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7/25/2019 Pyrogen Test (QFRIR)
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PYROGEN TESTQFRIR AND LAL METHOD
PRESENTED BY:
GONZALES, GREPO, HERNANDEZBSP4D
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PYROGEN
• Pyrogen is defined as “fever producingagent”
• products of metabolism of microorganisms. Mostbacteria, viruses and many molds
• Pyrogen are substances that initiate febrile reactions whensufficient numbers enter the circulatory system, therefore thehistory of intravenous therapy and the history of pyrogenresearch are closely related
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PYROGEN
• “pyrogen” is derived (Greek pyro= fire andgen =beginning
• Pyrogen test: became official !uality control testfor parenterals in "#$% in the &' harmacopeia(&', "%th edition
• ))"#$*+ the code of federal regulation re!uired antibiotics tobe tested for pyrogens ))
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-/-01'
/o2ins that are releasede2tracellularly as theorganism grows. 2oto2insmay travel from a focus ofinfection to distant part of thebody and cause damage.
.g. 1euroto2in (botulinumto2in, tetanus to2in,nteroto2in (cholera to2in,3ytoto2in
14/-01'
are lipopolysaccharides
to2in produced by Gram1egative bacteria. /hename endoto2in isderived from the fact
that these to2ins aregenerally cell bound andreleased only when thecell lyses.
% 5road 3lasses of yrogens
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BASIC PROPERTIES AND DIFFERENCES OF
EXOTOXINS AND ENDOTOXINS
roperty 2oto2ins ndoto2ins
5iomolecule63hemistry roteins 7ipopolysaccharide87ipoproteincomple2,
7ocation of genes lasmid or bacteriophage 5acterial chromosome
'ource 2creted by certain gram positive orgram negative bacteria
3ell wall of Gram 1egative bacteria,released only after lysis of cells
9eat 'tability 4estroyed rapidly at :;o3 (e2ceptstaphylococcal enteroto2in
'table at ";;o3 for one hour
Mode of <ction('ymptoms
'pecifi c, either cytoto2in, enteroto2inor neuroto2in with defined action oncells or tissues
General, fever, diarrhea, vomiting
/o2icity 9ighly to2ic, often fatal (fatal dose onthe order of " g
>eakly to2ic, rarely fatal (fatal dose onthe order of hundreds of micrograms
0mmunogenicity 9ighly immunogenic, stimulate theproduction of neutrali?ing antibody(antito2ins
@elatively poor immunogenicity
/o2oid potential6Aaccines /reatment of to2in with formaldehydewill destroy to2icity, but treated to2insremain immunogenic. /o2ids used asvaccines
1o to2oid formed and no vaccineavailable
/ypical disease /etanus, diphtheria, botulism Meningococcemia, sepsis by gramnegative rods
ndoto2ins
/hermostable
>ater soluble
&naffected by common bactericides
1on8volatile
0nsensitive to p9 change
/hese are the reasons whypyrogen are difficult to destroyonce produced in the product
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CHEMICAL NATURE OF
ENDOTOXIN
- Endotoxin has a polysaccharide
structure that is covalently bound to a
lipid component called, LIPID A
- Contain lipid, protein andcarbohydrate
- olecular si!e: vary "###-$"###
dalton to several million dalton
- Called Lipopolysaccharide or LP%
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BIOLOGICAL PROPERTIES OF
ENDOTOXIN
Pyrogen elevate the circulatory levels o& in&lammatory
cyto'ines (hich may be &ollo(ed by &ever, blood
coagulation, hypotension
Lo( doses o& pyrogen: asymptomatic in&lammation reaction
oderate doses o& pyrogen: &ever and changes in plasma
composition
)igh doses o& pyrogen: cardiovascular &unction, vasodilation,
vasoconstriction, endothelium dys&unction, multiple organ &ailure
*nset and extent o& in+ection &ever depends on the
total amount o& pyrogen delivered to patient and not
conc o& pyrogen per ml 'g L.P/%.P0
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SOURES OF PYROGEN
Any(here (here moisture is present
%olvents, drugs, additives, apparatus in manu&acturing, containers
ethod,preparation 1 storage
". !uipment+
%. 'olvent+
B. 'olute+
Glass
Metal
lastic
Aolatile
1onvolatile
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2 E3uipment:Glass
Metal
lastic
•9eating to %*;o3 for $* min•9eating to :*;o3 for " min•9eating with dil. acid, dil. <lkali or mild o2idi?ing agent
•>ash with detergent
$ %olvent:
Aolatile1onvolatile4istillation
<dsorption
B. 'olute+ @ecrystalli?ation
ELIMINATION OF PYROGEN
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RABBIT TEST (SHAM TEST)
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GENERAL DESCRIPTION OF USP PYROGEN TEST
(FROM USP 85)
4he test involves the measuring rise in
temperature o& rabbits &ollo(ing intravenous
in+ection o& a test solution and is designed &or
products that can be tolerated by the test rabbitin a dose not exceeding 2#ml per 'g in+ected
intravenously (ithin the period o& 54 2#
minutes
4he 6abbit Pyrogen 4est in an in vivo test to
detect pyrogens 3ualitatively
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APPLICATION
Pyrogen test is designed to limit the
ris' o& a &ebrile reaction in the patient
to the administration, by in+ection to
the product concerned
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FACTORS INFLUENCING THE TESTS
2 5ature o& the sample
4rugs or solutions that cause,
decrease or arrest fever. (eg.
<cetanilide, acetophenetidin,
and acetylsalicylic acid. 4rugs that mask pyrogenicity by
decreasing fever. (eg. 'ome
phenothia?ine derivatives,
hypnotics, local anesthetics
(procaine, strophanthin
hosphate and other specificbuffers solutions elicit pyrogenic
responses in rabbits even
though theyCre free from
pyrogens or bacterial endoto2in thers steroid and antibiotics
$ Degree o& restraint
8 0t is necessary to restrain pyroge test
rabbits if rectal thermometer probes
remain in place.
@abbits tend to become hypothermic
when they are restrained
7 tolerance
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LIMITATION OF RABBIT PYROGEN TEST
6epeated use o& animals lead to endotoxin
tolerance
Lo( reactivity to the endotoxin produced by
certain species ex Legionella0 .ariability on control results (hen identical
standardi!ed endotoxins preparations are used
Certain drugs may themselves elicit a rise in
temperature on administration diclo&enac0
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<dvantages
0t can be replicated anddemonstrate the production of
fever in humans
4etects all kinds of inDectablepyrogen unlike 7<7 test
4isadvantages
/ime consuming and e2pensive
ass and fail test
0t cannot be used to test certaindrugs that depresses the fever
/olerance to certain class of drugscan develop in rabbits and
biological variations are observed
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INSTRUMENTATION
aterials:
A 8lass(ares, syringes and needles
4horoughly (ash all glass(are, syringes and needles
(ith (ater &or in+ections and heat in a hot-air oven at $"#
9C &or 7# min or at $## 9C &or 2 h
6estraining boxes
C 4hermometer
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RESTRAINING BOXES
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THERMOMETERS
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TEST ANIMALS
;se healthy, adult rabbits o& either sex (eighing not less
than 2" 'g, &ed a complete and balanced diet not
containing antibiotics, and not sho(ing loss o& body mass
during the (ee' preceding the test
Do not use animals &or pyrogen tests more &re3uently than
once every <= hours A&ter a pyrogen test in the course o&
(hich a rabbit>s temperature has risen by #" 9C or more,or a&ter a rabbit has been given a test substance that (as
ad+udged pyrogenic, at least $ (ee's must be allo(ed to
elapse be&ore the animal is used again
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STRAIN
5e( !ealand (hite
(commonly used in&'<
asy handle E trainedfor pyrogen testing
procedure
ars are slightly shorterthan belgian whites(commonly used ineurope and canada
4isadvantage+ sufferseveral intestinal andrespiratory infections
Dutch belts
9ave a highresistance to
intestinaldisease
'maller si?eand tendency
toward slowweight gain
igmented skin
Chinchillas
Good healthand emotional
stability butgain weight
rapidlyigmented
skin
Fey factors inselecting rabbits
". <nimalbreeder
%. @abbitresistance to
diseaseB. 'ufficient si?e
for easehandling,large ears
$. @ate of weightgain
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ANIMALS’ QUARTER
4he ;%P re3uires that rabbits be individually housed in an area (ith
uni&orm temperature o& bet(een $# 9C and $7 9C and &ree &rom
disturbances li'ely to excite them 6oom temperature must not vary
more than ?7 9C &rom the selected temperature
4he pyrogen test area must be separted &rom the housing area but
maintained under similar environmental condition It is more
convenient to locate the pyrogen test lab ad+acent to the housing
area 6abbits can be transported easily &rom one location to the
other, (ith minimal excitement to animals
%ham test must be per&ormed (ithin seven days o& the actual test
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TEST LABORATORY
;%P re3uires that tests be per&ormed
in an area designated solely &or
pyrogen testing and under
environmental condition similar to
those animals are housedI& laboratory uses clinical
thermometers &or temperature
reading, rabbits may be returned to
their cages bet(een recording
periods
I& electronic devices are used, rectal
temperature probes must remain in
place throughout the pyrogen test
and animals should be lightly
restrained
&' states that the rabbits must befitted with loose neck stocks thatallow the rabbits to assume anatural resting posture
@estraining bo2es, constructed sothat the rabbitCs neck is placed in acomfortable, loose8fitting stock,may be placed on tables or shelves
/he test area should be kept calm
and peaceful with a minimumamount of activity. 'trangersshould not be allowed into the areaduring testing (can often results infalse8positive rabbit temperatureresponse
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TEMPERATURE READING
;se an accurate thermometer graduated in #2 9C or thermistor that
has been tested to determine the time necessary to reach the
maximum reading is reached in less than " minutes
Insert the temperature-sensing device into the rectum o& the testanimal to a depth o& 5L4 @"cm I& the temperature-sensing device is to
remain inserted throughout the sensing period, restrain the rabbit (ith
a lightly-&itting nec' stoc' that allo(s it to assume a natural resting
posture hen a thermometer is used, allo( su&&icient time &or it to
reach a maximum temperature, as previously determined, be&ore
ta'ing the reading
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INJECTION
PROCEDURE
A depyrogenation syringe is &illed either by aspiration or by
pouring the test solution directly into the syringe barrel
ith the syringe plunger in place, the needle is put into place
and all air bubbles are expelled &rom the barrelBor in+ection o& large volumes a $#g x 2 inch needle may be
used 4hin or brittle rabbit veins may necessitate the use o&
$$ to $"8 needles
4hree rabbits are in+ected &or each ;%P test
4he marginal ear vein area o& each rabbit is preparedappropriately
4he needle is inserted, bevel up as close to the tip o& the ear
as possible to prolong the use&ul li&e o& the vein lumen
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PRELIMINARY
TESTING
/o the rabbit, inDect intravenously "; m7of pyrogen free saline solution
@ecord the temperature of animalsbeginning at least #; minutes before
inDection and continuing for B hours afterinDection of the solution being e2amined.
<ny animal showing a temperaturevariation of ;.: degrees celcius or more
must not be used in the main test
erform the test in the areawhere the animals are
housed or under similarenvironmental conditions.
Hor % hours before the testand during the test,
withhold all food from theanimals being used
/he animals should beplaced under the conditions
of the test at least " hour
before the inDection.
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MAIN TEST
3arry out test using group of 7 rabbits.reparation of inDection of the product.
>arming the product, dissolving ordilution
@ecord the temperature of eachanimal. 1ot more than $; minutes
immediately preceding the inDection ofthe test dose
@ecord the initial temperature of eachrabbit. (not I ;.%
4o not use rabbit having the temperature higherthan 7= degrees celcius, and lo(er than 7=
degrees celcius
0nDect the solution being e2amined slowly in themarginal vein o& the ear o& each rabbit over a
period not e2ceeding <minutes
/he volume of inDection is 5L4 #" ml per 'gand 54 2#mL per 'g of body weight. @ecords
the temperature of each animal at half8hourlyintervals for B hours after the inDection
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