EN 17122-2019 化學(xué)消毒劑和防腐劑 用于評估獸醫(yī)領(lǐng)域使用的化學(xué)消毒劑和防腐劑的殺病毒活性的定量無孔表面試驗 試驗方法和要求 Phase2 step2

EUROPEANSTANDARDNORMEEUROPEENNEEUROP?ISCHENORM

EN17122

November2019

ICS11.080.20

EnglishVersion

Chemicaldisinfectantsandantiseptics-Quantitativenon-

poroussurfacetestfortheevaluationofvirucidalactivity

ofchemicaldisinfectantsandantisepticsusedinthe

veterinaryarea-Testmethodandrequirements-Phase2,

step2

Antiseptiquesetdésinfectantschimiques-Essai

quantitatifdesurfacesnonporeusespourI'évaluation

deI'activitévirucidedesdésinfectantsetantiseptiques

chimiquesutilisésdansledomainevétérinaire-

Méthoded'essaietprescriptions-Phase2,étape2

ChemischeDesinfektionsmittelundAntiseptika-

QuantitativerOberfl?chenversuchzurBestimmungder

viruzidenWirkungchemischerDesinfektionsmittel

undAntiseptikafürdenVeterin?rbereichaufnicht-

por?senOberfl?chen-Prüfverfahrenund

Anforderungen-Phase2,Stufe2

ThisEuropeanStandardwasapprovedbyCENon23September2019.

CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEuropeanStandardthestatusofanationalstandardwithoutanyalteration.Up-to-datelistsandbibliographicalreferences

concerningsuchnationalstandardsmaybeobtainedonapplicationtotheCEN-CENELECManagementCentreortoanyCENmember.

ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).AversioninanyotherlanguagemadebytranslationundertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCEN-CENELECManagementCentrehasthesamestatusastheofficialversions.

CENmembersarethenationalstandardsbodiesofAustria,Belgium,Bulgaria,Croatia,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,

Poland,Portugal,RepublicofNorthMacedonia,Romania,Serbia,Slovakia,Slovenia,Spain,Sweden,Switzerland,TurkeyandUnitedKingdom.

EUROPEANCOMMITTEEFORSTANDARDIZATION

COMITEEUROPéENDENORMALISATION

EUROPAISCHESKOMITEEFüRNORMUNG

CEN-CENELECManagementCentre:RuedelaScience23,B-1040Brussels

◎2019CENAllrightsofexploitationinanyformandbyanymeansreservedRef.No.EN17122:2019E

worldwideforCENnationalMembers.

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EN17122:2019(E)

ContentsPage

EuropeanForeword 3

Introduction 4

1Scope 5

2Normativereferences 5

3Termsanddefinitions 5

4Requirementsforvirucidalactivityonsurfaces 6

5Testmethod 6

5.1Principle 6

5.2Materialsandreagents,includingcellcultures 7

5.3Apparatusandglassware 10

5.4Preparationoftestorganismsuspensionsandproducttestsolutions 12

5.5Procedureforassessingthevirucidalactivityoftheproduc 13

5.6Experimentaldataandcalculatio 17

5.7Verificationofthemethodology 17

5.8Expressionofresults 18

5.9Testreport 18

AnnexA(informative)Referencedstrainsofnationalcollections 20

AnnexB(informative) 21

AnnexC(informative)Calculationofvirusinfectivitytitre 24

AnnexD(informative)Exampleofatypicaltestreport 31

AnnexE(informative)Fullvirucidalactivityandactivityagainstenvelopedvirusesonly 33

Bibliography 34

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EN17122:2019(E)

EuropeanForeword

Thisdocument(EN17122:2019)hasbeenpreparedbyTechnicalCommitteeCEN/TC216"Chemicaldisinfectantsandantiseptics",thesecretariatofwhichisheldbyAFNOR.

ThisEuropeanStandardshallbegiventhestatusofanationalstandard,eitherbypublicationofanidenticaltextorbyendorsement,atthelatestbyMay2020,andconflictingnationalstandardsshallbewithdrawnatthelatestbyMay2020.

Attentionisdrawntothepossibilitythatsomeoftheelementsofthisdocumentmaybethesubjectofpatentrights.CENshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.

AccordingtotheCEN-CENELECInternalRegulations,thenationalstandardsorganisationsofthefollowingcountriesareboundtoimplementthisEuropeanStandard:Austria,Belgium,Bulgaria,Croatia,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France,Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal,RepublicofNorthMacedonia,Romania,Serbia,Slovakia,Slovenia,Spain,Sweden,Switzerland,TurkeyandtheUnitedKingdom.

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EN17122:2019(E)

Introduction

ThisdocumentdescribesasurfacetestmethodforestablishingwhetheraproductproposedasadisinfectantinthefieldsdescribedinClause1hasordoesnothavevirucidalactivityonnon-poroussurfaces.

Thelaboratorytestcloselysimulatespracticalconditionsofapplication.Chosenconditions(contacttime,temperature,organismsonsurfacesetc.)reflectparameterswhicharefoundinpracticalsituationsincludingconditionswhichmayinfluencetheactionofdisinfectants.Eachuseconcentrationfoundfromthistestcorrespondstodefinedexperimentalconditions.

Theconditionsareintendedtocovergeneralpurposesandtoallowreferencebetweenlaboratoriesandproducttypes.Eachutilizationconcentrationofthechemicaldisinfectantfoundbythistestcorrespondstodefinedexperimentalconditions.

However,forspecialapplicationstherecommendationsforuseofaproductcandifferandthereforeadditionaltestconditionsmightbeneeded,whichcannotbecoveredbythisdocument.

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EN17122:2019(E)

1Scope

Thisdocumentspecifiesatestmethodandtheminimumrequirementsforvirucidalactivityofchemicaldisinfectantsthatformahomogeneousphysicallystablepreparationwhendilutedwithhardwater,or-inthecaseofready-to-use-products-withwater.

Thisdocumentappliestoproductsthatareusedfordisinfectingwithoutmechanicalactionnon-poroussurfacesintheveterinaryarea-i.e.inthebreeding,husbandry,production,veterinarycarefacilities,transportanddisposalofallanimalsexceptwheninthefoodchainfollowingdeathandentrytotheprocessingindustry.

EN14885specifiesindetailtherelationshipofthevariousteststooneanotherandto“userecommendations".

NOTE1Themethoddescribedisintendedtodeterminetheactivityofcommercialformulationsoractivesubstancesundertheconditionsinwhichtheyareused.

NOTE2ThismethodcorrespondstoaPhase2Step2test.

NOTE3Usingthisdocument,itispossibletodeterminethevirucidalactivityoftheundilutedproduct.

NOTE4ThisdocumentusesPorcineParvovirusbecauseBovineEnterovirusE(formerBovineEnterovirusType1(ECBO))virususedinthesuspensiontestEN14675cannotbeusedforsurfacetestingbecauseofitslossoftitreduringdrying.PorcineParvovirushascomparableresistancetoECBOvirus.

2Normativereferences

Thefollowingdocumentsarereferredtointhetextinsuchawaythatsomeoralloftheircontentconstitutesrequirementsofthisdocument.Fordatedreferences,onlytheeditioncitedapplies.Forundatedreferences,thelatesteditionofthereferenceddocument(includinganyamendments)applies.EN12353,Chemicaldisinfectantsandantiseptics-Preservationoftestorganismsusedforthedeterminationofbactericidal(includingLegionella),mycobactericidal,sporicidal,fungicidalandvirucidal(includingbacteriophages)activity

EN14675,Chemicaldisinfectantsandantiseptics-Quantitativesuspensiontestfortheevaluationofvirucidalactivityofchemicaldisinfectantsandantisepticsusedintheveterinaryarea-Testmethodandrequirements(Phase2,step1)

EN14885,Chemicaldisinfectantsandantiseptics—ApplicationofEuropeanStandardsforchemicaldisinfectantsandantiseptics

EN10088-2,Stainlesssteels-Part2:Technicaldeliveryconditionsforsheet/plateandstripofcorrosionresistingsteelsforgeneralpurposes

3Termsanddefinitions

Forthepurposesofthisdocument,thetermsanddefinitionsgiveninEN14675andEN14885applyISOandIECmaintainterminologicaldatabasesforuseinstandardizationatthefollowingaddresses:

·IECElectropedia:availableat

/

·ISOOnlinebrowsingplatform:availableat

/obp

EN17122:2019(E)

6

4Requirementsforvirucidalactivityonsurfaces

Theproductshalldemonstrateatleastadecimallog(lg)reductionof3invirustitreoftheparvovirusandcoronavirusteststrainswhentestedinaccordancewithTable1andClause5.ToclaimvirucidalactivityagainstenvelopedvirusestheproductshallpassthisstandardwiththecoronavirusteststrainandtoclaimfullvirucidalactivitytheproductshallpassbothEN14675withthebovineenterovirusteststrainandthisstandardwiththeporcineparvovirusteststrain.

NOTESeeAnnexEforfurtherinformationontheappropriatenessofclaimsofvirucidalactivityagainstenvelopedvirusesandfullvirucidalactivity

Table1-Minimumandadditionaltestconditions

Minimumspectrumoftestorganisms

Fullvirucidalactivity

PorcineParvovirus,StrainNADL2

Virucidalactivityagainstenvelopedviruses

FelineCoronavirus,StrainMunich

Testtemperature

10℃±1℃

Additionaltemperatures

4℃±1℃;20℃±1℃;40℃±1℃

Contacttime

Thecontacttime(s)shallbeselectedfromthevaluesgivenbelowa

Minimumcontacttime

1min±5s

Othercontacttimes

5min±10s,15min±10s,30min±10s,60min±10s

Maximumcontacttime

120min±10s

Interferingsubstances-lowlevelsoiling

3,0g/lbovinealbumin

Interferingsubstances-highlevelsoiling

10g/lyeastextractand10g/lbovinealbumin

Additionalconditionsb

Furthercontacttime(s),interferingsubstance(s)orvirus(es)

aThecontacttimesforsurfacedisinfectantsstatedinthistablearechosenonthebasisofthepracticalconditionsoftheproduct.Therecommendedcontacttimefortheuseoftheproductiswithintheresponsibilityofthemanufacturer.

bWhereappropriate(specificpurposes),additionalspecificvirucidalactivityshallbedeterminedunderotherconditionsoftime,temperature,andinterferingsubstances(see)inaccordancewith5.5,inordertotakeintoaccountintendedspecificuseconditions.Additionalvirus(es)canbetested,ifrelevant.Fortheadditionalconditions,theconcentrationdefinedasaresultcanbelowerthantheoneobtainedundertheminimumtest

conditions.

Thedeterminedvirucidalconcentrationofthetestproductissuggestedasbeingsuitableforpracticalsituationsofuse.

5Testmethod

5.1Principle

5.1.1Outline

Atestsuspensionofvirusesinasolutionofinterferingsubstancesisinoculatedontoatestsurfaceanddried.Apreparedsampleoftheproductundertestisappliedinamannerwhichcoversthedriedfilm.

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EN17122:2019(E)

Thetestsurfaceismaintainedataspecifiedtemperatureforadefinedperiodoftime.Thetestsurfaceistransferredtocellmaintenancemediumsothattheactionofthedisinfectantisimmediatelyneutralized.Thetitreofthevirusrecoveredfromthetestsurfaceisdetermined.

Thetitreoftheinoculumonatestsurfacetreatedwithhardwaterinplaceofthedisinfectantisalsodeterminedandthereductioninvirustitreattributedtotheproductiscalculatedbydifference.

5.1.2TestOrganisms

ThetestisperformedusingthetestorganismsasspecifiedinClause4,Table1.

5.1.3Variations

OthercontacttimesandtemperatureswithinthelimitsspecifiedinClause4,Table1maybeused.Additionalinterferingsubstancesandtestorganismsmaybeused.

5.2Materialsandreagents,includingcellcultures

5.2.1Testorganisms

ThevirucidalactivityshallbeevaluatedusingthefollowingstrainsastestorganismsselectedaccordingtoClause4,Table1

a)Non-envelopedDNAvirus

PorcineParvovirusstrainNADL2(PPV)

b)EnvelopedRNAvirus

FelineCoronavirus,strainMunich(FeCoV)

NOTEVirusstrainscanbeobtainedfromanationalorinternationalculturecollection.PPVandFeCoVcanbeobtainedfromtheFriedrich-Loeffler-Insitut,BundesforschungsinstitutfürTiergesundheit,HauptsitzInselRiemsSüdufer10,17493,Greifswald-InselRiems1.

Therequiredincubationtemperatureforthesetestorganismsis(36±1)℃or(37±1)℃(

2).The

sametemperature(either36℃or37℃)shallbeusedforallincubationsperformedduringatestanditscontrolandvalidation.

Ifadditionaltestorganismsareused,theyshallbekeptandusedunderoptimumgrowthconditions(temperature,time,atmosphere,media)notedinthetestreport.Iftheseadditionaltestorganismsarenotclassifiedatareferencecentre,theiridentificationcharacteristicsshallbestated.Inaddition,theyshallbeheldbythetestinglaboratoryornationalculturecollectionunderareferenceforfiveyears.

5.2.2Culturemedia,reagentsandcellcultures

General

Allweightsofchemicalsubstancesgiveninthisdocumentrefertotheanhydroussalts.Hydratedformsmaybeusedasanalternative,buttheweightsrequiredshallbeadjustedtoallowforconsequentmolecularweightdifferences.

Thereagentsshallbeofanalyticalgradeand/orappropriateformicrobiologicalpurposes.Theyshallbefreefromsubstancesthataretoxicorinhibitorytothetestorganisms.

1ThisinformationisgivenfortheconvenienceofusersofthisstandardanddoesnotconstituteanendorsementbyCENofthisinstitute.

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EN17122:2019(E)

Toimprovereproducibility,itisrecommendedthatcommerciallyavailable-dehydratedifappropriate

-materialisusedforthepreparationofculturemedia.Themanufacturer'sinstructionsrelatingtothepreparationoftheseproductsshouldberigorouslyfollowed.

Foreachculturemediumandreagent,atimelimitationforuseshouldbefixed.

AllspecifiedpHvaluesaremeasuredat(20±1)℃.

Water

Thewatershallbefreshlyglass-distilledwaterandnotdemineralizedwater.Ifdistilledwaterofadequatequalityisnotavailable,waterforinjections(seebibliographicreference[1])maybeused.

Sterilizeintheautoclave[

a].Sterilizationisnotnecessaryifthewaterisusede.g.forpreparation

ofculturemediaandsubsequentlysterilized.

See

fortheproceduretopreparehardwater.

Phosphatebufferedsaline(PBS)

Sodiumchloride(NaCl)

Potassiumchloride(KCI)

Disodiumhydrogenphosphate,12-hydrate(Na?HPO4x12H?O)Potassiumphosphate,monobasic(KH?PO4)

Water(

)

NeutralRed(1:1000solution)

8,00g

0,20g2,89g

0,20gto1000,0ml

Prepareneutralred(SigmaN7005)2stocksolutionat0,1mg/mlinwater(

).Filter

througha0,44μmporesizefilterandstoreat4℃inthedark.

Foetalcalfserum(FCS)

FCSshallbecertifiedfreeofvirusesandmycoplasma.Extraneousvirusesandmycoplasmamayinterferewithcellandvirusgrowthresultinginfalseresults.

Trichloroaceticacid(10%solution)(TCA)

Dissolve10gofTCAcrystalsin80mlofwater(

),andthenadjustthevolumeto

100mlwithwater.Stirtocompletesolution.

Hardwaterfordilutionofproducts

Forthepreparationof1lofhardwater,theprocedureisasfollows:

-preparesolutionA:dissolve19,84gmagnesiumchloride(MgCl2)and46,24gcalciumchloride(CaCl2)inwater(

)and

diluteto1000ml.Sterilizebymembranefiltration(

c)orintheautoclave[

a].Autoclaving-ifused-maycausealossofliquid.Inthiscasemakeupto

1000mlwithwater(

)under

asepticconditions.Storethesolutionintherefrigerator(

)for

nolongerthanonemonth;

2SigmaN7005isanexampleofasuitableproductavailablecommercially.ThisinformationisgivenfortheconvenienceofusersofthisstandardanddoesnotconstituteanendorsementbyCENofthisproduct.

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EN17122:2019(E)

一preparesolutionB:dissolve35,02gsodiumbicarbonate(NaHCO?)inwater(

)and

diluteto1000ml.Sterilizebymembranefiltration(

c).Storethesolutionintherefrigerator(

)

fornolongerthanoneweek;

-place600mlto700mlofwater(

)in

a1000mlvolumetricflask(

)and

add6,0mlofsolutionA,then8,0mlofsolutionB.Mixanddiluteto1000mlwithwater(

).ThepH(

)

ofthehardwatershallbe7,0±0,2.Ifnecessary,adjustthepHbyusingasolutionofapproximately40g/l(about1mol/l)ofsodiumhydroxide(NaOH)orapproximately36,5g/l(about1mol/l)ofhydrochloricacid(HCl).

Thehardwatershallbefreshlypreparedunderasepticconditionsandusedwithin12h.

NOTEWhenpreparingtheproducttestsolutions(5.4.2),theadditionoftheproducttothehardwaterproducesdifferentfinalwaterhardnessineachtesttube.Inanycase,thefinalhardnessinthetesttubeexpressedascalciumcarbonate(CaCO?)islowerthan375mg/L.

Interferingsubstance

.1General

Theinterferingsubstanceshallbechosenaccordingtotheconditionsofuselaiddownfortheproduct.

Theinterferingsubstanceshallbesterileandpreparedat10timesitsfinalconcentrationinthetest.

Theioniccomposition(e.g.pH,calciumand/ormagnesiumhardness)andchemicalcomposition(e.g.mineralsubstances,protein,carbohydrates,lipidsanddetergents)shallbedefined.

"Diluent"isgenerallyusedintheotherEuropeanStandardsintheveterinaryareatopreparetheinterferingsubstance.Sincethereisnoexperienceinvirucidaltestingwithdiluent,water(

)isused

instead.

NOTETheterm"interferingsubstance"isusedevenifitcontainsmorethanonesubstance.

.2Lowlevelsoiling(Bovinealbuminsolution)

Bovineserumalbuminshallbeusedascommerciallyavailableorshallbepreparedasfollows:

Dissolve3gofbovinealbuminfractionV(suitableformicrobiologicalpurposes)in90mlofwater(

)ina

100mlvolumetricflask(

).Makeuptothemarkwithwater(

).

Sterilizebymembranefiltration(

c).Keepinarefrigerator(

)andusewithinonemonth.

Thefinalconcentrationofbovinealbumininthetestprocedure(5.5)is3g/L.

.3Highlevelsoiling(mixtureofbovinealbuminsolutionwithyeastextract)

Dissolve50gyeastextractpowderin150mlofwater(

)in

a250mlvolumetricflask(

)and

allowfoamtocollapse.Makeuptothemarkwithwater(

).Transfer

toacleandrybottleandsterilizeintheautoclave(

a).Allowtocoolto20℃±1℃.

Pipette25mlofthissolutionintoa50mlvolumetricflask(

)and

add10mlofwater(

).

Dissolve5gofbovinealbuminfractionV(suitableformicrobiologicalpurposes)inthesolutionwithshakingandallowfoamtocollapse.Makeuptothemarkwithwater(

),sterilize

bymembranefiltration(

c),keepinarefrigerator(at2℃to8℃)(

)and

usewithinonemonth.

Thefinalconcentrationinthetestprocedure(5.5)is10g/lyeastextractand10g/lbovinealbumin.

Mediumforcellcultures

Eagle'sminimalessentialmedium(MEM)orequivalent,supplementedwithFCS(

),antibiotics,and

othergrowthfactorsasneededshallbeused.

EN17122:2019(E)

10

a)Agrowthmediumforcellmultiplicationissupplementedwith10%FCS.Add10partsofFCS(

)

to90partsofMEM.

b)Amaintenancemediumtomaintainthecellculturemetabolismwithoutstimulationofcellproliferationissupplementedwith2%FCS.Add2partsofFCS(

)to98partsofMEM.

Othermediamaybeusedifappropriateforcertaincelllines.

Seealsobibliographicreference[2],EN14675andEN12353formoredetaileddescriptions.

0

Cellcultures

Cellmonolayersshallbe>90%confluentbeforeinoculation.Celllinesareselectedinaccordancewiththeirsensitivitytothetestorganisms(5.2.1).Cellsforvirustitration,ifusedassuspensionsinquantaltests,shallbeaddedtothedilutionsofthetestmixture(5.5.2)insuchadensityastoenabletheformationofamonolayernolongerthan2daysinthecellcontrol.Cellculturescanbeusedascellmonolayersorinsuspensionsforquantaltests.Fordetailsofcelllinessee5.5.1e).

5.3Apparatusandglassware

5.3.1General

Sterilizeallglasswareandpartsoftheapparatusthatwillcomeintocontactwiththeculturemediaandreagentsorthesample,exceptthosewhicharesuppliedsterile,byoneofthefollowingmethods:

a)bymoistheat,intheautoclave[

a];

b)bydryheat,inthehotairoven[

b].

5.3.2Usualmicrobiologicallaboratoryequipment3

And,inparticular,thefollowing:

Apparatusforsterilization(moistanddryheat)

a)Formoistheatsterilization,anautoclavecapableofbeingmaintainedat(121±3)℃foraminimumholdingtimeof15min;

b)fordryheatsterilization,ahotairovencapableofbeingmaintainedatforaminimumholdingtimeof30min,at(170+5)℃foraminimumholdingtimeof1horatCforaminimumholdingtimeof2h.

c)formediasterilization,usesuitablemembranefiltrationapparatuswithfiltersofdiameter47mmto50mmandmembraneswith0,22μmporesize.

Waterbaths,capableofbeingcontrolledat(20±1)℃,andatadditionaltesttemperatures±1℃(5.5.1).

Invertedmicroscopeforreadingcellculturesmicroscopically

pHmeter,havinganaccuracyofcalibrationof0,1pHunitsat20℃.

Stopwatch

3Disposablesterileequipmentisanacceptablealternativetoreusableglassware.

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EN17122:2019(E)

Refrigerator,capableofbeingcontrolledat2℃to8℃

Electromechanicalagitatore.g.Vortex@mixer4

Containers:Petriplates,steriletesttubes,culturebottlesorflasksofsuitablecapacity.

5.3.2.9Volumetricflasks,calibratedat20℃.

0

Microtitreplatesortubes,petridishesandflasksforcellcultureuse.

1

Membranefiltrationapparatusforfiltration

2

CO2incubator(95%air,5%CO2),capableofbeingcontrolledat(36±1)℃,forincubationofcellcultures.Anincubatorat(37±1)℃maybeusedifanincubatorat(36±1)℃isnotavailable.

3

Graduatedsterilepipettesofnominalcapacities10mland1mland0,1ml.NOTECalibratedautomaticpipettescanbeused.

4

Magneticstirrerforkeepingcellsinsuspensionbeforeseeding

5

Iceproducingmachineorcommerciallyavailableicetocoolthecellmaintenancemediumandthereactionmixturesduringthetest(see5.5.2and5.5.4)

6

Basinasicebathwithiceandwater

7

Mechanicalshaker

8

Centrifuge

9

Biologicalsafetycabinet,classⅡ

0

Freezer,-70℃orless

1

Temperaturecontrolledcabinet,capableofbeingcontrolledat10℃±1℃

2

VacuumDiaphragmPump(e.g.throughputmax.3,8m3/hfinalvacuum<75mbar)

3

Cryotubes

5.3.3Testsurface

Theseshallbe1.4301(EN10088-1)stainlesssteeldiscs(2cmdiameterdiscs)withGrade2Bfinishonbothsides,inaccordancewiththerequirementsofEN10088-2.Thediscsshallbeasflataspossibleandthisisbestachievedbyusingstainlesssteelofagaugeof1,2mmor1,5mm.Thediscsshallbehandledonlywithforcepsandusedonlyonce.

4Vortexisanexampleofasuitableproductavailablecommercially.ThisinformationisgivenfortheconvenienceofusersofthisstandardanddoesnotconstituteanendorsementbyCENofthisproduct.

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EN17122:2019(E)

Priortousethediscsshallbeplacedinacontainerwithanappropriatequantityof5%pervolumeDecon900,orof1%Blanisol-Pur?for60min,inamannersothattheydonotsticktogetherandthesurfaceisnotdamaged.Rinsethediscswithrunningfreshlydistilledwater(

)or

demineralizedwaterfor10s,preventingthemfromdryingtoanyextent.

Rinsethediscswithwater(

)for

afurther10stoensurecompleteremovalofthesurfactant.Tosupplyasatisfactoryflowofwater,asterilizedfluiddispensingpressurevesselwithsuitablehoseandconnectorsorothersuitablemethodcanbeusedandregulatedtosupplyapproximately2000mlpermin.Dipthediscinabathcontainingisopropanol(IPA)for15min.Removethediscsandrinsethemwithwater(

)for

atleast10s.Sterilizebyautoclaving.

NOTESuitablestainlesssteeldiscscanusuallybepurchasedfromlocalengineeringcompanies.

5.4Preparationoftestorganismsuspensionsandproducttestsolutions

5.4.1Testorganismsuspensions(testvirussuspension)

ThetestorganismsandtheirstockculturesshallbepreparedandkeptinaccordancewithEN12353.

Thestockvirussuspensionismultipliedinanappropriatecelinethatproduceshightitresofinfectiousviruses.Thecelldebrisisseparatedbycentrifugation(400gnfor15min).Thispreparationiscalledthe"testvirussuspension".

Itissuggestedthattheminimumtitreofthevirussuspension-determinedbyaquantaltest[5.5.2a]orbyaplaquetest(5.5.2b)-isatleast108TCID50/ml.Inanycase,itshallbesufficientlyhightoenableatleastatitrereductionof3lgtoverifythemethod.

Ifnecessarythetestsuspensionmaybeconcentratedbyappropriatemethods(e.g.ultracentrifugation).

Thetestsuspensioniskeptinsmallvolumesbelow-70℃orpreferablybetween-135℃and-196℃inliquidnitrogenvaporphaseorliquidnitrogen(withsuitabletightcryotubes,

3).

Duetosafetyreasons,and-insomecases-toavoidthepossibilityofgeneticmutations,only10passagesfromtheoriginalseed(e.g.virusfromculturecollection)areallowed.

Thetestsuspensionisusedundilutedforthetestprocedure(5.5.2or5.5.3).

5.4.2Producttestsolution

Detailedsampleinformationoftheproduct,asreceivedfromtheproducerorfromanyothersource,shallberecorded.

Producttestsolutionsshallbepreparedinhardwater(

)at

aminimumofthreedifferentconcentrationstoincludeoneconcentrationintheactiverangeandoneconcentrationinthenon-activerange.Theproduct,asreceived,maybeusedasoneoftheproducttestsolutions.

Dilutionsofready-to-useproducts,ductswhicharenotdilutedwhenap