USP-51抗菌效力測(cè)試

FILENAMEUSP_51.doc第1頁(yè)，共8頁(yè)51ANTIMICROBIALEFFECTIVENESSTESTING抗菌的效力測(cè)試Antimicrobialpreservativesaresubstancesaddedtononsteriledosageformstoprotectthemfrommicrobiologicalgrowthorfrommicroorganismsthatareintroducedinadvertentlyduringorsubsequenttothemanufacturingprocess.Inthecaseofsterilearticlespackagedinmultiple-dosecontainers,antimicrobialpreservativesareaddedtoinhibitthegrowthofmicroorganismsthatmaybeintroducedfromrepeatedlywithdrawingindividualdoses.抗菌防腐劑是實(shí)際被添加到有菌的劑量配方里的物質(zhì)，使他們不受微生物滋長(zhǎng)或是被無(wú)意中引進(jìn)到生產(chǎn)過(guò)程中的微生物侵害。在無(wú)菌包裝多劑量配方的容器里，抗菌防腐劑被添加用來(lái)抑制由重復(fù)添加單劑量所引進(jìn)的微生物的滋長(zhǎng)。Antimicrobialpreservativesshouldnotbeusedasasubstituteforgoodmanufacturingpracticesorsolelytoreducetheviablemicrobialpopulationofanonsterileproductorcontrolthepresterilizationbioburdenofmultidoseformulationsduringmanufacturing.AntimicrobialpreservativesincompendialdosageformsmeettherequirementsforAddedSubstancesunderIngredientsandProcessesintheGeneralNotices.抗菌防腐劑不應(yīng)被作為在良好生產(chǎn)操作中的替代方式使用，或僅是用來(lái)減少一個(gè)有菌產(chǎn)品的微生物群數(shù)量，或是在生產(chǎn)時(shí)控制多劑量的微生物量。在劑量表里抗菌防腐劑與添加的要求必需符合規(guī)定。Allusefulantimicrobialagentsaretoxicsubstances.Formaximumprotectionofpatients,theconcentrationofthepreservativeshowntobeeffectiveinthefinalpackagedproductshouldbebelowalevelthatmaybetoxictohumanbeings.Theconcentrationofanaddedantimicrobialpreservativecanbekeptataminimumiftheactiveingredientsoftheformulationpossessanintrinsicantimicrobialactivity.Antimicrobialeffectiveness,whetherinherentintheproductorwhetherproducedbecauseoftheadditionofanantimicrobialpreservative,mustbedemonstratedforallinjectionspackagedinmultiple-dosecontainersorforotherproductscontainingantimicrobialpreservatives.Antimicrobialeffectivenessmustbedemonstratedformultiple-dosetopicalandoraldosageformsandforotherdosageformssuchasophthalmic,otic,nasal,irrigation,anddialysisfluids(seePharmaceuticalDosageForms(1151).所有有用的抗菌藥劑都是有毒物質(zhì)。為了最大的保護(hù)使用者，防腐劑的含量應(yīng)被有效地顯示在產(chǎn)品的最終包裝上，并應(yīng)會(huì)處在一個(gè)可能對(duì)人體有毒的水平線下。添加的抗菌防腐劑含量能夠被保持在一個(gè)最小的濃度，如果配方的活性成分具有一個(gè)固定的抗菌力?？咕男ЯΓ还苁钱a(chǎn)品里固有的或者是生產(chǎn)中添加的抗菌防腐劑，都必須在所有注入包裝的多劑量容器里或其它包含抗菌防腐劑的產(chǎn)品上顯示?？咕栏瘎┍仨毐伙@示在多劑量和口頭劑量型和給其它劑量型，如眼藥，耳藥，鼻藥，沖洗的和透析液。(看PharmaceuticalDosageForms(a1151).Thischapterprovidesteststodemonstratetheeffectivenessofantimicrobialprotection.Addedantimicrobialpreservativesmustbedeclaredonthelabel.Thetestsandcriteriaforeffectivenessapplytoaproductintheoriginal,unopenedcontainerinwhichitwasdistributedbythemanufacturer.本章提供的測(cè)試用來(lái)顯示抗菌保護(hù)的效力。添加的抗菌防腐劑必須顯示在標(biāo)簽上。測(cè)試的效力和標(biāo)準(zhǔn)適用于一個(gè)產(chǎn)品在原有的、未打開(kāi)過(guò)的經(jīng)由制造商銷(xiāo)售的容器里。

PRODUCTCATEGORIES產(chǎn)品分類(lèi)Forthepurposeoftesting,compendialarticleshavebeendividedintofourcategories(seeTable1).Thecriteriaofantimicrobialeffectivenessfortheseproductsareafunctionoftherouteofadministration.為了測(cè)試的目的，配制產(chǎn)品分開(kāi)為四個(gè)分類(lèi)（看表1）?？咕ЯΦ臉?biāo)準(zhǔn)對(duì)于這些產(chǎn)品是一個(gè)管理的作用。Table1.CompendialProductCategories表1:配制藥品分類(lèi)Category類(lèi)別ProductDescription產(chǎn)品概述1Injections,otherparenteralsincludingemulsions,oticproducts,sterilenasalproducts,andophthalmicproductsmadewithaqueousbasesorvehicles.針劑，其它非腸胃藥劑，包括乳劑、無(wú)菌耳用劑、鼻用劑和眼用劑含水的或其它介質(zhì)的。2Topicallyusedproductsmadewithaqueousbasesorvehicles,nonsterilenasalproducts,andemulsions,includingthoseappliedtomucousmembranes.局部使用產(chǎn)品，含水的或其它介質(zhì)，如未滅菌耳用劑產(chǎn)品、軟膏，包括那些涂在黏膜上的。3Oralproductsotherthanantacids,madewithaqueousbasesorvehicles.口服產(chǎn)品，除了以水為基底或其它的介質(zhì)的解酸劑。4Antacidsmadewithanaqueousbase.含水解酸劑。

TESTORGANISMS測(cè)試生物體Useculturesofthefollowingmicroorganisms1:Candidaalbicans(ATCCNo.10231),Aspergillusniger(ATCCNo.16404),Escherichiacoli(ATCCNo.8739),Pseudomonasaeruginosa(ATCCNo.9027),andStaphylococcusaureus(ATCCNo.6538).TheviablemicroorganismsusedinthetestmustnotbemorethanfivepassagesremovedfromtheoriginalATCCculture.Forpurposesofthetest,onepassageisdefinedasthetransferoforganismsfromanestablishedculturetofreshmedium.Alltransfersarecounted.Inthecaseoforganismsmaintainedbyseed-lottechniques,eachcycleoffreezing,thawing,andrevivalinfreshmediumistakenasonetransfer.使用以下微生物的培養(yǎng)基：白念珠菌（ATCCNO.10231）,黑曲霉菌（ATCCNO.16404）,大腸桿菌（ATCCNO.8739）,綠膿桿菌(ATCCno.9027),和金黃色葡萄球菌(ATCCNO.6538).在測(cè)試中使用活的微生物從原始的培養(yǎng)基移轉(zhuǎn)次數(shù)不得超過(guò)五次。為了測(cè)試的目的，「移轉(zhuǎn)」的定義是從一個(gè)已確定的培養(yǎng)基移到一個(gè)新鮮的媒介里。所有的移轉(zhuǎn)均應(yīng)被計(jì)算在內(nèi)的。一旦有機(jī)體被以種子庫(kù)(seed-lot)技術(shù)保存下來(lái)時(shí)，每個(gè)在新鮮媒介里冰凍的，融化的和再生的循環(huán)周期被認(rèn)為是一次轉(zhuǎn)移。Aseed-stocktechniqueshouldbeusedforlong-termstorageofcultures.CulturesreceivedfromtheATCCshouldberesuscitatedaccordingtodirections.Ifgrowninbroth,thecellsarepelletedbycentrifugation.Resuspendin1/20ththevolumeoffreshmaintenancebroth,andaddanequalvolumeof20%(v/vinwater)sterileglycerol.Cellsgrownonagarmaybescrapedfromthesurfaceintothe10%glycerolbroth.Dispensesmallaliquotsofthesuspensionintosterilevials.種子存儲(chǔ)技術(shù)將會(huì)被使用到培養(yǎng)的長(zhǎng)期存儲(chǔ)中。從ATCC得到的培養(yǎng)基將會(huì)依照操作方向蘇醒。如果在液體培養(yǎng)基里生長(zhǎng)，細(xì)胞會(huì)通過(guò)離心法的方式被聚集成塊。懸浮在1/20容量的新鮮的液體培養(yǎng)基里，并添加一個(gè)同等容量的20%（V/V水里）的無(wú)菌的甘油。從表面到10%的甘油液體培養(yǎng)基里可能得到在瓊膠培養(yǎng)基上的細(xì)胞生長(zhǎng)。分配小部分的懸浮液到無(wú)菌的小瓶里。Storethevialsinliquidnitrogenorinamechanicalfreezeratnomorethan-50.Whenafreshseed-stockvialisrequired,itmayberemovedandusedtoinoculateaseriesofworkingcultures.Theseworkingculturesmaythenbeusedperiodically(eachdayinthecaseofbacteriaandyeast)tostarttheinoculumculture.把小瓶子放在液化氮里或一個(gè)不超過(guò)-50攝氏度的機(jī)械冷藏室里。當(dāng)要求一個(gè)新鮮的種子存儲(chǔ)瓶時(shí)，它可能被移除和使用來(lái)給一系列的有效的培養(yǎng)菌做接種。這些有效的培養(yǎng)基到時(shí)可以被定期地（每天，當(dāng)測(cè)試細(xì)菌和酵母時(shí)）使用來(lái)啟動(dòng)接種培養(yǎng)基。

MEDIA媒介Allmediausedinthetestmustbetestedforgrowthpromotion.UsethemicroorganismsindicatedaboveunderTestOrganisms.測(cè)試中所有媒介的使用，必須做促生長(zhǎng)測(cè)試。在有機(jī)體測(cè)試下使用上面指定的測(cè)試微生物。

PREPARATIONOFINOCULUM接種體的準(zhǔn)備Preparatorytothetest,inoculatethesurfaceofasuitablevolumeofsolidagarmediumfromarecentlyrevivedstockcultureofeachofthespecifiedmicroorganisms.ThecultureconditionsfortheinoculumculturearedescribedinTable2inwhichthesuitablemediaareSoybean–CaseinDigestorSabouraudDextroseAgarMedium(seeMicrobialEnumerationTests(61)andAbsenceofSpecifiedMicroorganisms62)).準(zhǔn)備測(cè)試，由最近再生的培養(yǎng)基的表面適當(dāng)?shù)慕臃N特定的微生物。接種培養(yǎng)基的描述請(qǐng)參考表2，表2里的適合的培養(yǎng)基是黃豆-消化酪蛋白(soybean–caseindigest)或沙保羅氏葡萄糖瓊脂培養(yǎng)基。（參看MicrobialEnumerationTests(61)和AbsenceofSpecifiedMicroorganism62）ToharvestthebacterialandC.albicanscultures,usesterilesalineTS,washingthesurfacegrowth,collectingitinasuitablevessel,andaddingsufficientsterilesalineTStoobtainamicrobialcountofabout1×108colony-formingunits(cfu)permL.ToharvestthecellsofA.niger,usesterilesalineTScontaining0.05%ofpolysorbate80,andaddsufficientsterilesalineTStoobtainacountofabout1×108cfupermL.為了獲得細(xì)菌和白色念珠菌(C.albicans)培養(yǎng)基，使用干凈的鹽水溶液(SalineTS，注:9克氯化鈉溶于1000mL.的水)，清洗表面的生成物，把它收集在一個(gè)合適的容器里，并添加足夠的鹽水溶液以獲得一個(gè)每毫升大約1×108cfu的微生物計(jì)數(shù)。為了獲得黑曲霉菌(A.niger)細(xì)胞，使用干凈的鹽水溶液含0.05%的聚山梨醇酯80(polysorbate80)，并添加足夠的鹽水溶液來(lái)獲得一個(gè)大約1×108cfu每毫升的計(jì)數(shù)。Alternatively,thestockcultureorganismsmaybegrowninasuitableliquidmedium(i.e.,Soybean–CaseinDigestBrothorSabouraudDextroseBroth)andthecellsharvestedbycentrifugation,thenwashedandresuspendedinsterilesalineTStoobtainamicrobialcountofabout1×108cfupermL.[NOTE—Theestimateofinoculumconcentrationmaybeperformedbyturbidimetricmeasurementsforthechallengemicroorganisms.Refrigeratethesuspensionifitisnotusedwithin2hours.]或者，一個(gè)合適的液體的培養(yǎng)液（如，黃豆-消化酪蛋白液soybean–caseindigestbroth或沙氏瓊脂培養(yǎng)液sabourauddextrosebroth）可能生長(zhǎng)出培養(yǎng)菌微生物并通過(guò)離心法得到細(xì)胞，然后以無(wú)菌的鹽水溶液清洗和重新懸浮以獲得一個(gè)大約1×108cfu每毫升的微生物計(jì)數(shù)。（注意–預(yù)計(jì)的接種濃度可能通過(guò)濁度計(jì)測(cè)量來(lái)挑戰(zhàn)微生物。如果懸浮液沒(méi)有在兩個(gè)小時(shí)內(nèi)被使用，請(qǐng)冷凍它）DeterminethenumberofcfupermLineachsuspension,usingtheconditionsofmediaandmicrobialrecoveryincubationtimeslistedinTable2toconfirmtheinitialcfupermLestimate.Thisvalueservestocalibratethesizeofinoculumusedinthetest.Thebacterialandyeastsuspensionsaretobeusedwithin24hoursofharvest,butthefungalpreparationmaybestoredunderrefrigerationforupto7days.測(cè)出每個(gè)懸浮液的每毫升的菌落數(shù)量，使用在TABLE2里的媒介和微生物的恢復(fù)培養(yǎng)期的情況來(lái)確認(rèn)估計(jì)每毫升的原始cfu。這個(gè)值可以用在測(cè)試中做接種尺寸的比對(duì)。細(xì)菌和酵母菌的懸浮液在24小時(shí)收獲期里應(yīng)被使用，但真菌的準(zhǔn)備可在冷凍情況下儲(chǔ)存7天。

PROCEDURE程序Thetestcanbeconductedeitherinfiveoriginalcontainersifsufficientvolumeofproductisavailableineachcontainerandtheproductcontainercanbeenteredaseptically(i.e.,needleandsyringethroughanelastomericrubberstopper),orinfivesterile,cappedbacteriologicalcontainersofsuitablesizeintowhichasufficientvolumeofproducthasbeentransferred.測(cè)試可以在五個(gè)干凈的容器里操作，如果在每個(gè)容器里有足夠容量的產(chǎn)品且產(chǎn)品容器能被無(wú)菌地裝入（如，使用針和注射器穿過(guò)一個(gè)人造的橡膠瓶塞），或者在五個(gè)無(wú)菌的，加蓋的、尺寸合適的，能移轉(zhuǎn)產(chǎn)品的充足容量的細(xì)菌容器里。Inoculateeachcontainerwithoneofthepreparedandstandardizedinoculum,andmix.Thevolumeofthesuspensioninoculumusedisbetween0.5%and1.0%ofthevolumeoftheproduct.Theconcentrationoftestmicroorganismsthatisaddedtotheproduct(Categories1,2,and3)aresuchthatthefinalconcentrationofthetestpreparationafterinoculationisbetween1×105and1×106cfupermLoftheproduct.ForCategory4products(antacids)thefinalconcentrationofthetestpreparationafterinoculationisbetween1×103and1×104cfupermLoftheproduct.給每一個(gè)準(zhǔn)備好的標(biāo)準(zhǔn)的接種體的容器做接種注射，并混合。接種懸浮液的使用量在產(chǎn)品量的0.5%到1.0%之間。被添加到產(chǎn)品的測(cè)試細(xì)菌濃度（類(lèi)型1.2.3）到最終的測(cè)試準(zhǔn)備濃度在接種之后是每毫升1×105and1×106cfu之間。對(duì)于類(lèi)型4產(chǎn)品（解酸劑）測(cè)試準(zhǔn)備的最終濃度在接種后是每毫升產(chǎn)品1×103到1×104cfu之間。Theinitialconcentrationofviablemicroorganismsineachtestpreparationisestimatedbasedontheconcentrationofmicroorganismsineachofthestandardizedinoculumasdeterminedbytheplate-countmethod.在每次測(cè)試準(zhǔn)備的活的微生物的原始濃度是預(yù)計(jì)是基于每個(gè)標(biāo)準(zhǔn)接種體的微生物濃度，通過(guò)皿測(cè)法來(lái)預(yù)計(jì)的。Incubatetheinoculatedcontainersat22.5±2.5.SampleeachcontainerattheappropriateintervalsspecifiedinTable3.Recordanychangesobservedinappearanceattheseintervals.Determinebytheplate-countprocedurethenumberofcfupresentineachtestpreparationfortheapplicableintervals(seeProcedureunderMicrobialLimitTests(61)).Incorporateaninactivator(neutralizer)ofthespecificantimicrobialintheplatecountorintheappropriatedilutionpreparedforplating.TheseconditionsaredeterminedinthevalidationstudyforthatsamplebasedupontheconditionsofmediaandmicrobialrecoveryincubationtimeslistedinTable2.UsingthecalculatedconcentrationsofcfupermLpresentatthestartofthetest,calculatethechangeinlog10valuesoftheconcentrationofcfupermLforeachmicroorganismattheapplicabletestintervals,andexpressthechangesintermsoflogreductions.在22.5±2.5的溫度下將容器接種。如表3，在適當(dāng)?shù)臅r(shí)間間隔里把每個(gè)容器做樣品標(biāo)示。記錄下在每個(gè)間隔時(shí)間里出現(xiàn)的任何變化（參看MicrobialLimitTests(61)下的程序）。以平皿計(jì)數(shù)方式(platecount)在適當(dāng)?shù)臅r(shí)間間隔里計(jì)算出現(xiàn)的cfu數(shù)。使用特定的抗菌劑做為鈍化劑（中和劑）或是適當(dāng)?shù)南♂屃縼?lái)做平皿計(jì)數(shù)。在表2中規(guī)定出培養(yǎng)基與不同菌種的各種接種狀況與驗(yàn)收時(shí)間。在開(kāi)始測(cè)試時(shí)使用每毫升多少CFU來(lái)計(jì)算濃度，在適當(dāng)?shù)拈g隔測(cè)試對(duì)每個(gè)微生物計(jì)算每毫升CFU濃度的log10值的變化，并表述以log為單位的減少變化量。Table2.CultureConditionsforInoculumPreparation接種體準(zhǔn)備的培養(yǎng)基Organism微生物SuitableMedium適合的培養(yǎng)基Incubation

Temperature接種溫度Inoculum

IncubationTime接種時(shí)間MicrobialRecovery

IncubationTime驗(yàn)收時(shí)間Escherichiacoli大腸桿菌

(ATCCNo.8739)Soybean–CaseinDigestBroth;

黃豆-消化酪蛋白液Soybean–CaseinDigestAgar黃豆-消化酪蛋白基32.5±2.518to24hours18至24小時(shí)3to5days3至5天Pseudomonasaeruginosa綠膿桿菌

(ATCCNo.9027)Soybean–CaseinDigestBroth;

黃豆-消化酪蛋白液Soybean–CaseinDigestAgar黃豆-消化酪蛋白基32.5±2.518to24hours18至24小時(shí)3to5days3至5天Staphylococcusaureus金黃色葡萄球菌

(ATCCNo.6538)Soybean–CaseinDigestBroth;

黃豆-消化酪蛋白液Soybean–CaseinDigestAgar黃豆-消化酪蛋白基32.5±2.518to24hours18至24小時(shí)3to5days3至5天Candidaalbicans白念珠菌

(ATCCNo.10231)SabouraudDextroseAgar;沙氏瓊脂培養(yǎng)基

SabouraudDextroseBroth沙氏瓊脂培養(yǎng)液22.5±2.544to52hours44到52小時(shí)3to5days3至5天Aspergillusniger

黑曲霉菌(ATCCNo.16404)SabouraudDextroseAgar;

沙氏瓊脂培養(yǎng)基SabouraudDextroseBroth沙氏瓊脂培養(yǎng)液22.5±2.56to10days6至10天3to7days3至7天

CRITERIAFORANTIMICROBIALEFFECTIVENESS抗菌效力標(biāo)準(zhǔn)TherequirementsforantimicrobialeffectivenessaremetifthecriteriaspecifiedunderTable3aremet(seeSi