3) All needles and syringes are pyrogen free.

(4) Solutions are kept at 37°C.

(5) The drug solution is injected into the ear vein of 3 initial rabbits at a dose of 10 mL per kg body weight.

(6) Temperatures of injected rabbits are recorded every 30 minutes for 3 hours.

(7) If any of the rabbits in the initial group show a temperature rise >= 0.5°C after the injection then the remaining 5 rabbits are injected.


change in temperature in °C for a rabbit =

= (maximum temperature in °C) - (baseline temperature in °C)


number of rabbits showing temperature rise above threshhold =

= SUM(number of rabbits with change in temperature >= 0.5°C)


cumulative temperature rise in °C =

= SUM(change in temperature for all rabbits tested)


Criteria for a negative pyrogen test - one of the following:

(1) none of the first 3 rabbits have a temperature rise >= 0.5°C

(2) no more than 3 rabbits out of 8 have a temperature rise >= 0.5°C AND the cumulative temperature increase in all 8 rabbits is <= 3.3°C



• The presence of an occult infection can cause a false positive test.

• A drug may cause a temperature rise by impacting metabolism, cytokine release or CNS temperature control centers, resulting in a false positive.

• A false negative test may occur if the concentration of pyrogens is very low.


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