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1229.7

GASEOUSSTERILIZATION

INTRODUCTION

Theuseofsterilizinggasesforthepreparationofmaterialsandequipmentiscommonlyusedforitemsthatare
susceptibletodamagebyheatorradiationprocesses.Manypolymericmaterials,especiallymedicaldevices,are
surfacesterilizedinthismanner,asisnonpressureratedprocessequipment.Thesterilizationofdrypowders
usinggasesisinappropriateduetotheinabilityofgasestopenetratesolidmaterials.Themajorityofgas
sterilizationprocessesemployethyleneoxide(EO),andproceduresforusewithothergasesgenerallyare
patternedafterEOpractices.Ozone,mixedoxidesofnitrogen,andchlorinedioxidearesomeoftheother
gaseoussterilantsused.[Systemsthatcanexistinliquidandgasphaseattheoperatingtemperatures(e.g.,
hydrogenperoxide,peraceticacid,andparaformaldehyde)areexcludedfromconsiderationinthischapter.]EO's
abilitytopenetratethroughpolymers,cellulosics,andothermaterialsallowsittobeusedfortheterminal
sterilizationofmedicaldevicesintheirfinalpackaging.Theothersterilizinggasesmaybesuitableforsimilar
applications.
Processcontrolforgassterilizationequipmentisaccomplishedbycontrolofsterilantgasconcentration,relative
humidity,temperature,andsystempressure.Mixingofthegasinthesterilizationchambermaybebeneficial.EO
sterilizationmaybeusedforparametricreleaseasdescribedinTerminallySterilizedPharmaceuticalProducts
ParametricRelease 1222 .
Gassterilizationdiffersmarkedlyfromprocessesduringwhichtheagentusedcancondenseduringtheoperation.
VaporsterilizationprocesseswillbeaddressedseparatelyinVaporPhaseSterilization 1229.11 .
AsoutlinedinSterilizationofCompendialArticles 1229 ,analystsmusttakecareinensuringsterilityand
demonstratingthattheessentialqualityattributesofthematerialsarenotadverselyaffectedbytheprocess.With
respecttogasprocesses,keyconsiderationsincludetheimmediateeffectsofsterilizinggasonthematerialsor
equipmentbeingsterilized,residualsterilant,sterilantbyproducts,andpotentialchemicalreactions.Thecommon
gasprocessesdifferslightlywithrespecttoprocessexecutionandmaterialconcernsandthusaredescribed
individually.

ETHYLENEOXIDE
EOisapowerfulalkylatingagentthatdestroysmicroorganismsbychemicalreaction,primarilywithcellDNA.The
destructivemechanismlargelyfollowsfirstorderkineticsanddependsonconcentration,humidity,and
temperature.TheuseofEOformedicaldevicesintheirfinalpackaginghas,toalargeextent,shapedEO
sterilizationprocesses(and,toalesserextent,allgassterilization)forotherapplications(2,3).TheusualEO
processfollowsasequenceofprehumidification,airremoval,rehumidificationinthechamber,gasexposure,gas
removalfromthechamber,andpostexposureaeration.Thepreexposurestepsensurethatadequatemoistureis
presentonandwithintheitemsbeingsterilized.ThepostexposurestepsprovidetimeforthediffusionofEOand
itsbyproductsoutofthematerialsandpackaging.WhenEOisusedfornonporousequipmenttheprocesscanbe
streamlined,whicheliminatesmanyofthepreandpostexposurestepsbecauseoftheneedonlyforsurface
sterilization.DuringEOsterilizationthegasisintroducedatthebeginning,andonlyminimaladditionsare
necessarylatertomaintainpressureasthegasisabsorbedintothematerial/sterlizationloadwithinthevessel.
Humidityadjustmentduringtheprocessalsomayberequired.Insomeinstances,EOreactswithmaterialsinthe
loadtoformethylenechlorohydrinandethyleneglycol.Thesecompounds,includingEO,mustbereducedtosafe
levelsbeforetheitemscanbeusedbypatients(4,5).EOprocessingrequiresstrictworkersafetyand
environmentalcontrolsbecauseitisassociatedwithcarcinogenicity,mutagenicity,andneurotoxicity.Inaddition,
EOisexplosiveinconcentrationsofgreaterthan2.6%byvolumeinair,therefore,inertgasesareoftenusedto
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minimizeflammability.Thecommonlyacceptedbiologicalindicator(BI)strainisBacillusatrophaeus(formerlyB.
subtilisvar.niger).

OZONE
Ozoneisapotentoxidizingagentproducedbypassingastreamofoxygenorairthroughahighvoltageelectrical
field.Ozoneisaneffectivebiocidalagentfortreatmentofwatersuppliesandhasdemonstratedlethalityat
concentrationsfrom2%10%inair.Optimalmicrobialdestructionisaccomplishedwhentherelativehumidityis
above80%atroomtemperature.Ozonedegradestooxygeninthepresenceofmoistureandmetalsandtherefore
usuallyisgeneratedinsitu.OzonedoesnotpenetrateporousmaterialstothesameextentasEOdoes.Process
systemsthatuseozoneforgassterilizationhavetheadvantageofsimplicity.Itsgenerationanddestruction
(usingacatalyticconverter)areaccomplishedwithoutmovingpartsorconsumablesotherthanthesupplied
oxygen.Thesterilizationprocessusesasequenceofhumidification,injection,exposure,andventilationto
removetheozonefromthechamberattheendofthecycle.ThecommonBIsidentifiedforozoneareGeobacillus
stearothermophilusandBacillusatrophaeus.

CHLORINEDIOXIDE
Chlorinedioxideisaneffectivesterilizinggas.Purechlorinedioxideismetastableandthereforeisgeneratedas
needed.Chlorinedioxideisnoncarcinogenic,nonflammable,andeffectiveatambienttemperatures.Itsabilityto
penetratematerialsmaybelessthanthatofEO.
Atypicalchlorinedioxidesterilizationprocessusesasequenceofpreconditioning,conditioningdwellperiod,
charge,andexposure,followedbyaeration.
TheBImostcommonlyusedisBacillusatrophaeus.

NITROGENDIOXIDE
Nitrogendioxideisasterilizinggaseffectiveatambienttemperature.Liquidnitrogendioxideisconvertedtoagas
onintroductiontothetargetchamber.Nitrogendioxideisnonexplosiveanditsresiduesarenoncarcinogenic,
noncytotoxic,andnonteratogenic.IthasalimitedabilitytopenetratepolymericmaterialsincomparisontoEO,
whichmakespostcycleaerationmorerapid.Itisincompatiblewithcellulosicmaterialssuchaspaperand
cardboard.ThesuggestedBIsfornitrogendioxideareG.stearothermophilusandB.atrophaeus.

VALIDATIONOFGASSTERILIZATION
Thevalidationofgaseoussterilizationgenerallybeginswiththeestablishingofaminimumlethalprocessdwell
timethroughtheuseoffractionalexposurestudies.Thesefractionalstudiesestablishthatexposuretime,under
standardprocessconditions,wherethebiologicalindicatorisfullyinactivated.Thisminimumexposuretimethen
becomesthebasisfortheapplicationofthehalfcycleapproachforvalidatingthesterilizationcycle.Theabsence
ofinformationrelatingtheeffectofvaryinggasconcentration,humidity,andtemperatureonmicroorganisms
resultedinaconservativeassumptionthatthebioburdenisequalinantimicrobialresistanceandpopulationtothat
ofthebiologicalindicator.Thehalfcyclemethodcanbedefinedasfollows.
Thehalfcyclevalidationmethodrequiresthedestructionofahighconcentration(NLT106spores)ofaresistant
microorganismunderdefined,minimumconditionsforcompletekill.Thisestablishestheminimumlethalprocess
dwelltime.Inroutineoperation,theprocessdwellperiodisarbitrarilydoubledandsupportsatheoreticalreduction
ofthebiologicalindicator(andthusthebioburden)toaprobabilityofanonsterileunit(PNSU)of10 6(for
definitionsoftermsinthischapter,seeSterilizationofCompendialArticles 1229 ).
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ThehalfcyclemethodusedforgassterilizationisshowninFigure1.

Figure1.Halfcyclesterilizationvalidation.
Alternativeapproachestocyclevalidationareavailable.GillisandMosleydevelopedameansforparametric
evaluationofEOsterilizingconditionsthatmayresultingreateruseofothervalidationmethods(6).Abracketing
approach(seeFigure2)thatbettersupportstheprocessoperatingrangesforthecriticalparametersrelativetothe
halfcyclemethodhasalsobeenused.Inthebracketingmethod,oneevaluatesconditionsthatbracketthe
definedprocessconditioninordertoestablishparametersfortheminimumandmaximumeffectsonthematerials
andbioburden.Theminimumlethalprocessdwelltime(seehalfcycledescriptionabove)establishestheworst
caseformicrobialkill.Incrementalincreasesinprocessdwelltimebeyondtheminimumlethalprocessdwelltime
areusedtoestablishtheroutineandmaximumexposureperiods,thelatterofwhichimpartsthegreatesteffecton
materials.Inaddition,adjustmentstoagentconcentrationandrelativehumidityareutilizedtofurtherenhancethe
bracketingapproach.Bythismethod,theroutineprocessconditionsmaybeestablishedbetweentheminimum
andmaximumprocessconditionstoassurecompletemicrobialkillwhilemaintainingtheintegrityofthematerials.

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Figure2.Bracketingmethod.
EquipmentQualification
Theequipmentqualificationforgassterilizationshouldincludebothpreandpostcyclesystemstoconfirmthatthe
equipmenthasbeenproperlyinstalledandoperatesasintended.
EmptyChamberParameterDistribution
Despitetheuseoftruegases,evaluationofparameteruniformityacrossthechamberisacommonactivity.This
ensuresthatthegasandhumidityintroductionmethodsprovideconsistencythroughoutthechamberandcanbe
correlatedtotheroutinemonitoringlocation(s),whenpresent.Biologicalindicatorsarenotrequiredinthe
evaluationoftheemptychamberuniformity.
ComponentandLoadMapping
Componentandloadmappingusinginvasivesamplingarenotapartofgassterilizationbecausesamplingsystems
placedwithintheloaditemswouldaltergasandhumiditypenetration.Evaluationoflethalconditionswith
individualitemsandacrossloadingpatternsisbestprovidedbybiologicalindicatorsorprocesschallengedevices
placedwithintheloaditemsanddistributedwithintheload.Indicatorsorprocesscontroldevicesareplacedwithin
theitemsandloadatlocationsbelievedtobehardestforthegasandhumiditytopenetrate.
BiologicalIndicators
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Thebiologicalindicatorofchoiceforgassterilizationvaries,asnotedabove.B.atrophaeus(ATCC9372)isused
withEOandchlorinedioxide,andozonesterilizationismonitoredwithG.stearothermophilus(ATCC12980or
7953).Dvaluesforthebiologicalindicatorcanbeusedtoestablishexposureperiodsforthesterilizationprocess
toensureadequateprocessefficacy.Whenpositioningbiologicalindicatorswithinitemsitisimportanttoensure
thattheplacementoftheBIdoesnotoccludegaspassageorotherwiseinterferewiththedistribution/penetration
ofthesterilantwithintheitem.
ProcessConfirmationandMicrobiologicalChallenge
Thecoreofthevalidationactivityistheconfirmationofacceptableprocessparameterswithsimultaneousphysical
andchemicalmeasurementandmicrobialchallenge.Sensorsareplacedinthechamber,orbiologicalindicators
arepositionedwithintheloaditems.Proofofcycleefficacyisprovidedinreplicatestudiesinwhichthebiological
indicatorsarekilledandthephysicalmeasurementscorrespondtotheexpectedvalues.

ROUTINEPROCESSCONTROL
Gassterilizationissubjecttoformalcontrolsthatmaintainavalidatedstateovertime.Thepracticesoutlinedin
1229 includethegeneralrequirementsappropriateforallsterilizationsystems.Sterilizationisaccomplishedby
anumberofrelatedpracticesthatareessentialforcontinueduseoftheprocessoveranextendedperiodoftime.
Theessentialpracticestomaintainvalidatedstatusincludecalibration,physicalmeasurements,ongoingprocess
control,changecontrol,preventivemaintenance,periodicreassessment,andtraining.Whenparametricrelease
hasnotbeenestablished,biologicalindicatorspositionedwithintheloadareusedforroutinereleaseofeach
sterilizationload,alongwithareviewofdocumentationfromthesterilizercontrolsystem.

REFERENCES
1.USPGeneralChaptersMicrobiologyExpertCommittee.AnoutlineofplannedchangestoUSP 1211
SterilizationandSterilityAssuranceofCompendialArticles.PharmacopeialForum.201238(2).
2.ISO111351:2007SterilizationofHealthCareProductsEthyleneOxidePart1:Requirementsfor
Development,Validation,andRoutineControlofaSterilizationProcessforMedicalDevices.Geneva:
InternationalOrganizationforStandards(ISO)2007.
3.ISO111352:2008SterilizationofHealthCareProductsEthyleneOxidePart2:Guidanceonthe
ApplicationofISO111351.Geneva:InternationalOrganizationforStandards(ISO)2008.
4.ISO109937BiologicalEvaluationofMedicalDevices,Part7:EthyleneOxideSterilizationResiduals.
Geneva:InternationalOrganizationforStandards(ISO)2008.
5.Ethyleneoxide,ethylenechlorohydrin,andethyleneglycolproposedmaximumresiduelimitsand
maximumlevelsofexposure.FedRegist.197843(122):2747427483.
6.GillisJ,MosleyG.Validationofethyleneoxidesterilizationprocesses.In:AgallocoJ,CarletonFJ,eds.
ValidationofPharmaceuticalProcesses.3rded.NewYork:InformaUSA2007.
AuxiliaryInformationPleasecheckforyourquestionintheFAQsbeforecontactingUSP.

Topic/Question Contact
General
RadhakrishnaSTirumalai,
Chapter
Ph.D.
PrincipalScientificLiaison
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ExpertCommittee
(GCM2010)GeneralChapters
Microbiology

USP38NF33Page1482
PharmacopeialForum:VolumeNo.39(3)
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