SIST EN ISO 14729:2002

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.KLJLHQVNHJDAugenoptik - Kontaktlinsenpflegemittel - Mikrobiologische Anforderungen und Prüfverfahren für Produkte und Systeme zum Hygienemanagement von Kontaktlinsen (ISO 14729:2001)Optique ophtalmique - Produits d'entretien des lentilles de contact - Exigences microbiologiques et méthodes d'essai des produits et protocoles d'entretien des lentilles de contact (ISO 14729:2001)Ophthalmic optics - Contact lens care products - Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses (ISO 14729:2001)11.040.70Oftalmološka opremaOphthalmic equipmentICS:Ta slovenski standard je istoveten z:EN ISO 14729:2001SIST EN ISO 14729:2002en01-maj-2002SIST EN ISO 14729:2002SLOVENSKI
STANDARD



SIST EN ISO 14729:2002



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN ISO 14729April 2001ICS 11.040.70English versionOphthalmic optics - Contact lens care products - Microbiologicalrequirements and test methods for products and regimens forhygienic management of contact lenses (ISO 14729:2001)Optique ophtalmique - Produits d'entretien des lentilles decontact - Exigences microbiologiques et méthodes d'essaides produits et protocoles d'entretien des lentilles decontact (ISO 14729:2001)Augenoptik - Kontaktlinsenpflegemittel - MikrobiologischeAnforderungen und Prüfverfahren für Produkte undSysteme zum Hygienemanagement von Kontaktlinsen (ISO14729:2001)This European Standard was approved by CEN on 15 April 2001.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2001 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 14729:2001 ESIST EN ISO 14729:2002



Page 2EN ISO 14729:2001ForewordThe text of the International Standard ISO 14729:2001 has been prepared by TechnicalCommittee ISO/TC 172 “Optics and optical instruments” in collaboration with TechnicalCommittee CEN/TC 170 “Ophthalmic optics”, the secretariat of which is held by DIN.This European Standard shall be given the status of a national standard, either by publicationof an identical text or by endorsement, at the latest by October 2001, and conflicting nationalstandards shall be withdrawn at the latest by October 2001.According to the CEN/CENELEC Internal Regulations, the national standards organizationsof the following countries are bound to implement this European Standard: Austria, Belgium,Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UnitedKingdom.Endorsement noticeThe text of the International Standard ISO 14729:2001 was approved by CEN as a EuropeanStandard without any modification.SIST EN ISO 14729:2002



ReferencenumberISO14729:2001(E)©ISO2001INTERNATIONALSTANDARDISO14729Firstedition2001-04-15Ophthalmicoptics—Contactlenscareproducts—MicrobiologicalrequirementsandtestmethodsforproductsandregimensforhygienicmanagementofcontactlensesOptiqueophtalmique—Produitsd'entretiendeslentillesdecontact—Exigencesmicrobiologiquesetméthodesd'essaidesproduitsetprotocolesd'entretiendeslentillesdecontactSIST EN ISO 14729:2002



ISO14729:2001(E)PDFdisclaimerThisPDFfilemaycontainembeddedtypefaces.InaccordancewithAdobe'slicensingpolicy,thisfilemaybeprintedorviewedbutshallnotbeeditedunlessthetypefaceswhichareembeddedarelicensedtoandinstalledonthecomputerperformingtheediting.Indownloadingthisfile,partiesacceptthereintheresponsibilityofnotinfringingAdobe'slicensingpolicy.TheISOCentralSecretariatacceptsnoliabilityinthisarea.AdobeisatrademarkofAdobeSystemsIncorporated.DetailsofthesoftwareproductsusedtocreatethisPDFfilecanbefoundintheGeneralInforelativetothefile;thePDF-creationparameterswereoptimizedforprinting.EverycarehasbeentakentoensurethatthefileissuitableforusebyISOmemberbodies.Intheunlikelyeventthataproblemrelatingtoitisfound,pleaseinformtheCentralSecretariatattheaddressgivenbelow.©ISO2001Allrightsreserved.Unlessotherwisespecified,nopartofthispublicationmaybereproducedorutilizedinanyformorbyanymeans,electronicormechanical,includingphotocopyingandmicrofilm,withoutpermissioninwritingfromeitherISOattheaddressbeloworISO'smemberbodyinthecountryoftherequester.ISOcopyrightofficeCasepostale56CH-1211Geneva20Tel.+41227490111Fax+41227490947E-mailcopyright@iso.chWebwww.iso.chPrintedinSwitzerlandii©ISO2001–AllrightsreservedSIST EN ISO 14729:2002



ISO14729:2001(E)©ISO2001–AllrightsreservediiiContentsPageForeword.ivIntroduction.v1Scope.12Normativereferences.13Termsanddefinitions.14Principle.24.1General.24.2Stand-alonetest(Inoculumchallengetest).24.3Regimentest.35Performancerequirements.35.1Stand-alonetest:Primarycriteria(seealsoTable1).35.2Stand-alonetest:Secondarycriteria(seealsoTable1).45.3Regimentest:Regimencriteria(seealsoTable1).46Testmethods.56.1Materialsandreagents.56.2Preparationofmicrobialchallenge(Inoculum).66.3Stand-aloneprocedure.76.4Regimenprocedure.10AnnexA(informative)Testorganismsfromotherculturecollections.13AnnexB(informative)Exampleofamembranefiltrationprocedure.14AnnexC(informative)Technicalreport:Virustesting.16AnnexD(informative)Technicalreport:Acanthamoebatesting.17AnnexE(informative)Technicalreport:Artificialtears(organicsoil)inlaboratorytesting.18Bibliography.19SIST EN ISO 14729:2002



ISO14729:2001(E)iv©ISO2001–AllrightsreservedForewordISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingInternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollaboratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnicalstandardization.InternationalStandardsaredraftedinaccordancewiththerulesgivenintheISO/IECDirectives,Part3.DraftInternationalStandardsadoptedbythetechnicalcommitteesarecirculatedtothememberbodiesforvoting.PublicationasanInternationalStandardrequiresapprovalbyatleast75%ofthememberbodiescastingavote.AttentionisdrawntothepossibilitythatsomeoftheelementsofthisInternationalStandardmaybethesubjectofpatentrights.ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.InternationalStandardISO14729waspreparedbyTechnicalCommitteeISO/TC172,Opticsandopticalinstruments,SubcommitteeSC7,Ophthalmicopticsandinstruments.AnnexesAtoEofthisInternationalStandardareforinformationonly.SIST EN ISO 14729:2002



ISO14729:2001(E)©ISO2001–AllrightsreservedvIntroductionProductsforcontactlensdisinfectionbychemicalmeansareintendedtoreducemicrobialcontaminationintroducedduringlenswearandremoval,cleaningandstorageandarerequiredtocontainantimicrobialagentscapableofachievingthis.Itisessentialthatallliquidcontactlenscareproductsaresterileuntilopened.Dryproducts(tablets,granules,etc.)shouldbesubjecttocontrolofmicrobialcontaminationandshouldbedissolvedinasuitablediluentimmediatelypriortouse.Multidosecontactlenscareproductsmustbeadequatelypreservedorbepackagedinacontainerdesignedandlabelledtominimizetheriskofinjuryresultingfromin-usecontamination.Contactlensesarenormallysubjecttoaregimenofcleaningandcontactlensdisinfectionbetweenperiodsofwear.Aqueoussolutionscontainingcleaningand/ordisinfectingagentsarecommonlyusedforthispurpose.Theseproductsmaybemarketedassolutionsorastabletsfordissolutionimmediatelypriortouseinasuitablediluentsuchassaline.Thepast20yearsofexperienceintheuseandregulationofcontactlensdisinfectingproductshasshowndistinctdisinfectingantimicrobialcriteriaforthisclassofmedicaldevices.Oculartoxicologyconcerns,processconvenienceandproductcomfortontheeye,havemeantanevolutionofproductswhichmaintainalowincidenceofcontactlensassociatedocularinfectionwhenusedasinstructedbythemanufacturer.ThisInternationalStandardgivesthesedistinctcontactlensdisinfectingantimicrobialcriteriaalongwithannexestoexplainwhyviruses(annexC)andAcanthamoeba(annexD)arenotincludedaschallenges.Organicsoilisnotrequiredforevaluationofcontactlenscaredisinfectingproductsbutmaybeused;aninformativeannex(annexE)isincludedtodiscussorganicsoilinthecontextofcontactlensesandcontactlenscareproducts.SIST EN ISO 14729:2002



SIST EN ISO 14729:2002



INTERNATIONALSTANDARDISO14729:2001(E)©ISO2001–Allrightsreserved1Ophthalmicoptics—Contactlenscareproducts—Microbiologicalrequirementsandtestmethodsforproductsandregimensforhygienicmanagementofcontactlenses1ScopeThisInternationalStandardspecifiestwotestmethodsforevaluatingtheantimicrobialactivityofproductstobemarketedforcontactlensdisinfectionbychemicalmeansandforproductsthatarepartofacontactlenscareregimen.ThisInternationalStandardisnotapplicabletothehygienicmanagementoftriallenses.NOTEGeneraldisinfectionproductstandardsarenotapplicabletocontactlenscareproducts,e.g.EN1040:1997andEN1275:1997.2NormativereferencesThefollowingnormativedocumentscontainprovisionswhich,throughreferenceinthistext,constituteprovisionsofthisInternationalStandard.Fordatedreferences,subsequentamendmentsto,orrevisionsof,anyofthesepublicationsdonotapply.However,partiestoagreementsbasedonthisInternationalStandardareencouragedtoinvestigatethepossibilityofapplyingthemostrecenteditionsofthenormativedocumentsindicatedbelow.Forundatedreferences,thelatesteditionofthenormativedocumentreferredtoapplies.MembersofISOandIECmaintainregistersofcurrentlyvalidInternationalStandards.ISO8320-1:—1),Contactlensesandcontactlenscareproducts—Vocabulary—Part1:Contactlenses.ISO8320-2:—1),Contactlensesandcontactlenscareproducts—Vocabulary—Part2:Contactlenscareproducts.3TermsanddefinitionsForthepurposesofthisInternationalStandard,thedefinitionsgiveninISO8320applytogetherwiththefollowing.3.1contactlensdisinfectingproductproductthatpossessescidalactivity(kills,destroysand/orinactivates)meetingtheprimarycriteriaofthestand-alonetestspecifiedinthisInternationalStandard3.2contactlensdisinfectingregimencontactlenscareregimendesignedtomeetboththesecondarycriteriaofthestand-alonetestandtheregimentestasspecifiedinthisInternationalStandard1)Tobepublished.(RevisionofISO8320:1986)SIST EN ISO 14729:2002



ISO14729:2001(E)2©ISO2001–Allrightsreserved3.3contactlensdisinfectionchemicalorphysicalprocesstoreducethenumberofviablemicroorganismsasspecifiedintheperformancerequirementsectionsofthisInternationalStandard4Principle4.1GeneralThestand-alonetestisdesignedtoqualifyindividualsolutionswithasuitablelevelofantimicrobialactivityascontactlensdisinfectionproducts.Theregimentestisdesignedtoqualifyindividualsolutionsaspartofacontactlensdisinfectingregimen.Productsmeetingtheregimentestcriteriashallalsomeettheminimumperformancerequirementsofthestand-alonetest.Itisfundamentalthatsuchproducts(unopenedcontainers)arecapableofmeetingtherequirementsofthetestthroughouttheirlabelledshelflife.AsdescribedinFigure1,contactlenscaresolutionswhicharedesignedtopossessdisinfectingpropertiesshallbetestedinthestand-alonetestfirst.Iftherespectiveprimarycriteriaaremet(see5.1),theproductmaybelabelledasacontactlensdisinfectingproduct.Iftheproductfailstheprimarycriteriaofthestand-alonetest,theproductmustexhibitsufficientantimicrobialactivitytomeetthesecondarycriteriaofthestand-alonetestaslistedin5.2.Ifthesesecondarycriteriaaremet,theregimentestshallbeperformedinordertoqualifytheproductaspartofacontactlensdisinfectingregimenbymeetingtheregimencriteria(see5.3).Iftheproductmeetsboththesecondarycriteriaofthestand-alonetestandtheregimentestbutfailstheprimarycriteriaofthestand-alonetest,itshallbelabelledaspartofacontactlensdisinfectingregimen.Thedesignofcontactlenscareproductsforcleaningandcontactlensdisinfectionshalltakeintoconsiderationtheneedsofpatientcomplianceandtheprobabilityofnon-compliance.Forexample,disinfectingtimemustbeappropriateforcontactlenswear.NOTEUseofmultipleormixedmicrobialchallengescaninfluencetheapparentdisinfectingactivityofaparticularproduct.TheevaluationofthesevariablestogetherwithtestingagainstalargerpanelofmicroorganismsandtestingofsamplesfrompartiallyusedcontainersmaybeofvalueindevelopingacontactlenscareproductbutareexcludedfromthescopeofthisInternationalStandard.(SeeannexesCandD).4.2Stand-alonetest(Inoculumchallengetest)Thestand-alonetestchallengesadisinfectingproductwithastandardinoculumofarepresentativerangeofmicroorganismsandestablishestheextentoftheirviabilitylossatpre-determinedtimeintervalscomparablewiththoseduringwhichtheproductmaybeused.Thesizeofthemicrobialchallengechoseninthistestisnotintendedtoberepresentativeofthelikelychallengeinpracticebuttoprovidecountablenumbersfromwhichestimationoftherateandextentofviabilitylosscanbedetermined.Incarryingoutthetestforantimicrobialactivitythequalitativeandquantitativecompositionsoftheproducthavetobeknownatthetimeoftestingbyeitheranalyticaltestingorextrapolation.Appropriatemeasuresshallbetakentoinactivateorremoveresidualantimicrobialagentsduringculturingandcountingofchallengeorganismsurvivors,andtheeffectivenessofthesemeasuresshallbevalidated.Theactionofthisprocessduringthetestshallbedemonstratedbytheconstructionofsuitablecontrols.NOTEForinformationaboutvirustesting,seeannexC,andforAcanthamoebatesting,seeannexD.SIST EN ISO 14729:2002



ISO14729:2001(E)©ISO2001–Allrightsreserved3aProductmaybelabelledasacontactlensdisinfectingproduct.bProductshallbelabelledaspartofacontactlensdisinfectingregimen.Figure1—Flowchartforstand-alonetestandregimentest4.3RegimentestThisisatestwhichchallengesamultifunctionaldisinfectingregimenwithastandardinoculumofarepresentativerangeofmicro-organismsandwhichmeasurestheviabilitylossafterapredeterminedtimeinterval;theinoculumiscarriedthroughtheregimenbybeingappliedtoacontactlens.Thisprocedureisapplicabletomultifunctionaldisinfectingregimenswhichmayincludethestepsofcleaning,rinsingandsoaking.Incarryingouttheregimentestprocedure,theproductsareusedinthemannerandquantityrecommendedinproductlabellingand/orpatientinstructions.Thedisinfectingstageofanyproposedcontactlensdisinfectingregimenevaluatedbythistestshallsatisfytheminimumrequirementsofthestand-alonetestasdescribedinFigure1.Onlythoseproductsthatsatisfytheminimumperformancerequirementsforthestand-alonetestmaybeincorporatedintoadisinfectingregimen.Incarryingoutthetest,qualitativeandquantitativecompositionofallproductsusedinthetestregimenhavetobeknownatthetimeoftesting,eitherbyanalyticaltestingorextrapolation.Appropriatemeasuresshallbetakentoinactivateorremoveresidualantimicrobialactivityduringculturingandcountingofchallengeorganismsurvivors,andtheeffectivenessofthesemeasuresshallbevalidated.Theactionofthisprocessduringthetestshallbedemonstratedbytheconstructionofsuitablecontrols.NOTEForproblemsassociatedwiththeuseofhuman-wornlenses,seeannexE.5Performancerequirements5.1Stand-alonetest:Primarycriteria(seealsoTable1)5.1.1BacteriaThenumberofeachchallengeorganismrecoveredpermillilitreshallbereducedbyanaveragevalueofnotlessthan99,9%(3,0logs)withintheminimumrecommendedsoakingperiod.SIST EN ISO 14729:2002



ISO14729:2001(E)4©ISO2001–AllrightsreservedNOTEThevalueisdeterminedbytakingtheaverageofthelogreductionsforeachchallengeorganismfortheindividuallotstested.5.1.2MouldsandyeastsThenumberofeachchallengeorganismrecoveredpermillilitreshallbereducedbyanaveragevalueofnotlessthan90%(1,0log)withintheminimumrecommendedsoakingperiodwithnoincreaseatnotlessthanfourtimestheminimumrecommendedsoakingperiodwithinanexperimentalerrorof0,5logs.NOTEThevalueisdeterminedbytakingtheaverageofthelogreductionsforeachchallengeorganismfortheindividuallotstested.5.2Stand-alonetest:Secondarycriteria(seealsoTable1)Productsfailingtomeetthecriteriain5.1.1or5.1.2shallbeevaluatedbytheregimentestproceduredescribedin6.4,providedthesumoftheaveragesisaminimumof5,0logunitsreductionforthethreespeciesofbacteriawithintherecommendedsoakingperiodwithaminimumaverageof1,0logunitreductionforanysinglebacteria.Stasisfortheyeastandmouldshallbeobservedfortherecommendedsoakingperiodwithinanexperimentalerrorof0,5logs.5.3Regimentest:Regimencriteria(seealsoTable1)Foreachmicrobialspecies,theaverageregimenrecoverycount(foralllotstested)shallbenomorethan10cfuforeachlenstype/storagesolutioncombination.Datafrommorethanonelenstypeshouldnotbecombinedtocalculatetheaverage.NOTEWhenqualifyingalenscareproductregimenforusewithonelenstype,theaveragecountforeachspeciesrequiresaveragingthedatafromthe24inoculatedandtreatedlensesoftheonelenstype.Whenqualifyingalenscareproductregimenforusewithmorethanonelenstype,theaveragecountforeachspeciesbylenstypewouldrequireaveragingthedatafromthe12inoculatedandtreatedlensesforeachlenstype.SeeTable4fornumbersoflensestobeused.SIST EN ISO 14729:2002



ISO14729:2001(E)©ISO2001–Allrightsreserved5Table1—SummaryofperformancerequirementscriteriaforcontactlensdisinfectionproceduresAveragelogreductionatsoakingtimeFungiBacteriaTestFSaCAaSMaPAaSAaStand-alonetest:PrimaryCriteria11333Stand-alonetest:SecondaryCriteriabbcccRegimentest:Regimencriteriad4to54to54to54to54to5aPA=P.aeruginosaATCC9027;SA=S.aureusATCC6538;SM=S.marcescensATCC13880;CA=C.albicansATCC10231;FS=F.solaniATCC36031.bStasisatthesoakingtime.cTheminimumacceptablelogreductionforallthreebacteriacombinedis5.Theminimumacceptablelogreductionforanysinglebacterialtypeis1.dEquivalenttoanaverageofnotmorethan10cfuperlenstype/storagesolutioncombination.6Testmethods6.1MaterialsandreagentsThematerialsandreagents(i.e.thetestorganisms,mediaandreagents,equipmentandsamples)arecommontoboththestand-aloneprocedurefordisinfectingproductsandtheregimenprocedureforcontactlensdisinfection.6.1.1TestorganismsThestrainslistedinTable2shallbeused.NOTETestorganismsfromotherculturecollectionsthatmaybeusedarelistedinannexA.Table2—TestorganismsPseudomonasaeruginosaATCC9027StaphylococcusaureusATCC6538SerratiamarcescensATCC13880CandidaalbicansATCC10231FusariumsolaniATCC360316.1.2Culturemediaandreagents6.1.2.1Potatodextroseagar(PDA).SIST EN ISO 14729:2002



ISO14729:2001(E)6©ISO2001–Allrightsreserved6.1.2.2Tryptonesoyaagar(TSA).6.1.2.3SabouraudDextroseAgar(SDA).6.1.2.4Dulbecco'sPhosphateBufferedSaline,withoutcalciumchlorideandmagnesiumchloride(DPBS):200mg/lKCl,200mg/lKH2PO4,8000mg/lNaCl,and2160mg/lNa2HPO47H2Oorsuitablediluent.6.1.2.5Dulbecco'sPhosphateBufferedSaline,plus0,05%(mass/volume)polysorbate80(DPBST)orsuitablediluent.6.1.2.6Validatedneutralizingagents/mediaasrequired,forexample,Dey-EngleyNeutralisingBroth(DEB)andLetheenBroth2).6.1.3TestequipmentThefollowingcommonlaboratoryequipmentisrequired.6.1.3.1Sterilepipettes.6.1.3.2Swabs.6.1.3.3Tubes.6.1.3.4Petridishes(90mmto100mm20mm).6.1.3.5Incubator.6.1.3.6Spectrometer,fordeterminationofcelldensity.6.1.3.7Instrumentforcolonycounting.6.1.3.8Centrifuge.6.1.4TestsamplesTheproducttobetestedshallberepresentativeoftheproducttobemarketed.Aliquotsshouldbetakendirectlyfromthefinalproductcontainerimmediatelypriortotesting.Threelotsofproductshallbetested.Eachlotofproductshallbetestedwithaseparateinoculumpreparationforeachchallengeorganism.6.1.5CulturemaintenanceMaintainthetestculturesasrecommendedbythecuratoroftheappropriateculturecollection.Culturesshouldbenogreaterthan5passesremovedfromthedepositorystock(ATCC,NCIB,NCTC,NCPForotherrecognizedculturedepository;seeannexA).Eachpassisasubcultureofthepreviouspass.6.2Preparationofmicrobialchallenge(Inoculum)Thepreparationofthemicrobialchallengeorganisms(inoculum)iscommontoboththestand-aloneprocedurefordisinfectingproductsandtheregimenprocedureforcontactlensdisinfection.2)Dey-EngleyNeutralisingBroth(DEB)andLetheenBrothareexamplesofsuitableproductsavailablecommercially.ThisinformationisgivenfortheconvenienceofusersofthisInternationalStandardanddoesnotconstituteanendorsementbyISOofthisproduct.SIST EN ISO 14729:2002



ISO14729:2001(E)©ISO2001–Allrightsreserved7Fortheregimenprocedureforcontactlensdisinfection,organicsoilmaybeincludedaspartoftheinoculum.SeeannexEforanexample.CultureeachtestorganismonagarslopesundertheconditionsgiveninTable3.Table3—MediaandincubationconditionsforgrowthofchallengeorganismsOrganismMediumTemperature°CIncubationtimeP.aeruginosaTSA30to3518hto24hS.aureusTSA30to3518hto24hS.marcescensTSA30to3518hto24hC.albicanxSDA20to2542hto48horC.albicansSDA30to3518hto24hF.solaniPDA20to2510dto14dUsesterileDPBSTorsuitablediluenttoharvesteachculture;washthesurfacegrowth,transferittoasuitablevesselandvortex.FiltertheF.solanisuspensionsthroughsterileglasswool,cheeseclothorgauzetoremovehyphalfragments.Afterharvesting,theculturedorganismsmaybewashedusingcentrifugation.Thebacterialsuspensionsmaybefiltered(e.g.3µmto5µmporesize)toproduceasinglecelldispersion.ThenadjustallchallengecellsuspensionswithDPBSTorothersuitablediluenttoaconcentrationofbetween1107cfu/mland1108cfu/ml.Estimatetheapproximatecellconcentrationofeachsuspensionbymeasuringtheturbidityofthesuspensionoradilutionofthesuspensionusingaspectrophotometer.Theactualconcentrationofcolonyformingunitspermillilitreshallbedeterminedforeachsuspension,e.g.bytheplatecountmethod,atthetimeofthetest.Ifacentrifugeisused,eachcentrifugationshouldbeconductedat20°Cto25°Cfornolongerthantheequivalentof10minat4000gorless.Longercentrifugationtimesmayberequiredatlowerspeeds.Usebacterialandyeastcellsuspensionsonthedayofpreparation.Sporesuspensionsmaybeuseduptosevendaysfollowingpreparationifstoredunderrefrigeration(2°Cto8°C).6.3Stand-aloneprocedure6.3.1Inoculumchallengetestprocedure6.3.1.1Prepareoneormoretubes(foreachlottested)containingaminimumof10mloftestproductsolutionperchallengeorganism.NOTESampletubesareusedratherthanlenscasestoalloweffectivetechnicalexecutionofthetest.Sinceincompatibilitiescanexistbetweensolutioningredientsandtubematerials,tubesofanappropriatematerial,whicharecompatiblewiththeingredients,shouldbeused.Inoculatethesampletubeoftheproducttobetestedwithasuspensionoftestorganismssufficienttoprovideafinalcountofbetween1,0105and1,0106cfu/ml.Ensurethatthevolumeofinoculumdoesnotexceed1%ofthesamplevolume.Ensurecompletedispersionoftheinoculumbyadequatemixing.6.3.1.2Storetheinoculatedproductat20°Cto25°C.Thetemperatureshallbemonitoredusingacalibrateddeviceandthetemperaturedocumented.Iftheproductissensitivetolightitshouldbeprotectedduringtheperiodofthetest.SIST EN ISO 14729:2002



ISO14729:2001(E)8©ISO2001–Allrightsreserved6.3.1.3Take1,0mlaliquotsoftheinoculatedproductfordeterminationofviablecountat25%,50%,75%and100%oftheminimumrecommendeddisinfectingtimeforallorganisms,and,inaddition,notlessthan400%oftheminimumrecommendeddisinfectingtimeforyeastandmould.Ifovernightcontactlensdisinfectionisrecommended,useasoakingtimeof8h.6.3.1.4Subjecteachofthe1,0mlaliquots,removedatthespecifiedtimeintervals,toasuitableseriesofdecimaldilutionsinvalidatedneutralizingmedia.Mixthesuspensionwellbyvortexingvigorouslyandletstandtoallowneutralizationtobecompleted.Neutralizationconditionsshallbebasedonrecoverymediumcontroltesting(see6.3.2.2).Ifanantimicrobialagentintheformulationcannotbeadequatelyinactivatedorneutralized,eliminateitusingavalidatedmembranefiltrationprocedure(seeannexB).6.3.1.5Determinetheviablecountoforganismsinappropriatedilutionsbypreparationoftriplicateplates(unlessotherwisejustified)ofasuitablerecoverymedium(e.g.TSAforbacteriaandSDAformouldandyeast).Ifmembranefiltrationhasbeenemployedtoremoveorneutralizeantimicrobialagents,culturethemembranesonthesemediaasappropriate.Ifthepourplatemethodisutilized,keeptheagarforpourplatesbelow50Cpriortopouring.Theagarmediausedfordeterminationofviablecountsmayalsocontainantimicrobialinactivatorsorneutralizers,ifrequired.6.3.1.6Incubatebacterialrecoveryplatesat30Cto35C.Incubateyeastrecoveryplatesat20Cto25Cor30Cto35C.Incubatemouldrecoveryplatesat20Cto25C.Incubationtimesforoptimalrecoveryofbacteria,yeastandmouldsshallbedetermined.Minimumincubationtimesshallbebasedonrecoverymediumcontroltesting(see6.3.2).Recordthenumberofcfuobservedoncountableplates.NOTEPlatesshouldbeobservedperiodicallyduringincubationtopreventtheoccurrenceofuncountableplatesduetoovergrowth.6.3.1.7Determinetheaveragenumberofcolonyformingunitsoncountableplates.Calculatethemicrobialreductionatthespecifiedtimepoints.NOTECountableplatesreferto30cfu/plateto300cfu/plateforbacteriaandyeast,and8cfu/plateto80cfu/plateformoulds,exceptwhencoloniesareobservedonlyforthe100or10-1dilutionplates.6.3.1.8Theabsenceofmicroorganismsshallbedocumented,e.g.byrecordingan"0”or"NR”(norecovery),whenplatesforalldilutionsofasampleatasingletimepointhavezerocolonies.6.3.2Controls6.3.2.1InoculumControlPrepareaninoculumcountbydispersinganidenticalaliquotoftheinoculumintothesamevolumeasusedin6.3.1.1ofasuitablediluent(e.g.DPBST)toachieveafinalconcentrationof1,0105cfu/mlto1,0106cfu/ml.Ensurethatthevolumeofinoculumdoesnotexceed1%ofthesamplevolume.Ensuredispersionoftheinoculumbyadequatemixing.Evaluatethiscontrolsampleforcfu/mlatthebeginningofthetestinordertodemonstratethesuitabilityofthemediumusedforgrowthofthetestorganismandprovideanestimateoftheinitialinoculumconcentration.Platetheappropriatealiquotfromeachtubeontotherecoveryagarplatesintriplicate(unlessotherwisejustified).6.3.2.2RecoverymediumcontrolVortexa1/10dilutionofthedisinfectingproductinthevalidatedneutralizingbroth(1mlinto9ml).Letitstandtoallowneutralizationtobecompleted.Prepareasecondcontroltubewith10mlofasuitablediluent(e.g.DPBST).Inoculatethetubeswithsufficientinoculumtoresultin10cfu/mlto100cfuofchallengeorganismperplate.IncubateforanappropriateperiodoftimeatambienttemperaturebutnotlongenoughtoallowmultiplicationoftheSIST EN ISO 14729:2002



ISO14729:2001(E)©ISO2001–Allrightsreserved9inoculatedorganisms.Platetheappropriatealiquotfromeachtubeontotherecoveryagarplatesintriplicate(unlessotherwisejustified).Incubatebacterialrecoveryplatesat30°Cto35°C.Incubateyeastrecoveryplatesat20°Cto25°Cor30°Cto35°C.Incubatemouldrecoveryplatesat20°Cto25°C.Determineminimumincubationtimesforoptimalrecoveryofbacteria,yeastandmoulds.Checkthattherecoveryfromtheneutralizerbrothisatleast50%oftherecoveryinthesecondcontroltube.Performthiscontrolforeachchallengeorganism.Ifadilutionofgreaterthan1/10isrequiredforneutralization,thenmembranefiltrationshallbeused.Validatetheneutralizationoftheproductwitheachchallengeorganisminitiallyandasappropriate.6.3.2.3ControlspecificationIfanycontrolvalueisoutsidethatspecified,repeattheprocedureastheassociatedtestisinvalid.6.3.3TestreportThetestreportshallspecify:a)referencetothisInternationalStandard;
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