Test No. 478: Rodent Dominant Lethal Test [electronic resource]

2015·0.46 MB·English

by  Organisation for Economic Co-operation and Development

OECD/OCDE 478
28MAdyop2t0e1d5:
OECDGUIDELINEFORTESTINGOFCHEMICALS
RODENTDOMINANTLETHALTEST
INTRODUCTION
1. TheOECDGuidelinesfortheTestingofChemicalsareperiodicallyreviewedinthelightof
scientificprogress,changingregulatoryneeds,andanimalwelfareconsiderations.TheoriginalTest
Guideline478wasadoptedin1984.ThismodifiedversionoftheTestGuidelinereflectsmorethanthirty
yearsofexperiencewiththistestandthepotentialforintegratingorcombiningthistestwithothertoxicity
testssuchasdevelopmental,reproductivetoxicity,orgenotoxicitystudies;howeverduetoitslimitations
andtheuseofalargenumberofanimalsthisassayisnotintendedforuseasaprimarymethod,butrather
asasupplementaltestmethodwhichcanonlybeusedwhenthereisnoalternativeforregulatory
rtoexqiuciirteymetnesttss..CAomGbuiindianngcetoDxiocciutymetensttiongnhtahsetTheesptotGeunitdieallitnoesspoanregleanregteicnuTmobxeircsoloofgyaniismaclusrrfenrtolmyuusnedeinr
developmentandwillprovidesuccinctandusefulguidancetousers.
2. ThepurposeoftheDominantlethal(DL)testistoinvestigatewhetherchemicalagentsproduce
mutationsresultingfromchromosomalaberrationsingermcells.Inaddition,thedominantlethaltestis
relevanttoassessinggenotoxicitybecause,althoughtheymayvaryamongspecies,factorsofinvivo
mInedtuacbtoiloinsmo,fapDhaLrmmauctoaktiinoentaifctsereaxnpdosDuNrAe-troeapateisrtcphreomciecsaslesindairceateascttihvaettahnedchceomnitcrailbuhtaestaoffetchteedrgeesrpmoinnsael.
tissueofthetestanimal.
3. DLmutationscauseembryonicorfetaldeath.InductionofDLmutationafterexposuretoatest
chemicalindicatesthatthechemicalhasaffectedthegermcellsofthetestanimal.
4. ADLassayisusefulforconfirmationofpositiveresultsoftestsusingsomaticinvivoendpoints,
andisarelevantendpointforthepredictionofhumanhazardandriskofgeneticdiseasestransmitted
throughthegermline.However,thisassayrequiresalargenumberofanimalsandislabour-intensive;asa
result,itisveryexpensiveandtime-consumingtoconduct.Becausethespontaneousfrequencyof
dominantlethalmutationsisquitehigh,thesensitivityoftheassayfordetectionofsmallincreasesinthe
frequencyofmutationsisgenerallylimited.
©OECD,(2015) 1
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fromtheOECD,providedthesourceisdulymentioned.Anycommercialuseofthismaterialissubjectto
writtenpermissionfromtheOECD.
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5. DefinitionsofkeytermsaresetoutinAnnex1
INITIALCONSIDERATIONS
6m.ayinsTohmeetecsatsiessmobsetaopfptreonpcroiantdeucitfedsciinenmtiicfieca(l1l)y(j2u)st(i3f)iebdu.toDtLhsersgpeenceireasl,lysuacrheasthreatsre(s4u)lt(5)of(6)gr(o7s)s,
cbehreoxmcolsudoemda.lAabDerLramtiuotnasti(osntruisctaurmalutaantdionnumoecrciurcrailngabinnoramagleirtimesc)ell(8p)er(9)se,(1o0r),isbuftixgeednpeosmtutfaetritiolniszactiaonnnoitn
theearlyembryo,thatdoesnotcausedysfunctionofthegamete,butislethaltothefertilizedeggor
developingembryo.
7m.atingsfIonldliovwiidnugaltrmeaaltemsenatreismadteepdensdeequnetntoinaltlhyetoulvtiirmgaitnefpemuarlpeosseatoafpptrhoeprDiLatestiundteyrv(aPlas.raTghreapnhum23b)eranodf
shouldensurethatallphasesofmalegermcellmaturationareevaluatedforDLs(11).
8. Ifthereisevidencethatthetestchemical,oritsmetabolite(s),willnotreachthetestis,itisnot
appropriatetousethistest.
PRINCIPLEOFTHETESTMETHOD
9. Generally,maleanimalsareexposedtoatestchemicalbyanappropriaterouteofexposureand
matedtountreatedvirginfemales.Differentgermcelltypescanbetestedbytheuseofsequentialmating
intervals.Followingmating,thefemalesareeuthanizedafteranappropriateperiodoftime,andtheiruteri
areexaminedtodeterminethenumbersofimplantsandliveanddeadembryos.Thedominantlethalityofa
testchemicalisdeterminedbycomparingtheliveimplantsperfemaleinthetreatedgroupwiththelive
implantsperfemaleinthevehicle/solventcontrolgroup.Theincreaseofdeadimplantsperfemaleinthe
treatedgroupoverthedeadimplantsperfemaleinthecontrolgroupreflectsthetest-chemical-induced
post-implantationloss.Thepost-implantationlossiscalculatedbydeterminingtheratioofdeadtototal
implantsinthetreatedgroupcomparedtotheratioofdeadtototalimplantsinthecontrolgroup.Pre-
implantationlosscanbeestimatedbycomparingcorporaluteacountsminustotalimplantsorthetotal
implantsperfemaleintreatedandcontrolgroups.
VERIFICATIONOFLABORATORYPROFICIENCY
10. Competenceinthisassayshouldbeestablishedbydemonstratingtheabilitytoreproduce
dominantlethalfrequenciesfrompublisheddata(e.g.(12)(13)(14)(15)(16)(17))withpositivecontrol
chemicals(includingweakresponses)suchasthoselistedinTable1,andvehiclecontrolsandobtaining
negativecontrolfrequenciesthatareconsistentacceptablerangeofdata1(seereferencesabove)orwiththe
laboratory’shistoricalcontroldistribution,ifavailable.
DESCRIPTIONOFTHEMETHOD
Preparations
Selectionofanimalspecies
a1r1e.commoCnolmymousneldybuustedratlsabmoaraytoarlysostbraeinaspporfohperaialtteh.yAsenxyualoltyhemratapuprreoparniiamtaelsmasmhomualldibaenesmppelcoiyeesdm.aMyicbee
used,ifscientificjustificationisprovidedinthereport.
1SeeupcomingGuidanceDocumentonGenotoxicity.
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Animalhousingandfeedingconditions
12. Forrodents,thetemperatureintheanimalroomshouldbe22°C(±3°C).Althoughtherelative
humidityideallyshouldbe50-60%,itshouldbeatleast40%andpreferablynotexceed70%,otherthan
duringroomcleaning.Lightingshouldbeartificial,thesequencebeing12hourslight,followedby12
hoursdark.Forfeeding,conventionallaboratorydietsmaybeusedwithanunlimitedsupplyofdrinking
water.Thechoiceofdietmaybeinfluencedbytheneedtoensureasuitableadmixtureofatestchemical
whenadministeredbythisroute.Priortotreatmentormating,rodentsshouldbehousedinsmallgroups
(nomorethanfive)ofthesamesexifnoaggressivebehaviourisexpectedorobserved,preferablyinsolid
cageswithappropriateenvironmentalenrichment.Animalsmaybehousedindividuallyifscientifically
justified.
Preparationoftheanimals
13. Healthyandsexuallymaturemaleandfemaleadultanimalsarerandomlyassignedtothecontrol
andtreatmentgroups.Theindividualanimalsareidentifieduniquelyusingahumane,minimallyinvasive
method(e.g.,byringing,tagging,micro-chipping,orbiometricidentification,butnottoeandearclipping)
andacclimatedtothelaboratoryconditionsforatleastfivedays.Cagesshouldbearrangedinsuchaway
thatpossibleeffectsduetocageplacementareminimized.Crosscontaminationbythepositivecontroland
thetestchemicalshouldbeavoided.Atthecommencementofthestudy,theweightvariationofanimals
shouldbeminimalandnotexceed+20%ofthemeanweightofeachsex.
Preparationofdoses
14. Solidtestchemicalsshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesor
admixedindietordrinkingwaterpriortodosingoftheanimals.Liquidtestchemicalsmaybedosed
directlyordilutedpriortodosing.Forinhalationexposures,testmaterialscanbeadministeredasgas,
vapour,orasolid/liquidaerosol,dependingontheirphysicochemicalproperties.Freshpreparationsofthe
testchemicalshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorageanddefine
theappropriatestorageconditions.
TestConditions
Solvent/vehicle
15. Thesolvent/vehicleshouldnotproducetoxiceffectsatthedosevolumesused,andshouldnotbe
suspectedofchemicalreactionwiththetestchemical.Ifotherthanwell-knownsolvents/vehiclesareused,
theirinclusionshouldbesupportedwithreferencedataindicatingtheircompatibility.Itisrecommended
thatwhereverpossible,theuseofanaqueoussolvent/vehicleshouldbeconsideredfirst.Examplesof
commonlyusedcompatiblesolvents/vehiclesincludewater,physiologicalsaline,methylcellulosesolution,
carboxymethylcellulosesodiumsaltsolution,oliveoilandcomoil.
Positivecontrols
16. Concurrentpositive control animals should always beused unless thelaboratoryhas
demonstratedproficiencyintheconductofthetestandhasusedthetestroutinelyintherecentpast(e.g.
withinthelast5years).However,itisnotnecessarytotreatpositivecontrolanimalsbythesamerouteas
animalsreceivingthetestchemical,orsampleallthematingintervals.Thepositivecontrolchemicals
shouldbeknowntoproduceDLsundertheconditionsusedforthetest.Exceptforthetreatment,animals
inthecontrolgroupsshouldbehandledinanidenticalmannertoanimalsinthetreatedgroups.
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17.
Thedosesofthepositivecontrolchemicalsshouldbeselectedsoastoproduceweakormoderate
effectsthatcriticallyassesstheperformanceandsensitivityoftheassay,butwhichconsistentlyproduce
positivedominantlethaleffects.Examplesofpositivecontrolchemicals,andappropriatedoses,are
includedinTable1.
Table1.ExamplesofPositiveControlChemicals.
Chemical[CASno.](referenceno.) EffectiveDoserange Administration
(mg/kg) Time(days)
(rodentspecies)
Triethylenemclamine[51-18-3](14) 0.25(mice) 1
Cyclophosphamide[50-18-0](18) 50-150(mice) 5
Cyclophosphamide[50-18-0](4) 25-100 1
(rats)
Ethyl methanesulphonate [62-50-0] 100-300 5
(12) (mice)
MonomericAcrylamide[79-06-1](16) 50(mice) 5
Chlorambucil[305-03-3](13) 25(mice) 1
Negativecontrols
w1a8.yasthNeetgraetaitvmeenctongtrrooulpsa,nismhaolusl,dtrbeeatiendclwuidtehdsfoolrveenvteroyrvseahmipcllienaglotniem,ea(n1d9)o.tIhnertwhieseabtsreenacteedoifnhtihsetosraicmael
orpublishedcontroldatashowingthatnoDLsorotherdeleteriouseffectsareinducedbythechosen
solvent/vehicle,untreatedcontrolanimalsshouldalsobeincludedforeverysamplingtimeinorderto
establishacceptabilityofthevehiclecontrol.
PROCEDURE
NumberofAnimals
i1n9t.ervals,IPnadriavgirdaupahlsm2a1le&s2a3r)epmraetfeerdabsleyquteonotinaellvyirgatinafpepmraolper.iaTtheepnreudmebteerrmoifnmedaleinsteprevralgsro(ue.pg.s,howueledklbye
ptorepdreotveirdmeitnheedsttoatbisetiscuaflfpicoiweentr(nienccesosmabriynattoidoentewcittahttlheeasntuamdboeurbloifngmaitneDdLfefmraelqeusenactye(aPcahrmagartaipnhg4i4n)t.erval)
c2a0l.culatioTnshetonpuemrbmeirtotfhefedemtaelcetsiopnerofmaattilnegasitntaerdvoaulblsihnogulidnatlhseoDbeLpfrreedqeuteenrcmyin(ei.de,bsyufsftiactiiestnitcaplrepgonwaenrt
femalestoprovideatleast400totalimplants)(19)(20)(21)(22)andthatatleastonedeadimplantper
analysisunit(i.e.,matinggroupperdose)isexpected(23).
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AdministrationPeriodandMatingIntervals
s21h.ouldensTuhreenthuamtbaelrlpohfasmeastionfgmianlteergvaelrsmfcoellllomwaitnugrtarteiaotnmeanrteeisvaglouvaeterdnefdorbDyLthiendturcetaitomnen(t11s,ch2e4d).uleFoarnda
singletreatmentuptofivedailydoseadministrations,thereshouldbe8(mouse)or10(rat)matings
conductedatweeklyintervalsfollowingthelasttreatment.Formultipledoseadministrations,thenumber
ofmatingintervalsmaybereducedinproportiontotheincreasedtimeoftheadministrationperiod,but
maintainingthegoalofevaluatingallphasesofspermatogenesis(e.g.,aftera28-dayexposure,only4
weeklymatingsaresufficienttoevaluateallphasedofspermatogenesisinthemouse).Alltreatmentand
matingschedulesshouldbescientificallyjustified.
22. Femalesshouldremainwiththemalesforatleastthedurationofoneoestruscycle(e.g.,one
weekcoversoneoestruscycleinbothmiceandrats).Femalesthatdidnotmateduringaone-weekinterval
canbeusedforasubsequentmatinginterval.Alternatively,untilmatinghasoccurred,asdeterminedby
thepresenceofsperminthevaginaorbythepresenceofavaginalplug.
2t3h.egoalisThtoedeextpeorsmuirneeawnhdetmhaetrinaggrievgeinmcehnemuisceadlisinddeupceensdeDnLtomuntathteiounlstipmeartesep,utrhpeonsethoefatchceeDptLedstmuedtyh.oIdf
wweoeukl)dabnedtomaetxepoosneceanatentthiereenrdo.unHdowoefvesrp,ermifattohgeegnoeaslisis(et.og.i,de7ntwiefeyktsheinsetnhseitmivoeusgee,rm5-c7eltlretaytpmeenftosrDpeLr
induction,thenasingleor5dayexposurefollowedbyweeklymatingispreferred.
DoseLevels
24. Ifapreliminaryrange-findingstudyisperformedbecausetherearenosuitabledataalready
availabletoaidindoseselection,itshouldbeperformedinthesamelaboratory,usingthesamespecies,
strain,sex,andtreatmentregimentobeusedinthemainstudy(25).Thestudyshouldaimtoidentifythe
maximumtolerateddose(MTD),definedasthehighestdosethatwillbetoleratedwithoutevidenceof
study-limitingtoxicity,relativetothedurationofthestudyperiod(forexample,abnormalbehaviouror
reactions,minorbodyweightdepressionorhematopoieticsystemcytotoxicity),butnotdeathorevidence
ofpain,sufferingordistressnecessitatinghumaneeuthanasia(26).
25. TheMTDmustalsonotadverselyaffectmatingsuccess(20).
26. Testchemicalswithspecificbiologicalactivitiesatlownon-toxicdoses(suchashormonesand
mitogens),andchemicalswhichexhibitsaturationoftoxicokineticpropertiesmaybeexceptionstothe
dose-settingcriteriaandshouldbeevaluatedonacase-by-casebasis.
27. Inordertoobtaindoseresponseinformation,acompletestudyshouldincludeanegativecontrol
groupandaminimumofthreedoselevelsgenerallyseparatedbyafactorof2,butnotgreaterthan4.Ifthe
testchemicaldoesnotproducetoxicityinarange-findingstudy,orbasedonexistingdata,thehighestdose
ftooxricaitsyi,ngtlheeaMdmTinDistsrhaotuilodnbsehouthledhbiegh2e0s0t0dmogse/kagdmbiondiystweeriegdhta.ndHotwheevedors,eifletvheelstesutsecdhesmhiocuallddporeesfecraaubslee
coverarangefromthemaximumtoadoseproducinglittleornotoxicity.Fornot-toxicsubstances,the
limitdoseforanadministrationperiodof14daysormoreis1000mg/kgbodyweight/day,andfor
administrationperiodsoflessthan14daysthelimitdoseis2000mg/kgbodyweight/day.
AdministrationofDoses
28. Theanticipatedrouteofhumanexposureshouldbeconsideredwhendesigninganassay.
Therefore,routesofexposuressuchasdietary,drinkingwater,subcutaneous,intravenous,topical,
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inhalation,oral(bygavage),orimplantationmaybechosenasjustified.Inanycase,therouteshouldbe
chosentoensureadequateexposureofthetargettissue(s).Intraperitonealinjectionisnotnormally
recommendedsinceitisnotanintendedrouteofhumanexposure,andshouldonlybeusedwithspecific
scientificjustification.Ifthetestchemicalisadmixedindietordrinkingwater,especiallyincaseofsingle
dosing,careshouldbetakenthatthedelaybetweenfoodandwaterconsumptionandmatingshouldbe
sufficienttoallowdetectionoftheeffects(paragraph31).Themaximumvolumeofliquidthatcanbe
administeredbygavageorinjectionatonetimedependsonthesizeofthetestanimal.Thevolumeshould
n2omtLn/olrmOaOlglymaexycbeeedus1edm.L/TlhOeOugseboodfyvwoeliugmhetsegxrceeaptterinthtahnetchaisse(oiffpaeqrumeiotutesdsbolyutainoinmsalwhweerlefaaremalexgiismlautmioon)f
shouldbejustified.Variabilityintestvolumeshouldbeminimizedbyadjustingtheconcentrationtoensure
aconstantvolumeinrelationtobodyweightatalldoselevels.
Observations
29. Generalclinicalobservationsofthetestanimalsshouldbemadeandclinicalsignsrecordedat
leastonceaday,preferablyatthesametime(s)eachdayandconsideringthepeakperiodofanticipated
effectsafterdosing.Atleasttwicedailyduringthedosingperiod,allanimalsshouldbeobservedfor
morbidityandmortality.Allanimalsshouldbeweighedatthebeginningofthestudyandatleastoncea
weekduringrepeateddosestudies,andatthetimeofeuthanasia.Measurementsoffoodconsumption
shouldbemadeatleastweekly.Ifthetestchemicalisadministeredviathedrinkingwater,water
consumptionshouldbemeasuredateachchangeofwaterandatleastweekly.Animalsexhibitingnon-
lethalindicatorsofexcesstoxicityshouldbeeuthanisedpriortocompletionofthetestperiod(26).
TissueCollectionandProcessing
30. Femalesareeuthanisedinthesecondhalfofpregnancyatgestationday(GD)13formiceandGD
14-15forrats.Uteriareexaminedfordominantlethaleffectstodeterminethenumberofimplants,liveand
deadembryos,andcorporalutea.
31. Theuterinehornsandovariesareexposedforcountingofcorporalutea,andfetusesareremoved,
counted,andweighted.Careshouldbetakentoexaminetheuteriforresorptionsobscuredbylivefetuses
andtoensurethatallresorptionsareenumerated.Fetalmortalityisrecorded.Thenumberofsuccessfully
impregnatedfemalesandthenumberoftotalimplantations,pre-implantationlosses,andpost-implantation
mortality(includedearlyandlateresorptions)alsoarerecorded.Inaddition,thevisiblefetusesmaybe
preservedinBourn'sfixativeforatleast2weeksfollowedbyexaminationformajorexternal
malformations(27)toprovideadditionalinformationonthereproductiveanddevelopmentaleffectsofthe
testagent.
DATAANDREPORTING
TreatmentofResults
32. Datashouldbetabulatedtoshowthenumberofmalesmated,thenumberofpregnantfemales,
andthenumberofnon-pregnantfemales.Resultsofeachmating,includingtheidentityofeachmaleand
female,shouldbereportedindividually.Thematinginterval,doselevelfortreatedmales,andthenumbers
ofliveimplantsanddeadimplantsshouldbeenumeratedforeachfemale.
33. Thepost-implantationlossiscalculatedbydeterminingtheratioofdeadtototalimplantsfrom
thetreatedgroupcomparedtotheratioofdeadtototalimplantsfromthevehicle/solventcontrolgroup.
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34. Pre-implantationlossiscalculatedasthedifferencebetweenthenumberofcorporaluteaandthe
numberofimplants,orasareductionintheaveragenumberofimplantsperfemaleincomparisonwith
controlmatings.Wherepre-implantationlossisestimated,itshouldbereported.
35. TheDominantLethalfactorisestimatedas:(post-implantationdeaths/totalimplantationsper
female)x100.
36. Dataontoxicityandclinicalsigns(asperParagraph29)shouldbereported.
AcceptabilityCriteria
37. Thefollowingcriteriadeterminetheacceptabilityofatest.
a)Concurrentnegativecontrolisconsistentwithpublishednormsforhistoricalnegativecontrol
data,andthelaboratory'shistoricalcontroldataifavailable(seeParagraphs10and18).
b)Concurrentpositivecontrolsinduceresponsesthatareconsistentwithpublishednormsfor
historicpositivecontroldata,orthelaboratory’shistoricalpositivecontroldatabase,ifavailable,
andproduceastatisticallysignificantincreasecomparedwiththenegativecontrol(see
Paragraphs17and18).
c)Adequatenumbertotalimplantsanddoseshavebeenanalyzed(Paragraph20).
d)ThecriteriafortheselectionoftopdoseareconsistentwiththosedescribedinParagraphs24
and27.
AcceptablerangesofcontrolvalueswillbedescribedintheupcomingGuidanceDocumenton
Genotoxicitytesting.
EvaluationandInterpretationofResults
38. Atleastthreetreateddosegroupsshouldbeanalysedinordertoprovidesufficientdatafordose-
responseanalysis.
39. Providingthatallacceptabilitycriteriaarefulfilled,atestchemicalisconsideredaclearpositive
if:
a)atleastoneofthetestdosesexhibitsastatisticallysignificantincreasecomparedwiththe
concurrentnegativecontrol;
b)theincreaseisdose-relatedinatleastoneexperimentalcondition(e.g.aweeklymatinginterval)
whenevaluatedwithanappropriatetest;and,
co)fatnhyeolfatbhoerarteosruyl’tssahriestoourtiscialdenoefgathteivaeccceopnttarbollerdaantage(oe.fg.n,egPaotiisvseocno-nbtarsoeldda9t5a2%,orcotnhterodlistlriimbiutt)ioinf
available.
Thetestchemicalisthenconsideredabletoinducedominantlethalmutationsingermcellsofthetest
animals.RecommendationsforthemostappropriatestatisticalmethodsaredescribedinParagraph44;
2SeeupcomingGuidanceDocumentonGenotoxicity.
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otherrecommendstatisticalapproachescanalsobefoundintheliterature(19)(20)(21)(23)(28).
Statisticaltestsusedshouldconsidertheanimalastheexperimentalunit.
40. Providingthatallacceptabilitycriteriaarefulfilled,atestchemicalisconsideredaclearnegative
if:
a)noneofthetestdosesexhibitsastatisticallysignificantincreasecomparedwiththeconcurrent
negativecontrol;
b)thereisnodose-relatedincreaseinanyexperimentalcondition;and
cn)egaaltlivreesuclotnstraorledawtiath(ien.g.a,cPceopitsasbolne-braasnegde9o5f%necgoanttirovlelciomnitt)r,olifdaavtaail1,abolre.thelaboratory’shistorical
Thetestchemicalisthenconsideredunabletoinducedominantlethalmutationsingermcellsofthetest
animals.
41. Thereisnorequirementforverificationofaclearpositiveoraclearnegativeresponse.
42. Iftheresponseisnotclearlynegativeorpositive,andinordertoassistinestablishingthe
biologicalrelevanceofaresult(e.g.aweakorborderlineincrease),thedatashouldbeevaluatedbyexpert
judgmentand/orfurtherinvestigationsusingtheexistingexperimentaldata,suchasconsiderationwhether
tnheegaptoisvieticvoentrreosluldtatias(o2u9t)s.idetheacceptablerangeofnegativecontroldata3,orthelaboratory'shistorical,
43. Inrarecases,evenafterfurtherinvestigations,thedatasetwillprecludemakingaconclusionof
positiveornegativeresults,andwillthereforebeconcludedasequivocal.
44. Statisticaltestsusedshouldconsiderthemaleanimalastheexperimentalunit.Whileitis
possiblethatcountdata(e.g.numberofimplantsperfemale)maybePoissondistributedand/or
proportions(e.g.proportionofdeadimplants)maybebinomiallydistributed,itisoftenthecasethatsuch
dataareoverdispersed(30). Accordingly,statisticalanalysisshouldfirstemployatestforover-
underdispersionusingvariancetestssuchasCochran’sbinomialvariancetest(31)orTarone’sC(a)test
forbinomialoverdispersion(30,32). Ifnodeparturefrombinomialdispersionisdetected,trendsin
proportionsacrossdoselevelsmaybetestedusingtheCochran-Armitagetrendtest(33)andpairwise
comparisonswiththecontrolgroupmaybetestedusingFisher’sexacttest(34).Likewise,ifnodeparture
fromPoissondispersionisdetected,trendsincountsmaybetestedusingPoissonregression(35)and
pairwisecomparisonswiththecontrolgroupmaybetestedwithinthecontextofthePoissonmodel,using
pairwisecontrasts(35). Ifsignificantoverdispersionorunderdispersionisdetected,nonparametric
methodsarerecommended(22,30).Theseincluderank-basedtests,suchastheJonckheere-Terpstratest
fortrend(36)andMann-Whitneytests(37)forpairwisecomparisonswiththevehicle/solventcontrol
group,aswellaspermutation,resampling,orbootstraptestsfortrendandpairwisecomparisonswiththe
controlgroup(30,38).
45. ApositiveDLassayprovidesevidenceforthegenotoxicityofthetestchemicalinthegermcells
ofthetreatedmaleofthetestspecies.
46. Considerationofwhethertheobservedvaluesarewithinoroutsideofthehistoricalcontrolrange
canprovideguidancewhenevaluatingthebiologicalsignificanceoftheresponse(39).
3SeeupcomingGuidanceDocumentonGenotoxicity.
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TestReport
47. Thetestreportshouldincludethefollowinginformation.
Summary.
Testchemical:
source,lotnumber,limitdateforuse,ifavailable;
stabilityofthetestchemicalitself,ifknown;
solubilityandstabilityofthetestchemicalinsolvent,ifknown;
measurementofpH,osmolality,andprecipitateintheculturemediumtowhichthetestchemical
wasadded,asappropriate.
Mono-constituentsubstance:
physicalappearance,watersolubility,andadditionalrelevantphysicochemicalproperties;
chemicalidentification,suchasIUPACorCASname,CASnumber,SMILESorInChlcode,
structuralformula,purity,chemicalidentityofimpuritiesasappropriateandpracticallyfeasible,
etc.
Multi-constituentsubstance,UVBCsandmixtures:
characterizedasfaraspossiblebychemicalidentity(seeabove),quantitativeoccurrenceand
relevantphysicochemicalpropertiesoftheconstituents.
Testcjhuesmtiicfiaclaptiroenpaforratcihoonic:eofvehicle;
solubilityandstabilityofthetestchemicalinthesolvent/vehicle,ifknown;
preparationofdietary,drinkingwaterorinhalationformulations;
analyticaldeterminationsonformulations(e.g.,stability,homogeneity,nominalconcentrations)
whenconducted.
Testanimals
species/strainusedandjustificationforthechoice;
number,ageandsexofanimals;
source,housingconditions,diet,etc.;
methodofuniquelyidentifyingtheanimals;
fsotrudsiheosrlto-tnegremrstthuadnieosn:eiwndeievki:duianldibvoiddyuawlebiogdhytowefitghhetmsadluerianngimtahlesstatudtyheanstdarftoaonddceonnsduomfpttihoent.esBt;ofdoyr
weightrange,meanandstandarddeviationforeachgroupshouldbeincluded.
Testconditions'.
positiveandnegative(vehicle/solvent)controldata;
datafromtherange-findingstudy;
rationalefordoselevelselection;
detailsoftestchemicalpreparation;
detailsoftheadministrationofthetestchemical;
rationaleforrouteofadministration;
methodsformeasurementofanimaltoxicity,including,whereavailable,histopathologicalor
hematologicalanalysesandthefrequencywithwhichanimalobservationsandbodyweightswere
taken;
methodsforverifyingthatthetestchemicalreachedthetargettissue,orgeneralcirculation,if
negativeresultsareobtained;
actualdose(mg/kgbodyweight/day)calculatedfromdiet/drinkingwatertestchemicalconcentration
(ppm)andconsumption,ifapplicable;
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detailsoffoodandwaterquality;
detailsoncageenvironmentenrichment;
detaileddescriptionoftreatmentandsamplingschedulesandjustificationsforthechoices;
methodofanalgesia
methodofeuthanasia;
proceduresforisolatingandpreservingtissues;
sourceandlotnumbersofallkitsandreagents(whereapplicable);
methodsforenumerationofDLs;
matingschedule;
methodsusedtodeterminethatmatinghasoccurred;
timeofeuthanasia
criteriaforscoringDLeffects,including,corporalutea,implantations,resorptionsandpre-
implantationlosses,liveimplants,deadimplants.
Results'.
animalconditionpriortoandthroughoutthetestperiod,includingsignsoftoxicity;
malebodyweightduringthetreatmentandmatingperiods;
numberofmatedfemales;
dose-responserelationship,wherepossible;
concurrentandhistoricalnegativecontroldatawithranges,meansandstandarddeviations;
concurrentpositivecontroldata;
tabulateddataoreachdamincluding:numberofcorporaluteaperdam;numberofimplantationsper
dam;numberofresorptionsandpre-implantationlossesperdam;numberofliveimplantsperdam;
numberofdeadimplantsperdam;fetusweights;
theabovedatasummarizedforeachmatingperiodanddose,withDominantLethalfrequencies;
statisticalanalysesandmethodsapplied.
Discussionoftheresults.
Conclusion.
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©OECD,(2015)