This document discusses pyrogen testing, which involves measuring the rise in body temperature of rabbits injected with a substance to test for fever-causing pyrogens. Pyrogens include any substance capable of causing a fever response and can be endogenous or exogenous. The test procedure involves using healthy adult rabbits, recording their baseline temperatures, injecting the test substance, and monitoring temperatures for 3 hours. The test substance passes if the summed temperature rise across rabbits does not exceed specified limits.

1. PYROGEN TESTING
SAURAV BHANDARI
ASSISTANT PROFESSOR
DEPT. OF QUALITY ASSURANCE
ISF COLLEGE OF PHARMACY
WEBSITE: - WWW.ISFCP.ORG
EMAIL: BHANDARISAURAV89@GMAIL.COM
ISF College of Pharmacy, Moga
Ghal Kalan, GT Road, Moga- 142001, Punjab, INDIA
Internal Quality Assurance Cell - (IQAC)

2. INTRODUCTION
Pyrogens include any substance capable of eliciting a febrile (or fever) response
upon injection or infection
ŠEndotoxin is a subset of pyrogens that are strictly of gram- negative bacterial
origin; they occur (virtually) nowhere else in nature.
Lipopolysaccharide (LPS)is a part of endotoxin, or, endotoxin is the natural
complex of LPS occurring in the outer layer of the bilayered gram-negative
bacterial cell
2

3. 3CLASSIFICATION OF PYROGENS

4. 4PYROGRN TEST

5. 5PYROGRN TEST
The test involves measurement of the rise in body temperature 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.

6. 6
Use healthy, adult rabbits of either sex, preferably of the same variety,
weighing not less than 1.5 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 temperature (± 2º),
preferably with uniform humidity, and free from disturbances likely to
excite them.
TEST ANIMALS

7. 7
 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 days before testing
the substance being examined, by injecting i.v into them 10 ml per kg of body
weight of a pyrogen free saline solution.
Carry out the test in a room where there is no risk of disturbance of exciting
the animals and in which the room temperature 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 the test.
PRELIMINARY TEST (SHAM TEST)

8. 8
With hold food from the animals overnight and until the test is completed;
withhold water during the test.
Record the temperature of the animals beginning at least 90 min. before
injection and continuing for 3 hrs after injection of the solution being
examined.
Any animal showing a temp. variation of 0.6° or more must not be used in
the main test.
PRELIMINARY TEST (SHAM TEST)

9. 9
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 examination to approximately 38.5º before injection.
MAIN TEST

10. 10PROCEDURE
Inject the solution(38.5º) 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 temperature of each animal at half-hourly intervals for 3 hours
after the injection.
The difference between the “initial temperature” and the “maximum
temperature” which is the highest temperature recorded for a rabbit is
taken to be its response.

11. 11
The amount of sample to be injected varies according to the preparation
under examination and is prescribed in the individual monograph. The
volume of injection is not less than 0.5 ml per kg and not more than 10 ml
per kg of body weight.
PROCEDURE

12. Healthy, adult rabbits of either
sex
Healthy, adult rabbits of either
sex
Healthy, mature rabbits of
either sex
Not less than 1.5 kg Not less than 1.5 kg Not less than 2-4 kg
± 2o
C within 3o
C Not more than ±3°
Do not use animals for pyrogen
tests more frequently than
once every 48 hours
Not used (a) during the preceding 3
days or (b) during the preceding 3
weeks unless the material being
examined passed the test.
Do not use a rabbit for
pyrogen testing more
frequently than once every 48
hours
0.6o
C or more mean rise exceed 1.2 0.6o
C or more
Hot air oven at 250o
C for 30
minutes or at 200o
C for 1 hour
Hot air oven at 250o
C for 30
minutes or at 200o
C for 1 hour
Hot air oven at 250o
C for not
less than 30 minutes
1 hour before the test 1 hour before the test Not mentioned
ANIMAL
BODY WT
CONDITION BEFORE
TEST
CONDITION FOR
ANIMAL TO USE
FAIL TEST
APPARATUS
PREPARATION
ANIMAL IN
INSTRUMENT

13. 13
•If the sum of the responses of the group of three rabbits does not exceed
1.4o
C and if the response of individual rabbit is less than 0.6o
C, the
preparation under examination passes the test
•If exceeds continue the test using five other rabbits
•If not more than three of the eight rabbits show individual responses of
0.6o
C or more, and if the sum of responses of the group of eight rabbits
does not exceed 3.7o
C, the preparation under examination passes the test

14. 14
•If no rabbit shows an individual rise in temperature of 0.5° or more
above its respective control temperature, the product meets the
requirements for the absence of pyrogens.
•If exceeds continue the test using five other rabbits.
•If not more than three of the eight rabbits show individual rises in
temperature of 0.5° or more and if the sum of the eight individual
maximum temperature rises does not exceed 3.3°, the material under
examination meets the requirements for the absence of pyrogens.

15. 15
Number of Rabbits Material passes if
summed response
doesn’t exceed
Material fails if
summed response
exceeds
3 1.15 2.65
6 2.80 4.3
9 4.45 5.95
12 6.60 6.60