1. PYROGEN & ENDOTOXIN TESTING
COMPARISON AS PER IP, BP & USP
PRESENTED BY- SAURAV BHANDARI
DEPT. OF QUALITY ASSURANCE

2. • Pyrogens include any substance capable of eliciting a febrile (or
fever) response upon injection or infection
• Š Endotoxin is a subset of pyrogens that are strictly of gramnegative 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

3. PYROGEN TESTING

4. • 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.
Test Animals
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.

5. 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 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.

6.  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.

7. 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
examination to approximately 38.5º before injection.

8. PROCEDURE
• 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.
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.

9. TEST ANIMALS
ANIMAL
Healthy, adult rabbits of
either sex
Healthy, adult rabbits of
either sex
Healthy, mature rabbits of
either sex
BODY WT
Not less than 1.5 kg
Not less than 1.5 kg
Not less than 2-4 kg
CONDITION BEFORE
TEST
± 2oC
within 3o C
Not more than ±3°
CONDITION
FOR ANIMAL
TO USE
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
0.6oC or more
mean rise exceed 1.2
0.6oC or more
FAIL TEST
APPARATUS
PREPARATION
Hot air oven at 250oC for Hot air oven at 250oC for Hot air oven at 250oC for
30 minutes or at 200oC for 30 minutes or at 200oC for not less than 30 minutes
1 hour
1 hour
ANIMAL IN
INSTRUMENT
1 hour before the test
1 hour before the test
Not mentioned

10. INTERPRETATION OF RESULTS
I.P
• 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 0.6oC 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

11. INTERPRETATION OF RESULTS
U.S.P
• 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.

12. INTERPRETATION OF RESULTS
B.P.
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

13. LAL (Limulus Amebocyte Lysate)
 LAL (Limulus Amebocyte Lysate) test is used for detecting
endotoxin
 based on clotting reaction of horseshoe crab lysate by
endotoxin
 The specimen is incubated with LAL of a known senstivity.
Formation of a gel clot is positive for endotoxin.
 Types of LAL test
 Gel Clot
 Turbidimetric
 Colorimetric
 Equipment used in test must be endotoxin free

14. The test is applied using a lysate derived from the hemolymph cells or
amoebocytes of the horseshoe crab, Limulus polyphemus. Other
species of horseshoe crab namely Tachypleus gigas also yield
amoebocyte lysate having similar activity.
• The following methods can be used to monitor the endotoxin
concentration in a product official in the Pharmacopoeia and to
determine whether the product complies with the limit specified
in the monograph.
• Method A. Gel-Clot Limit Test Method
• Method B. Semi-quantitative Gel-Clot Method
• Method C. Kinetic Turbidimetric Method
• Method D. Kinetic Chromogenic Method
• Method E. End-Point Chromogenic Method

15. • Before carrying out the test for endotoxins in the preparation
under examination it is necessary to verify
• (a) in the case of gel-clot methods, the sensitivity of the lysate;
• (b) in the case of quantitative methods, the linearity of the
standard curve;
• (c) the absence of interfering factors, which inhibit or enhance
the reaction or otherwise interfere with the test on the
preparation under examination.

16. Special Reagents
The Endotoxin Reference Standard (ERS) is the freeze-dried, purified
endotoxin of Escherichia coli, which is calibrated in Endotoxin Units
(EU) by comparison with the International Standard.
The Endotoxin Reference Standard (ERS) or any other suitable
preparation the activity of which has been determined in relation to
the ERS or the International Standard using a gel-clot or other
suitable method is maintained by the Central Drugs Laboratory,
Kolkata.

17. Sensitivity of the lysate
Confirm the labelled sensitivity of each new batch of lysate prior to
use in the test using at least one vial of each batch of lysate. Prepare
a series of dilutions of CSE to give concentrations of 2l, l, 0.5l and
0.25l, where l is the labelled sensitivity of the lysate in EU per ml.
Perform the test as given under Method on these four standard
concentrations in duplicate and include a negative control consisting
of water BET.

18. Test for interfering factors
There is possibility of interference with the bacterial endotoxins test by
certain factors.
Dilution of the test preparation with water BET is the easiest method for
overcoming inhibition.
The allowable dilution level or Maximum Valid Dilution (MVD) is dependent
on the concentration of the product, the endotoxin limit for the product
and the lysate sensitivity. It is calculated by the following expression:

19. Method A. Gel-Clot Limit Test Method
Preparation of test solutions
Unless otherwise prescribed, prepare the solutions and dilutions with
water BET. If necessary, adjust the pH of the solution under examination to
6.0 to 8.0 using sterile 0.1M hydrochloric acid BET , 0.1M sodium
hydroxide BET or a suitable buffer prepared with water BET .
Prepare the sample solution at any dilution at or below MVD. Use water
BET as negative control (NC) and two positive controls. One of the positive
controls consists of the CSE at a concentration of 2 λ and the other consists
of the test solution spiked with CSE to give a concentration of 2 λ (PPC).
Methods A and B depend on the formation of a firm gel when a solution
containing bacterial endotoxins is incubated after mixing with the lysate.
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20. Interpretation of results
The product under examination complies with the bacterial endotoxin test
if the positive product control is positive and the negative control as well
as the test solutions are negative.
The test is not valid if the positive product control is negative or if the
negative control is positive.
The product under examination meets the requirements of the test if the
endotoxin content is less than the endotoxin limit stated in the individual
monograph.
Retests
If a positive result is found for one of the test solution duplicates and a
negative result for the other, the test may be repeated as described above.
The results of the retest should be interpreted as for the initial test.
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21. The kinetic turbidimetric method is a photometric assay measuring the
increase in turbidity caused by the reaction of the endotoxin with the
lysate. The kinetic turbidimetric assay is a method measuring either the
time (onset time) needed to reach a predetermined absorbance of the
reaction mixture or the rate of turbidity development.
The kinetic chromogenic method is a photometric assay measuring the
colour developed by the chromophore released from a chromogenic
substrate by the reaction of the endotoxin with the lysate. The kinetic
chromogenic assay is a method measuring either the time (onset time)
needed to reach a predetermined absorbance of the reaction mixture or
the rate of colour development.
The end-point chromogenic method is a photometric assay measuring
the colour developed by the chromophore released from a chromogenic
substrate by the reaction of the endotoxin with the lysate. The end-point
assay is a method measuring the colour intensity at the end of an
incubation period after the reaction is stopped by the addition of a
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suitable acid.

22. LAL Assay
Gel clot
Colorimetric
Turbidometric
Semi-quantitative
Quantitative
Quantitative
Simple, Least expensive
Requires incubating plate
or tube reader
Requires incubating plate
or tube reader
Manually read and
recorded
Can be automated
Can be automated
Sensitive down
to 0.03 EU/ml
Sensitive down
to .005 EU/ml
Sensitive down
to .001 EU/ml *

23. Limitations –LAL assay
• Disturbed by endotoxin-binding components like
blood components
lipids
• difficult to correlate with rabbit test
• False-positive for cellulose and many herbal preparations
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