Subchronic inhalation toxicity [electronic resource] : 90-day study

2009·0.46 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE
413
Adopted:
7September2009
OECDGUIDELINEFORTHETESTINGOFCHEMICALS
SubchronicInhalationToxicity:90-DayStudy
SUMMARY
ThisrevisedTestGuideline413(TG413)hasbeendesignedtofullycharacterizetestarticletoxicity
bytheinhalationrouteforasubchronicduration(90days),andtoproviderobustdataforquantitative
inhalationriskassessments.Groupsof10maleand10femalerodentsareexposed6hoursperday
duringa90day(13week)periodtoa)thetestarticleatthreeormoreconcentrationlevels,b)filtered
waiere(knebguattievxepocosnutrreolf)o,ra7ndd/aoyrsc)petrheweveehkicilseal(svoehailclloewceodn.trMola)l.eAsniamnadlfsemaarelegsenaerrealallywaeyxspotsesetded5,dbauytsthpeeyr
maybeexposedatdifferentconcentrationlevelsifitisknownthatonesexismoresusceptibletoa
giventestarticle.Thisguidelineallowsthestudydirectortheflexibilitytoincludesatellite
(reversibility)groups,interimsacrifices,bronchoalveolarlavage (BAL),neurologictests,and
additionalclinicalpathologyandhistopathologicalevaluationsinordertobettercharacterizethe
toxicityofatestarticle.
INTRODUCTION
1.OECDGuidelinesareperiodicallyreviewedinthelightofscientificprogress,animalwelfare
considerations,andchangingregulatoryneeds.TheoriginalsubchronicinhalationTestGuideline413
(TG413)wasadoptedin1981(1).TG413hasbeenrevisedtoreflectthestateofthescienceandto
meetcurrentandfutureregulatoryneeds.
2.Subchronicinhalationtoxicitystudiesareprimarilyusedtoderiveregulatoryconcentrationsfor
assessingworkerriskinoccupationsettings.Theyarcalsousedtoassesshumanresidential,
transportation,andenvironmentalrisk.Thisguidelineenablesthecharacterizationofadverseeffects
followingrepeateddailyinhalationexposuretoatestarticlefor90days(approximately10%ofthe
lifespanofarat).Thedataderivedfromsubchronicinhalationtoxicitystudiescanbeusedfor
quantitativeriskassessmentsandfortheselectionofconcentrationsforchronicstudies.ThisTest
Guidelineisnotspecificallyintendedforthetestingofnanomaterials.Definitionsusedinthecontext
ofthisTestGuidelinecanbefoundintheGuidanceDocument39(2).
INITIALCONSIDERATIONS
c3o.nAdlulctavianiglatbhleeisntfuodmyiaitnioonrdoenrtthoeteensthaanrctieclethsehoquuladlibteycoofnstihdeersetdudbyyathnedtmesitniinmgilzaeboraantoirmya-lpruisoarget.o
Informationthatwillassistintheselectionofappropriatetestconcentrationsmightincludethe
identity,chemicalstructure,andphysico-chemicalpropertiesofthetestarticle:resultsofanyinvitro
orinvivotoxicitytests;anticipateduse(s)andpotentialforhumanexposure:available(Q)SARdata
andtoxicologicaldataonstructurallyrelatedsubstances;anddataderivedfromotherrepeated
exposurestudies.Ifneurotoxicityisexpectedorisobservedinthecourseofthestudy,thestudy
directormaychoosetoincludeappropriateevaluationssuchasafunctionalobservationalbattery
©OECD,(2009)
tYforoowumriattrtheeenfOrpEeeeCrmtDoi,suspsireoontvhifidrseodmmattthehereisaOolEufCrocDre.peisrsdounlayl,mennotni-ocnoemdm.eArnciyaclopmumreprocsieaslwuistehoouftthsiesekmiantegriparlioriscsounbsjeenctt
413 OECD/OCDE
(FOB)andmeasurementofmotoractivity.Althoughthetimingofexposuresrelativetospecific
examinationsmaybecritical,theperformanceoftheseadditionalactivitiesshouldnotinterferewith
thebasicstudydesign.
4dc.eosnDicirelenudttrdiaoetngisroenoesfoscfhootrourxloisdciivbteeyo[lrroewfierrreinttooatuGignDghtt3eo9stn(o2at)r]tic.caWluehsseemnmaaeyrxkpboeesditnepsgatieandniaamntadlcosdnitscoterntethsrsea,steyieomtantsseurtfihfaailtcsiw,eintlthletyotiaeerlxgdteettnehdde
theconcentration-responsecurvetolevelsthatreachtheregulator)'andscientificobjectiveofthetest.
Theseconcentrationsshouldbeselectedonacase-by-casebasis,preferablybaseduponanadequately
designedrange-findingstudythatprovidesinformationregardingthecriticalendpoint,anyirritation
threshold,andthetimeofonset(seeparagraphs11-13).Thejustificationforconcentrationselection
shouldbeprovided.
5.Moribundanimalsoranimalsobviouslyinpainorshowingsignsofsevereandenduringdistress
shouldbehumanelykilled.Moribundanimalsareconsideredinthesamewayasanimalsthatdieon
ttehset.reCcroigtneirtiiaofnoorfmparkeidnicgtatbhleedoerciismipoenntdoinkiglldemaotrhi,baurnedthoerssuebvjeercetlyofsuafnfeOrEinCgDanGiumaildsa,ncaendDoguciudmaenncetoonn
HumaneEndpoints(3).
DESCRIPTIONOFTHEMETHOD
SelectionofAnimalSpecies
6.Healthyyoungadultrodentsofcommonlyusedlaboratorystrainsshouldbeemployed.The
preferredspeciesistherat.Justificationshouldbeprovidedifotherspeciesareused.
PreparationofAnimals
y7.ouFnegmaaldeulstssh7otuold9bweeenkulsliopfaargoeu.sBaonddynowne-ipgrhetgsnasnhto.ulOdnbtehewidtahyino±f2ra0n%doomifztahteiomne,aanniwmeailgshtshfoourledabceh
sex.Theanimalsarerandomlyselected,markedforindividualidentification,andkeptintheircages
foratleast5dayspriortothestartofthetesttoallowforacclimatizationtolaboratoryconditions.
AnimalHusbandry
8.Animalsshouldbeindividuallyidentified,preferablywithsubcutaneoustransponders,tofacilitate
observationsandavoidconfusion.Thetemperatureoftheexperimentalanimalmaintenanceroom
shouldbe22±3°C.Therelativehumidityshouldideallybemaintainedintherangeof30to70%,
thoughthismaynotbepossiblewhenusingwaterasavehicle.Beforeandafterexposures,animals
generallyshouldbecagedingroupsbysexandconcentration,butthenumberofanimalspercage
shouldnotinterferewithclearobservationofeachanimalandshouldminimizelossesdueto
cannibalismandfighting.Whenanimalsaretobeexposednose-only,itmaybenecessary'forthemto
beacclimatedtotherestrainingtubes.Therestrainingtubesshouldnotimposeunduephysical,
thermal,orimmobilizationstressontheanimals.Restraintmayaffectphysiologicalendpointssuchas
bodytemperature(hyperthermia)and/orrespiratoryminutevolume.Ifgenericdataareavailableto
showthatnosuchchangesoccurtoanyappreciableextent,thenpre-adaptationtotherestrainingtubes
isnotnecessary.Animalsexposedwhole-bodytoanaerosolshouldbehousedindividuallyduring
exposuretopreventthemfromfilteringthetestaerosolthroughthefuroftheircagemates.
Conventionalandcertifiedlaboratorydietsmaybeused,exceptduringexposure,accompaniedwithan
unlimitedsupplyofmunicipaldrinkingwater.Lightingshouldbeartificial,thesequencebeing12
hourslight/12hoursdark.
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OECD/OCDE 413
InhalationChambers
9.Thenatureofthetestarticleandtheobjectofthetestshouldbeconsideredwhenselectingan
inhalationchamber.Thepreferredmodeofexposureisnose-only(whichtermincludeshead-only,
nose-only,orsnout-only).Nose-onlyexposureisgenerallypreferredforstudiesofliquidorsolid
aerosolsandforvapoursthatmaycondensetoformaerosols.Specialobjectivesofthestudymaybe
betterachievedbyusingawhole-bodymodeofexposure,butthisshouldbejustifiedinthestudy
raeenxpipomorast.lusrTeostheoecunhlsnudirqneuoeatstemaxoncsdepehtdeheri5er%sptaaorbtfiiltcihutelyarwchhaademvnabneutsraignvegoslauamwnehd.odliePs-raibdnovcdaiynptlcaehsgaeomsfbaetrrhe,eatndhodesreet-sotosanelldyvionalnGudDmewh3o9ofl(te2h)e.botedsyt
TOXICITYSTUDIES
LimitConcentrations
10.Unlikewithacutestudies,therearenodefinedlimitconcentrationsinsubchronicinhalation
toxicitystudies.Themaximumconcentrationtestedshouldconsider:1)themaximumattainable
concentration,2)the“worstcase"humanexposurelevel,3)theneedtomaintainanadequateoxygen
supply,and/or4)animalwelfareconsiderations.Intheabsenceofdata-basedlimits,theacutelimits
oftheUnitedNationsGloballyHarmonizedSystemofClassificationandLabellingofChemicalsmay
bpliepmmiutssfeodwrh(giea.nsee„stu)ep;sttrieonfgaergmatasoexGsiDmoru3m9hicg(o2hn)l.cyenJvtuorsltaaittfiiilocenattoiefosnt5samrhgtoi/uclLledsfboe(re.apger.roovrseiofdlrsei,dge2ir0fanimttsgi)s/.LneTfcohersesvaalripymoiuttrosc,eoxnaccneednetd2r0ta,ht0ei0soe0n
shouldelicitunequivocaltoxicitywithoutcausingunduestresstotheanimalsoraffectingtheir
longevity(3).
Range-FindingStudy
s1t1u.dyB.efAorreancgoe-mfmienndcinigngstuwdiythistmheormeaicnompstruedhye,nsitiviesgtehnaenraalsliyghnteicnegsssatruydytobepcearufsoenintiasnroatnglei-mfiitneddintgo
cstoundcye.ntAratriaonnges-efleicntdiionng.sKtnudoywlmeadyg,efloerarenxeadmpflreo,mparroavnigdee-ftiencdhinnigcasltuidnyfocramnatlieoandtroegaarsduicncgessafnuallymtiacianl
maentdheosdtsi,maptairotincsleofsiwzhinagt,mdiasycobveerNyOoAfEtoLxiacnmdecMhTanCiscmosn,cecnltirnaitcialonpsatinhoalmogayinansdtuhdiys.toTphaethsotluodgiycdailredcattao,r
maychoosetousetherange-findingstudytoidentifythethresholdofrespiratorytractirritation(e.g.
withhistopathologyoftherespiratory'tract,pulmonaryfunctiontesting,orbronchoalveolarlavage),
theupperconcentrationwhichistoleratedwithoutunduestresstotheanimals,andtheparametersthat
willbestcharacterizeatestarticle’stoxicity.
12.Arange-findingstudymayconsistofoneormoreconcentrationlevels.Dependingonthe
elenvdeplo.iAntsracnhgoes-efni,ndtihnrgeesttoudsyixsmhaolueldsalansdttahrmeientiomsuixmfoefma5ledsaysshoaunlddgbeeneerxaplolsyednoatmeoarcehtchoannce2n8tradtayiso.n
Therationalefortheselectionofconcentrationsforthemainstudyshouldbeprovidedinthestudy
report.Theobjectiveofthemainstudyistodemonstrateaconcentration-responserelationshipbased
onwhatisanticipatedtobethemostsensitiveendpoint.Thelowconcentrationshouldideallybeano-
observed-adverseeffectconcentrationwhilethehighconcentrationshouldelicitunequivocaltoxicity
withoutcausingunduestresstotheanimalsoraffectingtheirlongevity(3).
13.Whenselectingconcentrationlevelsfortherange-findingstudy,allavailableinfonnationshould
b3)e.coAnsirdaenrgeed-fiinncdliundgingstsutdryuctmuraey-actvievriitfyy/rreelfauttieonswhhiaptsaanrdedactoansfiodresriemdilatrochbeemitchaelsm(osseetpasrenasgirtaipvhe
©OCDE,(2009)
413 OECD/OCDE
mechanisticallybasedendpoints,e.g.cholinesteraseinhibitionbyorganophosphates,methaemoglobin
nfeourtmraotpihoinlsbyinerbyrtohnrcohcyotaoltvoexoilcaragleantvsa,gethfyorroiidnalnohcouromuosneposo(rTly3,Tso4l)ufbolrethpyarrtoitcolxeiscaonrtsp,uplrmootenianr,yLDirHr,itaonrt
aerosols.
MainStudy
14.Themainsubchronictoxicitystudygenerallyconsistsofthreeconcentrationlevels,andalso
concurrentnegative(air)and/orvehiclecontrolsasneeded(seeparagraph18). Allavailabledata
shouldbeutilizedtoaidselectionofappropriateexposurelevels,includingtheresultsofsystemic
toxicitystudies,metabolismandkinetics(particularemphasisshouldbegiventoavoidinghigh
concentrationlevelswhichsaturatekineticprocesses).Eachtestgroupcontains10maleand10female
rodentsthatareexposedtothetestarticlefor6hoursperdayona5dayperweekbasisforaperiodof
13weeks(totalstudydurationofatleast90days).Animalsmayalsobeexposed7daysperweek(e.g.
whentestinginhaledpharmaceuticals).Ifonesexisknowntobemoresusceptibletoagiventest
article,thesexesmaybeexposedatdifferentconcentrationlevelsinordertooptimizethe
ncoosnec-eonntlrya,tiomna-xreismpuonmseexapsosduesrceridbuerdatiinonpsarmaagyrapbhea15d.juIsfterdodteontmisnpiemciiesseostpheecrietsh-asnpecriatfsicardeisterxepsso.seAd
rnaetcieosnsaalreysthoouclodndbuectpraolvoindgedduwrhateinonus(ei.ngg.2a2nheoxupross/udraey)duwrhaotlieo-nbloedsysetxhpanos6urheousrtsu/ddyay(r,efoerrtwoheGnDit39i)s
(2).Feedshouldbewithheldduringtheexposureperiodunlessexposureexceeds6hours.Watermay
beprovidedthroughoutawhole-bodyexposure.
15.Thetargetconcentrationsselectedshouldidentifythetargetorgan(s)anddemonstrateaclear
concentration-response:
• Thehighconcentrationlevelshouldresultintoxiceffectsbutnotcauselingeringsignsorlethality
whichwouldpreventameaningfulevaluation.
• Theintermediateconcentrationlevel(s)shouldbespacedtoproduceagradationoftoxiceffects
betweenthatofthelowandhighconcentration.
• Thelowconcentrationlevelshouldproducelittleornoevidenceoftoxicity.
InterimSacrifices
16.Ifintenmsacrificesareplanned,thenumberofanimalsateachexposurelevelshouldbeincreased
bythenumbertobesacrificedbeforestudycompletion.Therationaleforusinginterimsacrifices
shouldbeprovided,andstatisticalanalysesshouldproperlyaccountforthem.
Satellite(Reversibility)Study
17.Asatellite(reversibility)studymaybeusedtoobservereversibility,persistence,ordelayed
occurrenceoftoxicityforapost-treatmentperiodofanappropriatelength,butnolessthan14days.
Satellite(reversibility)groupsconsistof10malesand10femalesexposedcontemporaneouslywith
theexperimentalanimalsinthemainstudy.Satellite(reversibility)studygroupsshouldbeexposedto
thetestarticleatthehighestconcentrationlevelandthereshouldbeconcurrentairand/orvehicle
controlsasneeded(seeparagraph18).
©OCDE,(2009) 4
OECD/OCDE 413
ControlAnimals
18.Concurrentnegative(air)controlanimalsshouldbehandledinamanneridenticaltothetestgroup
animalsexceptthattheyarcexposedtofilteredairratherthantestarticle.Whenwateroranother
substanceisusedtoassistingeneratingthetestatmosphere,avehiclecontrolgroup,insteadofa
negative(air)controlgroup,shouldbeincludedinthestudy.Watershouldbeusedasthevehicle
wheneverpossible.Whenwaterisusedasthevehicle,thecontrolanimalsshouldbeexposedtoair
withthesamerelativehumidityastheexposedgroups.Theselectionofasuitablevehicleshouldbe
basedonanappropriatelyconductedpre-studyorhistoricaldata.Ifavehicle’stoxicityisnotwell
known,thestudydirectormaychoosetousebothanegative(air)controlandavehiclecontrol,but
thisisstronglydiscouraged.Ifhistoricaldatarevealthatavehicleisnon-toxic,thenthereisnoneed
foranegative(air)controlgroupandonlyavehiclecontrolshouldbeused. Ifapre-studyofatest
articleformulatedinavehiclerevealsnotoxicity,itfollowsthatthevehicleisnon-toxicatthe
concentrationtestedandthisvehiclecontrolshouldbeused.
EXPOSURECONDITIONS
AdministrationofConcentrations
19.Animalsareexposedtothetestarticleasagas,vapour,aerosol,oramixturethereof.Thephysical
statetobetesteddependsonthephysico-chemicalpropertiesofthetestarticle,theselected
concentrations,and/orthephysicalformmostlikelypresentduringthehandlinganduseofthetest
article. Hygroscopicandchemicallyreactivetestarticlesshouldbetestedunderdryairconditions.
Csuabrjeecstheodutlodmbeechtaankiecnaltopraovcoeisdsesgetnoerdaetcirnegaseextphleospiavrteiccloencsieznet.raFtuirotnhse.rguPairdtainccuelaitsepmraotveirdieadlsinmGaDyb3e9
(2).
Particle-SizeDistribution
20.Particlesizingshouldbeperfonnedforallaerosolsandforvapoursthatmaycondensetofonn
ma(eoergdo)isoailnns.taheeTroordaanylgnleaomowifcf1od.ri5aetmxoept3oe.sr0usrae(reMorMfeAaclDlo)mrmerelaennvdgaeintndgr(fe4)gr.ioomAnls1thtooofu3tghhpemarerwseipatishroanataogbrelyeomtreeaftcfrto,irtcasesrhtooasunoldldasrbdweidtemvhaidametaisotsno
mfeuemtetphairstisctlaensdwairldl,beexpsemratljluerdgtehmanentthisshsotualnddabred,parnodvicdheadrgifeditpcaarntnicoltesbeanadchfiiebvreeds.maFyorexecxeamepdliet.,metal
TestArticlePreparationinaVehicle
21.Ideally,thetestarticleshouldbetestedwithoutavehicle.Ifitisnecessarytouseavehicleto
generateanappropriatetestarticleconcentrationandparticlesize,watershouldbegivenpreference.
Wheneveratestarticleisdissolvedinavehicle,itsstabilityshouldbedemonstrated.
MONITORINGOFEXPOSURECONDITIONS
ChamberAirflow
22.Theflowofairthroughtheexposurechambershouldbecarefullycontrolled,continuously
monitored,andrecordedatleasthourlyduringeachexposure.Thereal-timemonitoringofthetest
atmosphereconcentration(ortemporalstability)isanintegralmeasurementofalldynamicparameters
andprovidesanindirectmeanstocontrolallrelevantdynamicinhalationparameters.Ifthe
concentrationismonitoredreal-time,thefrequencyofmeasurementofairflowsmaybereducedto
5 ©OCDE,(2009)
413 OECD/OCDE
onesinglemeasurementperexposureperday. Specialconsiderationshouldbegiventoavoiding
rebreathinginnose-onlychambers.Oxygenconcentrationshouldbeatleast19%andcarbondioxide
concentrationshouldnotexceed1%.Ifthereisreasontobelievethatthisstandardcannotbemet,
oxygenandcarbondioxideconcentrationsshouldbemeasured.Ifmeasurementsonthefirstdayof
exposureshowthatthesegasesareatproperlevels,nofurthermeasurementsshouldbenecessary.
ChamberTemperatureandRelativeHumidity
23.Chambertemperatureshouldbemaintainedat22±3°C.Relativehumidityintheanimals'
breathingzone,forbothnose-onlyandwhole-bodyexposures,shouldbemonitoredcontinuouslyand
recordedhourlyduringeachexposurewherepossible.Therelativehumidityshouldpreferablybe
maintainedintherangeof30to70%,butthismayeitherbeunattainable(e.g.whentestingwater
basedformulations)ornotmeasurableduetotestarticleinterferencewiththetestmethod.
TestArticle:NominalConcentration
24.Wheneverfeasible,thenominalexposurechamberconcentrationshouldbecalculatedand
recorded.Thenominalconcentrationisthemassofgeneratedtestarticledividedbythetotalvolume
ofairpassedthroughtheinhalationchambersystem.Thenominalconcentrationisnotusedto
characterizetheanimals’exposure,butacomparisonofthenominalconcentrationandtheactual
concentrationgivesanindicationofthegenerationefficiencyofthetestsystem,andthusmaybeused
todiscovergenerationproblems.
TestArticle:ActualConcentration
25.Theactualconcentrationisthetestarticleconcentrationassampledattheanimals'breathingzone
inaninhalationchamber.Actualconcentrationscanbeobtainedeitherbyspecificmethods(e.g.,
directsampling,adsorptiveorchemicalreactivemethods,andsubsequentanalyticalcharacterisation)
orbynon-specificmethodssuchasgravimetricfilteranalysis. Theuseofgravimetricanalysisis
acceptableonlyforsinglecomponentpowderaerosolsoraerosolsoflowvolatilityliquidsandshould
besupportedbyappropriatepre-studytestarticle-specificcharacterisations.Multi-componentpowder
aerosolconcentrationmayalsobedeterminedbygravimetricanalysis.However,thisrequires
analyticaldatawhichdemonstratethatthecompositionofairbornematerialissimilartothestarting
material.Ifthisinformationisnotavailable,areanalysisofthetestmaterial(ideallyinitsairborne
mstaatye)evataproergautlearorinstuebrvlailmsatdeu,riitnsghtohueldcobuerssehoofwnthtehasttuadllypmhaaysesbewenerceescsolalreyc.teFdobryaetrheosmoelitsheoddacgheonstesnt.hat
26.Onelotofthetestarticleshouldbeusedthroughoutthedurationofthestudy,ifpossible,andthe
testsampleshouldbestoredunderconditionsthatmaintainitspurity,homogeneity,andstability.
Priortothestartofthestudy,thereshouldbeacharacterizationofthetestarticle,includingitspurity
and,iftechnicallyfeasible,theidentity,andquantitiesofidentifiedcontaminantsandimpurities.This
canbedemonstratedby.butisnotlimitedto,thefollowingdata:retentiontimeandrelativepeakarea,
molecularweightfrommassspectroscopyorgaschromatographyanalyses,orotherestimates.
Althoughthetestsample’sidentifyisnottheresponsibilityofthetestlaboratory,itmaybeprudentfor
thetestlaboratorytoconfirmthesponsor’scharacterizationatleastinalimitedway(e.g.colour,
physicalnature,etc.).
27.Theexposureatmosphereshouldbeheldasconstantaspracticable.Areal-timemonitoringdevice,
suchasanaerosolphotometerforaerosolsoratotalhydrocarbonanalyserforvapours,maybeusedto
demonstratethestabilityoftheexposureconditions.Actualchamberconcentrationshouldbe
measuredatleast3timesduringeachexposuredayforeachexposurelevel.Ifnotfeasibledueto
©OCDE,(2009) 6
OECD/OCDE 413
limitedairflowratesorlowconcentrations,onesampleperexposureperiodisacceptable.Ideally,this
sampleshouldthenbecollectedovertheentireexposureperiod.Individualchamberconcentration
samplesshoulddeviatefromthemeanchamberconcentrationbynomorethan±10%forgasesand
vapours,andbynomorethan±20%forliquidorsolidaerosols.Timetoattainchamberequilibration
iG(tsu95ig)ednsaehnrocauetledfdo.breTehscitasilmctauatlkiaentsgeditngat5nocdaancrecbpoeournftteodut.nhedTtihinemGedsDurra3etq9iuoi(2nr)e.odftaonatetxapionscuhraemsbpearnseqtuhielitbirmaetitohnat(tt^h)eatensdtdaretciacyl.e
28.Forverycomplexmixturesconsistingofgases/vapoursandaerosols(e.g.combustionatmospheres
andtestarticlespropelledfrompurpose-drivenend-useproducts/devices),eachphasemaybehave
differentlyinaninhalationchamber.Therefore,atleastoneindicatorsubstance(analyte),normallythe
principalactiveinthetestedproductformulation,ofeachphase(gas/vapourandaerosol)shouldbe
selected.Whenthetestarticleisamixture(e.g.aformulation),theanalyticalconcentrationshouldbe
rAedpdoirttieodnaflorintfhoermtaottailonforremgualradtiinogn,acatunadlncootncjeunsttraftoirotnhsecaacntibveefionugnrdediinenGtDor39th(e2)c.omponent(analyte).
TestArticle:ParticleSizeDistribution
29.Theparticlesizedistributionofaerosolsshouldbedeterminedatleastweeklyforeach
concentrationlevelbyusingacascadeimpactororanalternativeinstrumentsuchasanaerodynamic
particlesizer(APS).Ifequivalenceoftheresultsobtainedbyacascadeimpactorandthealternative
instrumentcanbeshown,thenthealternativeinstrumentmaybeusedthroughoutthestudy.
30.Aseconddevice,suchasagravimetricfilteroranimpinger/gasbubbler,shouldbeusedinparallel
totheprimaryinstrumenttoconfirmthecollectionefficiencyoftheprimaryinstrument.Themass
cccooonnnccceeennntttrrraaatttiiiooonnnsootbbettsaatiiendneeiddnbtbyhyefieplaatrretlriycalpneahlaysssieizseof[astnehaeelyGsstiDusdy3s,9hto(hu2el)nd].fbuIerftehwqeirutihcvioannlfeirnrecmaeastoconaranyblbmeeealdsiemumirotensmsetonrfattstehmedaamytasablsel
omitted. Forthesakeofanimalwelfare,measuresshouldbetakentominimizeinconclusivedata
whichmayleadtoaneedtorepeatastudy.
m3a1.yParretsiucllteisniztihnegsfhoorumladtiboenpoefrfaonrmaeerdosfoolr,voarpoiufrsparitfitchleerseairseandyetpeocstseidbiilnitaytvhaaptovuarpoautrmocsopnhdeernesawtiitohn
potentialformixedphases.
OBSERVATIONS
32.Theanimalsshouldbeclinicallyobservedbefore,during,andaftertheexposureperiod. More
frequentobservationsmaybeindicateddependingontheresponseoftheanimalsduringexposure.
Whenanimalobservationishinderedbytheuseofanimalrestrainttubes,poorlylitwholebody
chambers,oropaqueatmospheres,animalsshouldbecarefullyobservedafterexposure.Observations
beforethenextday’sexposurecanassessanyreversibilityorexacerbationoftoxiceffects.
33.Allobservationsarerecordedwithindividualrecordsbeingmaintainedforeachanimal.When
animalsarekilledforhumanereasonsorfounddead,thetimeofdeathshouldberecordedasprecisely
aspossible.
34.Cage-sideobservationsshouldincludechangesintheskinandfur,eyes,andmucousmembranes;
changesintherespiratoryandcirculatorysystems;changesinthenervoussystem;andchangesin
somatomotoractivityandbehaviourpatterns.Attentionshouldbedirectedtoobservationsoftremors,
convulsions,salivation,diarrhoea,lethargy,sleep,andcoma.Themeasurementofrectaltemperatures
7 ©OCDE,(2009)
413 OECD/OCDE
mayprovidesupportiveevidenceofreflexbradypneaorhypo/hyperthenniarelatedtotreatmentor
confinement.Additionalassessmentsmaybeincludedinthestudyprotocolsuchaskinetics,
biomonitoring,lungfunction,retentionofpoorlysolublematerialsthataccumulateinlungtissue,and
behaviouralchanges.
BODYWEIGHTS
35.Individualanimalweightsshouldberecordedshortlybeforethefirstexposure(day0),twice
weeklythereafter(forexample:onFridaysandMondaystodemonstraterecoveryoveranexposure-
freeweekend,oratatimeintervaltoallowassessmentofsystemictoxicity),andatthetimeofdeath
oreuthanasia.Iftherearenoeffectsinthefirst4weeks,bodyweightsmaybemeasuredweeklyfor
theremainderofthestudy. Satellite(reversibility)animals(ifused)shouldcontinuetobeweighed
weeklythroughouttherecoveryperiod.Atstudytermination,allanimalsshouldbeweighedshortly
beforesacrificetoallowforanunbiasedcalculatedoforgantobodyweightratios.
FOODANDWATERCONSUMPTION
36.Foodconsumptionshouldbemeasuredweekly.Waterconsumptionmayalsobemeasured.
CLINICALPATHOLOGY
37.Clinicalpathologyassessmentsshouldbemadeforallanimals,includingcontrolsandsatellite
(reversibility)animals,whentheyaresacrificed.Thetimeintervalbetweentheendofexposureand
bloodcollectionshouldberecorded,particularlywhenthereconstitutionoftheaddressedendpointis
rapid.Samplingfollowingtheendofexposureisindicatedforthoseparametersw'ithashortplasma
half-time(e.g„COHb.CHE,andMetHb).
38.Table1liststheclinicalpathologyparametersthataregenerallyrequiredforalltoxicologystudies.
Urinalysisisnotrequiredonaroutinebasis,butmaybeperformedwhendeemedusefulbasedon
expectedorobservedtoxicity.Thestudydirectormaychoosetoassessadditionalparametersinorder
tobettercharacterizeatestarticle’stoxicity(e.g.,cholinesterase,lipids,hormones,acid/bascbalance,
methaemoglobinorHeinzbodies,creatinekinase,myeloid/erythroidratio,troponins,arterialblood
gases,lactatedehydrogenase,sorbitaldehydrogenase,glutamatedehydrogenase,andgammaglutamyl
transpeptidase).
©OCDE,(2009)
OECD/OCDE 413
Table1.StandardClinicalPathologyParameters
Haematology
Erythrocytecount Totalleukocytecount
Haematocrit Differentialleukocytecount
Haemoglobinconcentration Plateletcount
Meancorpuscularhaemoglobin Clottingpotential(selectone):
Meancorpuscularvolume Prothrombintime
Meancorpuscularhaemoglobin Clottingtime
concentration Partialthromboplastintime
Reticulocytes
ClinicalChemistry
Glucose* Alanineaminotransferase
Totalcholesterol) Aspartateaminotransferase
Triglycerides Alkalinephosphatase
Bloodureanitrogen Potassium
Totalbilirubin Sodium
Creatinine Calcium
Totalprotein Phosphorus
Albumin Chloride
Globulin
Urinalysis(optional)
Appearance(colourandturbidity) Totalprotein
Volume Glucose
Specificgravityorosmolality Blood/bloodcells
pH
*Becausealengthyfastingperiodcanintroducebiasinglucosemeasurementsforthe
treatedversuscontrolanimals,thestudydirectorshoulddeterminewhetheritis
athpeprsopperciiaetseutosefda;stftohretahenimraatlst.hiIsfmaafyastbieng1p6erhio(doviesrnuisgehdt,iftassthionug)l.dDbeeteapnpnrionpartiiaotneotof
fastingglucosemaybecarriedoutafterovernightfastingduringthelastexposure
week,orafterovernightfastingpriortonecropsy(inthelattercasetogetherwithall
otherclinicalpathologyparameters).
39.Whenthereisevidencethatthelowerrespiratorytract(i.e.,thealveoli)istheprimary'siteof
depositionandretention,thenbronchoalveolarlavage(BAL)maybethetechniqueofchoiceto
quantitativelyanalysehypothesis-baseddose-effectparametersfocusingonalveolitis,pulmonary
inflammation,andphospholipidosis.Thisallowsfordose-responseandtime-coursechangesof
alveolarinjurytobesuitablyprobed.TheBALfluidmaybeanalysedfortotalanddifferential
leukocytecounts,totalprotein,andlactatedehydrogenase.Otherparametersthatmaybeconsidered
airneflatmhmoasteioinndwihciactihvemaoyfinlcylsuodseomthaeldeitnejunrnyi,natpihoonspohfolpirpoi-dionsfilsa,mmaftiobrroys'isc,ytaonkidnesir/rcihtaenmtokiornesa.llBerAgiLc
mtheeams.urGeumiednatnscgeenoenrahlolwytcoompeprlfeomremntlutnhgelraevsaulgtescfarnobmehfiostuonpdaitnhoGloDgy39e.xa(2m)inationsbutcannotreplace
9 ©OCDE,(2009)
413 OECD/OCDE
OPHTHALMOLOGICALEXAMINATION
40.Usinganophthalmoscopeoranequivalentdevice,ophthalmologicalexaminationsofthefundus,
refractivemedia,iris,andconjunctivaeshouldbeperformedforallanimalspriortotheadministration
ofthetestarticle,andforallhighconcentrationandcontrolgroupsattermination.Ifchangesinthe
eyesaredetected,allanimalsintheothergroupsshouldbeexaminedincludingthesatellite
(reversibility)group.
GROSSPATHOLOGYANDORGANWEIGHTS
41.Alltestanimals,includingthosewhichdieduringthetestorareremovedfromthestudyfor
animalwelfarereasons,shouldbesubjectedtocompleteexsanguination(iffeasible)andgross
necropsy.Thetimebetweentheendofeachanimal'slastexposureanditssacrificeshouldbe
recorded.Ifanecropsycannotbeperfonnedimmediatelyafteradeadanimalisdiscovered,theanimal
shouldberefrigerated(notfrozen)atatemperaturelowenoughtominimizeautolysis.Necropsies
shouldbeperfonnedassoonaspossible,normallywithinadayortwo.Allgrosspathologicalchanges
shouldberecordedforeachanimalwithparticularattentiontoanychangesintherespiratory'tract.
42.Table2liststheorgansandtissuesthatshouldbepreservedinasuitablemediumduringgross
necropsyforhistopathologicalexamination.Thepreservationofthe[bracketed]organsandtissuesand
anyotherorgansandtissuesisatthediscretionofthestudydirector.Theboldedorgansshouldbe
trimmedandweighedwetassoonaspossibleafterdissectiontoavoiddrying. Thethyroidand
epididymides should only be weighed ifneeded because trimming artefacts may hinder
histopathologicalevaluation.Tissuesandorgansshouldbefixedin10%bufferedformalinoranother
suitablefixativeassoonasnecropsyisperformed,andnolessthan24-48hourspriortotrimming
dependingonthefixativetobeused.
©OCDE,(2009) 10