This document summarizes information about pyrogens and pyrogenicity. It defines pyrogens as agents that cause an increase in body temperature when injected. The most common pyrogens in pharmaceuticals are bacterial endotoxins from gram-negative bacteria. There are two classes of pyrogens - endogenous pyrogens produced within the body and exogenous pyrogens from external sources. Common sources of pyrogens in pharmaceutical products include water, materials, and equipment. The rabbit pyrogen test involves injecting a substance into rabbits and measuring temperature changes, while the LAL test detects endotoxins using lysate from horseshoe crab blood.

1. SEMINAR ON
PHARMACEUTICS-IX
TOPIC: Pyrogen & pyrogenicity
PRESENTED BY:
Udit borah
B.Pharm 4th year
DEPARTMENT OF PHARMACEUTICAL SCIENCES
DIBRUGARH UNIVERSITY
Assam

2. Introduction
• Pyrogens are the agents which are responsible for increase in
body temperature when injected into the body.
• Most common pyrogens which are of significant in
pharmaceutical industry are Gram negative bacterial endotoxin.
• Endotoxin are part of the outer membrane of the cell wall of
gram negative bacteria.
• These bacteria constantly shed endotoxin into environment as
they grow & multiply ,as well as when they die & disintegrate.
• Bact. Endotoxin can either exist in a cell-associated state or in a
free state.

3. Classification
Pyrogens have been divided into two classes:
• Endogenous pyrogen is a low-molecular-weight protein that is
produced by phagocytic leukocytes in response to stimulation
by exogenous pyrogens and released into the circulation.It
induces fever by acting on the preoptic area of the
hypothalamus to raise the set-point of the hypothalamic
thermostat..
• Exogenous pyrogen is a fever-producing agents of external
origin, e.g., bacterial endotoxins and other microbial products,
antigen-antibody complexes, viruses and synthetic
polynucleotides, incompatible blood and blood products, and
androgen breakdown products such as etiocholanolone.

4. Source of pyrogen
The sources of pyrogens in parenteral products include;
• water used at the end stages of the purification.
• crystallization of the drug or excipients : water used during
processing.
• packaging components; chemicals, raw materials or
equipment used in the preparation of the product.
• The presence of endotoxins on devices may be attributed to
water in the manufacturing process.
• washing of components such as filter media (filters).
• Washing/rinsing of tubing or other plastic devices prior to
their sterilization.

5. Physiological effects of pyrogens
Pyrogens elevate the circulating levels of inflammatory cytokines .They
produces fever, blood coagulation ,hypotension, lymphopenia,
neutrophilia, elevated levels of plasma, acute phase proteins
Low doses of pyrogens induce asymptomatic inflammatory reactions and
moderate doses induce fever and changes in plasma composition
High pyrogenic doses results in :
• Shock
• characterized by cardiovascular dysfunction
• Vasodilation
• vasoconstriction
• endothelium dysfunction
• multiple organ dysfunction or failure

6. Pyrogenicity
This is the ability to cause a change in body temperature. In humans
endotoxins cause an increase in temperature (i.e. fever response).The
pyrogenic effect is indirect: the active chemical agent that causes the
temperature change is an endogenous pyrogen that is released from the
blood leukocytes under the influence of the endotoxins. The pyrogen affects
the hypothalamus of the brain, which regulates body temperature.
Rabbits are used to perform this test because their body temperature
increases when pyrogen are introduced into their bodies by parenteral route.
Three healthy adult rabbits of either sex, each weighing not less than 1.5 kg
should be selected. Rabbit having a temperature higher than 39.8o c and
showing temperature variation >0.2o c between two successive reading in the
determination of initial temperature should not be slected.
Pyrogen test :(Rabbit test)

7. Method of Rabbit test
• Dissolve the substances being examined in, or dilute it with a pyrogen
free saline solution. Warm the liquid being examined to approx. 38 o c
temperature before injection. The volume of injection is not less than
0.5ml/kg & not more than 10ml/kg of body weight. Withhold water
during test. Clinical thermometer is inserted into the rectum of rabbit to
record body temperature. 2 normal reading of rectal temperature
should be taken prior to the test injection at an interval of half an hour
and its mean is calculated- initial temperature.
• The solution under test is injected through an ear vein. Record the
temperature of each rabbit in an interval of 30 min for 3 hours . The
difference between initial temperature and maximum temperature is
recorded- taken as response.

8. Interpretation of results
• If the sum of response of the group of 3 rabbits does not exceed 1.4 o c and
if the response of individual rabbit is less than 0.6 o c, the preparation being
examined passes the test.
• If the response of any rabbit is 0.6o c or more / if the sum of response of 3
rabbits exceed 1.4 o c, continue the test using 5 other rabbits.
• If not more than 3 rabbits show individual response of 0.6 o c or more, and if
the sum of response of 8 rabbits does not exceed 3.7o c, the preparation
being examined passes the test.
Advantages Disadvantages
can be replicated and demonstrate the
production of fever in humans
Time consuming
Detects all kinds of injectable Pyrogen
unlike LAL test.
Expensive Procedure
It is pass/fail test than assay
cannot be used to test certain drugs
that depresses the fever

9. Bacterial Endotoxin Test
Limulus Amoebocyte Lysate Test (LAL Test) : It depends on gelling
property of lysate of amoebocyte of limulus (horseshoe crab). LAL reacts
with the bacterial endotoxin or lipopolysaccharides which is a membrane
component of gram negative bacteria and forms the basis of LAL test
which is used for detection and quantification of bacterial endotoxin.
The lysate is mixed with an equal volume of test solution in a
depyrogenated container. The tube is then incubated at 37 o c for 60 min.
The positive reaction is indicated by formation of gel/clot.
Now a days LAL Test is done according to three basic prescribed
method :
Gel Clot Method : It is the most commonly used method. The gel-clot
technique allows detection or quantification of endotoxins and is based
on clotting of the lysate in the presence of endotoxins .

10. Cont……..
Turbidometric Method : It is based on the fact that increase in endotoxin
concentration will cause increase in turbidity. The optical density is read
spectrophotometrically either at fixed time or constantly for kinetic assay
as turbidity develops.
Chromogenic Method : It utilizes colourimetric method. The colour
produced is proportional to the amount of endotoxin.
Application of LAL Test :
• Pharmaceuticals: Parenteral dosage form, Large volume Parenterals and
Small volume Parenterals
• Biologicals : In blood products & plasma fractions Vaccines
• Medical Device :Nebulizers used in Respiratory therapy
• Diagnosis of disease caused by Gram negative bacteria
• In food & drinking water
• Others: For validation of dry heat sterilization

11. Other Pyrogen Tests
Invitro Pyrogen Test (Ipt):
• This test exploits the reaction monocytes/macrophages detection of pyrogens
• Human whole blood taken from healthy volunteers is incubated in presence of test
sample, pyrogenic contamination initiate the release of “the endogenous pyrogen”
• Interlukin 1-β determined by ELISA after incubation.
(A)Removal of Pyrogens by physical Methods :
• Dilution
• Ultra-filtration
• Reverse osmosis
• Distillation
• Adsorption on Charcoal and Column Chromatography
(B) Inactivation of pyrogens :
• Dry heat sterilization
• Moist heat sterilization
• Use of dilute acids & Bases
• Oxidation and Alkylation
Depyrogenation

12. References
• Microbiology by J. Pelczar , JR. E.C.S. Chan and Noel R. Krieg, page no. 897 and
699.
• Miller-Keane Encyclopedia and Dictionary of Medicine, Nursing, and Allied
Health, Seventh Edition. © 2003 by Saunders, an imprint of Elsevier, Inc.
• Pharmaceutics II by R.M Mehta, page no.232 and 245.
• www.slideshare.net
ThankYou