pyrogen test (qfrir)

7/25/2019 Pyrogen Test (QFRIR)

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PYROGEN TESTQFRIR AND LAL METHOD

PRESENTED BY:

GONZALES, GREPO, HERNANDEZBSP4D



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



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 ))



-/-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



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



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%



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



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



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



RABBIT TEST (SHAM TEST)



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



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



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



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



<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



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



RESTRAINING BOXES



THERMOMETERS



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



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



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



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



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



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



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.



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

pyrogen test (qfrir)

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