EFPIANOTETHISISANINITIALDRAFTOFTHEPAPERWHICHHASNOWBEENPUBLISHEDINTHEFOLLOWINGJOURNALEFPIA/ECVAMpaperongoodpracticeinadministrationofsubstancesandremovalofblood,JApplToxicol2115-23,2001.ThispublicationistheresultofaninitiativebetweenEFPIA(theEuropeanFederationofPharmaceuticalIndustriesAssociations)andECVAM(theEuropeanCenterfortheValidationofAlternativeMethods).Itsobjectivesaretoprovidetheresearcherinthesafetyevaluationlaboratorywithanup-to-date,easytousesetofdatasheetstoaidinthestudydesignprocesswhilstatthesametimeaffordingmaximumwelfareconsiderationstotheexperimentalanimals.AlthoughthispublicationistargetedatresearchersintheEuropeanPharmaceuticalIndustryitisconsideredthattheprinciplesunderpinningthedatasetsandrefinementproposalsareequallyapplicabletoallthosewhousethesetechniquesonanimalsintheirresearchwhetherinResearchInstitutes,Universitiesorothersectorsofindustry.TheimplicationsofthisdocumentmayleadtodiscussionwithRegulatorssuchasthoseresponsibleforPharmacopoeialtesting.Therearenumerouspublicationsdealingwiththeadministrationoftestsubstancesandremovalofbloodsamplesand,additionally,manylaboratoriesalsohavetheirown‘in-house’guidelineswhichhavebeendevelopedbycustomandpracticeovermanyyears.WithintheEuropeanUnionDirective86/609EEC(EU,1986)wehaveanobligationtorefineexperimentstocausetheminimumamountofstress.Wehopethisguidewillprovidebackgrounddatausefultothoseresponsibleforprotocoldesignandreview.Thisdocumentisbasedonpeerreviewedpublicationswheneverpossible,but,wherethatisnotpossible,wehaveused‘in-house’dataandtheexperienceofthoseontheworkingparty(aswellashelpfulcommentssubmittedbytheindustry)forafinalopinion.Thedocumentalsoaddressesthefile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第1／22頁(yè)）2008-6-2510:52:35EFPIAcontinuingneedtorefinethetechniquesassociatedwithadministrationofsubstancesandwithdrawalofbloodandsuggestswaysofdoingso.Datasharingbetweenlaboratoriesshouldbeencouragedtoavoidduplicationofanimalwork,aswellassharingpracticalskillsconcerninganimalwelfareandscientificproblemscausedby‘overdosing’somewayoranother.Therecommendationsinthisdocumentrefertothe‘normal’animalandspecialconsiderationisneeded,forinstance,duringpregnancyandlactation.Interpretationofstudiesmaybeconfoundedwhenlargevolumesareadministeredorexcessivesamplingemployed,particularlyifanaestheticsareused.1.IntroductionDosingofexperimentalanimalsisnecessaryforavarietyofscientificinvestigationsandtomeetregulatorydemands.Thepharmaceuticalindustry,inparticular,hasinvestigatedthelevelsofdosingcompatiblewithanimalwelfareandvalidscience(Hull1995).Inthepreclinicalstageofthesafetyevaluationofnewdrugsitisnormalpracticetousemultiplesofthe‘effectivedose’inordertoattempttoestablishthenecessarysafetymargins.Wherechemicalsareoflowtoxicityorareonlypoorlysolubleinacceptableformulations,alargevolumemayberequiredtobegiventoindividualanimalstosatisfybothscientificandregulatoryrequirements.Theintendedclinicalusemayalsoimpactontheacceptabilityoflargerthanusualdosevolumes,egimagingagentsorplasmaexpandersforintravenousapplication.TheobjectivesoftheWorkingGroupwereasfollows:a.Toprovideaguideonadministrationvolumesforuseincommonlaboratoryspeciesusedintoxicitystudiesrequiredbyregulatoryauthorities.b.Toprovideconsensusdosagelevelsforroutineusewhichrepresentgoodpracticeintermsofanimalwelfareandpracticality.c.Toproduceaguidetodosagelevelsrepresentingtheupperlimitofcommonpractice.Thisleavesscopetomakethecaseforspecialinvestigations.AdministrationvolumesTable1presentsadministrationvolumesforthecommonlyemployedroutesinthemostfrequentlyusedspecies.Theyareconsensusfiguresbasedonpublishedliteratureandinternalguidelines.ThemarmosetandminipigarenowconsideredwithinthiscategorysincetheyarebeingusedincreasinglyinEurope.Table1AdministrationVolumesConsideredGoodPracticefile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第2／22頁(yè)）2008-6-2510:52:35EFPIA(andpossiblemaximaldosevolumes)SpeciesRouteandVolumes(ml/kgexcept*ml/site)Oralscipimivbolusiv(slowinj)(25)(20)(10)(5)(-)(10)(5)MouseRatRabbitDogMacaqueMarmosetMinipig10(50)10(40)10(15)5(15)5(15)10(15)10(15)10(40)5(10)1(2)1(2)2(5)2(5)1(2)20(80)10(20)5(20)1(20)-(10)-(20)1(20)0.05*(0.1)*0.1*(0.2)*0.25(0.5)0.25(0.5)0.25(0.5)0.25(0.5)0.25(0.5)5522.522.52.5Notestobeusedinconjunctionwithtable:(-)datanotavailableFornon-aqueousinjectatesconsiderationmustbegiventotimeofabsorptionbeforere-dosing.Nomorethan2intramuscularsitesshouldbeusedperday.Subcutaneoussitesshouldbelimitedto2to3sitesperday.Twosetsoffiguresareshownineachcolumn.Figuresintheleftsideofcolumnsareintendedasaguideto‘goodpractice’dosevolumesforsingleormultipledosing.Thesecondbracketedsetoffigures,wheregiven,arethepossiblemaximalvalues.Ifexceeded,animalwelfareorscientificimplicationsmayresultandreferencetotheresponsibleveterinarysurgeonshouldbemade.InsomeinstancesfiguresaretheretoaccommodatePharmacopoeialrequirements.file:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第3／22頁(yè)）2008-6-2510:52:35EFPIASomeofthesesuggestedmaximumvalueshavebeenobtainedfromrecentliterature(Flecknell1996;Wolfensohn&Lloyd1998),butappearhighwhencomparedwith‘goodpractice’values.Theneedforcarefulattentiontoanimalwelfare,andformulationofmaterialusedathighdosevolumes,isemphasised,particularlyifrepeatdosingisintended.Studydurationcouldberestrictedandscientificvaliditycompromisedbyphysiologicalreactiontohighdosevolumes.Itisthereforeessentialfromanethicalstandpointthattheseissuesarefullyconsidered,egbyinspectorateorethicalcommittee,beforeprotocolsarefinalisedandworkcommences.Itisalsostronglyrecommendedforethicalaswellasscientificreasonsthatphysicochemicalcompatibilitystudies(invitro)andsmallscalepilotstudies(smallgroupsofanimals)arecarriedoutonanynewformulationbeforecommittingtolargerscalestudies.Dosevolumesshouldbetheminimumcompatiblewithcompoundformulationandaccuracyofadministration.AdministrativeRoutesOralRouteOnoccasions,itmaybenecessarytorestricttheanimals’foodintakebeforedosing.Thisfactormayaffectabsorption.Largedosevolumes(40ml/kg)havebeenshowntooverloadthestomachcapacityandpassimmediatelyintothesmallbowel(Hejgaardetal1999).Largervolumesmayalsorefluxintotheoesophagus.Thedurationoffastingwilldependuponthefeedingpatternofthespecies,thestartingtimeforfoodrestriction,physiologyofthespecies,lengthoftimeofdosing,dietandlightcycle(Vermeulenetal1997).Itisrecommendedthatforaccuracyofdosing,andtoavoiddosingaccidents,thatliquidsareadministeredbygavage.ParenteralRoutesForsubstancesadministeredparenterally,thedosevolumeused,stabilityoftheformulationbeforeandafteradministration,thepH,viscosity,osmolality,bufferingcapacity,sterilityandbiocompatibilityoftheformulationarefactorstoconsider.Thisisparticularlyimportantformultipledosestudies.ThesefactorsarereviewedinsomedetailbyClaassen(1994).Thesmallestneedlesizeshouldbeusedtakingintoaccountthedosevolume,viscosityofinjectionmaterial,speedofinjectionandspecies.a.SubcutaneousThisrouteisfrequentlyused.Therateandextentofabsorptiondependonformulation.b.IntraperitonealThisrouteisusedinfrequentlyformultipledosestudiesbecauseofpossiblecomplications.Thereisapossibilityofinjectingintotheintestinaltractandirritantmaterialsmaycauseperitonitis.Drugabsorptionfromtheperitonealcavityaftertheadministrationofthecompoundasasuspensionisdependentonthepropertiesofthedrugparticlesandthevehicle,andmaybeabsorbedintobothsystemicandportalcirculations.c.IntramuscularIntramuscularinjectionsmaybepainful,becausemusclefibresarenecessarilyplacedundertensionbytheinjectedmaterial.Sitesneedtobechosentominimisethepossibilityofnervefile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第4／22頁(yè)）2008-6-2510:52:35EFPIAdamage.Sitesshouldberotatedformultipledosestudies.Adistinctionneedstobemadebetweenaqueousandoilyformulations(speedofabsorption,oilyformulationslikelytoremainasadepotfor>24hours).Withmultipledosestudiesthereisaneedtoconsidertheoccurrenceofinflammationanditssequelae.d.IntravenousadministrationForthisroute,distinctionsaremadebetweenbolusinjection,slowintravenousinjection,andintravenousinfusion.ThevaluesinTable1relatetobolusinjectionandslowintravenousinjection.Bolusinjection:Inmoststudiesusingtheintravenousroutethetestsubstanceisgivenoverashortperiod,approximately1minute.Suchrelativelyrapidinjectionsrequirethetestsubstancetobecompatiblewithbloodandnottooviscous.Whenlargevolumesarerequiredtobegiventheinjectionmaterialshouldbewarmedtobodytemperature.Therateofinjectionisanimportantfactorinintravenousadministrationanditissuggestedthat,forrodents,therateshouldnotexceed3ml/min.Nodetectablechangesinhaematocritorheartratewereobservedindogsfollowingrapidintravenousinjectionof6ml/kgsaline,but20ml/kgwasassociatedwith15%haemodilutionandatransienttachycardia(up46%over1min)(Zeolietal1998).Slowintravenousinjection.Becauseoftheexpectedclinicalapplicationofthecompound,orbecauseoflimitingfactorssuchassolubilityorirritancy,itmaybenecessarytoconsideradministeringsubstancesbyslowintravenousinjection.Typically,differenttechniqueswouldbeappliedforslowinjectiontominimisethepossibilityofextravascularinjectionofmaterial.Forslowintravenousinjectionoverthecourseof5–10minutesastandardorbutterflyneedlemightbeused,orbetterstillanintravenouscannulamaybetapedinplaceinasuperficialvein(shortterm),orsurgicallyplacedsometimepriortouse(longerterm,ormultipleinjections).Ithasbeenshownthatratsmaybegivendailyintravenousinjectionsofisotonicsalineatdosagesupto80ml/kgat1ml/minfor4dayswithoutsignificantsignsofdistressorpulmonarylesions(Mortonetal1997a).However,pulmonarylesionsincreasedinincidenceandseveritywhenthedurationoftreatmentincreasedto30daysandtheinjectionwasadministeredateither0.25,0.5or1.0ml/min(Mortonetal1997b).Theremaywellhavebeenadverseeffectsatanearliertimepointbutthepathologyhadnothadtimetodevelop.Continuousinfusion.Forsimilarreasonsofsolubilityorclinicalindicationitmaybenecessarytoconsidercontinuousinfusion,butcarefulconsiderationisneededifinfusionsareprolonged.Thevolumeandrateofadministrationwilldependonthesubstancebeinggivenandtakeaccountoffluidtherapypractice.Asaguide,thevolumeadministeredonasingleoccasionwillbelessthan10%ofthecirculatingbloodvolumeovertwohours.InformationoncirculatingbloodvolumesisavailableinTable3ofthisguide.Minimaleffectiverestraintofanimalswithleaststressisakeyfactortoconsiderforprolongedinfusions.Thetotaldurationofaninfusionisalsoafactor.Table2presentsrecommendeddosefile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第5／22頁(yè)）2008-6-2510:52:35EFPIAratesandvolumesfordiscontinuous(4hourperday)andcontinuous(24hour)infusion.(Furtherdataarerequiredtocompletethistable.)Table2RepeatedIntravenousInfusion-DoseVolumes/Rates(andpossiblemaximalvolumes/rates)DailyInfusionPeriodTotalDailyVolumes(ml/kg)4Hour24Hour-96(192)2060(96)-24(72)Rate(ml/kg/h)4Hour24Hour-4(8)52.5(4)-1(3)(-)datanotavailable*basedonteratologystudiesFornonaqueousinjectatesseetextInsomecases,twosetsoffiguresareshowninacolumn.Thesecondbracketedsetoffiguresarethepossiblemaximalvalues.MouseRatRabbit*DogMacaqueMinipig2024(96)-60-2451(4)-2.5-1Volumesandratesfortherabbitarebasedondataderivedfromembryotoxicitystudieswhichshowednoeffectsonthefoetusbutperivasculargranularleukocytecuffingandproliferativeendocarditisindamsreceiving2ml/kg/handabove(McKeonetal1998).Infusionratesinratsaretypicallyintherangeof1to4ml/kg/h(Caveetal1995;Barrow&Heritier1995;Logetetal1997)butideallyshouldnotexceed2ml/kg/hinembryotoxicitystudies.Valuesforthemouse(vanWijk1997),dogandmacaque(Perkin&Stejskal1994)andminipig(unpublisheddata)arebasedonrepeateddosestudiesofonemonthduration.Otherlimits,indicatingtheimportanceofthevehicleformulationathighdosevolumesarefile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第6／22頁(yè)）2008-6-2510:52:35EFPIAhighlightedinfourpublications(Corneliusetal1978;Concannonetal1992;Manentietal1992;Mann&Kinter1993).Thesedataindicatethattherearelargedifferencesintoleratedvolumebyivinfusion,dependentuponthevehicleused.Thelong-termeffectsonotherphysiologicalsystemshavenotbeeninvestigated.e.IntradermalThissiteistypicallyusedforassessmentofimmune,inflammatory,orsensitisationresponse(Leenaars1997;Leenaarsetal1998).Materialmaybeformulatedwithanadjuvant.Volumesof0.05to0.1mlcanbeuseddependentuponthethicknessofskin.VehiclesforAdministrationVehicleselectionisanimportantconsiderationinallanimalinvestigations.Vehiclesthemselvesshouldofferoptimalexposurebutshouldnotinfluencetheresultsobtainedforthecompoundunderinvestigationand,assuch,theyshouldideallybebiologicallyinert,havenoeffectonthebiophysicalpropertiesofthecompoundandhavenotoxiceffectsontheanimals.Ifacomponentofthevehiclehasbiologicaleffects,thedoseshouldbelimitedsuchthattheseeffectsareminimisedornotproduced.Simplevehiclesusedtoadministercompoundsincludeaqueousisotonicsolutions,bufferedsolutions,co-solventsystems,suspensionsandoils.Fornon-aqueousinjectatesconsiderationshouldbegivenfortimeofabsorptionbeforere-dosing.Whenadministeringsuspensionstheviscosity,pHandosmolalityofthematerialneedstobeconsidered.Theuseofco-solventsystemsneedscarefulattentionsincethevehiclesthemselveshavedoselimitingtoxicity.Laboratoriesareencouragedtodevelopastrategytofacilitateselectionofthemostappropriatevehiclebasedontheanimalstudybeingperformedandthepropertiesofthesubstanceunderinvestigation.2.IntroductionBloodremovalisoneofthemostcommonproceduresperformedonlaboratoryanimalsandmethodsforlaboratorymammalsandbirdswerereviewedinthefirstreportoftheBVA/FRAME/RSPCA/UFAWJointWorkingGrouponRefinement(1993).Thiscurrentpublicationaimstoprovideaneasytouseguidebasedonthelatestavailableinformation,andaddressestheneedsoftoxicokinetic(pharmacokinetic)andtoxicologystudies.Thepracticeofbloodsamplingfromavarietyofrodentsusingtheretrobulbarvenousplexustechniqueisstillincommonuseandsuggestionsforalternativeroutesaredescribedbecauseofconcernsoverthesequelaeofusingthismethod.Circulatingbloodvolumesfile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第7／22頁(yè)）2008-6-2510:52:35EFPIAThecalculationoflimitvolumesforbloodsamplingreliesonaccuratedataoncirculatingbloodvolumes.Reviewoftheliteratureindicatesthatthereisconsiderablevariationinthesevalues,probablyrelatingtothetechniquesused,thestrainandsexofanimal,etc.Thetechniquesmostfrequentlycitedareradio-labellederythrocytes(Smith1970;Sluiteretal1984;Fujiietal1993),radiolabelledtransferrin(Argentetal1994),radiolabelledserumalbumin(Carvalho1989;Gillenetal1994;Callahanetal1995),markerdyes(Schadetal1987)enzymedilution(Visseretal1982;Holmes&Weiskopf1987);fibreoptics(Kischetal1995)anddextran-70(VanKreeletal1998).Table3givesthecirculatingbloodvolumesofthespeciescommonlyusedinsafetyevaluationstudies.Dataonthemarmosetandminipig,whicharebecomingmorefrequentlyusedintoxicology,havenowbeenincluded.Thevaluesshownhavebeenadaptedfromdifferentsourcesassumingtheanimalismature,healthyandonanadequateplaneofnutrition(Altman&Dittmer1974;Swenson1977;Jain1986;McGuill&Rowan1989;FirstreportoftheBVA/FRAME/RSPCA/UFAW1993)Table3:CirculatingbloodvolumeinlaboratoryanimalsSpeciesMouseRatRabbitDog(Beagle)Macaque(Rhesus)Macaque(Cynomolgus)MarmosetMinipigBloodvolume(ml/kg)Recommendedmean*7264568556657065Rangeofmeans63-8058-7044-7079-9044-6755-75-8261-68*Therecommendedmeancorrespondstothemid-pointoftherangeofmeans.BloodSamplingVolumesOurrecommendationsarebasedonpublishedwork,recentworkcarriedouttoinformtheworkingpartyaboutcertainissuesandwhichisbeingsubmittedforpublication,andinformationfrom‘in-house’standardoperatingprocedures.file:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第8／22頁(yè)）2008-6-2510:52:35EFPIAAnimalwelfareisaprimeconsiderationwhenbloodsamplingisapproachinglimitsbutthescientificimpactofananimal’sphysiologicalresponsemustalsobeconsideredasthismayaffectdatainterpretationandvalidity.Assessmentofclinicalsignsshownbytheanimals,withreferraltosupervisoryorveterinarystaffindoubtfulcases,priortosampling,isanexpectedprerequisite.WorkofScipionietal(1997)indicatedthatremovalofupto40%ofarat’stotalbloodvolumeover24hoursandrepeated2weekslatercausednogrossilleffects.Byandlargethereislittledataoncriticalaspectsofanimalwellbeingafterremovalofbloodsuchasheartrate,respiratorypatterns,varioushormonallevels,andbehaviouralaspectssuchasactivitiesandtimespentcarryingthemout.Allthesemaychangeinresponsetoexcessivebloodremovalbutitwouldrequireconsiderableeffortandresourcetoinvestigatethem.However,haematologicalparameterscanbeeasilymeasuredandinasmallproject(Nahasetalsubmitted)redbloodcellcount(RBC),haemoglobinlevel(HGB),haematocrit(HCT),meancorpuscularvolume(MCV),andredcelldistributionwidth(RDW)weremeasuredaftertheremovalofvaryingbloodvolumes.Volumesof7.5%,10%,15%and20%ofcirculatingbloodvolumes(as0.3mlaliquots)wereremovedfrommaleandfemaleSpragueDawleyrats(N=7)weighingapproximately250govera24hourperiodtomimicakineticstudy.Animalswerethenfollowedforupto29days.Theresultsshowedthattherewasconsiderablevariationinthetimestakenforalltheseparameterstoreturntobaselinelevels(andinthe15%and20%groups,someoftheparameters(MCV,RDW)didnotreturntobaselineevenafter29days).Therecoverytimerecommendedinthispaperformultiplesampling,therefore,isthetimetakenforALLratsina‘volume’grouptoreturnto‘normal’(thestartinglevelforeachanimalplusorminus10%).Singlesampling(suchasthatrequiredforroutinetoxicitystudies)beyond15%isnotrecommendedsincehypovolaemicshockmayensueifitisnotdoneveryslowly.Multiplesmallsamplesareunlikelytoproducesuchacuteeffects.Thefollowingguideforlimitvolumesandadequaterecoveryperiodtakesintoaccountthestressofmultiplesamplinginadditiontootherproceduresinassessingoverallseverity.Thetableaddressesbothsingleandmultiplesamplingregimes.Additionalrecoverytimeisproposedforanimalsontoxicitystudiessinceacriticalevaluationofhaematologicalparametersisrequiredinsuchstudies.Table4:LimitvolumesandrecoveryperiodsSinglesampling(egtoxicitystudy)%circulatorybloodvolumeremovedApproximaterecoveryperiodMultiplesampling(egtoxicokineticstudy)%circulatorybloodvolumeremovedin24hApproximaterecoveryperiodfile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第9／22頁(yè)）2008-6-2510:52:35EFPIA7.5%10%15%1week2weeks4weeks7.5%10-15%20%1week2weeks3weeksThehighervolume(20%)isintendedtofacilitateserialbloodsamplingfortoxico-orpharmacokineticpurposeswheremultiple,smallsamplesareusuallyrequired.However,itshouldberememberedthattheconsequentialhaemodynamiceffectoftakingsuchlargevolumesmaywellaffectthecalculatedhalf-life.Assessmentofterminalhalf-lifeshouldbepossibleiffinalsamplesaretakenwithin24hoursofthekillingofananimal.Thesefiguresdonotincludeaterminalsamplewhichcanbetakenwhentheanimalisterminallyanaesthetised.Bloodreplacementhasnotbeenconsideredsincethevolumesproposeddonotwarrantsuchintervention.UsingthevaluesfromTable4,aneasyreferenceguideforthevolumeswhichcanberemovedwithoutsignificantdisturbancetoananimal’snormalphysiologyispresentedinTable5.Table5:TotalbloodvolumesandrecommendedmaximumbloodsamplevolumesforspeciesofgivenbodyweightSpecies(Weight)Mouse(25g)Bloodvolume(ml)1.8Rat(250g)16Rabbit(4kg)224Dog(10kg)850648512717017223443.27.5%(ml)0.110%(ml)0.215%(ml)0.320%(ml)0.4file:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第10／22頁(yè)）2008-6-2510:52:35EFPIAMacaque(Rhesus)(5kg)Macaque(Cynomolgus)(5kg)Marmoset(350g)2803252521284256243249652.02.53.55Minipig(15kg)9757398146195SamplingsitesSitesforvenepunctureandvenesectionhavebeenconsideredmainlyinrodentsandrabbit(FirstReportoftheBVA/FRAME/RSPCA/UFAW1993).ThisinformationhasbeenreviewedinthelightoftechnicaladvancesinbloodsamplingproceduresandtheadvantagesanddisadvantagesofsitesforeachspeciesareshowninTable6withtherecommendedonesforrepeatedsamplingsummarisedinTable7.Table6:SummaryoftheadvantagesanddisadvantagesofthevariousmethodsofbloodsamplingROUTE/VEINJugularCephalicSaphenous/lateraltarsalnononolowlowlowGeneralAnaesthesiaTissue(1)damageRepeatbleedsyesyesyes++++++++(+)rat,dog,rabbitmacaque,dogmouse/ratmarmoset/macaquedogVolumeSpeciesfile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第11／22頁(yè)）2008-6-2510:52:35EFPIAMarginalearFemoralSub-lingualLateraltailCentraleararteryCranialvenacavaTailtipamputation(<1to3mm)RetrobulbarplexusCardiac(2)no(local)noyesnono(local)noyesyesyeslowlowlowlowlowlowmodmod/highmodyesyesyesyesyesyeslimitedyesno+++++++++++(+)++++++++++++++rabbitminipigmarmoset/macaqueratratmouse/marmosetrabbitminipigmouse/ratmouse/ratmouse/rat/rabbit(1)Thepotentialfortissuedamageisbasedonthelikelyincidenceofitoccurringandtheseverityofanysequelaeeginflammatoryreaction,histologicaldamage(2)OnlycarriedoutasterminalprocedureundergeneralanaesthesiaTable7:RecommendedsitesforrepeatedbloodsamplingSpeciesMouseRatRabbitDogMacaqueMarmosetMinipigRecommendedsiteSaphenous,lateraltailSaphenous,lateraltail,sub-lingualMarginalear,centralearartery,jugularCephalic,jugular,saphenousCephalic,saphenous,femoralFemoral,saphenousCranialvenacavafile:///D|/animal/bloodsample/AGoodPracticeGuidetotheAdministrationofSubstancesand.htm（第12／22頁(yè)）2008-6-2510:52:35EFPIAItisimportanttonotethatsamplestakenfromdifferentsitesmayshowdifferencesinclinicalpathologyvaluesandhaveimplicationsforhistoricaldatabases.Forthemoretraditionalroutes,adescriptionofthemethodologycanbeobtainedfromthestandardliterature.However,othermethodsrequireaspecialmentionandhavebeenreviewedbelow:Lateraltarsal(saphenous)veinThistechniquehasbeenusedinmanylaboratoryanimalsincludingrats,mice,hamster,gerbil,guineapig,ferret,mink(Hemetal1998),aswellaslargeranimals,andvolumessuchas5%ofcirculatingbloodvolumemaybetaken.Itdoesnotrequireananaestheticandsoisparticularlysuitableforrepeatedbloodsamplingasinpharmacokineticstudies.Thesaphenousveinisonthelateralaspectofthetarsaljointandiseasiertoseewhenthefurisshavedandtheareawipedwithalcohol.Theanimalisplacedinasuitablerestrainer,suchasaplastictube,andtheoperatorextendsthehindleg.Theveinisraisedbygentlepressureabovethejointandthevesselpuncturedusingthesmallestgaugeneedlethatenablessufficientlyrapidbloodwithdrawalwithouthaemolysis(eg25gto27gforratsandmice).Forsmallvolumes,asimplestableadstoadropofbloodformingimmediatelyatthepuncturesiteandamicrohaematocrittubecanbeusedtocollectastandardvolume.Afterbloodhasbeencollected,pressureoverthesiteissufficienttostopfurtherbleeding.Removalofthescabwillenableserialsampling.Thereappeartobenocomplicationsreportedotherthanpersistent(minor)bleedingandthemethodhastheadvantagethatanaesthesiaisnotrequired.Eventhoughnostudieshavebeendoneonanimalwelfareintermsofbodyweightgain,diurnalrhythm,behaviouretc,itseemsunlikelythatthisroutewillseriouslyaffectananimal’swellbeing.Marginalearvein/centraleararteryBloodsamplingfrommarginalearveiniscommonlyusedinrabbitsandguinea-pigs.Thisroutemayalsobechoseninminipigs,oftencombinedwiththeuseofanintravenouscannula.Goodrestraintisnecessaryandtheapplicationoflocalanaestheticcreamsome20to30minutesbeforebleedinghelpspreventananimalshakingitsheadastheneedleispushedthroughtheskin.Bleedsmayalsobetakenby