Test No. 436: Acute Inhalation Toxicity - Acute Toxic Class Method [electronic resource]

2009·0.63 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 436
Adopted:
7September2009
OECDGUIDELINEFORTHETESTINGOFCHEMICALS
AcuteInhalationToxicity-AcuteToxicClassMethod
INTRODUCTION
1. OECDGuidelinesfortheTestingofChemicalsareperiodicallyreviewedinthelightof
scientificprogress,changingregulatoryneedsandanimalwelfareconsiderations.Thefirstacute
inhalationTestGuideline403wasadoptedin1981,andhassincebeenrevised(1).Developmentofan
IaabndeshoisapneltgsaistomuinesonentdofAofcfoutrththeeCelraTAesoTvsxiiCisfcietcdeaCstltoiaromsanelsta(hAnAodTdTCCLfa)obmremealetclthuitnhoegoddipn(uh(Tra2plG)aots(4i3e2o)sn3)((t46o))x(.i5wc)Tiahtsiyenscioh2nn0ohs0wai1led.adetriAteohdnatraAetpthTprerCoosmppeTreteichsatittovedeGfuisopildesleruolifwtioainrbnemlgaewntifchloeelr
allowtheuseofserialstepsoffixedtargetconcentrationstoprovidearankingoftestarticletoxicity.
Lethalityisusedaskeyendpoint,however,animalsinseverepainordistress,sufferingorimpending
dtheeatOhEsChoDulGduibedahnucmeaDneolcyumkeilnltedNtoo.m1i9ni(7m)i.zesuffering.Guidanceonhumaneendpointsisavailablein
2. GuidanceontheconductandinterpretationofthisTestGuidelinecanbefoundinthe
GuidanceDocumentNo.39onAcuteInhalationToxicityTesting(GD39)(8).
3. DefinitionsusedinthecontextofthisGuidelineareprovidedinGD39(8).
4. Themethodprovidesinformationonthehazardouspropertiesandallowsthesubstancetobe
rankedandclassifiedaccordingtotheUnitedNations(UN)GloballyHarmonizedSystemof
ClassificationandLabellingofChemicals(GHS)fortheclassificationofchemicalsthatcauseacute
tTsoeexlsietccitGtiuyoind(9ec)la.innIbene4cf0aos3uen(p1d)oiininstGteDhset3ia9mpap(tr8eo)sp.rToihfaitLseCTTe5e0ss-tvtaGGluuuiiedsdeelolirinneceointscoennuostter.saptFeicuoirnft-ihrceearsllpgyouniisndetaenancndeaeldoynsfeosTretashrteeGtrueeisqtduieinlrgienode,f
specializedmaterials,suchaspoorlysolubleisometricorfibrousmaterialsormanufactured
nanomaterials.
INITIALCONSIDERATIONS
5. BeforeconsideringtestinginaccordancewiththisTestGuidelineallavailableinformation
onthetestarticle,includingexistingstudieswhosedatawouldsupportnotdoingadditionaltesting
shouldbeconsideredbythetestinglaboratoryinordertominimizeanimalusage.Informationthat
mayassistintheselectionofthemostappropriatespecies,strain,sex,modeofexposureand
appropriatetestconcentrationsincludetheidentity,chemicalstructure,andphysico-chemical
propertiesofthetestarticle;resultsofanyinvitroorinvivotoxicitytests;anticipateduse(s)and
potentialforhumanexposure;available(Q)SARdataandtoxicologicaldataonstructurallyrelated
©OECD,(2009)
YftoroowumriattrtheeenfOrpEeeeCrmtDoi,suspsireoontvhifidrseodmmattthehereisaOolEufCrocDre.peisrsdounlayl,mennotni-ocnoemdm.eArnciyaclopmumreprocsieaslwuistehooufttshiesekmiantegriparlioriscsounbsjeenctt
436 OECD/OCDE
ssuebvsetraenlcyeisr.ritaCnotncacetnitornast,isohnosutlhdatnoatrebeetxepsetcetdewdittohcthaiussTeesstevGeuriedeplaiinnea[nsdeediGsDtre3ss9,(d8)u]e.tocorrosive1or
PRINCIPLEOFTHETEST
6. Itistheprincipleofthetestthatbasedonastepwiseprocedure,sufficientinformationis
obtainedontheacuteinhalationtoxicityofthetestarticleduringanexposureperiodof4hoursto
enableitsclassification.Otherdurationsofexposuremayapplytoservespecificregulatorypurposes.
Atanyofthedefinedconcentrationsteps,3animalsofeachsexaretested.Dependingonthemortality
and/orthemoribundstatusoftheanimals,2stepsmaybesufficienttoallowjudgementontheacute
toxicityofthetestarticle.Ifevidenceisprovidedthatonesexismoresusceptiblethantheother,then
thetestmaybecontinuedwiththemoresusceptiblesexonly.Theoutcomeofthepreviousstepwill
determinethefollowingstepsuchthat:
a)Nofurthertestingisneeded,
b)Testingofthreeanimalspersex,or
c)Testingwith6animalsofthemoresusceptiblesexonlyi.e.thelower
boundaryestimatesofthetoxicclassshouldbebasedon6animalspertest
concentrationgroup,regardlessofsex.
7. Moribundanimalsoranimalsobviouslyinpainorshowingsignsofsevereandenduring
distressshouldbehumanelykilled,andareconsideredintheinterpretationofthetestresultsinthe
samewayasanimalsthatdiedontest.Criteriaformakingthedecisiontokillmoribundorseverely
sufferinganimals,andguidanceontherecognitionofpredictableorimpendingdeath,arethesubject
ofGuidanceDocumentNo.19onHumaneEndpoints(7).
DESCRIPTIONOFTHEMETHOD
Selectionofanimalspecies
8. Healthyyoungadultanimalsofcommonlyusedlaboratorystrainsshouldbeused.The
preferredspeciesistheratandjustificationsshouldbeprovidedifotherspeciesareused.
Preparationofanimals
9. Femalesshouldbenulliparousandnon-pregnant. Ontheexposureday,animalsshouldbe
youngadults8to12weeksofage,andbodyweightsshouldbewithin±20%ofthemeanweightfor
eachsexofanypreviouslyexposedanimalsatthesameage.Theanimalsarerandomlyselected,
markedforindividualidentification.Theanimalsarekeptintheircagesforatleast5dayspriortothe
startofthetesttoallowforacclimatizationtolaboratoryconditions.Animalsshouldalsobe
acclimatisedtothetestapparatusforashortperiodpriortotesting,asthiswilllessenthestresscaused
byintroductiontothenewenvironment.
Animalhusbandry
10. Thetemperatureoftheexperimentalanimalmaintenanceroomshouldbe22±3°C. The
relativehumidityshouldideallybemaintainedintherangeof30to70%,thoughthismaynotbe
1esTixhmpeielraciroernsrcuoebs,sivteiaxtniycsetesivnoagrlu(aaintnyivoointthrceoor)upldeadrttabi,eneen.bgta.sdeaTtdGa.son4e3x0pe(r1t0)j,u4d3g1men(1t1)usoirng43s5uc(h12e)v,ipdeHncvealause:s,huimnfaonrmaantidonanfirmoaml
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possiblewhenusingwaterasavehicle. Beforeandafterexposures,animalsgenerallyshouldbe
cagedingroupsbysexandconcentration,butthenumberofanimalspercageshouldnotinterfere
withclearobservationofeachanimalandshouldminimizelossesduetocannibalismandfighting.
Whenanimalsaretobeexposednose-only,itmaybenecessaryforthemtobeacclimatedtothe
restrainingtubes.Therestrainingtubesshouldnotimposeunduephysical,thennal,orimmobilization
stressontheanimals.Restraintmayaffectphysiologicalendpointssuchasbodytemperature
(hyperthermia)and/orrespiratoryminutevolume.Ifgenericdataarcavailabletoshowthatnosuch
changesoccurtoanyappreciableextent,thenpre-adaptationtotherestrainingtubesisnotnecessary.
Animalsexposedwhole-bodytoanaerosolshouldbehousedindividuallyduringexposuretoprevent
themfromfilteringthetestaerosolthroughthefuroftheircagemates. Conventionalandcertified
laboratorydietsmaybeused,exceptduringexposure,accompaniedwithanunlimitedsupplyof
municipaldrinkingwater.Lightingshouldbeartificial,thesequencebeing12hourslight/12hours
dark.
Inhalationchambers
s1e1l.ectingTanheinnhaatlautrieonofchtahmebetre.stTarhteiclpereafenrdretdhemoodbejecotfiveexpoofsutrheeistenstosseh-oounlldy(bwehiccohnstiedremreidncwluhdeens
head-only,nose-onlvorsnout-only).Nose-onlyexposureisgenerallypreferredforstudiesofliquidor
solidaerosolsandforvapoursthatmaycondensetoformaerosols.Specialobjectivesofthestudymay
bebetterachievedbyusingawhole-bodymodeofexposure,butthisshouldbejustifiedinthestudy
raeenxpipomorast.lusrTeostheoecunhlsnudirqneuoeatstemaxoncsdepehtdeheri5er%pstaoarbtfiiltcihutelyarwchhaademvnabneutsraignvegoslauamwnehd.odliePs-raibdnovcdaiynptlcaehsgaeomsfbaetrrhe,edtnehosecsreti-obtoaenldlyvinoalGnudDmew3ho9ofl(t8e)h.ebotedsyt
EXPOSURECONDITIONS
Administrationsofconcentrations
12. Afixeddurationofexposureforfourhours,excludingequilibrationtime,isrecommended.
phOrotouhvesireddeddiunridanitvitiohdneusaslmtlyuadytyorpberepeovnretenet[dsieendegeGtsotDiomn3e9eotf(8t)seJsp.tecAairnftiiiccmlaerledsquueeixrtpeoomgesnretodso,imnihnwoghwoeolvfeec-rab,goedjuymsatctiehfsiac.mabtFeieorensdssshhhooouuullldddbbbeee
withheldduringtheexposureperiod.Watermaybeprovidedthroughoutawhole-bodyexposure.
13. Animalsareexposedtothetestarticleasagas,vapour,aerosol,oramixturethereof.The
physicalstatetobetesteddependsonthephysico-chemicalpropertiesofthetestarticle,theselected
concentration,and/orthephysicalformmostlikelypresentduringthehandlinganduseofthetest
article.Hygroscopicandchemicallyreactivetestarticlesshouldbetestedunderdry'airconditions.
Careshouldbetakentoavoidgeneratingexplosiveconcentrations.
Particle-sizedistribution
14. Particlesizingshouldbeperformedforallaerosolsandforvapoursthatmaycondenseto
fmoerdmiaaneraoesorlosd.yTnaomailclodiwafmoerteerxspo(sMurMeAoDf)allrarnegleivnagntfrroemgio1ntsoo4fpthmewreistphiraatgoeroymetrtarcit,casetraonsdoalrsdwdietvhiamtaisosn
mm(eoagtd)aelintfotuhmmeeeerstantmghaeisyosftba1en.d5astrmoda,l3l.ee0xrpaerrtetharjneucdtgohmmisemnestntdasnehddoaur(ld8d,)b(ea1n3pd)ro(cv1hi4a)d.regdAelidtfhiptoacurtaginhcnlaoetsr,ebaefsiaobcnrehasib,elveeadenf.dfoFrhotyrgserhxooasumclpodplbieec,
materials(whichincreaseinsizeinthemoistenvironmentoftherespiratorytract)mayexceedthis
standard.
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436 OECD/OCDE
Testarticlepreparationinavehicle
15. Avehiclemaybeusedtogenerateanappropriateconcentrationandparticlesizeofthetest
articleintheatmosphere.Asarule,watershouldbegivenpreference.Particulatematerialmaybe
subjectedtomechanicalprocessestoachievetherequiredparticlesizedistribution,however,care
shouldbetakennottodecomposeoralterthetestarticle.Incaseswheremechanicalprocessesare
believedtohavealteredtestarticlecomposition(e.g.extremetemperaturefromexcessivemillingdue
tofriction),thecompositionofthetestarticleshouldbeverifiedanalytically.Adequatecareshouldbe
twahkiecnhtaorenopturcpoonsteafumlilnyatfeontnhuelatetsetdmtaotebreiauln.-iInthiaslanbolte.neAcnessaattrryi'titoontteessttsnhono-uflrdiabbeleusgerdantuoladremmoantsetrriaatles
thatrespirableparticlesarenotproducedwhenthegranularmaterialishandled.Ifanattritiontest
producesrespirablearticles,aninhalationtoxicitytestshouldbeperformed.
Controlanimals
16. Aconcurrentnegative(air)controlgroupisnotnecessary.Whenavehicleotherthanwater
isusedtoassistingeneratingthetestatmosphere,avehiclecontrolgroupshouldonlybeusedwhen
historicalinhalationtoxicitydataarenotavailable.Ifatoxicitystudyofatestarticleformulatedina
vehiclerevealsnotoxicity,itfollowsthatthevehicleisnon-toxicattheconcentrationtested;thus,
thereisnoneedforavehiclecontrol.
MONITORINGOFEXPOSURECONDITIONS
Chamberairflow
17. Theflowofairthroughthechambershouldbecarefullycontrolled,continuouslymonitored,
andrecordedatleasthourlyduringeachexposure.Themonitoringoftestatmosphereconcentration
(orstability)isanintegralmeasurementofalldynamicparametersandprovidesanindirectmeansto
controlallrelevantdynamicatmospheregenerationparameters.Specialconsiderationshouldbegiven
toavoidingre-breathinginnose-onlychambersincaseswhereairflowthroughtheexposuresystem
areinadequatetoprovidedynamicflow'oftestarticleatmosphere.Thereareprescribedmethodologies
thatcanbeusedtodemonstratethatre-breathingdoesnotoccurundertheselectedoperation
conditions(8)(15).Oxygenconcentrationshouldbeatleast19%andcarbondioxideconcentration
shouldnotexceed1%.Ifthereisreasontobelievethatthesestandardscannotbemet,oxygenand
carbondioxideconcentrationsshouldbemeasured.
Chambertemperatureandrelativehumidity
18. Chambertemperatureshouldbemaintainedat22±3°C.Relativehumidityintheanimals'
breathingzone,forbothnose-onlyandwhole-bodyexposures,shouldbemonitoredandrecordedat
leastthreetimesfordurationsupto4hrs.andhourlyforshorterdurations.Therelativehumidity
shouldideallybemaintainedintherangeof30to70%,butthismayeitherbeunattainable(e.g.when
testingwaterbasedformulations)ornotmeasurableduetotestarticleinterferencewiththetest
method.
Testarticle:nominalconcentration
19. Wheneverfeasible,thenominalexposurechamberconcentrationshouldbecalculatedand
recorded.Thenominalconcentrationisthemassofgeneratedtestarticledividedbythetotalvolume
ofairpassedthroughthechambersystem.Thenominalconcentrationisnotusedtocharacterizethe
©OCDE,(2009) 4
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OECD/OCDE 436
animals’exposure,butacomparisonofthenominalandtheactualconcentrationgivesanindicationof
thegenerationefficiencyofthetestsystem,andthusmaybeusedtodiscovergenerationproblems.
Testarticle:actualconcentration
20. Theactualconcentrationisthetestarticleconcentrationattheanimals'breathingzoneinan
isnahmapllaitnigo,nacdhsaomrbpetirv.eAocrtucahlemicconaclenrteracattiivoensmectahnodbse,oabntdaisnuebdseeqiutehnertbanyalsypteicciaflicchmareatchtoerdissat(ei.ogn)doirrebcyt
non-specificmethodssuchasgravimetricfilteranalysis.Theuseofgravimetricanalysisisacceptable
onlyforsinglecomponentpowderaerosolsoraerosolsoflowvolatilityliquidsandshouldbe
supportedbyappropriatepre-studytestarticle-specificcharacterisations.Multi-componentpowder
aerosolconcentrationmayalsobedeterminedbygravimetricanalysis.However,thisrequires
analyticaldatawhichdemonstratethatthecompositionofairbornematerialissimilartothestarting
material.Ifthisinfonnationisnotavailable,areanalysisofthetestmaterial(ideallyinitsairborne
state)atregularintervalsduringthecourseofthestudymaybenecessary.Foraerosolisedagentsthat
mayevaporateorsublimate,itshouldbeshownthatallphaseswerecollectedbythemethodchosen.
Thetarget,nominal,andactualconcentrationsshouldbeprovidedinthestudyreport,butonlyactual
concentrationsareusedinstatisticalanalysestocalculatelethalconcentrationvalues.
21. Onelotofthetestarticleshouldbeused,ifpossible,andthetestsampleshouldbestored
underconditionsthatmaintainitspurity,homogeneity,andstability.Priortothestartofthestudy,
thereshouldbeacharacterizationofthetestarticle,includingitspurityand,iftechnicallyfeasible,the
identity,andquantitiesofidentifiedcontaminantsandimpurities.Thiscanbedemonstratedby,butis
notlimitedto,thefollowingdata:retentiontimeandrelativepeakarea,molecularweightfrommass
spectroscopyorgaschromatographyanalyses,orotherestimates.Althoughthetestsample'sidentity
issponnostotrh’esrcehsaproancstiebriilziattyioonfatthleeatsetstinlaabolriamtiotreyd,wiatyma(ye.gb.ecoplrouudre,npthfyosrictahlentaetsutrlea,beotrca.)t.orytoconfirmthe
22. The exposure atmosphere shall be held asconstant aspracticable andmonitored
continuouslyand/orintermittentlydependingonthemethodofanalysis.Whenintermittentsampling
isused,chamberatmospheresamplesshouldbetakenatleasttwiceinafourhourstudy.Ifnotfeasible
duetolimitedairflowratesorlowconcentrations,onesamplemaybecollectedovertheentire
exposureperiod.Ifmarkedsample-to-samplefluctuationsoccur,thenextconcentrationstestedshould
usefoursamplesperexposure.Individualchamberconcentrationsamplesshouldnotdeviatefromthe
meanchamberconcentrationbymorethan±10%forgasesandvapours,andbynomorethan±20%
tfdoiurmraeltsiiqroueniqdouoifrraesndolteioxdpaaotetsrauoirsneoUlsss.paGTnuisimdtehaentcoteicmhfeoarmthbeasettritmehaqetutiielnsigtbtra9ar5ttciicoalnne(bites95)gfeosnuhenorudaltidendbGeaDncad3lt9chui(ls8a)t.teadkeasndinrteocoarcdceodu.ntTthhee
23. Forverycomplexmixturesconsistingofvapours/gases,andaerosols(e.g.combustion
atmospheresandtestarticlespropelledfrompurpose-drivenend-useproducts/devices),eachphase
maybehavedifferentlyinaninhalationchambersoatleastoneindicatorsubstance(analyte),normally
theprincipalactivesubstanceinthetestedproductformulation,ofeachphase(vapour/gasandaerosol)
shouldbeselected.Whenthetestarticleisamixture(e.g.aformulation),theanalyticalconcentration
s(ahnoaullydte)b.eArdedpiotritoendalfoirnftohnenatottiaolnfroergmaurldaitnigonacatunadlncootncjeunsttraftoirotnhsecaacntibveefionugnrdediinenGtDor39th(e8).component
Testarticle:particlesizedistribution
24. Theparticlesizedistributionofaerosolsshouldbedetenninedatleasttwiceduringeach4
hourexposurebyusingacascadeimpactororanalternativeinstrumentsuchasanaerodynamic
particlesizer.Ifequivalenceoftheresultsobtainedbyacascadeimpactororanalternativeinstrument
5 ©OCDE,(2009)
436 OECD/OCDE
canbeshown,thenthealternativeinstrumentmaybeusedthroughoutthestudy.Aseconddevice,
suchasagravimetricfdteroranimpinger/gasbubbler,shouldbeusedinparalleltotheprimary
instrumenttoconfirmthecollectionefficiencyoftheprimaryinstrument.Themassconcentration
toohbbettaaiisntnueedddy,bbyythfieplantretrfiucarlnteahleysrsiizcseon[asfneiaerlmyGsaitDsor3ys9hom(ue8l)aJds.uIbrfeeemqweuinittvhsailnmenarceyeascboaennaboblmeeitdtleeimdm.oitnssFtoorrfatatenhdiemianmlatshweselefcaaorrnleycepnrhtearaasstoeinoso,nf
measuresshouldbetakentominimizeinconclusivedatawhichmayleadtoaneedtorepeatan
exposure.Particlesizingshouldbeperformedforvapoursifthereisanypossibilitythatvapour
condensationmayresultintheformationofanaerosol,orifparticlesaredetectedinavapour
a26t.mospherewithpotentialformixedphases(seeparagraph14).
PROCEDURE
Maintest
Threeanimalspersex,orsixanimalsofthemoresusceptiblesex,areusedforeachstep.If
rodentspeciesotherthanratsareexposednose-only,maximumexposuredurationsmaybeadjustedto
minimisespecies-specificdistress.Theconcentrationleveltobeusedasthestartingdoseisselected
fromoneoffourfixedlevelsandthestartingconcentrationlevelshouldbethatwhichismostlikelyto
p(rinocdluucdeedtoixnicAintnyeixnesso1m-e3)orfetphreesdeontsetdheantiemstailnsg.wTihtehttehsetcinugt-sofcfhevmaleusesfoorfgtahseesG,HvSapocuatresgoarnidesae1r-o4so(l9)s
forgases(100,500,2500,20000ppm/4h)(Annex1),forvapours(0.5,2,10,20mg/L/4h)(Annex2)
andforaerosols(0.05,0.5,1,5mg/L/4h)(Annex3).Category5relatestoconcentrationsabovethe
respectivelimitconcentrations.Foreachstartingconcentration,therespectivetestingschemeapplies.
Dependingonthenumberofhumanelykilledordeadanimals,thetestprocedurefollowstheindicated
arrowsuntilacategorisationcanbemade.
26. Thetimeintervalbetweenexposuregroupsisdeterminedbytheonset,duration,andseverity
orefatsooxniacblseigcnos.nfEixdepnocseurientohfeasnuirmvailvsalaotftthheenperxetvicoounscleynttreasttieodnanliemvaells.shAoulpderbieoddoeflatyherdeeuontrilfotuhrerdeayiss
betweentheexposuresateachconcentrationlevelisrecommendedtoallowfortheobservationof
delayedtoxicity.Thetimeintervalmaybeadjustedasappropriate,e.g.incaseofinconclusive
responses.
Limittest
27. Thelimittestisusedwhenthetestarticleisknownorexpectedtobevirtuallynon-toxic,i.e.
elicitingatoxicresponseonlyabovetheregulatorylimitconcentration.Informationaboutthetoxicity
ofthetestmaterialcanbegainedfromknowledgeaboutsimilartestedcompoundsorsimilartested
mixturesorproducts,takingintoconsiderationtheidentityandpercentageofcomponentsknowntobe
oftoxicologicalsignificance.Inthosesituationswherethereislittleornoinformationaboutits
tgouxiidcaitnyc,eocranthbeefteosutndmaitnerGiDal3i9s(e8)x]p.ectedtobetoxic,themaintestshouldbeperformed[further
28. Usingthenormalprocedure,threeanimalspersex,orsixanimalsofthemoresusceptible
sex,arcexposedatconcentrationsof20000ppmforgases,20mg/Lforvapoursand5mg/Lfor
tdeussttisn/gmiasetrso,sorless,petchteivperliyma(rifyagcohailevsahboluel),dwbheitcohacsheirevevseaasrtehsepilriambiltetpeasrttifcolretshiizseT(ei.set.GaunidMeMliAneD.oWfhe1-n4
tGphmHa)nS.2Tdhimisgsc/oiuLsrapsgohesossuiltbdelsetoinwnligythibnemeoxascttetsetsmepsottfeadartiilfcilmaeistreacstopniacreacnbotlnrecaeptnaitrortnaitcflioeornsaionzfiem2caalmngw/ebLle.faaArceehriroeesvaeosdlont[sesse(te9i)nG.gDTaets3tg9irne(ag8t)ie].nr
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OECD/OCDE 436
GHSCategory5shouldonlybeconsideredwhenthereisastronglikelihoodthatresultsofsuchatest
wouldhavedirectrelevanceforprotectinghumanhealth(9),andjustificationprovidedinthestudy
report. Inthecaseofpotentiallyexplosivetestarticles,careshouldbetakentoavoidconditions
favourableforanexplosion.Toavoidanunnecessaryuseofanimals,atestrunwithoutanimalsshould
beconductedpriortothelimittesttoensurethatthechamberconditionsforalimittestcanbe
achieved.
Observations
29. Theanimalsshouldbeclinicallyobservedfrequentlyduringtheexposureperiod.Following
exposure,clinicalobservationsshouldbemadeatleasttwiceonthedayofexposure,ormore
frequentlywhenindicatedbytheresponseoftheanimalstotreatment,andatleastoncedaily
thereafterforatotalof14days.Thelengthoftheobservationperiodisnotfixed,butshouldbe
detenninedbythenatureandtimeofonsetofclinicalsignsandlengthoftherecoveryperiod.The
timesatwhichsignsoftoxicityappearanddisappearareimportant,especiallyifthereisatendency
forsignsoftoxicitytobedelayed.Allobservationsaresystematicallyrecordedwithindividual
recordsbeingmaintainedforeachanimal.Animalsfoundinamoribundconditionandanimals
showingseverepainand/orenduringsignsofseveredistressshouldbehumanelykilledforanimal
welfarereasons.Careshouldbetakenwhenconductingexaminationsforclinicalsignsoftoxicitythat
initialpoorappearanceandtransientrespiratorychanges,resultingfromtheexposureprocedure,are
notmistakenfortreatment-relatedeffects.TheprinciplesandcriteriasummarisedintheHumane
EndpointsGuidanceDocumentshouldbetakenintoconsideration(7).Whenanimalsarekilledfor
humanereasonsorfounddead,thetimeofdeathshouldberecordedaspreciselyaspossible.
30. Cage-sideobservationsshouldincludechangesintheskinandfur,eyesandmucous
membranes,andalsorespiratory,circulatory,autonomicandcentralnervoussystems,andsomato-
motoractivityandbehaviourpatterns.Whenpossible,anydifferentiationbetweenlocalandsystemic
effectsshouldbenoted.Attentionshouldbedirectedtoobservationsoftremors,convulsions,
salivation,diarrhoea,lethargy,sleepandcoma.Themeasurementofrectaltemperaturesmayprovide
supportiveevidenceofreflexbradypneaorhypo/hyperthermiarelatedtotreatmentorconfinement.
Bodyweights
dao3tfa1yt.thooexfitceiixmtpeIyonosdafuinvrddieedaauptnarhiliomoararnltiseomuaetelxhxaphwnioebasiisugtirihaentigs(fdeasaxhycosueu0s)eltddaaiibnnnedgedradteadcyleoerca1sdr.teeBdmooenodnnydtcaewyoesfdiug>1rh.2it3n0g%ias,tnhrdeecc7oaomcg(cpnalainirsdmeeawddteiaeztsakotlaipyorcnretih-tpesiertcreauialdofytdie,nrvd)oai,lncuaatetnshod,er
shouldbecloselymonitored.Survivinganimalsareweighedandhumanelykilledattheendofthe
post-exposureperiod.
Pathology
32. Alltestanimals,includingthosewhichdieduringthetestorareeuthanizedandremoved
fromthestudyforanimalwelfarereasons,shouldbesubjectedtogrossnecropsy.Ifnecropsycannot
beperformedimmediatelyafteradeadanimalisdiscovered,theanimalshouldberefrigerated(not
frozen)attemperatureslowenoughtominimizeautolysis.Necropsiesshouldbeperformedassoonas
possible,normallywithinadayortwo.Allgrosspathologicalchangesshouldberecordedforeach
animalwithparticularattentiontoanychangesintherespiratorytract.
33. Additionalexaminationsincludedaprioribydesignmaybeconsideredtoextendthe
interpretivevalueofthestudy,suchasmeasuringlungweightofsurvivingratsand/orproviding
7 ©OCDE,(2009)
436 OECD/OCDE
evidenceofirritationbymicroscopeexaminationoftherespiratorytract.Examinedorgansmay
includethoseshowingevidenceofgrosspathologyinanimalssurviving24ormorehours,andorgans
knownorexpectedtobeaffected.Microscopicexaminationoftheentirerespiratorytractmayprovide
usefulinformationfortestarticlesthatarereactivewithwater,suchasacidsandhygroscopictest
articles.
DATAANDREPORTING
Data
34. Individualanimaldataonbodyweightsandnecropsyfindingsshouldbeprovided.Clinical
observationdatashouldbesummarizedintabularform,showingforeachtestgroupthenumberof
animalsused,thenumberofanimalsdisplayingspecificsignsoftoxicity,thenumberofanimalsfound
deadduringthetestorkilledforhumanereasons,timeofdeathofindividualanimals,adescription
andtimecourseoftoxiceffectsandreversibility,andnecropsyfindings.
Testreport
35. Thetestreportshouldincludethefollowinginformation,asappropriate:
Testanimalsandhusbandry
Descriptionofcagingconditions,including:number(orchangeinnumber)ofanimalsper
cage,beddingmaterial,ambienttemperatureandrelativehumidity,photoperiod,and
identificationofdiet;
Species/strainusedandjustificationforusingaspeciesotherthantherat;
Number,age,andsexofanimals;
Methodofrandomization;
Detailsoffoodandwaterquality(includingdiettvpe/source,watersource);
Descriptionofanypre-testconditioningincludingdiet,quarantine,andtreatmentfor
disease;
Testarticle
Physicalnature,purity,and,whererelevant,physico-chemicalproperties(including
isomerization);
IdentificationdataandChemicalAbstractServices(CAS)Registry'Number,ifknown;
Vehicle
Justificationforuseofvehicleandjustificationforchoiceofvehicle(ifotherthanwater);
Historicalorconcurrentdatademonstratingthatthevehicledoesnotinterferewiththe
outcomeofthestudy;
Inhalationchamber
Descriptionoftheinhalationchamberincludingdimensionsandvolume;
Sourceanddescriptionofequipmentusedfortheexposureofanimalsaswellas
generationofatmosphere;
©OCDE,(2009)
;
OECD/OCDE 436
Equipmentformeasuringtemperature,humidity,particle-size,andactualconcentration;
Sourceofair.treatmentofairsupplied/extractedandsystemusedforconditioning;
Methodsusedforcalibrationofequipmenttoensureahomogeneoustestatmosphere;
Pressuredifference(positiveornegative);
Exposureportsperchamber(nose-only);locationofanimalsinthesystem(whole-body);
Temporalhomogeneity/stabilityoftestatmosphere
Locationoftemperatureandhumiditysensorsandsamplingoftestatmosphereinthe
chamber;
Airflow1rates,airflowrate/exposureport(nose-only),oranimalload/chamber(whole-
body);
Informationabouttheequipmentusedtomeasureoxygenandcarbondioxide,if
applicable;
Timerequiredtoreachinhalationchamberequilibrium(ty);
Numberofvolumechangesperhour;
Meteringdevices(ifapplicable);
Exposuredata
Rationalefortargetconcentrationselectioninthemainstudy;
Nominalconcentrations(totalmassoftestarticlegeneratedintotheinhalationchamber
dividedbythevolumeofairpassedthroughthechamber);
Actualtestarticleconcentrationscollectedfromtheanimals'breathingzone;fortest
mixturesthatproduceheterogeneousphysicalforms(gases,vapours,aerosols),eachmay
beanalysedseparately;
Avlolluaimrec(oen.cge.nptrpamt,iopnpsb)shmoaulydablesorbeeporretpeodritnedunpiatrsenotfhemtaiscasll(ye.;g.mg/L,mg/m3,etc.),unitsof
Particlesizedistribution,massmedianaerodynamicdiameter(MMAD),andgeometric
standarddeviation(ag),includingtheirmethodsofcalculation. Individualparticlesize
analysesshouldbereported;
Testconditions
Detailsoftestarticlepreparation,includingdetailsofanyproceduresusedtoreducethe
particlesizeofsolidmaterialsortopreparesolutionsofthetestarticle. Incaseswhere
mechanicalprocessesmayhavealteredtestarticlecomposition,includetheresultsof
analysestoverifythecompositionofthetestarticle;
Adescription(preferablyincludingadiagram)oftheequipmentusedtogeneratethetest
atmosphereandtoexposetheanimalstothetestatmosphere;
Detailsofthechemicalanalyticalmethodusedandmethodvalidation(including
efficiencyofrecoveryoftestarticlefromthesamplingmedium);
Therationalefortheselectionoftestconcentrations;
Results
Tabulationofchambertemperature,humidify',andairflow;
Tabulationofchambernominalandactualconcentrationdata;
9 ©OCDE,(2009)
436 OECD/OCDE
Tdiasbturliabuttiioonn,oafnpdarctailccluelastiizoensdaotfathiencMluMdiAngDanaanldytoigc;alsamplecollectiondata,particlesize
Tabulationofresponsedataandconcentrationlevelforeachanimal(i.e.animalsshowing
signsoftoxicityincludingmortality,nature,severity,anddurationofeffects);
Individualbodyweightsofanimalscollectedonstudydays,dateandtimeofdeathifprior
toscheduledeuthanasia;timecourseofonsetofsignsoftoxicity,andwhetherthesewere
reversibleforeachanimal;
Necropsyfindingsandhistopathologicalfindingsforeachanimal,ifavailable;
TheGHScategoryclassificationandtheLC50cut-offvalue;
Discussionandinterpretationofresults
ParticularemphasisshouldbemadetothedescriptionofmethodsusedtomeetthisTest
Guideline’scriteria,e.g.thelimitconcentrationortheparticlesize;
Therespirabilityofparticlesinlightoftheoverallfindingsshouldbeaddressed,especially
iftheparticle-sizecriteriacouldnotbemet;
Theconsistencyofmethodsusedtodeterminenominalandactualconcentrations,andthe
relationofactualconcentrationtonominalconcentrationshouldbeincludedintheoverall
assessmentofthestudy;
Thelikelycauseofdeathandpredominantmodeofaction(systemicversuslocal)should
beaddressed;
Apaninexoprlsahnaotwiionngsshiogunlsdobfesepvreorveidaenddiefntdhuerriengwadsistarensese,dbatsoehduomnantehleycrsiatcerriifaicientahneimOaElsCiDn
GuidanceDocumentonHumaneEndpoints(7).
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