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 37oC.(sample
does not have endotoxins or toxic)
Rabbit temperature out off 37oC.(sample have
endotoxins or toxic)
37oC =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.
