Avian acute oral toxicity test [electronic resource]

2010·0.74 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 223
Adopted:
22July2010
OECDGUIDELINESFORTHETESTINGOFCHEMICALS
AvianAcuteOralToxicityTest
INTRODUCTION
ntTd1oeeh.epebdieernLddds.D,o5a0nT-nShsedwilqhosiuetpetetpnehrtsaeotinvragdiluodireLtdseenDslott5ithi0nr-neaegoenddlpeteyrsefosciotncripiienttbdgiieuvosorenpepstsmrieooatdcnraeiser:dagusne(ert1qed)ustoelhsndieetmeiisp(taillLgdaDnoctes5ee0desm)tettiaonennsgtted,spot(srfi2lom)ocadpeLtoedesDueort5sfe0hs-ets.ahalnecoTdpudhetoemestaeeotst-rtecar,slhteastnmptodeohxteni(csh3iep)otrdyLcecuoDsirfes5vl0sie-eouconbatnsreltoedyafbntwoectistehlthsel.
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2. DevelopmentofthistestguidelinebeganattheSETAC/OECDWorkgrouponaviantoxicity
tSeEstTiAngCfoalnldocwlionsgeadwOoErCksDhoExppheerltdGirnouPepnsmaeceotlian,gsFlionriEduar,oUpneitaenddtShtaeteUsn,itiend1S9t9a4te(s1t)owdietvhelsoupbsaenqdueonpttiompiesne
thesequentialtestingdesign.Thesequentialtestingdesignhasbeendevelopedwithextensivestatistical
validation(2).
INITIALCONSIDERATIONS
3. Theinformationrequiredbydifferenthazardassessmentschemesmayvaryconsiderably. To
satisfythesevariousneeds,thefollowingthreetestsaredescribed(seeAnnex2fordefinitionsoftheterms
usedinthisguideline):
• Limitdosetest-Thisisthepreferredtestwhentoxicityisexpectedtobelowandlethalityis
unlikelyatthelimitdose. Thelimitdoseshouldbeadequateforassessmentpurposes,anditis
usually2000mg/kg-bwt.Fiveortenbirdsaretestedatthelimitdoseinadditiontoacontrolgroup
(Figure1).
• LD5o-slopetest-Thisisthepreferredtestwhenregulatoryorotherrequirementsdeterminethat
te(hsFetiigsmularotepe2e)o.fof1thteheLdDo5s0e.-rTehsipsoinssea3c-urovre4-asntda/goerttehstewciotnhfi2d4enocre3i4ntbeirrvdasliinsardedqiutiiroendtoinaacdodnittrioolngtroouapn
• ocplLeanoDrblnceyf5eloiln-dtitoheninengllceypmeueotrdfiepisnoattasene-rssvp.alTelehcTtiihhfesaioslsrisdstteoetshnshteseeihtLapiissrvDiertt5fe0yweqrouidrsiisesrtdtrearedgtiqeebussbuit,utrtwwieoidhnn.teehinoTtf1hhr4eiLersbgDiutrml5hd0aeasstyosiarlnnyobadpedoedrttioohtofeitpohrtneaohrpevtpoirrddeaooeqpscureoiiinnratrfetreomeosrelpmntaogettnssrtisooedutnepoctfu(eeorFrrsvimtegipiunmrroeaoertdte2uth)cha.teat
W85h0e.2n10a0n).estIinmattheeoifntselroepsteiosfraeqnuiimraeld,waelcflaarses,icatlestdomsee-trheosdpsonwsheitcehstucsoeulmdobreecaonnidmuacltse,dsiuncshteaads(UUSSEEPPAAOOPPPPTTSS
850.2100,aregenerallynotpreferred.
©OECD,(2010)
Youarefreetousethismaterialforpersonal,non-commercialpurposeswithoutseekingpriorconsentfrom
ptehremOiEssCiDo,npfrroovmitdheedOthEeCDso.urceisdulymentioned.Anycommercialuseofthismaterialissubjecttowritten
223 OECD/OCDE
4S.tagesareThdeefiLnDed50-asslpoepreiaodnsddLuDri5n0-gonalnyetxepsetsricmoennstisitnowfhsitcahgebsirtdhsataarreedpoesrefdosrimmeudltsaenqeuoenutsilayllaynd(Foibgsuerrev2e)d.
AfLonDrn5ae0-xpsel1ro.ipoedaonfdtLimDe5.0-Tohnulsy,tetshtes.teArmnsesxepqluaennattiiaolnteosfttahnedlosgeiqcueanntidalstadteissitigcnalarbeasuisseodfbtehleodwestiogrnefisergitovebnotihn
5. TheLD50-slopetestdesignhasbeenshowntohaveperformancepropertiesadequateforhazard
easstsiemsastmienngtthpeurLpDos5e0,scotnhfrioduegnhceexitnetnersviavles,caonmdpudtoeser-rseismpuolnatsieosnlo(p2e)s..Theseperformancepropertiesinclude
6. AteachstageineithertheLD50-slopeortheLD50-onlytestdesigns,oneormorebirdsaregivena
swisiontrgyklpieincogarlaellsytdbioamssaeeted(oomfng/tokhbegs-LebrDwvt5e)0d.omBfoirrttdhaselitaterysetaosnbudsbetsrotvxaiencdcietfyourssii1gn4ngsdadayofsst,eersbtuththraesteeladeraceytsie.oxnTpoheifcstdeoidnstetesorvfiaonlrcmsluuadbyesebtqehueerneetdvuoscltevaidgneigsf
mortalityorsignsofrecoveryoccursquickly,ortheintervalmaybeextendedifdelayedmortalityis
expectedorobserved.
7f.ailedlimiTthdeosseeqtueesntti(aolneteosrtsm(oLrDe50m-osrltoapleitioers)LDor50f-ronolmy)exctaenrnbaleiinnfitoiramtaetdiouns.inFgorincfoormmpaotuinondsgaoifnesdusfpreoctmeda
hdtwUShoiitsesgtairehhgnesegtSio2ctstxoahiauvgecseiewoitroyn2ur.,gttkhctTeiMeohnsabmegtexeisnepitogsrmftoamuicvSamaemtaiysatlsgLaebeibfeolko1feeri,lnteatiihshthewteioiaoLotmLrdeaDkdDti5Eei50ns0ngota-fsviSelamttsoiaahtpltgeieaiemboLtal1neteDsewt(f5h0oMrcefLo(oreEmnt.et)hgaie,enafLubaacDeainhs5lsde0eotdfdoaitlsfoiSodemntueiaartttcgehhebdreiom3orsridfeonsadetnteideedsns.nttgp,Tdiooohtvsrsheeseeoinstdb,shaoleesyqodreuintsefbeonfiaterSbrirdtieearanlsddgtpeteeeditcss4oeitsredes(mmo,’sisatnesyLoedop.dDsptti5fha0naorI)gtrt.f
rulesareoutlinedanddetailedinparagraphs40through43).
8. DefinitionsusedinthisguidelinearegiveninAnnex2.
Figure1:Limitdosetestprocedure;figuredoesnotincludecontrolbirds
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Figure2:Sebiqrudesn.tiFaulrtDheesridgentaPilrsocoendusrteop(piLnDg50r-uslleospearaenpdrLovDid50e-donilnyptaersatgs)r;apfihgsur4e0dtohersoungoht4i3n.cludecontrol
3 ©OECD,(2010)
223 OECD/OCDE
PRINCIPLEOFTHETEST
9. Thetestisdividedintoanumberofdiscretestages. Ateachstage,birdsaresimultaneously
givenadose(mg/kg-bwt)ofthetestsubstanceintothecroporproventriculus. Dependingonthetest
stage,individualbirdsmayreceivedifferentdosesormorethanonebirdmayreceivethesamedose{i.e.
thedoseisreplicatedwithinthestage).Therecommendedstrategyfortestingmaterialsthatareunlikelyto
presentasignificanthazardistoperformatestwithmultiplebirdsdosedatthelimitdose.Iftoxicityis
expected,therecommendedstrategyistouseasequentialdesignratherthanthelimitdoseapproach.
eSrsstautanbiggsmeeeasqtuoi1efonandtnoodsfset2tsahgreeiessLqubDaiarr5see0eanddnoendto-entrrhemetpihlsneilecobadpetesebtdoafsdaeotvsdhaeeislod,anobwsltheeh-ierleeesmstoSiprtmotaanagtsleeeistcio3eufsravtnohedebas4rLeerrDvce5aeq0rudri(ireiee.ndgr.ao,eluplttlhipsecriaemrtvueoilddoteudansontseeosLstu.asDgl5eyI0s.)n,.SstoDaogtsehea1st,ttfhhoeer
10. Afterdosing,thebirdsareobservedfora14-dayperiodinordertomeasuremortality.Itmaybe
necessarytoextendtheobservationperioddependingonevidenceofdelayedeffects. Thestagedtest
designiseasiesttoapplytochemicalsthatproducedeathwithinafewdays. Insuchcasesitisnot
necessarytowait14daysbeforestartingthenextstage,althoughobservationofallbirdsshouldcontinue.
Mortalityobservedafterashorterintervalmaybeusedtodeterminedosesforthefollowingstage(three
daysmayoftenbeused).Birdsthatareobviouslyinpainorshowsignsofdistressshouldbeeuthanized.
Thedatacollectedinthefirstthreedaysofastageusuallysupplysufficientinformationtodetermine
whetherbirdsarelikelytorecoverfromeffectsencountered,orwhetheradditionalmortalitywilloccur(2).
CalculationoftheworkingestimateonDay3ofateststage,allowsthetestandalldosingtobecompleted
ottehvseetrcbaialrcsduhsloiarsttieeovrnitdoiefmntet.hferIawnmoser.okmieInfgcDaeasseytsi3miatitmnefaooyrfmbtaehteinoeLncDeis5n0sdaivrcayaltuteeoswmtahaiayttffbouerrtduhepelratomyoe1rdt4audlnaitiytlysrmbeeacfyoovroeercymcuoorvfiitnnhgeatrtoeestmthaesitnnaiegnxegt,
stage.Finalcalculationisbasedontheaccumulated14-dayoutcomesfromallstages.
11. Mortalityistheprimaryendpointinthisstudyandbackgroundmortalityispresumedtobe
negligible.Controlsarerequiredtomonitorthehealthandhusbandryoftestbirdstoensurethattheability
ofthestudytoprovidereliableresultsisnotcompromised.Proceduresandtimelinesforcontrolbirdsare
ddiisstcruessssedshionulpdarbaegreaupthhsan4i4zetdo.46.AsDfuarrinasgptohesstiebslte,atnhiemuaslesoobfvhiouumsalnyeinenpdapioninotrsshasowdiensgcrsiibgendsionfOseEvCerDe
GuidanceDocumentNo.19ontheuseofhumaneendpointswillbetakenintoaccountwhenperforming
thistest(10).
VALIDITYOFTHETEST
12. Thetestisinvalidifthereisonenon-incidentaldeathormorethanonedeathfromanyother
cause.Ifthereisoneincidentaldeathintheinitialfive-birdcontrolgroup,fivemorecontrolbirdsare
addedtothetest,sothatthetotalnumberofcontrolbirdsusedinthetestisten. Incidentaldeathsare
definedasthosedeathsthatdonotreflectuponthehealthofthetestpopulationandresultfromself-
inflictedinjuriessuchasbrokenlegsorabrasions. Thisisincontrasttonon-incidentaldeathsamong
controlbirdsdefinedasanindicationofpoorhealthofthetestpopulationorconductofthestudy,i.e.
diseaseormishandlingofanimals.
DESCRIPTIONOFTHETESTMETHODS
Selectionofbirds
13. Captivebredspecieswithlowbackgroundmortalityandalowpropensitytoregurgitateare
preferred.Frequentlyusedspecieswhichfittheserequirementsarethenorthernbobwhitequail,Colinus
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avivrogiidneidaniufsataanlldpJoasspiabnlees,eoqwuianilg,toCoatniummailxwjealpfoanrieccaon(sGiadlelriaftoiromn)s.aTnhdediusseeasoefcwointlrdo-lc.aughtbirdsshouldbe
14. Itmaybenecessarytotestadditionalspeciestodevelopadistributionofspeciessensitivity.In
a((CdPsdoiilttltiuaomcnibfitoforomr)qmu)a)i)m,la,zyetbhbreeamfuaislnelcdha.r{dPTohdeiupschkillisa{tAginusatstnaopttalai(tnPytaresnhsdyeenrdcihftooosrml(i)A)m,nitsaentrhdiefborurdemgc)e)o,rmimgfeearrnadl{eMpdeilgosepposenciitet{saCcobulustuumrnbadatuhlelaritvutisoa
provideguidanceonsomerelativelyrobustandlaboratory-bredspeciesfromdifferentOrders.
15. Birdsshouldbeinmatureplumagebutnotinbreedingcondition.Wildphenotypesarepreferred,
wherepossible. Captive-bredbirdsshouldbefromthesamesourceandbreedingpopulation,andwhen
possible,breedinghistoryshoulddemonstrateperiodicout-breedingtomaintaingeneticheterogeneity.If
wildphenotypescannotbeused,birdsshouldbeselectedfromamixtureofdifferentphenotypestolimit
inbredtraits.
16. Birdsshouldbeallocatedtotreatmentatrandomfromreproductivelyquiescentmixedorsingle
sexgroups.Theuseofreproductivelyquiescentbirdsmayhelptominimizedifferencesinsusceptibilityto
acuteoraladministrationofpesticidesduetosexofthebird(3,4).Forsomechemicalsorchemical
classes,evidenceofsensitivityduetosexofthebirdsmayexist(5).Thefrequencyoftoxicitydifferences
pdeurefotormseedxtoacarlolsoswaelslticmhaetmiiocnaolfsthiseLunDce50rtfaoirn.eacIfhsseenxs.itivityduetosexissuspected,testingshouldbe
Housingandtestconditions
17. Thisguidelineisdesignedtohavesinglebirdsperconcentrationintheearlystagesofthestudy.
Inordertoobservetheeffectsandrecordif/whenregurgitationtakesplace,itishighlyrecommendedthat
thebirdsbehousedindividuallyatleastforthefirstthreedaysofthestudy.Thismakesitpossibleto
observebirdsindividuallyandnotedowndetailsoftheirbehaviour(includingregurgitation).Forsociable
speciessuchaszebrafinchitisessentialandforotherspeciesitishighlyrecommendedthatbirdsbe
placedincagesnexttoeachothersotheyhavevisualandauditivesocialinteractionswitheachother.
Slmiaimlnilctaesrdtfh,oirs1ts0ht0eu0dtycesmits2sdpfeeoscriigeqnsueaiadln,tdoamhnaidvnei50mbi0urmdcsmrh2eocfuoosrmemdbeuidnngddeievrdiidgsuaaprlalcayen,dpheozruesbbiirnradgfci3on3nc3dhi.3ticLoamnrs2gefsorhrocupaligdgeeboesni,wzei2st0h0wi0intochpmtm2iomfraoelr
spaceperbirdshouldbeusedwhenrequiredorrecommendedbyregionalorindividualcountryguidelines
faanedc/eosrtroegfualllattihornosugfhor,abnuitmnaoltwreelsftarircet2i.veCtaogetheflboiorrds’sshmoouvledmbeentcson.stPriugcetoends,ofzembersah,filnacrhgeeseannodubguhdgteorailglaorws
needperchesaspartofthehousingconditionsforahealthyenvironment.
18. Thetestenvironmentmaybeundercontrolledconditionsoratambienttemperatureand
humidity.Temperatureswithintherange15-27°Caresuitableforquailandduckbutshouldfluctuateas
littleaspossibleduringtheteststages.Ventilationshouldbesufficienttosupplyatleasttenchangesofair
perhour.Thephotoperiodforquailandmallardshouldbeeighthourslightand16hoursdark.Forother
speciesitmaybenecessarytoincreasethelightphasetotenhours.Freshfoodandwatershouldbe
providedadlibitum.Commercialgamebirddietsandvitaminsupplementscanbeused,buttheyshouldbe
nutritionallyappropriateforthespeciesused. Medicationshouldbeavoidedwithin14dayspriorto
dosing,duringdosingandduringtheobservationperiod.Dietsandwatershouldbeperiodicallyanalysed
tocheckforimpuritiesthatmayinfluencethebirds’health.
2ForexampletheRecommendationoftheEuropeanCommission(2007/526/EC)
5 ©OECD,(2010)
223 OECD/OCDE
Preparationofbirds
19. Birdsshouldbeuniquelyidentified.Acclimatisationtotestconditionsanddietpriortodosing
psehroiuoldds.beAlaltlbeiarsdts1s4hodualydsbfeojrucdaggeed-rteoarbeedibnihrdesa.lthNyorcmoanldliyti,onwialndd-csahuoguhltdbniortdsbeneuesdedloinfggerreaatcecrlitmhaatni5sa%tioonf
cage-rearedorgreaterthan10%ofwildtestbirdsdieduringtheacclimatisationperiod.Ifwildbirdsdo
notacclimatetheyshouldbereleasedclosetotheirplaceofcapture.Theuseofwild-caughtbirdsshould
beavoidedifatallpossible,owingtoanimalwelfareconsiderationsanduncontrolledvariablesasage,
geneticmake-up,andhealthstatus.Capture,transportationandconfinementareassociatedwithextreme
stressandmayleadtohighermortalityofthebirds.Cage-rearedbirdsshouldbeapproximatelythesame
age.
Preparationandadministrationofdoses
20. Thetestsubstanceshouldbeadministeredinacapsuleordissolvedorsuspendedinasuitable
vehicleandthenadministeredbygavage. Ifthesubstanceisdissolvedorsuspendeditisrecommended
tcvheoahntis,cildweehsr.eanteiFovonerrovfpeohasiscsilobellsuet,oitothnheerorutsheeamnuolfwsaaitneorn,aqitnuheeooiultosx(eis.coiglt.uyctiooofmntohoreil)sv,uehsaipncedlnestihsoehnnoubbleydcpbooenssskiindboelwreendd,isfaisrnosdlt,utitfioosnlhloiounwledodtnhboeytr
causeregurgitation. Thedoseforeachbirdisdeterminedbasedonitsbodyweightmeasuredwithin
24hoursofdosing.
c2o1n.stantwTihtehrteesstpescutbsttoabnocedyiswaedimgihntisatnedresdhoiunladsniontgleexcdeoesdeb1y0mglav/akggeb.odTyhewediogshitn.gvBiorldusmsehsohuoludlbderfeamsatiend
for12-15hoursovernightimmediatelypriortodosing.Fastingovernightpriortodosingforlargerbirds
suchasNorthernbobwhiteandJapanesequailiscommended. Shorterfastingperiodsoftwohoursare
suitableforbirdsofaround50gorless. Fastingovernightmightbedeleterioustobirdsofsmallbody
mass(11).
22. Regurgitationcompromisestheevaluationoftoxicityandshouldberecorded.Theadditionofa
enaosni-ltyoxrieccocgonliosuerded(e.fg.o,odFDdy&eCthBaltuec#olnt,raFsotrsmuwiltah7t7h3e38c9o,loCurASof5f7a-e5c5e-s6)w.ilRlegaulrlgoiwtarteigounrgiistaatfieoantutroeboefamcourtee
oraltoxicitytestinginbirdsandmayberelatedtothedosingtechniqueorcharacteristicsofthetest
substance.Thefrequencyofregurgitationmaybereducedbyloweringthedosevolumeorbychanging
carriers.Itisadvised,especiallywhentestingpasserinespecies,toperformsomerange-findingworkto
evaluatethepropensityofthespeciestoregurgitate.Theoccurrenceorfrequencyofregurgitationmaybe
reducedbyloweringthedosevolume,usingcapsulesorbychangingcarriers.Ifregurgitationremainsa
problem,itmaybenecessarytoconsidertestinganotherspecies.Iftheonlyremainingoptionistoperform
tawhnheiacschtcuundroyatrdeeegsLupriDgti5e0tatvthaieloupner.oorpTemhnoesrittoaylnilotyfyctoohcneccuslrpuresecidio.ensptoossriebgulregimtaatyebtehethdaotset,heitLmDay50niostabbeovpeostshieblleowtoesdtetleevremlinaet
Observationofbirds
23. Birdsareobservedcontinuouslyduringthefirsttwohoursafterdosingforregurgitationandfor
theonsetofclinicalsigns,onatleastthreeevenlyspacedadditionaloccasionsduringthedaylightperiod
ofexposureforclinicalsigns,andatleastdailythereafterforatotalof14days.However,thedurationof
theobservationperiodshouldnotbefixedrigidly.Observationsarecontinueduntiltheprogressionof
clinicalsymptomsandmortalitycease.Iftheobservationperiodisextendedforanystage,itisalso
extendedforallsubsequentstages.
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24. Observationsmadeoneachindividualincluderegurgitation,signsofintoxicationandremission,
abnormalbehaviour,bodyweight,mortalityandtimetodeath.
2e5x.cludedfObrsoemrvcaaltciuolnastioonfs.deIaftthhseitnhcatideanrtealcldeeaartlhyoncocturtsreianttmheentcornetlraotledgr(oeu.pg,apdhdyistiiocnaallicnojnutrryo)lsbhirodusldmabye
needtobeadded(seeparagraph46).
26. Birdsshouldbeweighedbeforedosing,andthenat3,7and14daysafterdosing(orlater
dependingonthedurationofthestudy)todetermineweightchange. Foodconsumptionshouldbe
measureddailyuntilday3,thenfortheperiods3-7and7-14daysafterdosing. Attheendofthetest,
grosspathologyshouldbeundertakenonallbirdsfromeachtreatmentgroup,includingcontrolsandbirds
thatdidnotdieduringthestudy,tohelpidentifyincidentalmortalitiesandobvioussymptomsoftoxicity.
Ifnomortalitiesorclinicalsignsofsignificancearerecordedforanybird,nonecropsyisnecessary.
27. Duringthetest,animalsobviouslyinpainorshowingsignsofdistressshouldbeeuthanized(10).
PROCEDURE
Limitdosetest
28. Thelimitdosetestdesignconsistsofdosingfiveanimalssimultaneouslyatthelimitdosein
additiontosham-dosingthecontrolbirds.Controlbirdproceduresandtimelinesarediscussedin
paragraphs44to46.Birdsarethenobservedfor14days.Figure1describestheproceduretobefollowed
accordingtothemortalityobserved.Euthanizingabirdtopreventsufferingfromtoxiceffectsshouldbe
consideredequivalenttoobservingtreatmentrelatedmortality.
2cisn9oh.nowfuhiliddcemhnociIevtfelcneaotvneonlmeo,oitrttthhbaaeeltritctShoytenaicgLnleDut1hd5e0eodirdsoSaasttbeatodghveebei29rt5dohsef%otlcchimeocinustfreisdqdoufseeoennr.ctei1aT4llhedetvaeesytltes.stthaafittsetcrhodemopsLliDentg5e0,dii.stAcaabnfoavibeleetdchoelnimlciiltmuidtdoedsdoesatteestathneids9oo5nn%ee
30. Ifonetreatmentrelateddeathisobserved,andnosignsoftoxicityareobservedinotherbirds,
thenfivemorebirdsmaybedosedatthelimit,oratthediscretionofthestudydirectorandsponsor,the
testmayproceedtoStage2ofthesequentialdesign.Additionaldosingcanbeginbeforethe14-day
observationperiodiscomplete. Ifthelimitdosetestisexpanded,andthereisonlyonedeathandno
ccloinnfiicadlensciegnlsevoefltthoexiLciDty50airseaobbosveertvheedliinmitthdeostoetaalndofthteentobxiircdist,ytshteundyitisccaonmpbleetceodn.cludedthatatthe95%
31. Iftheobservedtreatmentrelatedmortalityisoneoutoffivebirdsandtherearesignsoftoxicity
inotherbirdsoriftherearetwotofourmortalitiesamongfivebirds,oriftherearetwoormoremortalities
amongtenbirds,usethesequentialdesigndescribedbelowandinFigure2,startingwithStage2.
32. Ifmortalityiscomplete(i.e.,allbirdshavedied,includingeuthanizedbirds),usethesequential
designshowninFigure2startingwithStage1.Additionaldosingcanbeginbeforethe14-dayperiodof
observationiscomplete.
tLwp3e3ahDs.reta5r0agetriaastpnhnhyeTe4leol0idi.mpemiirdttAodctodoemsoeedesdteeihtfseorwdrdoaemmssifcontr2rehi0ecbd0aelo0lidscmmeuiisgtlna/dAtfkoniogsorn-neebSowxtteftas4,gtt.ehtteo2h.weSotlraUokgsewiinn2gagnoefdtshttehihimewgasohtereqkduoioefsnnegttshieaelscLtadinDems5abi0tgeefno,colaflaloctwwuhoilernakgtLieaDdng5f0aaesifsltreddioemsmlacitrTmeiiatbboelfddeotshi1een,
7 ©OECD,(2010)
223 OECD/OCDE
34.
anobtovaelwtahyesItlbismehiotpuodlsosdsiebb.elenBtoeotceeasdutstihemaatttheienrtehseoarmLeeDc5co0inrfsoctrruamsislntitagshntcolenys,ttohtxehiecusLseuDbos5f0tavcneacrneynso.htigbhedeossteismaotfedtewstitshuobusttaunscien,gitdomsaeys
Table1:deWroirvkeidnfgroesmtmiomrattaeloitfyLiDn5a0lfiomriutsdeoisneSttesatgeat22o0f00thmegs/ekqgue-nbtwitaldesign
Mortality(%) 10 20 30 40 50 60 70 80 90
WorkingEstimate 3606 2944 2541 2244 2000 1782 1574 1358 1109
ofLD50
LD50-onlyandLD50-slopetests
Generalities
d3e5t.ailedfDleotwacihlasrtofofdtohseesdeequteenrtmiianlatdieosnigfnorfoeradcohsesdtabgierdosfitshiencsleuqdueendtiinalFidgeusriegn2.arAesdaemscprliebetdimebleilnoew.forAa
designwithfourstages,eachwitha14-dayobservationperiod,isincludedinFigure3. Proceduresand
timelinesforcontrolbirdsarediscussedinparagraphs44to46.
(s3et6.u.gd.y,,mIadmosTmeha,elhidianoinsteita,loxaeinscdtiitsymtaettpeesstosh)foutolhrdeobLneDort5e0hcecaralcncuolbmaetpebodausinneddosrodinenrptrthioeodrseaktmneeorwmcilhneeedmgtiehceaoldfoctslheaessst.eoqxuFiecoinrtcyeeaofcfohrthstethaactghesetomafigect.ahle
37. MaximumLikelihoodEstimation(MLE)isaprocedurewhichsolvesforthevaluesofmodel
parametersthatgivethegreatestlikelihoodofseeingthedataastheywereobserved.Forthistest
ttgawthuwhieoildecLerahlseDitvan5erte0re,wsaaountlsdhsree)esdvalpetnoaropdsreaacalamttsleocht(uibeslgereahseteeepdrsaettsrhtiteameagargLtrmeeDaisdpn5(he0ib.dfy3Ia8nat)tr.holeeeradItMsfehtrLetthtEwesoolcufoospprnaeeordmtMiaitnaLtilhdoEenkitsrilhtelaessirspeioonrnmnteeasecttree,cslseesupiaatsnsrteysottoftwufodotithhehreaseevvpaseerterosqaSbatuitletlasne)gta.meisosatldt1edwlaeo,nsidptaghr2neti(waivilfalllakutialelllseslaoooswtrf
38. Determinationofreversalsandpartialkillsisessentialtoidentifyingthenextstageofthetest
correctly.Reversals(i.e.,instanceswhenpercentmortalityisloweratthenexthigherdosethanpercent
mortalityatthegivendose)andpartialkills(i.e.,multiplebirdsaregivenasingledose,andmortalityis
between0and100%atthatdose)areusedascriteriaforcontinuingtothenextstageandstopping
(completing)thetest.Itisrecommendedtocountthenumberofpartialkillsfirst,then,ifnecessary,count
thenumberofreversals.Examplesofhypotheticaltestoutcomeswithpartialskillsandreversalsidentified
areprovidedinAnnex3.
Detaileddescriptionofthesequentialdesign
Foranoverviewofstoppingrules,seeparagraphs7-8andFigures1and2.Fordescriptionofthe
sequentialdesign,seeAnnex1.Fordefinitions,seeAnnex2.
©OCDE,(2010)
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OECD/OCDE 223
Stage1-FourdosesequallyspacedonalogscalearoundtheinitialestimateoftheLD5o
39. ThecalculationsforIdose,hdose,andstepforStage1areasfollows:
(1)CovdaafollstcuehueelosatLfhtDeerro5t0mthheiTsaalnbboa2lws0eea0dn10dofmonhgri/LgakhDgft5a-0irb’lesweatdtatmwlteiaanmsitintduedsdoeosfdse)res:outmessirtondg(eAtnnhtneeofrxolo4ltohceworinntsgapieencqsiueaestsittoionmxsaictwiethsyefrtoeersttLshDeor5i0niiwttihiaselbneassateildmiamotinet
Ihddoossee==ddoossee1==07..1047114xx((iinniittiiaalleessttiimmaatteeooffLLDD5500))and
(2)Ifhdoseisgreaterthan3330thensethdose=3330(maybesetloweriflimitedbyphysical
constraints)andrecalculatethelowestdoseasIdose=hdose/50
(3)Calculate:step=501'3=3.679
(4)Calculatethesecondandthirddoses:
dose2=Idosexstep and
dose3=dose2xstep=Idosexstepxstep
(5)Giveonedose{dose1,dose2,dose3,dose4)toeachofthefourbirds.
(6)Observebirdsforaperiod(typicallythreedays),andnotewhethereachbirdisdeadoralive.
Computea"workingestimateoftheLD50"whichisdefinedhereasthegeometricmeanofthe
dLoDse50swtihlaltbperuosdeudcetoaetsrtaanbsliitsihondofsreosmfosrurSvtiavgael2t.odeath(seeTable2).Thisworkingestimateofthe
(7)Continuetoobservethebirdsforelevenadditionaldays(14daysintotal).
(8)IteIffvhDietdahewyenotro.3kbiIisnnenfrgsovoraemmtsaiettoiincmoaasnpteeeisrn,iodofiidtctamphtareeiysoLtrbDheat5to0nfecmucaraeltscyhsuelarbaretmyiodntrogetalwltaaihyiteetydwumouparntyktioilonc1gr4ceuLcdroaDvywe5si0rtybhieisfoneoftrxhtetihesmentodervesetidmnsafgtioanrgtieont,htgichsaetlescnstutealxgabteti,irsodtianstgoeii.fss
extendedforallsubsequentstages.Ifthereisevidenceofdelayedeffectsafter14days,thelength
ofthestagemaybefurtherextended.
9 ©OECD,(2010)
5 2 2) 2 4 222 4
223 OECD/OCDE
Table2:CalculationoftheworkingLD50computedfromthefourdosesinStage1a,b
dose1 dose2 dosei dose4 WorkingLDS0fromStage1data
0 0 0 0 (dose4xdosei)1'2
0 0 0 X {dose3xdose4)1/2
0 0 X 0 {dose2xdose3xdose4xdose5)1/4={dose3xdose4)1/2
O X 0 0 {dose1xdose2xdose4xdose5)1/4=dose3
X 0 0 o {dose0xdose1xdose4xdose5)1/4={dosexdose3)1/2
0 0 X X {doseixdose3)1/2
0 X X 0 {dose1xdose2xdoseAxdose5)1/4=dose3
X X 0 0 (dose0xdose1xdose4xdose5)l,4=(dose2xdose3)l/2
0 X 0 X {dose1xdosexdose3xdose4)1/4={dosexdose3)1/2
X 0 X 0 {=d(o<dsoes0exdxodsoes1ex3)d1o/2sexdose3xdose xdose5)17
X 0 0 X {doseOxdoseixdose3xd0se4)l/4=dose
0 X X X {doseixdose2)1/2
X 0 X X {dose0xdoseixdoseixdose3)1/4=(doseixdose2)1/2
X X 0 X {doseOxdoseixdose3xdose4)1/4=dosei
X X X 0 {dose0xdoseixdose4xdose5)l/4={dose!xdoseO)112
X X X X {doseOxdose11/2
a Survivalisrepresentedby0anddeathbyX.
b Eventhoughonlyfourdoses{dose1throughdose4)areusedinthetest,calculatedvaluesfor
dose0anddose5areusedinthetable.Thevaluesthatshouldbeusedforthesedosesareone
sslttoeewppe.sutDptoeossrte0ddoosswheo,nualfndrdobmdeotashdeedeaidcsttauoadldthetedesctwohdmeopsnuetsta.hteirToehniasotfsuitsrh,veidvwoaoslreak0tin=thgedLohisDge5h01ews/thsetteensptmdaoornstdea.ldiotsyeo5cc=urdsosatethxe
Stage2-TendosesequallyspacedonalogscalearoundtheworkingestimateoftheLD50obtainedfrom
Stage1orfromalimitdosetestwithpartialmortality
40. ThecalculationsofIdose,hdoseandstepforStage2areasfollows:
(1)CisalbcausleadteotnheStlaogwean1drehsiuglths,trbeaastemdentondovsaelsueussifnrgotmheTafbollelow1infgoreqauafatiiloendslwihmietredotsheewteosrtk(inAgnnLeDx540
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