What is pyrogens? Sources of pyrogens and its elimination methods Tests for pyrogens- 1. In Vitro Test / LAL Test 2. In Vivo Test / Rabbit Test. Objective Principle Requirements Procedure Observation table Result and interpretation

1. Experiment No. 9
Test for pyrogens (rabbit method)
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5

2. Overview of Discussion
• What is pyrogens?
• Sourcesof pyrogens and its elimination methods
• Tests for pyrogens-
1. In Vitro Test / LAL Test
2. In Vivo Test / Rabbit Test.
• Objective
• Principle
• Requirements
• Procedure
• Observationtable
• Resultand interpretation
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3. What is pyrogens?
Pyrogen
In Greek Pyro = fire, gen = beginning.
A Pyrogen is a substance that is products of the growth of
microorganisms or may be parts of dead cells or metabolic
products which cause febrile reactions like fever, chills, back
pain etc.
It is also referred to as endotoxins.
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4. Sources of pyrogens and its eliminationmethods
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Sr. No. Source Eliminationmethod
1 Equipment Glass Heating to 250°C for 45 min/
650°C for 1 min/ washed with
dilute acid or dilute Alkali
Metal
2 solvent Volatile Distillation
Non-
volatile
Adsorption
3 Solute Re-crystallization

5. Tests for pyrogens
The Pyrogen test is designed to limit the risk of febrile reaction
following parenteral administration of drugs. It includes
both In vitro and In vivo tests.
1. In Vitro Test / LAL Test
2. In Vivo Test / Rabbit Test.
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6. In Vitro Test / LAL Test
LAL Test: Limulus Amoebocyte Lysate Test (In Vitro Test)
In Vitro assay used to detect the presence and concentration
of bacterial endotoxins in drugs and biological products.
Limulus Amoebocyte Lysate (LAL) is an aqueous extract of
blood cells (amoebocytes) from the horseshoe crab, Limulus
polyphemus.
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7. In Vitro Test / LAL Test
LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS),
which is a membrane component of Gram negative bacteria
and forms gel which is then used for the detection and
quantification of bacterial endotoxins.
Limitationsof LAL Test:
1. Disturbed by endotoxin binding components like lipids, blood
components etc.
2. Difficult to correlate with rabbit test.
3. False positive for cellulose and many herbal preparations.
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8. Objective
To perform the test for pyrogens (In-vivo/ SHAM test/ rabbit
method) to measure the rise in body temperature of rabbits
following IV injection of sterile solution of substance being
examined.
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9. Principle
Pyrogens are the fever inducing organic substances (metabolic
products of microorganisms) responsible for many febrile
reactions. Pyrogen testing should be done to every batch of
pharmaceutical product (particularly parenterals) for which
water is the usual vehicle. The best animal model for
pyrogen test is the rabbit as it generates reproducible
results that are similar to threshold response to humans and
also economic.
The test involves the measurement of rise in body temperature
of rabbits following IV injection of sterile solution of
substance being examined.
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10. Requirements
 Animals: Healthy matured rabbits of either sex. Select same
variety of healthy mature rabbits weighing less than 1.5Kg
and should maintain balanced diet. They should not show
any loss of body weight during the preceding week of test.
 Instruments used: Pyrogen free syringes, needles,
glassware. Accurate temperature sensing devices such as
Clinical Thermometer graduated in 0.1°C / Thermister / Probes
are used to measure the temperature of rabbit. Insert the
thermometer into the rectum of rabbit to a depth of not less
than 6 cm and record the temperature.
 Reagents: 0.9% sodium chloride injection as diluent.
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11. Procedure
 Select healthy matured rabbits of either sex for the study. House
them individually in the place that is free from disturbances that
likely to excite them and maintain the room temperature at 20-23°c.
Make all the materials and equipment pyrogen free either by
heating the same at 25°c for not less than 30 minutes or any other
method. Standardized clinical thermometer with precision of ±0.1°c
is used to measure rectal temperature of the rabbit. Test them to
determine that maximum reading is attaining in <5 minutes or not.
 Pyrogens testing of solutions should done in two steps-
a. Preliminary test (Sham test), and
b. Main test
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12. a. Preliminary test (Sham test)
Conduct Sham test using fresh rabbits used for first time in pyrogen
testing or not been used during the two previous weeks.
Acclimatize the rabbits for 1-3 days before using for pyrogen testing of
sample.
Select three rabbits and fast them overnight with free access to water
and withhold water during the test.
Record the temperature of animals 90 minutes prior to the injection.
After 90 min., inject sterile pyrogen free saline solution intravenously at
a dose of 10 ml per kg of bodyweight.
Record the temperature of animals at an interval of 30 minutes after
injection and continued for 3 hours.
Exclude any animal showing temperature variation of 0.6°C or more for
main test.
Note: It is carried out in room without disturbances and temperature
variance must be ± 3°C.
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13. b. Main test
Select three rabbits that passed the Sham test.
Determine the initial body temperature of the rabbits and it should be
between 38-39.8°c.
Dilute solution to test with pyrogen free saline solution or any solution
prescribed in monograph.
Warm the test liquid to 38.5°c before injection. Inject the test solution to
the animal slowly into the marginal vein of the ear for a period of
not more than 4 minutes and the volume injected should not less
than 0.5 ml/kg and not more than 10 ml/kg of body mass.
Determine the temperature of each animal at every half an hour for 3
hours after injection.
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14. Interpretation of the result
Consider any decrease in final body temperature as rise in zero.
Case I) Consider the test solution as pyrogen free if the response in
individual rabbit showing a temperature less than 0.6°c or if the sum
of responses of three rabbits does not exceed 1.4°c.
Case II) If the temperature exceeds than the above in case I, then
continue the test using other five rabbits. If not more than 3 rabbits
out of eight shows individual rise in body temperature of 0.6°c or if
the sum of the responses of the group of 8 rabbits does not
exceed3.7°c then the preparation under test is passed and considers
it as pyrogen free.
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15. Observation table
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Table 1: Recording of initial and final body temperatures
No. of
Rabbit
Initial
Body
Temp.
(°C)
Final Body Temp. (°C)
30
min.
60
min.
120
min.
180
min.
240
min
300
min.
Rabbit 1
Rabbit 2
Rabbit 3

16. Observation table
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Table 2: Result of pyrogen test
No. of Rabbit
Individual
body temp
rise
(°C)
Body
temp. rise
in group
(°C)
Test
Rabbit 1
(Pass/ Fail)
Rabbit 2
Rabbit 3

17. Result and interpretation
Difference in initial and final body temperature of rabbits
individually or in group determines the presence or absence
of pyrogens in a test substance.
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18. ANY QUESTION???
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