This document provides an overview of pyrogen testing. It defines pyrogens as fever-producing agents that are metabolic byproducts of microorganisms. There are two main types of pyrogens - endogenous pyrogens produced within the body and exogenous pyrogens derived from outside the body, such as bacterial lipopolysaccharides. The key pyrogen tests discussed are the rabbit test, Limulus amebocyte lysate (LAL) test, and in vitro pyrogen test. The rabbit test involves measuring fever responses in rabbits after injection, while the LAL test detects gram-negative bacterial endotoxins and the in vitro pyrogen test uses human blood cells. The document compares the advantages and limitations of

1. PYROGEN TEST
PRESENTED BY: PARTH KHANDHERIA
M.PHARM SEM-2
DEPARTMENT OF PHARMACOLOGY
L.M. COLLEGE OF PHARMACY
AHMEDABAD.

2. CONTENTS
 DEFINITION
 TYPES
 STRUCTURE
 SOURCES
 PYROGEN TEST

3. PYROGENS
 A Pyrogen is defined as “a fever producing agent”
 Metabolic products of Microorganisms.
 They are
Soluble
Filterable
Thermostable
Non Volatile
 Having nature
Endogenous (inside body)
Exogenous (outside body)

4. Exogenous pyrogens
 They are foreign substance that are derived outside the host.
 Lipopolysaccharides and other substances produced by
pathogenic microorganisms.
 These substance becomes pyrogens when they are
administered parenterally to the host.
They can be subdivided into the two group;
Microbial pyrogens
Nonmicrobial pyrogens

6. Endogenous Pyrogens
 Produced by the immune cells that are activated by the
presence of infectious agents (e.g. bacteria, viruses )
 Endogenous pyrogens are usually cytokines, such
as interleukin-6, interleukin-1, tumour necrosis
factor, interferon-alpha, gp130 Receptor Ligands, and so on

7. How Endogenous Pyrogens cause FEVER

8. Bacterial endotoxin Pyrogen
 A toxin present inside a bacterial cell that is released when it
disintegrates.
 It is a high molecular weight complex which constitute an
integral components of the outer cell membrane of gram-
negative bacteria.
 It can exist in a cell-associated state or in a free state.
 Cell associated can be removed from a solution by micro
porous filters.
 But a free state easily passes through sterilizing filters, heat
stable.

9. Structure of endotoxin

10.  The basic structure of endotoxin (LPS) is that of a
Oligosaccharide covalently bound to a lipid component,
called lipid A.
 Oligosaccharide is composed of two parts;
 Core Oligosaccharide
 O-antigen
 The lipid A is the least variable components of Endotoxin, it
consist of a disaccharide of glucosamine which is highly
substituted with amide linked and ester linked long chain
fatty acids.
 Lipid A exerts its toxic effects when released from
multiplying cells or when bacteria are lysed as a result of
autolysis, ingestion and killing by phagocytes or certain
types of antibiotics.

11. Sources of Pyrogen
 Solvents, drugs, additives apparatus used in manufacture,
containers may be sources of pyrogens.
 The method of storage in between preparation and
sterilization also may cause the development of pyrogens.
 Hence every item must be apyrogenic and method of storage
must not allow any bacterial growth

12. Test for pyrogens
Rabbit test LAL Test
In Vitro
Pyrogen
Test(IPT)
Leucocyte
count
By measuring
Electrical
resistance
Pyrogen testing defines a process used by drug
manufacturers to determine if bacterial toxins are
present in vaccines and drugs that might cause fever
when used on humans.

13. Rabbit test
Qualitative fever response test
 The test consists of measuring the rise in body
temperature in healthy rabbits after the intravenous
injection of a sterile test solution.

14. Why the Rabbit?
 Other species not predictable
 Rabbit chosen for economic purposes
 Similar threshold pyrogenic response to humans
 Reproducible pyrogenic response

15. Requirements for the Rabbit
Pyrogen Test
 Rabbits must be healthy and mature
 New Zealand or Belgian Whites used mostly
 Either sex can be used
 Must be individually housed between 20 and 23°C
 Not varies more than ± 3º c.
 Free from disturbances likely to excite them.
 equipment and material used in test (glassware, syringes,
needles etc) must be free from Pyrogens by heating at 250º c
for not less then 30 minutes or any other method
 retaining boxes (comfortable for rabbits as possible)

16.  Rabbits selected for testing on any given test day must not
have been used for testing during two weeks.
 General health status should be evaluted.
Preinjection Procedure
May not vary
More than 3◦C.
To determine the correct dosage of test.

17. Procedure
Thermomistor probes or
clinical thermometers are
inserted rectally in the rabbits
If the probes remain throghout the
Test period the rabbits must be
Restrained with light fitting neck stocks.

18. A controlled temperature is determined
not more than 30 min prior to injection of
the test dose.
Inject the solution being examined
slowly into the marginal vein of the
ear of each rabbit.
Volume of injection is not less than 0.5
ml per kg and not more than 10 ml per
kg of body weight.

19. After injection rabbits
temperature are recorded at
30 min intervals between 1 &
3 hour
If rabbit shows an individual
temperature rise of 0.5◦C or
more the test is continued with
an additional 5 rabbits.

20. Interpretation of pyrogen test
<0.6◦C
Preparation being
examined passes
the test.
>0.5◦C
Continue with

21. 8
< Not >3.7◦C
Show
individual
>0.6 ◦C
Preparation passes the
test

22. Limitation of Rabbit test
 sensitivity variation
 For many drugs the volume tested is significantly smaller.
 The rabbit test gives only a pass/fail result and is not suitable
for the control of endotoxin limit.
 It is not a quantitative test and less-well standardized.
 A gap between the observed pyrogenicity in rabbits and the
expected pyrogenicity in humans.
 It is not suitable for all product categories.
 It is expensive.
 It involves the use of animals.
 Radiopharmaceuticals cannot be detected in rabbit tests.

23. LAL Test
Limulus amebocyte lysate test.
 To measure the concentration of
endotoxins of gram-negative bacterial
origin
Reagent: amoebocyte lysate from horseshoe
crab, Limulus polyphemus

24. Principle
 The addition of solution containing endotoxin to a solution
of lysate produce turbidity.
 The rate of reaction depends upon concentration of
endotoxin , the pH and the temperature.
 The endotoxin reference standard is the freeze dried.
 The test is also based on the primitive blood-clotting
mechanism of the horseshoe crab.

25. LAL Test
• There are mainly Six methods used;
Method A. Gel-clot method
Method B. Gel-clot method: semi-quantitative test
Method C. Turbidimetric kinetic method
Method D. Chromogenic kinetic method
Method E. Chromogenic end-point method
Method F. Turbidimetric end-point method

26. Gel clot method
• In presence of endotoxin a proenzyme in amoebocyte lyasate
is converted into an active form.
• The active enzyme then cleaves a clotting protein also found
within the Limulus Amoebocyte.
Proenzyme Active Enzyme
Endotoxin
Clotting Protein Clot
Active

27.  After incubation period reaction tubes are inverted.
 If clot remains intact after inversion, the test is positive.

28. Turbidimetric technique
 This technique is a photometric test to measure the increase
in turbidity .
 Based on the test principle employed this technique is
classified as ;
 The end point-turbidimetric test (method-F)
 The kinetic-turbidimetric test (method-C)

29. • It is based on development of turbidity after cleavage of an
endogenous substrate.

30. Method C :
The kinetic-turbidimetric test
• The kinetic-turbidimetric test is a method to measure either
the time needed for the reaction mixture to reach a
predetermined absorbance or the rate of turbidity
development.

31. Method F:
The end point-turbidimetric test
• The end-point turbidimetric test is based on the quantitative
relationship between the endotoxin concentration and
the turbidity of the reaction mixture at the end of an
incubation period.

32. Chromogenic technique
• This is technique is used to measure the chromophore released
from a suitable chromogenic peptide by the reaction of
endotoxins with the lysate.
• Depending on the test principle employed this technique is
classified as;
• The end-point chromogenic test (method E)
• The kinetc chromogenic test (method D)

33. • It is based upon the development of colour after cleavage of
a synthetic peptide chromogen complex.
• LAL reagent is formulated with a synthetic substrate gives
yellow colour when acted upon by endotoxin activated
enzyme.
• After cleavage of a synthetic peptide chromogen

34. Method E:
The end-point chromogenic test
• The end-point chromogenic test is based on the
quantitative relationship between the endotoxin
concentration and the quantity of chromophore released
at the end of an incubation period.

35. Method D:
The kinetic chromogenic test
• The kinetic chromogenic test measure either the time
needed for the reaction mixture to reach a predetermined
absorbance or the rate of colour development.

36. Applications of LAL test
1.Pharmaceuticals
 In parenteral dosage form
 Large volume parenteral
 Small volume parentral
2. Biological
 In blood product and plasma fraction
 Vaccine
 Injectables, biological products, surgical devices or medical
devices and renal dialysis fluids can be tested for absence of
pyrogens by LAL test.
 (1,3)-β-D-glucan, a material found in fungal cell walls, plant
tissue can be detected by LAL test.

37. 3. Diagnosis
 Disease caused by gram (-)ve bacteria
 urinary tract infections and spinal meningitis.
4. Medical device
• British pharmacopoeia(2007)-Appendix XIV-C-A-331
• Indian pharmacopoeia(1996)-VOL:II-A-24
• Encyclopedia of PHARMACEUTICAL TECNOLOGY: edited
by James swarbrick –(2007)- vol -5 pg no:3056.
• nebulizer used in respiratory therapy
5.The LAL test has also been used to assess food spoilage, air
and water quality.
6. For validation of dry heat sterilization

38. 7.LAL test is also assay of choice for researchers studying both the
clinical and environmental effects of endotoxin.

39. ADVANTAGES OF LAL TEST
 Less variable
 In vitro test
 Easier to perform
 More sensitive
 Less Expensive
 Less Time consuming
 Can give Quantitative result

40. LIMITATION OF LAL TEST
 Specific for gram (-)ve pyrogen only
 Clotting enzyme is heat labile, pH sensitive
 Possible interference problem

41. INVITRO PYROGEN TEST (IPT)
Test Technology
• The IPT is an ELISA assay that utilizes fresh or cryopreserved
human whole blood.
• Immune cells wlthin the blood will recognize exogenous
pyrogen and in response release fever-inducing signal
molecules that can then be measured.

42. Alternative of Rabbit Pyrogen Test
Limitatians of the rabbit pyrogen test include
 Sensitivity variations
 It is also not possible to test certain drugs such as cytokines,
antibiotics, certain sedatives/analgesics, plasma proteins and
radiopharma- ceuticals.
 The rabbit pyrogen test does not always identify human
pyrogens.

43. IPT ASSAY STEPS
1.Prepare Blood
Draw fresh blood in heparinized tubes. Blood must
be used within four (4) hours of collection.
2. Prepare Reagents
Prepare Standards, Positive & negative
controls and samples following package
inserts and Certificates of Analysis.
3. Toad samples, Standards and Controls
Using the tissue culture microplate, add 0.2 mL NaCl
to wells f‹›r standards, negative control
and 0.18 mL NaCI to wells for positive sample controls. Next
add 0.02 mL of samples and controls to designated wells.

44. 4. Load Blood and Incubate Sample
Add 0.02 ml of blood to each well. Mix tl›e welJs thoroughly
using a plate mixer and incubate overnight at 37°C. The next
day, remove from incubator and mix the plate until cells are
resuspended. Allow cells to settle for approximately 45
minutes.
5. Transfer to Coated plate
Using the antibody-coated microplate, add 0.1 mL of conjugate
to each well. Add 0.1 mL of supernatant to antibody plate.
Incubate the plate on a plate mixer at room temperature for 90
minutes.
6. Add Substrate
Wash the plate 4-5 times with wash solution until clear, tapping
plate between washes to remove excess wash solution. Add 0.2
mL of TMB substrate to each well.
Incubate at room temperature in the dark for 30 minutes. Add 0.1
mL of STOP solution to all wells. Read plate at 450 nm within 15
minutes.

45. Samples are considered pyrogenic if OD readings of Sample are
>
OD reading of 0.5 EU/mL Standard.

47. By leucocyte count
 Injection of pyrogens causes changes in the white cells
picture.
 IF Pyrogens present.
 Fall in small lymphocytes
 Rise in young neutruphills.

48. By measuring electrical resistance
Pure water has very high resistance.
If Pyrogens are present they will carry minerals and decrease
in resistance occurs . Resistance measured by conductivity
meter.

49. REFERENCES
 Indian pharmacopoeia 2007, Government of India: Ministry
of health & Family welfare, Vol-1, page no:34
 Williams Kevin L., Endotoxins Pyrogens, LAL testing and
Depyrogenation, Volume 167, 3rd edition.
 Sushruta Mulay et al, AN OVERVIEW OF LIMULUS
AMOEBOCYTE LYSATE (LAL) TEST, International
Research Journal Of Pharmacy 2011; 67-71.
 U.S.PHARMACOPEIA
 Pharmaceutical Formulations by M.E.Aulton, H.C.
Ansel.page no 195-196.