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Pyrogen Test

This document discusses pyrogen testing which involves measuring the temperature rise in rabbits after injection of a substance to test for fever-causing contaminants. It classifies pyrogens as substances that cause fever and notes endotoxins are gram-negative bacterial pyrogens. The test uses healthy rabbits injected intravenously, monitors their temperatures, and defines criteria for a positive test result based on temperature increases. The document provides details on test animals, procedures, interpretation of results, and references the Indian Pharmacopoeia for standards.

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Srijon
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218 views

Pyrogen Test

This document discusses pyrogen testing which involves measuring the temperature rise in rabbits after injection of a substance to test for fever-causing contaminants. It classifies pyrogens as substances that cause fever and notes endotoxins are gram-negative bacterial pyrogens. The test uses healthy rabbits injected intravenously, monitors their temperatures, and defines criteria for a positive test result based on temperature increases. The document provides details on test animals, procedures, interpretation of results, and references the Indian Pharmacopoeia for standards.

Uploaded by

Srijon
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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PYROGEN TESTING

Contents

Introduction
Classification of pyrogen
Pyrogen test
Test animals
Preliminary test (sham test)
Main test
Procedure
Conclusion
References
INTRODUCTION

 Pyrogen include any substance capable of elicting a febrile


(rrr fever) response upon injection or infection.

 Endotoxin is a subset of pyrogen that are strictly of gram-


negetive bacterial origin; they occur (virtually) nowhere
else in nature .

 Lipopolysaccharide (LPS) is a part of endotoxin, or,


endotoxin is the natural complex of LPS occuring in the
outer layer of the bilayered gram-negetive bacterial cell.
CLASSIFICATION OF PYROGEN
PYROGEN TEST

 The test involvs measurement of the rise in body


temperature rabbit of rabbits following the intravenous
injection of a sterile solution of the substance under
examination .It is designed for products that can be
tolerated by the test rabbit in a dose not exceeding 10 ml
per kg injected intravenously within a period of not more
than 10 minutes.
TEST ANIMALS

 Use healthy, adult rabits of either sex, preferably of the


same variety, weighing not less than kg, fed on a complete
and balanced diet and not showing loss of body weight
during the week preceding the test .House the animals
individually in an area of uniform temparature
(±2◦),preferably with uniform humidity, & free form
disturbnces likely to excite them.
PRELIMINARY TEST (SHAM TEST)

 If animals are used for first time in pyrogen test or have not
been used during the two previous weeks, condition them
one to three day before testing the substance being
examined ,by injection i.v into them 10 ml per kg body
weight of a pyrogen free saline solution.

 Carry out the test in a room where there is no risk of


disturbance of exciting animals and in which the room
temparature is within 3° of that of the area where the
animals are housed or in which the animals have been kept
for at least 18 hrs before test.
PRELIMINARY TEST (SHAM TEST)

 With hold food from the animals overnight & until the test
is completed;Withhold water during test.

 Record the temparature of the animals beginning at least


90 min. before injection & continuning for 3hrs after
injection of the solution being examined.

 Any animals showing a temp. variation of 0.6 or more must


not e used in the main test.
MAIN TEST

 Carry out the test using a group of three rabbits.

 Preparation of the sample


Dissolve the substance under examination in, or dilute
with, pyrogen-free saline solution or other solution
prescribed in the monograph.Warm the liquid under
examinationto approximately 38.5° before injection.
PROCEDURE

Inject the solution(38.5°) under examination slowly into the


marginal vein of the ear of each rabbit over a period not
exceeding 4 minutes, unless otherwise prescribed in the
monograph.

Record the temparature of each animal at half-hourly


inteervals for 3 hours after the injection.

The temparature between the “initial temparature” & the


“maximum temparature” which is the highest tempaature
recorded for a rabbit is taken to be its response.
PROCEDURE

 The amount of sample to be injected varies according to the


preparation under examination & is prescribed in the
individual monograph.The volume of injection is not less
than 0.5 ml per kg & not more than 10 ml per kg body
weight.
AS PER I.P
Healthy,adult rabbits of either sex.
ANIMAL
Not less than 1.5 kg
BODY WT

CONDITION ±2◦
BEFORE TEST
Do not use animals for pyrogen tests more
CONDITION FOR frequently than once every 48 hours
ANIMAL TO USE

FAIL TEST 0.6°c or more


APPARATUS Hot air oven at 250°c for 30 minutes or at
PREPARATION 200°c for 1 hour
ANIMAL IN
INSTRUMENT 1 hour before the test
Conclusion

 Interpretation of result:
• Sum of response of group of 3 rabbits does not exceed
1.4 degree & if the response of any individual rabbit is
less than 0.6°c.The preparation under examination
passes the test.
• If the response of any rabbit is 0.6 or more or the sum of
response of three rabbits exceed 1.4, continue the test
using five other rabbits
• If not, more than three of eight rabbit show individual
response of 0.6 or more & if sum of response of group of
eight rabbit does not exceed 3.7, the preparation under
test passes the test.
References

INDIAN PHARMACOPOEIA 2010, Published by-


Indian pharmacopoeia commission, Volume 1, 6th
edition(effective from 1st sept, 2010), Page number-
36-37
Thank you….

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