Document VJV6LzogR25R8YwYN9VYqXnag

Shepf LL J HE SENN. pipes Duecior September 19, 2001 r---- AR226 _ 1056 0 Core, Bang 2 Receiven SPiN Ss133.3220 npn ore OPPTEBIC ohnson@mmmcom ---- wIsEP26 mi: ss 51903 Document Processing Center (7407) Attn: Section 8(e) Coordinator 4U1S0EMnvSitrroenetm,enStWal Protection Agency Washinglon, DC 20460 EHC-090/-1445.2, RPee:rflTuSorCabAut8a(ne)sSulUfBonSyTlAFNluTorIiAdeL: CAS# 375.724 RISK NOTICE (SUPPLEMENTAL) ON: Dear Sir: resolved by the next day. The functional observational battery and hisopathologica f3lMuorhiadsec(oPnBdSuFc)teidn ara2t8s-adtacyonrceepntartadtoisoensionfha4l7a,ti1o6n2staunddy4w7i9thpppemrf(lhurosr/odbauyt,an5esulfonyl dayshweek). Animals exposed to PBSF exhibited clinical signs consistent with neurotoxicity at all levels. Clinical signs were observed early in the exposure and had krensouwlnt.s wePrBeSwFitahsisnocnioartmedalneluirmiottso.xiTcihtey mweacshraenpiosrmteodfiancatimoon ufsoretihnihsanleautriootnotxoixciictiytyis not screen (TSCA 8e Substantial Risk Notice, dated 30 July 1999) and ina rat acute inhalation study (TSCA 8e Substantial Risk Notice, dated September 28, 2000). f3oMr PhBaSsFe.st3abMliussheesd aPwBoSrFkpslafaecley eaxspaossiutreelilmimiitted(-inhtrerTmWedAi)ateofw0i.t1h mthge/fmo3ll(o0w.i0n0g8 ppm) cilnodtuhstirniga,lahnydgileocnael perxahctaiucsets:vesnutpipllaiteidonaidrurrienspgicrhaatrogryinpgr.otection, chemical protective Please contact me at 651.733-9218, for further information. Sincere . S7 aal i4B,ron Cong, Nocs LDairrreycRto.r -JohCnoon,rporaDt.eVT.oMx,i,cPohl.oDg.y, DABT 28 on z i= 1= 3385 E23 Enclosure: Complete Report: "T-7499 TOXICITY STUDY BY REPEAT DOSE INHALATION ADMINISTRATION TO CD RATS FOR 4 WEEKS" BEHG-35-14523 - 85010000317 CONFIDENTIAL 57903 MIN 252/004312 EORCITY STUDY BY REPEAqT DrOSE HALATION KOMINSTRATION TOC poner 3M Medical Department, ny 3M Center Building 220-2E-02, EiLrai St. Paul, N---- Huntingdon Life Sciences Ltd., fms Alconbury, CtA 1e e0 -- `Cambridgeshire, Page 1 of 251 MIN 252/004312 `COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS PrecThestudy arsand describedi mde gen n this reportwascondu 5wi ctedincompliancewiththefollowingGoodLaboratory TheUKGoodLaboPrr actaicetRegoulartioyns 1999(StatutoryInstrumentNo 3106). OECD Principles of GoodLaboratoryPractice (asrevisedin 1997), ENV/MC/CH(9E8M) 17. ECCommissionDirective, 1999/11/ECof 8 March 1999 (OfficialJournal No L77/8). hat 58 UPnairtte7d92,StaFteesdeErnavlirRoengimsetnetra,l P2r9otNeoctvieomnbAegren1c9y8,3(TaSndCAs)uTbisetqluee4n0t CaomdeendomfeFnetdeFredaelrRaelguRleagtiisotnesr Informationregardingtestsubstancecharacterisation,namelythebatchnumberandexpirydate, wasnot avaitolHuantbinlgde onLife SciencesforcompliancewiththeGoodLaboratoryPracticeregulationsgiven above. (] = 2b zl Derek W. Coombs, B.Se., M.Se., Date Study Director, Huntingdon Life Sciences Ltd. N/A -- Fe erectogy Seve 28HuseAsst Due `Submitter. Date 12: QUALITYASSURANCE STATEMENT MIN 252004312 "Thefollowinghavebeeninspectedar auditedin eltontothisstudy: `Study Phases Inspected DateofInspection. DateofReporting Protocol audit 31October2000 31October 2000 `Study basedinspections `Study preparation ) Exposure ) `Testsubstance control ) Sampling ) Clinical signs ) Recordsaudit ) 13 November 2000 13 November2000 Blood sampling 8December 2000 8 December 2000 Post mortems Recordsaudit ) 11 December 2000 ) 11 December 2000 Report audit 6April 2001 9April 2001 Protocol: An auditofthe protocol for this studywas conductedandreported to the StudyDirector and CompanyManag asie ndim cate edan bovte. StudybasedInspections: Inspectionsandaudits ofphase ofthis studywereconductedandreportedto `the StudyDirector and CompanyManagementasindicatedabove. Processbased inspections: At or about the time thisstudy was in progressinspectionsand audits of other routine andrepetitiveprocedures employedon this type of studywerecarriedout. Thesewere promptly reported to appropriate CompanyManagement. cRoenpdourctteAduadnidt:reTphoirstreedptoortthheaSstbudeyeDniraeucdtiotreadnbdyCtohmepQaunaylMiatynaAgsesumreanntcaesDienpdaircattmeedntb.oTveh.isauditwas `Ttohreegemsethodhse,rparwodcaead.uresandobservatiwoenrsefoundto beaccuratelydescribed andthereportedresults `TracyScarfe, Date. GroupManager, Depaofr QuatlitmyAsesurnancte, HuntLi ifn eScg iendceo s Ltnd. i3: CONTRIBUTING SCIENTISTS STUDY MANAGEMENT Derek W. Coombs, B.Sc., MSc, Study Director. ASnttudhyonSyupMer.viBsoowrd.en, B.Sc. (Hons), CLAT., TOXICOLOGY ColinJ. Hardy, B.Sc., Ph.D., M.LBiol,, C.Biol., Dip.R.C.Path. (Toxicology), Principal Toxicologist. AEROSOL TECHNOLOGY AND ANALYSIS IaSn. Gilkison, MA., PhD, Section Head, Aerosol Technology and Analysis. CLINICAL SCIENCES David Crook, BSc.(Hons), Ph.D, HeadofData Quality, Central Laboratory Services. STATISTICS GrahamF. Healey, B.Sc, MSc, ARCS., Head, DeparotfSmtateisnticts. PATHOLOGY `Sammuel McCormick Directorof Pathology. ta MIN 252/004312 CONTENTS MIN 252/004312 Page INTRODUCTION ccsnrmssmmmsssssmssoss, 8 RELEVANT STUDY DATES cccmrmsssssss. 9 TEST SUBSTANCE corerereemsmsmmmmmmmmmsmsssssmsmmmmsmmmrss 10 DISCUSSION ccm 29 REFERENCES vermeemosmssmomsssapmmsmsmsmsmmmesoses 30 FIGURES TABLES 21.. CClliinniiccaall ssiiggnnss pdousrtinEgXpeOxSpUoIsEur--eN--GgIrAoEuNpCdiSstUriTbuMtAioLn Yof.O.DSvEvIvVAvHvONvSrmcnvrirnosss 3332 43.. BFoodoydWceoingshutmsp--tiFonO--UgMroaunp mVeAIaUnESva(l8u)es.(/animal)......owevscens 35 36 65.. FFuunnccttiioonnaall oobbsseerrvvaattiioonnalbabtattetreyr:yAc--tgirvoiutypCSoUunmtMsr--ygFOfOOUBP SMeEaInVVAalIlO..R.S....vcs 37 42 7. 8 FFuunnccttiioonnaall oobbsseerrvvaattiioonn bbaatttteerryy:: RGerairpisntgrecnogutnht~s --GrG0rUopuMpemaenanVVaallueus.e.....c.vvvs 43 44 9. 10. FFuunnccttiioonnaall oobbsseerrvvaattiioonn bbaatttteerryy:: LTeamnpdeirnagtfuoroets~plFaOyU--pM1OeUaPnmVeAIaSnS.V..alues... 4456 1112.. FFuunnccttiioonnaall oobbsseerrvvaattiioonn bbaatttteerryy::BCooudlybWoeuirgnhtasct--ivirty0UmponMietaonrVina--glguroeuSp.m.ea.nvvealnuees.s. 47 48 5: MIN 252004312 Page 1134., BHlOoCoMdtcOheImOig--sYtrGyF--OBUIPOMUPEAMNEVRAVEASISr ... ss 4593 15. 16. MOarcgraonsWceoipgihctpsa--thGoFlOoUgDyE--aInnciVdAeNnEceSS(U)M.MrA.r.v.rorrevernscnnoris 6613 17. Microscopic pathology --expanded incidenceSURETY ..eevrvocrrncers 61 APPENDICES I. 2. CClliinniiccaall ssiiggnnss (pprreea-nedxppoossutree)xp--osinudrievi--idnuadlivwiedeukallydOabiSlyEIOVbASHeOrVNaStio ons ccwwe 6 10 34.. BFuondcytwieoingalhtosbs~erivnadtiivoindaulalbVatAtIeUrEyS--( individu8 al ObSCT) VALIONS .. ccs. rce. v 18 80 56.. TAodtdailtitoinmaelcspoemnmteinnltoscformoomtfournaccttiiovniatlyo~bsienrdviavtiidounalalvbaaltuteesr(y --5i6nd0ivMid)ua.lobservations 112202 78.. HBaleomoadtcohleomgisyt--ryI--nddiviivdiuadluValBIVaUlEUESS rccv c s, 116628 9. 10. 1A0bdsiovliudtuealorPAgtahnOwIOeGiIgCahltS--sIiNnGdSiv.idual VAIUEs (8)... 117768 ADMINISTRATION OF T-7499 BY INHALATION TO RAccT o S 219 16 SUMMARY MIN 252/004312 `6Thhorueresgraoduapysfoofrra5tcson(seaecchutoivfedmaaylsesaawnedek$ffore4mwaeleokefstsuhs)einCg rawhiol:BeR-CbsotDdryaienxpwoesruereesxypsotseemd.toTA-7f4ou9r9t,h group, acting a control, was exposed toair only. a`TnhdeHsitgudhydmoeseangraonuaplsysreedspceocntciveenltyr.ationsofT-7499 were 47, 162 and 459 ppm for the Low, Intermediate `Thefollowing commentsare madein summary: Clinical signs observed during exposure included circling movement and lethargy. wCallikniicnaglosnigtnoseosbs(earbvneodrmiamlmgeadiit)ataenldy phoysptereaxctpiovsiutrye.Tihncelsuedesdigvnocsawleisriengcaonndsaigisttatweiiontnhtwhaenneuhraontdolxeidc, effect and generally resolved prior to exposure the following day. a`Tthteainoivnegraalldemgreeaenofbsotdaytiwsetiigcahltsigganiinfsicfaonrcealflorteasltl treasttsm(alWeese.ks 0 to 4) were lower than controls, AreductioninfoodconsumptionwasevidentforGroups 4malerats. `bTlhoeordecwhaesmisntoryefpfaercatmoeftetrrse.atment on the functional observational battery or haematological and Ninecorrogpansyweriegvhetasl.ed notreatmentrelated macroscopic findings and notreatment.related differences Histopathological examinationofthe respiratory tract revealed no treatment.related findings. Conclusion A no observed consistent with aetfrfeacntsileenvteelff(eNcOtEoLn)thweasnenrovtouessstaybsltieshmehdaddugreinngertahlislysrteusdoyl.vedHopwreivoertr,o ecxlipnoiscaulrestighnes following day and there was no evidenceofsustained neurotoxicity in the functional observation battery. 17: INTRODUCTION MIN252/004312 "aTshseepsusrtphoesseyosftetmhiicssttouxidcyppoetrenftoiralmeidnartatHsuontrienpgedaotn aLdimfieniSsctireantcioens Lbiymiintheadla,tHiounn,tiinngwdhoonl,Ee-nbgoldayndexwpaossutroe chambers,ofthe testsubstanceT-7499, for5 consecutive daysaweekfor4 weeks. "Thestudywasperformedincompliance withtheguidelinesoftheOrganisation forEconomicCo-operation andDevelopment: TestingofChemicals (412). "wTahesttehsetsspuebssiteanscoefwcahsoiacdemidnuisetetorreedbguylianthoarlyartieoqnu,irapeomsesnitbslearnoduttheefsotrraacicindweantsasleelxepcotseudroeninacmcaonu.ntTohfetrahte availability of comprehensive background data, relating to clinical and pathological parameters, at our laboratories. Exapproesluirmienlaerveylisnwhealraetsieonletcotxeiidotynsttuhedbya(sHiLsoSsftcoundsyulntautmibonerwMitIhNth2e51S/p0o0n3s3o4r1a)n.dontheresults obtainedfrom "The ine experimental phaofstheestudywasundertakenbetween 13 Novemabnde3r0 January2001. 8: RELEVANT STUDY DATES Approvedby: StudyDirector HRC Management `Study Sponsor Animalsaivedat HRC: 26October2000 26 October 2000 30 October 2000 1 November2000 Allocation to groups: 1 November 2000 [Exposures commenced: 13 November 2000 Functional observation battery (FOB) and Motor activity testing (MAT): `WWeeeekk-11((SFhuolrlt FFOOBB/)MAT) `Wee2k (Short FOB) `Wee3k (Short FOB) `Week 4(FullFOB/MAT) 91a8nNdov1e0mNbeorv2e00m0b20e0r0 25 November2000 2December 2000 9and 10Decemb20e0r0 Haematologyblood chemistry: Week 4 8December 2000 Terminal ill: Weeks 11December 2000 Histopathology completed: 30 January 2001 MIN 252004312 19: Tradename: Chemical name: Other name: Intended use: Appearance: Storage conditions: Amount received: Batch number: Assay: Expiry date: Date received: Supplier: `TEST SUBSTANCE MIN 2520004312 T7499 Perfluorobutyl sulfonyl fluoride PBSF `None stated Clearcolourlessliquid(prienassteeel prnesstureveessdel) Ambient temperature or in a refrigerator, unless otherwise stated by the Sponsor Ca.35kg None stated PePrefrlfulouroroosbuultfyollsaunlefonyl fluoride 962-948%% None stated 26 April 2000 Sponsor 110: EXPERIMENTAL PROCEDURE MIN252004312 ANIMALS KsetFnrifatti,yn(oo2nf5S1mpNarolaevgeuamen-bdDea2rw52l0fe0ey0mo.arlieg)inr,atwse,raegoebdtaaipnperdofxirmoamtCehlayr6lewseReikvse,ro(fUtKhe) CLirmli:tCeDd, MBaRn,satocnaResoaaredaMnardegraitvee,d aOlnlocaartreivdalt,o a1oilf a4ngimarlsowuecraepchesoxfa,5minmeadlesforanadbn$orfmeamlailtesi.es Tahnedansiigmnaslsowfeorveetrhteinlunhieqalutehlyaindedntriafnideodmblyy tnruemabtmeernst.tattooed on the tail. The animals were acclimatisedforat least 7 days before commencement of `The identificationofindividualratsinthe 4 grouwperse as follows: Group 21 ((LCoontwrdool)se) 43((IHnitgehrddoossee)) Reserve MalReat numberFsemale 166--1200 3316--3450 11--15 226--320 AE [=] tIrnevaitemwenotfgrtohuepnse,ethdefaonritmhaelnFuumncbteiroinnalgOsbyssetrevmatwiaosnsaulBcahtttheartyittowbaesnpoertfeoarsmyetdowiidtehntoiuftykantorwelaetmdegnetogfrtohuep from theanimal numbers. `bTyhealreetmtaeirnwinrgainiotmnalttsh,eetamniallaensdwanedre5rfeetmaailneesda,wseproteeanstsiiaglnreepdtlaoctehmeernetssedruvreignrgotuhpe.acTclimhatwiese raetiodnsenpteirfeiioedd. Foltlhecoomwmeincenmegntofexposurtehse reserveswerekilled. ACCOMMODATION Tbhacekraantdswfelrooerhwoiutshesdtaoifntlhseetsesaeslmseheseetxs1i0deasc.aTgheiencasguesspweendreedsstuaisnlepssesotnenedrlacecakgdse.sPfliatstteidcwtirtahymsleisnhedfrwointt,h aEbascohrbceangtepahpaedrawceorleopulraecdeldabbeelliodwenctaicfyhicnaggtehetgocroolulpecatnadntihmealneuxmcbreertsaofantdhethaenpiampaelsrwcaonstacihnaedngweidthdianilyi.t `pTohseitraitosnwederoenkeapntiinndivsiidnugallercoagoem abantde,rya.dditEiaocnahllbya,tatfetreyrtwhasstianrtaofsetphaereatxepovseuntrielpaetrediocda,beinaecth gwriothuipnwathse Exhpolodsiunrgertoooomkpilnaocredienrtthoe savaomiedrtohoemp.ossibility of inhalationoftest material from the rats in other groups. san: MIN 252/004312 `TThhee tsetmupdeyrahtoulrdeinagndrroeolmatitveemhpuemriadtiutryeoafntdherehlaotlidvie.nghurmoiodmitwyerweerreecosretdetdoubseingmaainKteanitneCdlewairtshpiann rleicmoitrsdeor.f 2142C and 55 + 10% respectively. Recorded ranges were 19.5 to 21.0C and 39 to 58% relative hafufmeicdtietdy.theDseciveinattiifiocnsintfergormitythoefsteherastnugdeys. were of relatively short duration and considered not to have Lighting was controlled to give 12 hours light (0600 - 1800 hours) and 12 hours dark per 24 hours. DIET `fWohoidle(iSnDtSheRiartcaangdeMsoalu,lsreatNsoh.ad| aScQcCesmsotdoifaiweedimgahiendtqeunaanntcietydioeft,sStpaencdiaalrdDiqeutasliSteyr-vciocnetsr,olWlietdhlaamb,orEastsoerxy).rat `There was no information available toindicatethat any non-nutrient substance likely to influence the effect ofthe test compound could reasonabbley expected to be present in the diet. The analyticaldata have been lodged in Huntingdon Life Sciences Archives. Tap water was available from moulded polypropylene water botles at all times while the rats were in the cages. The water bottles were emptied and refilled daily and thoroughly cleaned at intervals during the study. Therewasnoinformationavailabletoindicatethatanysubstancelikelytoinfluencetheeffectof thetest system couldreasonablybeexpectedtobepresentinthedrinkingwater. RWeastuelrt)soasftchonedurcoutteidnebyphtyhseiscuaplplaincdr,cAhnegmliicaanl WaantaleyrseSserovifcweastLetrd,athasvoeurbceee(nsmamapdleinagvapiolianbtl,eGtroaHfuhnatminFgidnoanl Life Sciences. Anglian Watertakes its guidelines onwater quality from the EEC directive relatintog water for human consumption, viz. Council Directive 80/778/EEC. `The analytical data have been lodged in Huntingdon Life Sciences Archives. ADMINISTRATION "The vapteostsrubsotanucewsas administeredfor 6 houadrays, for S consecutive daayweesk, f4oweerks. "The rats were exposed to the controlest atmosphere in whole-body exposure chambers constructed from thsteatiensltesslistqeueild ainntdogsltaasisn,lwesisthstaenelicnotielmvalapvooulrugmeenoefra0t.o7r5s amn.d wTahse dtiesltutaetdmowsitphhecrleeawnaas prpordiuocteodtbhyemreetsuelrtianngt Vapour atmosphere passing into the exposure chamber. T45h0e ptparmge(tHicgonhcdeonster)a.tiCoonnstrfoorl reaxtpsorseucreeivweedraeir o5n0lyp.pm (Low dose), 150 ppm (Intermediate dose) and 2: MIN 252004312 Dpetairls eoifandsAmiDnMeiIstNrnIatSitTonRaAenTdIadOnNalyOsFisoT-f7t4h9e9tBeYstIatmoN spheH restoA TgeOthRL eArwTiSA thatphpT eenrdeesI dutlotstO hoisbrteapN ionretd are CLINICAL INVESTIGATIONS D`awetleldaastndotshieggnerdoruepcoobrsdesrovfaatliloancstaivnidteexsaremliantaitnigotnostohuetdlainyetdoidnatyhrisunpnrioncgeadunrdemwaeirnetreencaonrcdeoedfitnhetshteuSdtyu,days Daybook. recorded. In addition,observationsrelating toindividualanimalswere madethroughoutthestudywere Mortality d`aThyrfoourgdhoeuatdtohremsotruidyb,uanldlanciamgeaslwse. recheckedinthemomingandagainattheendofthe normalworking Clinicalsigns IDnadtiveiddaunaldasniigmnaeldrreeccoorrddswseorfemaappienatraainnceed,ocnhtahnegbeaasnidodfi:sappearanceofclinical signsweremaintained. - anyobservation,consideredtobeofpossibleimportance,madeatany timeduringthestudy; - (apnryioobrseorevxaptoisounr,ec)o,nosnidreerteudmttoobheolodfipnogscsiabgleesim(paofretreaxnpcoes,umrae)daendudrdiunrigntgradnasifelrytcoheexspo;surecages - sapceacriealfautlteexnatmiionnwaatisognimvaednteowteheekdletyecctoimomnoenfcaivnaiglab1lwereeeskpiprraitoorrtysootuhnedsst.artofexposureswhen rDeusrpionngdeixnpgossiumirleasrilygtnoswtheerteersetcsourbdsetdanaces. aIgnrtohuepcrliensipcoanlsseignwsheirnedaivlildvuiaslidbaltea,andiemaatlhscaopdpeea7rreedfterosbteo terminal all BODYWEIGHT E`acocmhmreantcwiansgwe1wiegehkepdroinoratriovtahlefotlalrotwoifnegxrpaonsduorem.alDluorciantigotnh.eTthreewatemiegnhttpoerfeioadc,hboradtywwaesirgehctowradesdrewceeokrldye,d beforeexpoosn tuherdaey. Bodway salsw orece ordei dpriog rtonhecrot psy. FOODCONSUMPTION s`tTahretoqufaenxtpiotsyourfefsouondtcilotnhseuemneddbofytheeascthudcya.geof atswasrecordedweekly, commencing 1weekprior tthe fc: MIN 252/004312 FUNCTIONAL OBSERVATION BATTERY Neurobebavioural screening Dusarmiengttihmeeosftuddayy,. afNuontctailornaailswobesreervtaetsitoendalibnoatnteedrayaynd(ombosterovraatcitoinvsimtyawdeereprpee-rtrfeoartmmeednattaanpdprinoxWiemeatke4l)y,tbhuet dtaiymsewohfetnesntointgexwpaossebda)laanncdepdriaocrrotsosstthuedygirnoiutpisa.tioOnb.servations were made during the treatment period (on AAfsuhlolrftuenncetdibonaatltoebrsyewravastipoenrfobratmteedrydwuraisnpgeWrefeorkmse1d,dur2ianngdt3he. prTeh-terefauntcmteinotnpaelrioobds,earnvadtdiuornianlbgaWteteerkyi4s. detailed below: t`Thheeabnaitmtaelr.ycoTmhperisseecdon4dssetestooffoobsbesrevravtaitoinosn.swTahsepfierrsftosretmewdaisnpetrhfeotremsetdawrheenanainnitditalhley fhiannadllsinegt (opbrsee-rtvraetaitomnesnwtearnedmaWdeeewkit4hotnhleyo)bcsoemrpvreirbsleidnhdatnodtlhinegt/rsepaetcimfeincttceosntdiitnigoonfotfhtehaenainmiamla.l.~Allthese Observations in the hand: Easeofremovingtheanimalfrom thecage. ROecaccutrirveintycetoofhcaonndvluilnsgi(ocnass,eotrfehmaonrdsl,itnwgi)tches Salivation/lacrimation Palpebral closure `PiElxooeprhetshtailonmus FVoucraalipspaetairoanncoen handling Observation in the arena: `Occurrenceofconvulsions, tremors, twitches Activity counts LReevaerlinogfacroouunstal GPirloooermeicntgion RAescsoersdsmpernetsoefncgaeiotf/fpeocstaulreboluses, rine P14 Manipulations: MIN 252/004312 Approach response Touch response: Auditory startle response. RTaiiglhtpiinngchrerfelesxp:onse Pupil reflex GLrainpdisntgrefnogotths(pfloarye and hindlimb) Body temperature ( C) Bodyweight (g) Atanypoint duringtheobservations,additionalcommentsweremadeasfreetextwhereconsidered appropriate. +The manipulationswereonly madeduringthepre-treatmentperiodandWeek 4. pArlestehnotuegdhibnotdhyiwsseeicgthitonwaassthriescohradsebd,eennocodivsecruesdseilosnoewfhaenryeipnostshiebrleepoerfftectsoftreatment on bodyweight is hFaosrbetheenoobmsietrtveatdifornosm,ipfraelslenatnaitmiaolns nitnhalelregproorutpaslftihloeudghtoitshhaosbweaengirveecnorsdiegdnosnucthhearsalwadcaritmaasthieoentt.his sign Mwoatsomroancittiovrietdy wusaisnpgearfCoorumlebdobuerfoIrnfirnai-tRieadtiAocntoivfittyreMaotnmietnotrainndgdSuyrsitenmg't.hefourthweekof treatmentand re`Tchoirsdesdy:sttehmeutsiemseasnpeinntfria-nrendodmetoevcteomrentot,molnoictoomrotaoctriavintyd.noTnhe-lfoollcowoinmgacocttaitvoeigtroyr.ieTshoef anctuivmitby eoarrfe olcoccuormroetnocresac(tevvienytsis)oprfeeseancthed.category is also recorded. For reporting this data, only the time spent in Fpolarcteedsitnitnogt,hdeesciaggneast,edthaenteismtaslesswsieornepprloagcreadsmimnegwlaysisnatroteodb.seTrhvaettieosntcsaegsessi.onOfnorceeaaclhlaaninmaliwmahsaad1 bhloeuesrn. Data was collected every 2 minutes and writen onto a floppy disk. `pTehreffournmcetdioinnaRlooobm 0s07.erbvattaertywiasopenrfoarmledia Room 011andthemotoractivitymonitoriwnags Analysis and presentation ofthe behavioural screening data sTthreengftohl,lohwiinndg ldiamtba swpelraye, rbooutdiynweeliyghstubjaencdtetdemtpoersattatuirset.icalThaenasleysdisa:tarweaerrienganaanldysaecdtiuvsitiyngcoaunotns,e-gwraiyp Praena-lryesaistmeofntdvaartiaanwceerefaonlalloywseedd bbyyanWaillylsiiasomsf' vatersitan(ceWiflollilaomwsed1b9y71S/t2u)defonrtsa tedsot.se-related response. 1 System supplied by Coulbou Instruments, Lehigh Valley, PA, US.A. Ds: MIN 252/004312 `Tmhaennerre.porTtihnegoofbtsehrevatciaotnegaolriecnadlpodianttas fsourcthheasobesaesrevaotfiohnaanldlbiantgt,erayrohuassalb,eeetnc,hahnadvleedbeienntthaebufloaltleodwifnogr tFreqeuoenfcrtyhoefmdoecgcsruereroenrcte foryoefarcpehsgprooeunps.e (Ail.thsotuagrthled:unroinrg erecaordciangtn,eisaormotewinrtec,shp,onasfelsinwcehr,eetccl.a)s,sfiofiretdhien pthuerproessepsonosferheapsorbteinegn,raesptohretreed awserbeeinnog reeitmhaerrkparbelseendtifofrearbesnecnets.between the groups, for a given endpoint As there were no remarkable differences in the incidence of observations, no statistical analyses were performed on the categorical data. "teTshte (CWoiulllbioaumrsn 1a9ct7i1v/i2t)y dfaortaawedorsee-arneallaytseeddruessipnongsae.oneP-rew-atyreaantamleynstisdoatfavawreiraencaenfaollylsoewdedbybyanWaillylsiiasmso'f variance followed by Student's? test. `daWthaenwtahse acnataelgyosreidcaulsdiantgaJsoungcgkehseteerdea-Tpeopssstirbaletdesitff(eHroelnlceanbdeetrwe&enWcoolnfter,ol1a9n7i3m;alSstaatnXdacttr,eat1e9d92g)r.oupAs,ttwhoe tailed test has been reported, untlheeressponsses were directionalin nature. Key to the functional observational battery Easeof2remeovaaslyf(riotmecargeesistance) 3 slightly awkward Easeof2handleiansgy litle resistance) 3 slightly awkward SalivatYion (onsliygnscoobrseedrivfepdresent) `The numbers associated with salivation indithcedaegtreeeofeffect (1,2, 3 increasing degreeofeffect) N signnot observed Arousa3l reduced alertness 4 5 alseormte(wnhoartmahli)gh (slight excitement, tense, over reatoecxtetrnailstoimulni) 116: Gait T walking on toes UHu nhauncbhetdolasseess - seeadditional comments A- anbonmoarlmaglait `(T1h,e2n,u3mibncerresasaisnsgodceiagtreedewoiftehfgfaeictti)ndicatethedegreeofeffect Mobility impaired: mobilityofthe rat impaired due to gait abnormalities Approach 21 noreaction sniffs only 43 afrpeperzoeasches and sniffs 5 backitums away O6 otwhaelrkrs paestapro-cbesetcadiditoionnal comments Touch 1 oreaction 23 ums walks away 4 freezes 65 tums to opposite side walks backwards otherrea-cseteadiditoionnal comments Startle1 noreaction 23 enaorrtmwailtfclhionnchly 4 5 enoxtaigcgeearbalteerderseposnpsoen.se O other reaction - ee additional comments MIN 252/004312 17: MIN 252/004312 "Tail pinIch noreaction 2 m2 s msimmediately 3 violenttum, 3 walksaway 45 jfurmepezsefsorward 6 Tunsaway other reaction -seeadditional comments ALwith aresponsethat isnot atun indicatestha theresponseincluded a tumsuchas walks awaywithatum Rightin1g refliemxmediatereaction VocalisYingsignobserved N signnotobserved `(T1h,e2n3,umibnecrresaassisnogclioautdednewsisothfvvooccaalliissiinngg)indicate the degreeof effect PupilreBflexreflexobsebrotvheeydes Urine N noneobserved MS smmodaelrlaatmeoaumnotuonbtseorbvseedrved L largeamountobserved Reaandractiivityncougnts Creoaurnitnsg wwearsecomuandteedoefvreeraryitnigmaentdhaecatniviimtaylwlhifetendtbhoetahnfiomraelfesewtecrleeiarnotfheaasruepnpao.rtiAncgosuunrftafcoer. mTahdefelwoohreonefvtehretahreeannaiwmaasl mmaorvkeeddaolfffionutrofe6eetiqnutaoloanreeaosf(t"hseqsuearseqsu"a)r.esA. coforuactin vitywtas D1: LABORATORY INVESTIGATIONS MIN 2521004312 Sample collection dBeplroiovdatsiaomnploefsfofoodr,hpareiomrattooleoxgpyosaunrdeobnloDoadyc2h6em.istry were collected from all animals following overnight Satasmpwleerseohfevlednuonudserbilsooofdluwreanreeawniaetshtdhreasiwan.from the etro-orbital sinus using sterile glas pipettes while the `The blood samples collected were put into tubes containing the following anticoagulants: EDTA Citrate --ffoorrchlaemoattotlteosgtiiscna0l.g5inmveIstwihgoaltieonbslo(o0d.5) ml whole blood) Heparin - for blood chemical investigations (0.7 mi whole blood) `aTbhberehvaiaetmeadtotltolgi(cfaolr uasnedinbalpopoedndcihceemsiacnadltaibnlveess)t,igmaettihoonsdspaenrdfotrhmeeudnitasroeflmiestaesdurbeemleonwt,.together with an Haematology `The following estimations were performed using aBayer.TechniconH*IE haematology analyser: Units Haematocrit (Het) wm Haemoglobin (Hb) gL Red cell count (RBC) 10% Mean corpuscular haemoglobin (MCH) " Mean corpuscular haemoglobin concentration (MCHC) ga Mean corpuscular volume (MCV) Total white celcount(WBC) OL Differential WBC count LNeyumtprhoopchyiltses (Neutrophil) ~} (Lymphocyte) } EBoassionpohpihlisls (Eosinophil) } (Basophi) ~} OL MoLnarogceyutnestsained cells ((MLoUnCo)cyte) }} 119: MIN 2520004312 Units Cell morphology: the most common morphological changes (anisocytosis, microfmacrocytosis, hyporhyperchromasia) were recorded as follows: - = no abnormalities detected + ++ == msoidgenrate = marked Platelet count (Pl) X10 "The following estimations were performed using the appropriate methodology, as described below: Reticulocyte count (Ret) ~ Sysmex R3000 Reticulocyte Counter B cel w "Thefollowingwereperformedusingan ACL 3000Plusanalyserusingthe appropriateIL.reagents: Prothrombintime(PT) -methodofQuick, HLA. (1942) sec ARcatpiavpaotretd, PSaIrt,ia(l19T6h1r)omboplastin Time (APT) - methodofProctor, RR and. sec Blood chemistry `Thefollowingparameterwas analysed with an Hitachi 917 clinical chemistry analyser using, except where: indicated, standardHitachi 917 methodologies: Triglycerides (Trig) mmol/L "Total protein (Total Prot) aL Protein electrophoretogram (Beckman Paragon system) Albumin (Alb) Alpha 1 globulin (s1 Glob) Alpha 2globulin (a2 Glob) Beta globulin (Beta Glob) `Gamma globulin (Gamma Glob) Albumin/globulin ratio (A/G Ratio) i200: gLand% PLand% PLand% PLand% PLand% Urea (Urea) Alkaline phosphatase (Ak. Phos) - reaction temperature 37C Bilirubin - total (Bil. Total) Creatinine (Creat) `Sodium (Na) Potassium (K) Calcium (Ca Total) Phosphorus (Phos) Chloride (CI) Cholesterol -total (Chol Total) Glucose (Glue) - hexokinase mediated Alanineaminotransferase (ALT) -reactiontemperature 37C Aspartateaminotr(ASaT)n- sreafctieontrempaerastuere 37C MIN 252/004312 Units mmol uL umolL mol mmol/L mmollL mmollL mmol, mmollL mmolL mmol ur uL SACRIFICE pAlelrfgorromuepdswoenrDekaiyl2l9edoffotlhleowsitnugdy.5consecutivedaysofexposure aweek,for 4weeks.Thetermikinllwaals Afrnoimmatlhse bwrearcehiaklilalretderbiyes.an intraperitoneal injectionofpentobarbitone sodium followed by exsanguination p21: MIN 252004312 MACROSCOPIC EXAMINATION AND ORGAN WEIGHTS All rats were subjected toa detailed macroscopic examination. The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighed: aedpriedniadlysmides lliuvnegrs heart testes Kidneys Bilateral organs were weighed together. FIXATION OF TISSUES Swaemrpelperse,soerrtvhede iwnhboulfef,eorfetdh1e0f%olfloromwailnign,oregxacne/tpitstsuheese,yteso,gewthheirchwiwtehraenpyremsaecrrvoesdcoipniDcaavlildysoanb'nsorfmixaaltievnet,iatineds testes and epididymides which were fixed in Bouin's solution and then transferred to 70% alcohol. abandormenmaallsi(tcioerst*ex and medulla)* alimentary tract soteosmoaphcahgus djueojudneunmum iclaecucmum colon arneicmatlumidentification mark braaoritna (thoracic) berpoindcihdiymides efyeemsur harderian glands hheeaardt*(including auricular and Kivdennetyrsic(uilnacrlruedgiinogncso)r*tex, la`cmherdyumlalla aglnadnpdaspilla regions) lliavreyrnaxl2l olbeevesl)s*) lungs (all obes)* ly`mmepshenntoedreics a(nmadndibular, `tmraamcmheaorbyroanrcehai(acla)udal) onapstailc nteurrbviensates (3 levels) opvaanrcireesas pituitary psraolsitvaatrey glands ssecimaitniaclnevrevseicsles sskkeilnetal muscle (thigh) spinal cord ssptleemeunm ttehsytmeuss tthoynrgouieds with parathyroids trachea (including bifircation)* uurtienraursywibltahdcdeerrvix vagina * Including nasal cavity, paranasal sinuses and nasopharynx in HISTOPATHOLOGY MIN 2520004312 aprnHoidscteo4s.psaTethdhaeolnsodegtsictaaliineexdsawwmieistnrhaehteuaimoenbmseaetdwodesxerydeliipnneparanfrdoarefmofesidinnwofanoxraaellxnasdmcsihenecadttuiilooenndsbtyaipspslruieogshxti(mmimacatrerolkysecd4o-pw5ei.tuhmS*t)ehifccokrtwcGeiurrotoefucrpnuosstm,1 the testes were stained with PAS-heamatoxylin. Fstoarintiensgtemsettihsosduoelsoognyltyo(raenmiomavle nnounm-bsepercsif1i-c5haaenmdat1o6x-y2l0)i,n s0t.a5in%ing(.v/v) hydrochioric acid was used in the Histopathological examination was only performed on any abnormal tissues arising from Groups2 and 3. STATISTICAL ANALYSIS All statistical analyses were performed separately for males and females FBoordyawlelipgahrtadmaettaewrserteheanaanlaylsyesdeussiwnegreweipgehrtfgoarimnesd.uTshiengfotlhleowiinndgisviedquuael nacneiomfasltataissttihcael teexsptesriwmaesntuasledunfiotr. bodyweight, organ weight and clinical pathology data. I7f5%th)e,tdhaetaprcoopnosirsttiopnroedfoamniinmanatllsywiotfhonvealpuaerstidciuflfaerrevnatlfureom(retlhateivmeofdreeqwueanscyaonafltyhseedmboydeapperxocpereidaetde methods. Otherwise: (sBatatarbttllheeevtat1r%iteasnltceveweaslts)rauhpcepttlueirreeodgcteonouelidtteybstefwooabrsthaefitoneuerndod.g,eaneliotgyaorfitvhamriicatnrcaensbfeotrwmeaetniotnrewaatsmenttrise;dwthoesreeesiifgn8ifmiocarnet dImofosrneeo-treshliaganntietfdiwcroaengstrpoohunepstseerw(oeWgrielnelcbiietaiymsnw,gac1so9m7dp1eatreaecndtd,ed1g9r(7oo2rui)p,fmoaerasinafstitswhfeearrceetowcrayosmtpreaavrnisedfdeonrucmseaitnfigoornWialwlanisoanmf-so'mutonendso)tt,fooanrnicda rSetsupdoennste',sDtuenstnewtats'susteeds.t (Dunnett, 1955 and 1964). For separate two-group comparisons, a trIreFassnpisogfnnoisrfemiac(taSinhotinr)hl,eetye,grroo1gu9ep7ns7e)i,wtyeoorrefivf caortmihaeprnaecreewdawsausseivpnrigedseSenhnciterl(efayon'rsd acnoonunol-ndp-amnrooatnmoebtteroinrciecmtoersvetesdpfoonbrsyea,adDlouosngea-nrr'iestlhatmteiescdt (wDausnuns,ed1.964). For separate two-group comparisons, a Wilcoxon rank sum test (Wilcoxon, 1945) Fo`bWorhdoeyrrwegeiaagpnphwrteoipwgrhihiatctdhea,mtaiang,athlthyseiinfsfiolnfuaeclnobcvoeadtryhiweaenoicrgeghawtnwawsaesuisugehsdse.dinasplcaocveaorfiaantaeilynsainsoaftvtaermipatntcoealilnotwhefoarbdoivfefesreeqnuceenscei. For microscopic findings Fisher's exact test was employed to detect reatment-related differences. 123: MIN 252/004312 LOCATION OF STUDY RECORDS oAflthreawSpodnatsao,r.samples and specimens arising from the performance of this study will remain the property aTrycpheisvionfg smaamyplbeeadnidspsopseecdimoefnaftthearttahreepuenrsiuoidtsabsltea,tebdyirneHausonntionfgdinosntabLiilfietyS,cfioerncleosn,gSttearnmdarredteOnpteiroantianngd. Procedures. Aalrlchoitvheefrorsaamppelreisodaonfdfisvpeecyiemaernssfarnodm atlhlerdaawtedoantawwhiilclhbtehereSttauidneydDibryecHtuonrtsiinggndsotnheLiffinealScrieeponrcte.s Aifntietrs osufcthhetimmaet,ertihaelS.poInfrseoqruewsitledb,e HcuonnttaicntgeddoannLdifhiesSacdiveincceessowuilglhtcoonntitnhueerettourrne,tadiinstphoesamlaotrerfiuarltshesurbrjeetcetnttiooan reasonable fee being agreed with the Sponsor. HthuentfiinnagldroenpoLritfienSictsiearnccheisvweilinlderfeitnaiitneltyh.e Quality Assurance records relevant to this study and a copy of 2: RESULTS MIN 2520004312 CHAMBER ATMOSPHERE CONDITIONS Chamber analysed concentroaftT-i7o49n9 The data are presented in ADMINISTRATION OF T-7499 BY INHALATION TO RATS `Thedataaresummaribesleowd: Group J2(uowedodsoed)) AGighdos) CThaarmgbeterconcentrAantailoynse(dppm) 50 Meaa7n sd5 41500 415692 22 Tchoenceancthriateivoends.chamber concentrationsfor Groups 2to 4werein good agreementwiththe target CLINICAL OBSERVATIONS Mortality Therewerenounschedudelatehds. Clinical signs `Thedataaepreseasnfotlleowds: TTaabbllee]2 -dpuorsitnegexxpp--ion~ocgisrdoesunpcudeiussturrmribmuaetreioynof observations AAppppeennddii|2xx p-prerandepos-te~xepiondsiuxvried--upianldwioeveikdsluyaoldbuasielryrvoabtsieeornvsations CDleiynicIoalfstihgenesxopbosseurrveesd. L1estahgarrgoyuwpraessepvaindsenedtfuroirnGgreoxupposs3uarneidn4clduudriendcgithrecleixnpgomsourveesm.entforGroup 4on aGClnridonui4pc.aslH2ys,pi3egnrasancotdbis4v.eirtyvWeawdlakisimnomgbesdoeinravtteeodleysfo(plsolbsortwiioenrxmgpaoltshugeraeeixtpionwcsalusurdeeesvdifdvoeronctGarlfioosuliplnosgwia3nnagdntdahgei4teadxtuiprooinsnugwrheWesenefokhrsaGn2drloaeundpdsfo33.r Thissignwasalso evident follthoeewxpiosnurgesforGroup 2rats during Week 3. fOe3nmftaehlmraeelereoartacptcrsaipsoririotonrostesoixegnpxswopesrooenusporDruneaseDyreaney1t7.pr19iV.orotWcoaalelkxiipnsogsiouanrnnegdt.oaAcggsiitta(tasitboinnoonwrhwmaehlnegnahihatan)dnwldaeldsedpwrweerasesenpptrrfeesoseernnGttrffooorurptawG4orfGoerumoapulpe3 raftolslporwiionrgtdoaey.xpoosnDuary2e6. Atalothrtimes,alloftheabovesignshadresolvedprior to exposurethe 125: MIN 252/004312 SAitgontshearrteicmoenssi,dderryedskiinnciadbernataslioannsdtonottairleslaitietdatbolterebaethmaevnito.ur and pale coloured teeth were observed. These Bodyweight `The data are presented as follows: Figure | - group mean values (5) TAapbpleend3ix 3 - girndoiuvpidmuealanvavlauleuse(sg()g) `Theoverallmeanbodyweightgainsforalltestrats (Weeks 0to 4)were lower thancontrols,attaining a degreeofstatistical significance for all test males. Food consumption `The data are presented as follows: Tabled - group mean values (g/animal) A reduction in food consumption was evident for Group 4 male rats. "Thefoodconsuomfopthteriesotrantswasconsidered nottobeaffectedby treatment. Functional observation battery `The data are presented as follows: TTaabbllees6 -acgtriovuiptyscuomumntasry--ofgroobuspermveaatniovnaslues Table7 rearing counts-- group mean values TTaabbllee8d - glrainpdisngtfroeon--tggsrto--puhgplrmoaeuypanmveaalnuevsalues Table 10 -tempe--grroaupmteaunvraluees TTaabbllee 1112 -- cbooduylwbeoiugmhtasct--ivgitryoumopnmiteoarninvga.lues A`Appppeennddiixx 45 tiontdailvtidiumaelsopbesnetrvinaltoicoonsmotoractivity ~individual values (seconds) `Appendi6x -additional comments ~ individual observations Fleuvnecltsiounpaltoob4se5r0vaptpiomn,alshbaotwteedrynfoinedvinigdsenfcoer oafninmeaulrsottorxeiactietdy.witThheT-f7o4l9l9owfionrgfcoourmmweenetkss,aarte emxapdoseurien summary. D2: MIN 252/004312 In the hand observations inthe hand observationswereunaffectedby treatment. Arena observations Aberterneaatombseenrtvraetliaotnesd.showed some inter-group variationbutthedifferences were not considered to in`CtohmepaarreednwaidtuhricnongtrWoel,ekfem2aolfestrienaatlmletnrte,aatledthgoruoguhpsscsohroewsefdorremdaulceedsacwteirveituynaanfdfecrteaerdi.ngIsnctorhees adibfsfeenrceencoesf wsiemrielacrornessipdoensreetodpabtetdeumestdourniantgurparlevcaerdiaitnigono.r subsequent weeks of treatment, these Manipulations During Weck 450 ppm were 4 of lower treatment, than those loafndcionntgrofloso.tspMleaaynmveaalsuuersemfoernttshesfeorgrfoeumpaslewserree,cehiovwienvger1,50alsoor somewhat considered rteodbueceadssboecfioarteedcwoimtmhetnrecaetmmeenntt.oftreatment and these differences were therefore not Motor activity, `cTohnettriolmeansipmeanltsidnulroicnogmWoeteorkac4tiovfittryefaotrmemnatlwehserreecaesi,viinngcon1t5r0aspt,pmfemwaalselsorweeceritvhianngt5h0atosr h1o50wnpbpmy. sviheowweodfithne lcackraoceftidvoaistasyget-iermeeldsat.iTohnsehsiepdainfdfetrheencoepspfoasiilnegdtdoiraecchtiieonvseosftacthiastnigceal,swiegrnieficcoannsciedaenrde,ditno be due to natural variation. LABORATORY INVESTIGATIONS `Haematology `The data are presented as follows: TAapbpleend1i3x7 --girnoduivpimdeuaalnvvalauleuses. There were no treatmeat-related findings. p27: Blood chemistry `The data ae presented as follows: Table 14 - group mean values Appendix8~~ - individual values There were no treatment-related findings. MIN 2520004312 TERMINAL STUDIES Macroscopic pathology "Thedataare presented as follows: Table 15 Appendix 10 -- iinndciivdiednucaelspuatmhmoalorgyical findings There were notreatment.related findings. Organ weights The data are presenteda follows: TAapbpleend1i6x --gindir vimdueaoalnvvaaluuleusep s(g()g) There werenotreatment.related findings. Microscopic pathology `The data are presented as follows: `ATpapbleend1i7x 10 -- einxdpiavniddueadlipnactihdoelnocgeicsaulmfmiandriyngs Treatment related findings `There were no microscopic findings that were considered to be related to treatment with T-7499. Incidental findings All microscopic findings were considered to be incidental andofno toxicological importance. 128: DISCUSSION MIN 252/004312 Icnthoissntudsdy,ayresaetasccwhewureeekte,xfpoiors4edvwbeyeeiknsh.aTlahteisotnutdoymtheeanvaepxopuosruorefTl-ev7e4ls9w9efroer476,ho1u62rsancdac4h5d9apyp,mf.or 5 TchoenosvuemprtailolnmweaasnalbsoodeyvwiediengthftogramianlfeoarnaimlalltseisntGgrroouupps4.waslowerthancontrols. Areducedfood Aexnpiomsaulrseepxrpoocsedeudreast.conCcleinnitcraaltisoingsnsofwe1r6e2 palpsmo n(oGtreodupfo3r)alalntdes4t5g9rpouppms (oGnroocucpas4i)ownesrpeosltetehxarpgoiscurdeurainndg nienucrloutdoexdivcoecfafleicstatainodngaennderaagliltyatrieosnolwvheednprhainodorleedxpaonsduhryepetrhaectfiovliltoyw.ingThdeasye. signs were consistent with a Tchheemriestwrayspnaroameeftfeercts.ofNterceraotpmsenytroevneatlheedfnunoctrieoantamlenotb.sreerlvaatteidonmalacbraotstceorpyiocrfhianedmiantgsolaongdicnalo tarnedstbmleonotdrterleaattemdendti-frfeelraetnecdesfinidninogrsg.an weights. Histopathological examinationofthe respiratory tract revealed fio Aconnsiostoebnsterwivtehd aefftercatnsileenvtelef(feNcOtEoLn) twhaesnenrovtouesstasbylsitsehmedhadudrignegnerthailslystruedsy.olveHdopwreivoerr,to celxipniocsaulresitghnes following day and there was no evidenceofsustained neurotoxicity in the functional observation battery. 129: REFERENCES MIN 252/004312 BARTLETT, MS. (1937) Propofesurffitciienceyansdstatisticaltest. Proc.Roy. Soc., A 160, 268. DACIE, J.V. and LEWIS, S.M. (1966) in: (EDS). PracticalHaematology. 3* ed. p. 28 and/or p. 140. DUNN, 0.J. (1964) Multiple comparisons using rank sums. Technometrics, 6, 241. DUNNETT, C.W. (1955) A multiple comparison procedure for comparing several treatments witha control. J. Am. Stat. Assoc. 50, 1096. DUNNETT, C.W. (1964) New tables for multiple comparisons with a control. Biometrics, 20, 482. FISHER, RA. (1950) in: (Eds).StatisticalMethodsfor Research Workers. Oliver and Boyd, Edinburgh. HOLLANDER M, WOLFE DA (1973) Nonparametric StalMethods. ohn Wiley& Son, New PROCTOR, RR. AND RAPAPORT, S.1. (1961) The partial thromboplastintimewith kaolin. Am. .. Clin. Path, 36,212. QUICK, A. (1942 in: EDS). The HoemorrhagcDisses andthe Physiology of Haemostasis. hl p SHIRLEY, E. (1977) A non-parametric equivalentofWilliams' test for contrasting increasing dose levels of a treatment. Biometrics, 33, 386. `StatXact-Turbo User Manual (1992), Cytel Software Corporation, Cambridge MA. WcoImLpLaIrAedMSwi,thDaA.zer(o1d9o7s1e) cAonttreoslt.fBoiromdeitfrfiecrse,nc2e7s,b1e0t3w-1e1e7n. treatment means when several dose levels are WILLIAMS, DA. (1972) The comparisonofseveral dose levels with a zero dose control. Biometrics, 28, 519-531 WRIGHT, BM. (1950) A new dust-feed mechanism. J. &. Inst. 27, 12. WILCOXON, F. (1945) Individual comparisons by ranking methods. Biometrics Bullen, 1, 80-83. 130: FiGuRE1 Body--greopmieagnvahsest 0) eaGno [ER | eSnagpomrosmh iE aiey | nie ; Lo gm eT ee [ = rR mmm mbna aneFosn TnY w g | : LT : trmmmeemsmermp-- r EE r TSeres gneg sboomulitiesdetected | Y AV VNAANA `yrA 4 A A AA AAA AA 8B | | sea | Chotepotpee may | be ] TT -- oPrNrn mms [5 595555595 505880050 od | aer, 1|i :: TABLE? (Clasigspost expose contianed) Gon TT [oem ECe EIC--TS a IIHR rH m r -- G ) Tw [5S SSS SSE 8s see (Couey (ow3er) |VANeoirsadrnoawihvesendnenrdicesedd [T T5ii 5 int 4 a s 41oa 4 tsn40t4P4 3A r402a43 T aa5 g4 2 Tope : (e5do)| [VANpioagswvonimsimieatsdsd [[(311313141305121 551 3:3 03 52 [E 44i 2a a 4 VTiokveingsoon tz [ 112} es 2230 aaa Gi|V(Wn aoktianmggoi prcnima) isdeot n ([53[5355553555115205553325s0Sa5s sdi' s oss ge Specie Hi ++ AAoepereetstaihhheepppdddrsrasesrcaaotoppoikhofoorsniCldr+bserrlG4F81d 7 | | | --_-- sniper | : i tapi --pasa MIN 252/004312 Funcom observational be`TyABgLrEoupsummary of observations Pre-dose Sex] ----Male---- ----Female-- | ss 5 sls sss oromiEmsRvbRToOnReSsesy | ss ss | sos | ss -- wa | s[0 s0 o0a aooss0 5o da EEwig | oo a ole 14 a cpaaims || 50 0ss0 s0] s oos oos 0a ----ww |a 2 23 aalee 5aeoe Gr wage | 0 1 1 3 2 1 3 (evemwroms | 0 0 r--ar--i || 2ss gem | +5 o_o ]o ssA|]: ss | oo a ios ds:s os oss SO ippoiame righting,immediately || 55sss5 ss5 s5ss|5s so5s ---- $ssss5 a (Functions observTaAtiBoLnsEb5aery continued) MIN 252/004312 Weeks --S a a a pae e we sss sls sss o l obiesem | a 4 ssn | 4s ] ss ww. || 05s0 40 oslse a0 oa2 rE REAvg | 0 oo ali 3a wpooms || 0ss0 0ss0] |e soosso w--we | 22 53 3a aallee eno e0 or amas | 0 2 2 2s 3 2s carmmesn || 00 00 0o_o]alo 0oo0 o SE -- ne TABLE5 Mvaszo0ss12 (Functional observational bat--t conetir nueyd) wea wo 13 a s] ear s a RED weeI sss ss 5-- 5% wommncsy | ss ss | ss ss ETwwims | 00 `handling,easy 5 050 500 2a0 33|e a23 oa5 3a4 mmaoods pre || 50 s0 0ss0 ET | s|o s0 sa os v ww 22 a ale 04 vvaomraateess || 00 |0 01 0 20 0 42 s|o oo 5oaos1 s4 J i TABLES (Functional observational battery -continued) MIN 2520004312 Week3 o --ae Gow| 1 2 3 4 Nemberl 5 5 5 8 INTHE HAND [removing from cage, easy handling, easy. salvation vocalsing \IF THE ARENA grooming arousal, alec defecation urine [GATT `Walking on Toes Unable to Asses Hunched +s ss sas 4 Too 1 0 oo 0 0 roo ss oo 23 00 ss 00 21 ro2 oo oo 33 00 11 `Numbers reflect the numofbaneimarlsshowingthe response --Femae-- 1 2 34 5 s ss s sss 5 33 0 21 3 soo 0 oo 0 s sas 0 oo 0 o oo 0 ` sss o oo 0 0 ro2 140: Tames Functoa aeration basry comand) snasaoossiz wes PS=o a wow ss ss |s ss 3 fE oomDresnse | 5 ss ss ss 3 dipaen | E 54 E ss | o4 5 --wis | 0 0 oo |i 2. a aPnE ---- E E E 0 1 0 ole oo a ar ww | 0 2 3 so oa a Pweaa | 0 0 eons | oo 01 o20o o1 a5 4a RAPoTOaRSn | 5 ss ss wi | 5s ssa sos ssss moses onde ||| s33s33ssssss||sssoss545s%s me | 3s ss |so4 3d SE-- a MIN 252/004312 Funan observationater: AcTABL6E cout groupmens vlus redo ww. FE: = " -w wet ww" : [rr=] >; 5 iow . ow we=a wks [ 5` | " Ww Wak Iett V--ins--Ts B Se u a redo oe 2: ;; we 2: ;. Mn2200632 TABLE 7 Functional observation battery: Rearing cou--ngrtousp mean values ;: :3 `; .s `: :; ., ;- ers wn - a , [3 vres 7 ;: `; ;: .;; wes wr 2r .: ;; :; ;: .. tet tansTe 143: MIN 252/004312 TABLE 8 Functional observation battery: Grip strength -- group mean values redo [~[ee] oSlo oawn oemm ea em ea RE. DSoo EoEe es ooee Cae wm Noiferenf sainsits005) ks I CC E s wowm Fa A m Tii wom 2So | ae m e EE oy rvzsznasiz Fanci ahervnion ey:TLAaBnLi9E ply -groupmssvie redo [Tee-- wwoaw "reoatrs Vwitiatm s was eTrno w0 eeom La Function proto D -- w Do oEwae +rots sud to Av 2s200es12 servationbae"yTA:BTLeEm1p0erature-gosp mess ves wet op wwee D -- mmsws Doom wm EE cw a sm s200612 Functional areasbates:Bdyweihts-rospmeanvalues redo = 2-- | wm a Cs l wme wep0Wilh0Tx wes [[TVEerFeae]) Wwoowws we a MIN 252/004312 TABLE 12 `Functional observation battery: Coulbourn activity monitorin-g group mean values Predose Tardugreimnogv1ehmoeunrtosbs(erv5a6t6i0on08) period Males Females ws Ex 46 686 589 7 m 465 No differencesofstatistical significance (p> 0.05) Week Group T 2 3 4 | Lardguermionvg1ehmoeunrtosbs(eirnvsaetcioonnds) period Males Females Ki E23 601 m 338 19 610 557 48: as - Tanz Hc gropmey as vies I Wills: +p<005 ; TABLETS (semao-clonotngucyd) ae a mes mee we we me om wm Da Ba Wha Boe meen men we ome 2 F Toomey ep ome op Willams test *p<005 : 8 [ tmattoTnABsLE-c1n3ionc) ee me wm we. www ww wie memn vm we a men Hen itnes: *p<003 TABLES (isema-tcolntoingneyd) en EE EE EE Sha ha Bn Tha ween men we me " FE " hem em ae ad am ad wa WDiulmleatm'ss"itsett: #+pp<<000055,, W*pp<<0000l1 Shileystest 1p<005 5 TABLE14 Bloodchem- grioupsmetanrvalyues | = anh Wd dE dm a a | Willams't p<00S Tae Bodchemin conn) mn mn Don Ea RR : Fopogop aa oso op oa BE Sain Williams'test: 1*p4<70.0050, **p<0.01 : : : : : TABLE 1 (Blood chemistry - continued) | Williams test: #4p<001 | TABL1E (Blood chem--icsonttiarneyd) 5 = . - [a DuWnilmleatm'ss' eestt #*4pp<<000051 5 | ----- (Bloodchemistry continued) TE ow on Di man wn man Si een Willan' est *p<005, +4p<001 TABL1E4 (Blood chem-icsonttinrueyd) ares x a mee mem ow ww Willams' est *p<005, +4p<001 taratare etiam 5 { TABL1E4 (Blood chemi--cosnttinruedy) = ES - 8 " Willams"est +p<005 | TaAnris -- urernscsst eit Organweights - groupmeanvalues(g) mam aR i Tams (Organ wei-gcohntitnuesd) eo SITTER meme i Tans Onno cntnsc lh Boh Yelk ok hob g TABLE16 (Organwei-gcohntitnuesd) B e--r------------ STO I, | | Wir iyTApBsLEd eens | Bored + ded ne To TEmmme TTT HERR | onSETA iii is m---- lll Toe respi pty -conisnt) Bh -- 3g mE 5 MIN 2520004312 APPENDI1X Clinical signs pre and post exposure individual daily observations [=TM Tau1mm6b-e2e0r |Nosbuomaliisdeiecied (Control) eww (Low2dose) 6-10 |Nosboormalitesdetected (ner3.mdose) 11-15 | No abnormalitiesdetected (Higahmdose)| 21 || NAogaboiommatantdiavtoetcsadleeistiedncgtwehden handled,Exposure | 34 || ANgoiaabtnoerdmaanlditvioecsaldiestiengcewdhenhandied,Exposure 11 5 | Noabaomlitesdetected (ConItrol) (Low2d8ose) (ne3rFdose) (High8dose) 31-35 |Noabnormalitiesdetected 36 |A1g5i.Vaotceadlswihnegnbwahnedinehda,nEdxlpeods,uErxepso3sur0e9s,31110,91,41a1anndd161t4o0202a0annddpprree--eesxppoossuurrees1135and 3378 39 ||AAggiittaatteeddasnnddvvooccaalliissiinnggwwhheennhhaannddlieedd,,EExxppoossuurrees |Hyperactive, Exposure 11 199, 11and 17. Hypecactive,Exposure 11 " Ee ba`iwheenhEanxdlped,oE5xr,p1ot5s1ur2seds 197,s1n1,1r5epanods17san1d5..pBroep-eerxcpoisvuer,eE1x5.poVnoeeeai1s1ing 26 |AHgyipeartaecdtivweh,enExbpaonsduireesd,Etxo p1o1.sWualr11ke.inVgoocnaltioessin(gabwnhoernmhalagnadilt)e,dE,xEpxopossuurrees14 11and 14 77 | EWalkxing posonuaroneesdsI(abnorgmial,Exposes 4,6,1210 14, 17,18,404 20. Hyperactive, 2298 ||AAgpiitaatteeddaannddvvooccaalliissiinngg wwhheennhhaannddlleedd,,ExEpxopossuurree11.6H,ypeWraacltkiivneg,oEnxtpoocssur(essb9oaonrmda1l0gait) 30 |AEgitaxted p1n2d,voo4c6al1in2s1i0n1gz5w,h1ee7n1h0adsnd1l0e.dH,ypExeprocsiurvees,E|xfpoo3s,es6a9n1dd11.10Walkingontoes (abnormal gai),Exposure 17 to 19. Hyperactive, Exposure 10 21 |gAagii)t,atEexdpaonsudrveosca2lts0i2n0gawnhdepnrbea-nedxipeod,sEu2xrp0eo.suBrypeesra1cttoive5,.ExWposuare lo9ntkoesi(sbnnorgmal 2 |gAagiit,teExdpaosnudrveosc2a1li0s8ingndwh1e0nh0an2d0alendd,pErxep-oesxuporseusre1t20o. ,HypeWractaivelo,Enxtkpooseusi(rseb9nnorgmal 23 |Ag13i.tWaateldkwihnegonhnatnodelse(ds,bEnxorpmaolgsaui)1r,teEox6sp,oVsoucraeliss2ing0w8ahnednh1a0ndtle2d0,aExnpdopsruer-eesxpo11s0ur7ea2n0.d 20 | AHydpgeraaicteidvew,henETxpaolseur,9eExpose 1105and 11. Vocaliingwhen bandied,Expos 1po01n1ea5n2d0,1,T3y.pWearlckiivneg,oEnxtpoeoss(sebo5ormalgai,Exposures 2to 8and 10to20andpre25 |EAx1gpiotsa02tue0rde1aswn1hd1e00n.5,h7a,npre-8dxposu,lre ed1,1aE2n0d.x1H6y.ppWeraa1locktiitvnseog,5ou,Enxp1tosour1eressaen(sdb1n9soa6rn.mdalg1V1ato),cExpaoswluhrieesns2hai0ndn8laegndd,. 169: APPENDIX Clinesigns (resposure) - dividualweekly observations so mmm || PR J | z Co oe ae 5 | APPENDIX? (Clea sgas (pre-exposure) contianed) raJ rer : | || Armia | [-- Ee -- : APPENDIX2 (Cleasigas (pre-exp-ocsonutirned)) Beemer SCT : | ssAgreeeasopmoxn-cte FR ------ re RESTR, aa ints : | | ves (Bodyw-eciongtihnuteds) | : | s MIN 252/004312 APPENDIX 4 Functional observational battery -- individual observations Pre-dose Group 1 Males Ca Tv 7 (TNOBTSHEERVATIOHAND NS RHaenmdolviinngg Vocadleigsrieneg. 22 22 22 22 22 N. N: N: N: N: INTHE ARENA Activity count ArBReooalurusisanlcgocuonutnt Urine present Gait 3 0 0 0 2 1 6 9 10 16 4 4 4 4 4 H 1 9 5 8 N N N N L - - - : - NIP! (TIONS Approach "Touch Startle Righting reflex `pTailohplisnceh s Pupil reflex TBeodmypweeirgahtture(8()C) 3 3 3 3 3 3 2 2 2 2 3 3 2 4 1 1 1 1 1 1 3: 33: 3:3 3: 3: B B B 376 378 380 B 38.1 B 383 248 249 238 253 238 GRIP STRENGTH (KG) T forelimb hindlimb 0.69 0.80 0388 0.80 0.71 0.80 0.85 0.52 0.69 0.61 TF Values represct the meaof nZ ls 180: APPENDIX 4 MIN 252004312 (Functional observational batter-y continued) Group 2 Males Pre-dose logge INTHE HAND Removing HVoacnadlliisnigng [TT] 2 2 2 2 22 22 22 N N N N N INTHE ARENA ArAcotuisviatly count Rearing count BUroilnues pcroeusnetnt GaNiItPULATIONS TAopupcrhoach RStiagrhttleing reflex Tail pinch 24 24 145 146 145 8 3 4 1 9 0 6 0 3 0 M - M - s - N TI M - 3 2 3 2 3 0 3 2 3 3 3 1 2 1 3 1 3 1 3 1 5 6 3 3 3 Puvpoiclarleifsleexs TBeomdpyewreaitguhrte((8)C) GRfIoPreSlTimRbENGTH (KG) hindlimb 2 2 1 B B B B . B 382 244 373 252 384 26 381 247 379 201 004578 00.7750 00.6518 007378 006897 T Values representthe meaonf2 trials 81: MIN 252/004312 APPENDIX 4 (Functional observational battery continued) Pre-dose Group 3 Males onssavaons TNTHE HAND RHeanmdolviinngg Vocadleigsrieneg INTHE ARENA AArcotuivsiatly count Rearing count UBroilnues pcroeusnetnt MANGaIiPtULATION: Approach STtoaurtclhe RTaiiglhtpiinngchreflex. m EE rr ara 2 2 2 2 2 2 2 2 2 3 N- N- N- N- N- 54 14s 14s n4 140 0 2 10 0 70 25 33 s - N Tl N- N- s- 3 2 3 0 23 32 32 3 1 3 1 31 21 21 3 3 3 3 3 Pupviolcarleiflseexs 2 B 2 B B B 2 B TBeomdpyewreaitguhrte((g)C) 32871 378 22 379 22 378 253 384 23 GRIF STRENGTH (KO) forelimb 082 0st 047 077 0.80 hindlimb 056 055 056 081 0.76 [CAT NDIVNaGlFueOsOrTepSrPesLenAtYtGhe mmeasn_of_2t|riDals l 12 96 B2 17 |] CE MIN 252/004312 APPENDIX 4 (Functional observational battery -- continued) Group 4 Mates Pre-dose opseTHERvHaATNIDONS Removing Handling. Vocalising TNActTHiE vAiyRcEouNnAt BAoRreloauurssianclgocuotunt Urine present GaNiItFULATIONS rT 2 2 2 2 2 2 3 2 2 3 N N N N N 16 wa s i 504 04` 048 45i 4i0 N N N N Ss TL Tl Tl - - Toon Approach RSiTtgaahirlttiilnengchreflex releies TePmuppielraretfulrexe (C) Bodyweight (g! RIP STREN( G)T 23 33 35 31 3> 33i 2 33:i 233i 33!i 23:i i 2 i sB2 wBs wBma sBmo wmB 228 21 259 244 240 Values represent te max TT als forelimb `hindlimb 0.62 0.54 0.68 0.71 0.84 0.79 0.65 058 0.66 0.79 iw APPENDIX 4 (Functional observational battery ~ continued) Group 1 Females `IONBTSHEERVATHAIND ONS RHVeaomncodavliisningngg INTHpE AyRENA A Activity count Rearing count GBUaroiiltnues pcroeusnetnt MANTAopIupPcrhUoaLcAhTIONS RSitagrhtlten.g efex `Taivlvaponisnlche. s TPeumppielrraetfulerxe (C) GRBIoPdyweSigThRtE(Ng)GTH) forelimb `hindlimb. Pre-dose IT = 5 N23: N2>: N23: 47 6i y13 2 2 6 0 Nv 0 N: 0 s: 23 23 32 3i 3i 31 3>2 6:3 5:: wBi wBs wBa 155 176 161 0.83 0.83 0.63 0.63 0.75 0.65 1 Valuesrepresent the meanof 2trials' MIN 252006312 73 N2>: x2>! J i 16 16 7 7 0 0 mNn ns 3 >3 31 3i 33: 33: aBme wBs 159 179 0.80 077 0.79 0.80 ue MIN 252/004312 APPENDIX 4 Prodose (Functional observational bat--ctone tinrueyd) `Group 2 Females. oIpNTsHeErHvaATNoDNs wml I A HaRnedmloviinngg 23 22 2> 32 32 TNVTopcHarElisiAnRgENA N2 N: N: N: N: Activ out 1 1 A u 16 `ARreoaurisnagl count 4> 45 4 4; 4 BGroilnuss pcroeusnetnt N0 N0 N0 N0 N0 VTAGoaA NiuTtcP.hULATIONS - - - - T1 33 23 33 2i 33 RSigthrtng reflex ii 3i 3i 31 3i hoy `Tail pinch 3: 3! 3! 3: 3: Pupviolcarleiisers 52 51 5- 52 5- Temperature (C) Bodyweight (g) 389 384 384 317 386 164 170 168 169 153 `GRIP STRENGTH (KG) T forelimb 0.82 0.75 073 0.64 0.63 (CAN`hDinIVdaNlilGmubFesOOrTeSPpLrAYetGhsemmeyantntSTZ|v1l0.1685110857 T0.i73a 52 0.63 9] 0.49 Jas: APPENDIX 4 (Functions! observational batter--y continued) Pre-dose Group 3 Females Tn OBSERVATIONS I RemoiEvHinAgND Handling Vocalising TNActiTHEvityAcRoEuNnAt Arousal Rearing count Bolus count Urine present MAGaNiItP.ULATIONS Approach S`aTonutceh re TRaiiglhtpiinngchreflex vocalises TPuepmiplerreaftluerxe (C) Bodyweight (g) GRIP STRENGTH (KG) forelimb hindlimb 2 2 2 3 3 2 N N N u4 14 140 2 6 3 0 0 0 N N N - TI = 3 3 2 33 02 2i 1 32 1 3: 1 25 1 2 3 mB1 mBo wBma 155 170 159 0.67 0.83 0.75 0.54 0.86 0.54 Vales repre themea of 76 MIN 252/004312 > To 2 2 2 2 N Y 24 ui 6 4 0 0 N N Tl TL 3 3 23 2z 1 1 3: 3: - 2 aBs 3Bs 171 175 0.86 0.64 0.83 0.68 it: MIN 252/004312 Group 4 Females APPENDI4X (Functional abservational bat--tconetirnueyd) Predose ---- IN THE HAND Removing Handling Vocalising ATcHtEiviAtyRcEoNunAt Arousal RBeoalruisnsgoucnotunt VGANUarTtiPnUeLprAeTseInOt NS pr `STtaorutcleh Righting reflex wvonralises `Tail pinch TePmuppielrraetfulerxe (C) Bodyweight(g) `GRIP STRENGTH (KG) forelimb hindlimb TT Valies reprtehesmeeannotF I 2 2 2 2 2 2 3 2 2 2 N N N N N inP 1 4 4 4 5 4 70 09 60 30 40 mN onN oNm s: TN 333 3 3 23 65 32 32 25 1 1 1 1 1 3i: 3? 3:i 3: 3>: sBs aBs aB1 seBa aBr 167 154 172 179 160 Tvl0.69 0.55 0.79 0.80 0.51 0.51 0.65 0.80 0.64 0.66 oe Group 1 Mates OBSERVATIONS N THE HAND Removing HSaanlidvlaitnigon Vocalising degree INTAHctEiviAtyRcEoNunAt ArRoeaursianlg count UBroilnues pcroeusnetnt Gait APPENDI4X (Functional observational battery - continued) Week 1 17 2 2 2 N2 N2 N2 N- N- N- u4 124 140 4 5 9 N2- M0- N4- MIN 252/004312 TM = 2 2 N2 N2 N. N 74 74 3 5 N4- M0- CH Group? Males onsexvarions INTHE HAND HRaenmdolviinngg Saldievgatrieoen Vocalising IANcTtHiEvityAcRoEuNnAt RAeraoruisngalcount UBroilnues pcroeusnetnt Gait APPENDI4X (Functional observational bat--tconetirnueyd) Week1 MIN 2520004312 [Fr 23 22 22 NNN N22 N22 N- N. N- N- 2 N 43 74 140 1s4 34 51 37 05 06 46 M s N N s : TL - TI - 189: Group 3 Males `IONBTSHEERHVAATNIDONS RHeamnodlviinngg Salivation TNVoTcHarlEiArsiRnEgNA arousal Activity count. Rearing count GaitUBroilnues pcroeusnetnt APPENDIX 4 (Functional observational battery - continued) `Week 1 TT = Ts 22 22 22 N N N N: N: N? i4 `12 i13 0 11 5 3 STs 0 N: 0 N: MIN 252/004312 5 22 23 N N N? N\ i9 7i 7 3 2 3 mNn N! (50: Group 4 Males onseyamons INTHE HAND Removing Handling Saldievagtrieone Vocalising THE ARENA AArcotuivsiatly count BRoelaurisncgocuonutnt GaUriitne present APPENDIX 4 (Functional observational batctonetinrueyd) Week 1 MIN 2520004312 [ror 2 2 2 2 2 3 2 2 2 3 piN N- N- N- N- N N N N N 12 4 7 4 9 4 n 4 9 4 06 40 60 30 20 ns N: TNl N: N- pon: Group 1 Females onseRvamoNs TRNemTHoEviHAnNgD HSaanldvlatiinogn degree Vocalising ri degree `AAcrtoiuvsiatly count BoRleaursicngoucnoutnt GaUriitne present APPENDIX 4 (Functional observational battery --continued) Week 1 MIN 252/004312 rorFF 223 2 2 N2 N2 N2 N2 N2 - - py k Y N N N N 2 - - - + u4 55 1s 24 145 70 40 09 110 60 N- N- mN nN TNn ER Group 2 Females OBSERVATIONS TN THE HAND HaRenmdoivningg en Salivation Vocalising TNTHdEegArReeENA AArcotuivsiatly count BoRUreliaunrseincpgroecusoetunntt Gait APPENDIX 4 (Functional observational battery -- continued) Week 1 I-- LT 36 3 3i MIN 252004312 -- 39 0) 2> 2> ;2 2> 25 N: Nj jNy N N N Y Y N N - 2 2 - s i16 4J i12 i14 :12 s40 N80 N20 N70 N50 - TL = Tl T io Group 3 Females `ROeBTSmHEoERvHViAAnTgNIDONS Handling `VSoalciavtpastiieonng TN THE ARENA Arcotvivsiaty count Rearing count UBroilnuespcroeusnetnt Gait APPENDIX 4 (Functional observational battery - continued) Week 1 MIN 252/004312 % == 0 222 2 2 2 2 2 2 3 NN: NN: NYi NN> Yv| J17 n4 i14 Y20 i9 N N N x N 6 7 7 9 5 0 0 0 0 0 - - Tl T : ios Group 4 Females oBseRvATIONS Removing Handling VSoaclaiddvleeawggtirrimeeoegn TNTHE ARENA Activity count Arousal Rearing count Bolus count Urine present Gait APPENDIX 4 (Functional observational batiery continued) Week MIN 252/004312 |T rE E 2 2 2 2 2 3 2 2 3 2 N N: NY1 jy pNN:y jNNy! NN?" 9 8 13 12 6 4 4 4 4 4 50 01 60 60 60 N N N N N T2HU1 "T2HU1 TI - - ios: Group 1 Males onsexyaTions INRHaeTnmHdoElviiHnnAggND Vocdaelgirseineg THE ARENA AArcotuivsiatly count BURroeilanureisnpcgroecusoneutnntt Gait APPENDIX 4 (Functional observational battery continued) Week MIN 252004312 [ 7 22 22 N- N- 142 14m 1N1.0 M4:0 FF me 22 22 22 N- N- Nt 49 124 144 N07 N42 TM41 - - : 196: ------------------ Group 2 Males oINnTsHeErvamoHANDns one HaVnodelsiingng degree IN AAcrTtoHiuEvsiaAtlyRcEoNunAt Rearing count UBroilnues pcroeusnetnt Gait APPENDIX 4 (Fanctona observational btier--y continued) Week 2 252008312 rrr] 222 2 2 N2 ow2 ~2 N2 N2 - - x - - n4 oon oin u4 1y0 9 5 5 3 9 v2 on5 N0 N0 35 - hv] = - - iors Group 3 Males OBSERVATIONS TN THE HAND RHaenmdolviinngg Vocalising IANcTtHiEvitAyRcEouNnAt RAeraoruisnaglcount Bolus count Urine present Gait APPENDIX 4 (Functional observational battery continued) Week TT = 5 N23 N23 N23 s 10 47 48 4 2 0 0 N N N Tl TI - MIN 252/004312 05 N23 N23 27 4 47 0 0 S N - - Jos: Group 4 Males JTXTH--E H--AND RHeamnodlviinngg Vocalising TANcTtoHiwvEsitAyRcEoNunAt Roaringcount Bolus count GaUriitne present APPENDIX 4 (Functional observational battery - continued) Week MIN 252/004312 rr:TF cl 23 23 25 32 23 N N N N N i4 1: 1i3 :5 1i0 5 10 i 5 0 0 0 0 0 nM omS mN m.s maNu Joo: Group 1 Females TN ns THE HAND Votes Handling INATcHtEidveigAtryeRceEouNnAt rover Rearing count St Bolus count Gait APPENDIX 4 (Functional observational battery -- continued) Week 2 MIN 252/004312 222 2 2 v2 v2 N2 N2 N2 2 2 - - - Wiw oion o:w uin z3 7 6 9 9 8 x0 N0 N0 N0 N0 T2 Tl TI TL T2 S100: Group 2 Females OBSERVATIONS INReTmHoEvHiAngND HVaoncdalliisnigng degree INATcHtiEviAtyRcEoNunAt Arousal R`Beoalruisncgocuonutnt UGariitne preseat APPENDIX 4 (Functional observational bat--tconetinrueyd) Week 3 37 2 2 2 Y2 Y2 3 N 2 1 - 8 2 14 4 3 4 5 64 0 0 0 N- TNl hvN) MIN 2520004312 3% 0 22 23 N- N- 5 7 24 43 0 0 Nu TNl S101: Group 3 Females OBSERVATIONS IN THE HAND Removing Handling Vocadleigrseisng TN THE ARENA Activitycount Arousal Rearing count Bolus count Urine present Gait APPENDIX 4 (Functional observational battery --continued) Week2 MIN 252/004312 2% 7 2% 5 30 2 2 2 2 2 2 2 2 2 2 N: N- N- N= N- 20 12 8 13 13 4 4 4 4 4 8 4 4 5 5 0 0 0 0 0 N N N N N T Tl hv] T T2HU 1102: Group 4 Females OBSERVATIONS IRN eTmHoEviHnAgND Vos Handling ActHidEveigeAryRecEeoNunAt Arousal Rearing count UGraBioilntues pcroeusnentt APPENDIX 4 (Functional observational battery ~ continued) Week 2 2 222 N ~ x 3 2 2 - - : un 10 s 4 4 4 5 9 2 o~0Y m N0 wN0 MIN 252004312 7= 2> x N 3 2 - : .1 4 4 5 5 N0 nN0 103: MIN 2521004312 APPENDIX 4 (Functional observational battery -- continued) Week 3 snyprons Group 1 Males INTHE HAND Removing [Fw 2 2 2 2 3 SHaalnidvlatiinogn degree Vodcealgirsieneg INTHE ARENA GAcrtoiovmitiyncgount ArReoaursianlg count : BUroilnues pcroeusnetnt Gait N2 Y2 N2 N2 N2 - 1 - N N N N : N : : - - N10 N9 Ni N3 Y12 4 9 4 4 4 4 4 4 4 9 0 0 0 0 0 N . bNl s: N: s> 104: ------------------------------------------ a Group 2Males OBSERiE VHAATNIDONS RHeamndolviinngg Salivation Vocalising INTHE ARENA GAcrtoiovmitiyncgount ArRoeuarsianlg count BUorliunsecporuensetnt Gait APPENDI4X (Functional observational battery continued) Week3 MIN 252/004312 A CC 5nm7 almm5 ee0r 22 22 22 22 23 N N N N N N N N N N 1N3 Ni Nn N9 1N6 47 47 43 34 34 0 0 0 0 0 s. s- N N msl 1105: Group 3 Males OBSERVATIONS TRNeTmHoEvHinAgND HaSanldvlaitrineogne Vocating TNGTrHoEomAiRngENA `oActitvity count RBeoalruisngcocouutnt Urine present Gait APPENDIX 4 (Functional observational bat--t conetir nueyd) Week3 TT T > >22 NN2 NN2 Nv2| N Ni iN ii 4 7 000 N S N TL TI - MIN 252006312 T > 2 NN02 NN2: 1N N9 i5 i3 0 S N Tl TIHUL 106: Group 4 Males OBSERVATIONS IN THE HAND Removing Handling Salivation Vocdaeligsrieneg TIN THE ARENA' Grooming. Activity count Arousal Rearing count BUorlinues pcroeusnetnt Gait APPENDI4X (Functional observational batery - continued) `Wee3k MIN 252006312 I T --7 TL -- 7 5 2 2 2 2 2 2 3 2 2 2 N N N N N - - - - . N N N N N N N N N N 12 6 14 13 9 4 4 4 4 4 4 1 3 6 4 s0 N0 N0 N0 N0 - hv] T2HUL TL - 107: Group 1 Females OBSERVATIONS IN THE HAND RHaenmdolviinngg Salidveagtrieone INVoTcdHaeElgiArseiRneEgNA GAArcrtooiouvsmiatiylncgount Rearing count UBroilnues pcroeusnetnt Gait APPENDIX 4 (Functional observational battery -- continued) Week3 MIN 252006312 T== 7 5 22 22 22 22 22 N N. N: NY N" Y2 Y' N- Y| N- Nn4 N14 25N4 piN4 2N4 7 5 11 6 8 N0 N0 N0 x0 N0 TL - Tl hv] kr] 0s: Group 2 Females OBSERVATIONS INRTeHmEorHnAgND SaHlavnadtliinogn degree TVRoIcaEElirAsRinEgNA AcGtriovoimtiyncgo.unt Arousal UBReotalerusisnpcgocruonuttnt Gait APPENDIX 4 (Functional observational ba--tcoentirnueyd) Week 3 MIN 252006312 5 Ty 3 w 222 2 2 N2 N3 v2 N2 v2 - - 1 . Y2 Y2 Y2 Y> Y2 N N N N N 15 14 10 16 16 4 5 4 4 4 N30 N07 nom N30 N06 om mw N80 om 1109: Group3 Females THE HAND OBSERVATIONS HRaenmodviinngg Salivation degree Vocalising INTHE ARENA GAraroovoumsyailnsg t Rearing count GBroilnusspcroeusnentt Gait. APPENDIX 4 MIN 252/004312 (Functional observational battery -- continued) `Wee3k w 5 ma l = ] 5 23 23 22 23 25 N- N- Y1 N: N. N N N N N 2Ny Nyi) N14 phN` NI4o io s 3 : 5 x0 x0 N0 N0 N0 T1 T hv] T2HU1L T2HUL os Group 4 Females OBSERVATIONS N THE HAND HRaenmdolviinngg Salivaativon or Vocalising IN THE ARENA Grooming. RAeAracotriuivsniatglyccoouunntt UrBionlues pcroeusnetnt Gait APPENDIX 4 (Functional observational bat--tconetirnueyd) Weeks MIN 2527004312 Ta 77 22 23 22 23 22 N: N. N. Y| N: N: jNy N NN NN N N N N N 15i7 i8 3i8 13i 5 1:4 i N0 N0 N0 N0 x0 mm om mn mo Ss APPENDIX4 (Functional observational battery -- continued) Week 4. Group 1 Males 6 T Ti `TROOBTSHEE RVHAATNIDONS RHeamnodvliinngg. Salivation 2 2 2 2 2 2 N N N Vocalising N INTHE ARENA sal Activity count i8 Rearing count 3 Bolus count 0 Urine present N Gait TL MANIPULATIONS Approach 3 "Touch 6 Teplohpiunchs 5: StRairgthlteing reflex 3 1 degree. - Pupil reflex B GRIF STRENGTHKGY "Temperature (C) 372 Bodyweight (8) 395 forelimb 114 `hindlimb 115 FT Values representth meanoT N N 9i J14 5 7 0 0 N N - TI 0 3 1 1 2 31 3 31 v: : 2 2 B B 379 38.1 362 398 132 151 093 136 MIN 252/004312 Ts w 2 2 2 2 N N N N 9i i17 5 8 0 0 N M - - 3 3 6 6 3 3 5:1: v31: - 2 B B 379 37.8 379 384 1.07 1.51 0.97 092 2 MIN 252/004312 APPENDIX 4 (Functions observational batery -- continued) `Week 4. Group 2 Mates oIpNsTeHREvaTIHANODNS Reming SHaalnvdaltiinogn Vocdaetgiroenes TNTHdEegArReEeNA |FT 5 7 5 5 2>2 2 2 N2 N2 N2 N2 ~3 N- N- N- N- N- - - = - - Activity count Arousal Rearing count Bolus count Urine present Gait TPoULnATIONS 3 3 3 3 2 "Touch Sere 3 3 3 2 3 Righting reflex `Tail pinch I | : : : : vocalises PaTpepmiprerroafmture (C) w5:s a53 w5:r m5:o a5:m Bodyweight RIP STRENGTH forelimb T Values repretsheemenatn of Tle hindlimb (KG) 9 14 13 17 13 4 4 4 4 4 6 S 7 7 6 4 0 0 0 0 s N N s N - TL - - Tl 5 6 3 3 3 1 1 i 1 1 6 3 3 3 3 - = - - - 366 3n 291 350 368 099 123 1.05 131 1.28 0.83 111 099 124 127 cs MIN 252/004312 APPENDIX 4 (Functional observational bat-tconetirnueyd) Week4 Group 3 Males [ Awmalmmber 7] OBSERVATIONS NRemToHEvHinAgND SHaalnidvlaitniogn Vocadleigsrieneg 2 2 2 2 2 2 2 2 2 2 N N N N N N- N- N+ N- N- IE ARENA Activity count ArRoeuarsianlg count UBroilnues cproeusnetnt MGANaIiPtULATIO! Approach STtoaurtclhe Righting reflex Tail pinch vdoecgarliesees Pupil reflex Temperature (C) Bodyweight (p) RfIoPrelimbSTRENGTH (KG) hindlimb 10 4 13 4 14 94 42 8 0 4 0 05 02 20 N - s - N- s- Nu 3 3 3 1 33 31 33 21 31 21 21 31 3 3 3 3 3 - - - Y 2 E - B 376 B 372 B 370 3B70 3B78 333 355 381 352 332 132 091 1.06 0388 116 091 1.40 1.30 123 117 Valuesrepresent the mean of 2tr fu APPENDI4X (Functional observational bat--tconetinrueyd) MIN 252/004312 Week 4. Group 4Males oTsRsEeRvHaAmNoDns |r HRaenmdolviinngg ar Salivation Sr Vocalising [N THE ARENA 22 23 23 23 2> N. Y| N N N: N: N! jNy jNy N prod Activity count Rearing count BUroilnues pcroeusnetnt MAGNaiItFULATIO ` 4 4 J i 12 11 18 17 9 3 5s 4 9 2 0 s 0 N 0 s 0 s 0 N HUI TIHUL - - Tl ATpopurcohach RSitgarhttlneg etex `Tailvopcnisanlchses PTuedpmeiplegreraetteuxre (C) RIBFodSyTmRaEghNtGT(3H ROT 33 03 33 35 33 2i 3i 3| 2i 3i 3: v32 v6: 3! 3! w5-s w51a w51s w5-s a5"m sow am ow 0 Values reprehsemeentoF Tal forelimb hindlimb 135 1.02 1.49 112 137 1.18 1.14 112 125 1.01 115: MIN 252004312 APPENDIX 4 (Functional observational batter--y continued) Week4 Group 1 Females oIpNsTeHrEvHaATNIODNs [r *= FF] RHeanmdolviinngg. 22 22 2 2 2 2 2 2 Salidveagtrieoen Vocadleigsrieng INTHE ARENA N- N- N- N; N2 Y| N: N: N: N: R`eAAacrrtoiiuvsniagtlyccoouunntt Boluscount 7s2 1453 1478 943 1h5i6 00 o MGAaNUriIitPneULprAeTseInOt ATpopurcohach TN TN mNn mN umN 32 31 63 33 33 Startle TRaiiglhtpiinngchreflex 3 1 2 1 41 31 31 3 6 3 6 3 vocalises : v TPeudpmipelegrrreaeftleuerxe (C) 42B mB3s 85-2 6B2 32B83 GRfIoBPordeySlwTieimRgbEhtNGT(gH) KC hindlimb 208 221 196 208 207 Ls 10s oss 116 12 095 1.10 0.54 122 1.06 lis represent the meas of Tl $116: MIN 252/004312 APPENDIX 4 (Functional observational batery - continued) Week 4 Group 2 Females onsrvaTIONs INTHE HAND [7 7 % Removing HSaalniddvleagiirnoegn Vocalising ActiTdEveigArteyR:cEoNunAt R`eAarroiunsaglcount BUroilnuespcroeusnetnt GaNiItPULATIONS T`oAuppcrhonch SRTtaaiirglthltpeiinngchreflex vomcaslises degree Pupil reflex `Temperature (C) Bodyweight (g) `GRIP STREM KOT 2 2 2 2 2 N2- 21 NY3| NN2: Nv21 2 i - - - 46 55 64 24 u4 20 60 02 20 05 nN __mmNu omN N nN 3` 30 33 03 33 431 231 3`1 3s1 351 Y: : : : v: 222 2 2 B B B B B 388 382 382 386 386 188 220 204 209 202 forelimb L115 1.06 1.08 L12 123 hindlimb 093 1.08 1.08 124 0.79 T Values represent Ge meas STZ 0H curs Group3 Females OTBXSTEHREVHAATNIDONS RHeanmdolviingn.g Salivation APPENDIX 4 (Functional observational battery continued) Weekd Ts 22 22 2 2 N N N MIN 252/004312 = El 2 2 2 2 N N Vocalising NATctEiviAtyRcEoNuAt ArRBeooauliusnsagclocuonutnt Urine present VAGaNiItP.ULATIONS TAopupcrohach StRairgthlteing reflex "pTailvhoprinaclhs.es dene Pupil reflex TBeomdpyewreaitguhrte (1C) `GRIP STRENGTH (KG) hfionredlliimmbb N N N N N 1s ' s is 5 480 i40 401 45' 405 N N N N N T TL T2HUL T T2HU1 33 33 33 30 3` 31 31 31 31 31 v3: 3.: v>2 3: v6: |i> 1 i B B 38Ww9 3a738 B B 3m80 am37.7 B 318939 00.95 1i2m1 uL1s0 iLInS om1.08 T alues represent the mean of 2 trials 18: MIN 252/004312 APPENDIX 4 (Functional observational bat--t conetir nueyd) Group 4 Females OBSERVATIONS `Wee4k. T RHeanmdolviinngg 32 >2 Salivation Vocdadehegugrireneege TNATcHEhycARoEuNmA pRreaiiogoount N N N:- N-: i5 .` ii CBorlouns cproeusnetnt N0 N0 MANGaIiPt en ON: Touch T3HUL 33 "T2HUL 33 SRTtiaagrfthlpteinngcehiex deognrely TPeumppierraetuxre (C) GRIBFodywSeTiRghEtNGTHTRO)T 23i 23i v3 v2 m52 m5a wm forelimb 1.03 0.99 (CTANhiDnIdVlNaiGlmubFesOOrTeSpPLrAetYGesmmeanntof|Tal7s04.83 650.81 oy 7 3 23 23 23 N N N N-: N-: N-: 0i 1 1`2 35 ~0 N0 N0 Tl Tl Tl >3 :3 03 23i 33i 53i v| : v3 wr w5a w5e maw 20 119 123 097 741.20 a1.609 --&11.06 | ne: MIN 252/004312 APPENDIX Total time spent in locomotor activity -- individual values (seconds) [Asimal 7 Prefese|Weekd | (Group 1M (Control) 76 7aT Ea I 24 426 18 09 1073 19 65 88 20 705 985 (Group 2M (Low dose) W 95 7 492 78 8 sas sas 9 550 1072 10 180 163 |Group 3M (inter. dose) 7 878 655 269 148 538 250 674 31s (Group 4M (High dose) 3 69 426 864 1198 649 987 517 m1 381 S120: APPENDIX (Total time spent in locomotoractivity -- continued) MN 252006512 37 Group IF (Control) Tr wT 532 7oonn86 25555s02 5 0 |Group 2bF5(Low dose) aosr 501757 p33s8 6TM51003 uamn1 Group 73F (Inter. dose) 7iw 5u5e7 22 o1r00s0 0717 5 0 1 (Group 422F (High dose) 5200 716 5pl a33s0 ow2 3 o 506 ns MIN 252/004312 APPENDIX 6 `Additional comments from functional observational battery - individual observations Pre-dose Group 1 Sex M No additional comments sa: MIN 252/004312 APPENDIX 6 (Additional comments from functional observational battery -- continued) Pre-dose Group 2 Sex M 70 SInligthhte barreonwan: naSscarlatcshtianigning 8 6) Touch response: Rears p13 MIN 252/004312 APPENDI6X (Additional comments from functional observational bat--ctonetinrueyd) Pre-dose Group 3 Sex u an) Slight brown nasal staining 12 az) Touch response: Rears uae) Slight brown nasal staining 15 as) During manipulations: Soft pale faeces D124 MIN 252/004312 APPENDI6X (Additional comments from functional observational bat~tconetirnueyd) Predose Group 4 Sex au Slight brown nasal staining 565) Temperature: Slight body tremors 12s: MIN 252/004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Pre-dose Group 1 Sex F n En) Slight brown nasal staining D126: MIN 2520004312 APPENDI6X (Additional comments from functional observational bat-tconetirnueyd) Pre-dose Group 2 Sex F 36 (36) LDuarnidnigngmafnoioptuslpaltayi:onsA:wkwVaorcadlitsoinhganmdloederately 3 61) Slight brown nasal staining 10 0) In the arena: Scratching 127: MIN 252/004312 APPENDI6X (Additional comments from functional observational bat~tconetinrueyd) Pre-dose Group 3 Sex F 21 (21) Touch response: Rears 30 (0) Slight brown nasal staining D128: MIN2520004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Pre-dose Group 4 Sex F 2 (2) Slight brown nasal staining 2 (22) In the arena: Scratching 1129: MIN 252004312 APPENDIX6 (Additional comments from functional observational battery --continued) Week1 Group 1 Sex M 16 a6) In the arena: Outside digit right hindlimb curled under 1 as) Slight brown nasal staining 130: MIN2521004312 APPENDI6X (Additional comments from functional observational bat-tconetirnueyd) Week1 Group 2 Sex M 6 (6) Slight brown nasal staining LINCS) Slight brown nasal staining 10 (10) SSlciagbhtlebftrowfnorneapaswal staining [REI MIN 252/004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week1 Group 3 Sex 12 a2) Slight brown nasal staining 15 as) Slight brown nasal staining s132 MIN 252/004312 APPENDI6X (Additional comments from functional observational bat~tconetirnueyd) Week 1 Grow 4 Sex M "uw SSlliigghhtt bhraoiwrnlonsassalfosrtealiimnbisng D133: MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week1 Group 1 Sex No additional comments D134 MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetinrueyd) Week1 Group 2 Sex F 7 a7) Slight brown nasal staining 40 (0) SIlnigthhte barreonwan: stCaliinmibnegd hoenadfraonntd eeadrgse of arena Ls: MIN 2520004312 APPENDI6X (Additional comments from functional observational battery -- continued) Week1 Group 3 Sax F 2 (2) Slight hair loss face 29 (29) SIlnigthhte haarienra:losCslinmebcekd on front edge of arena D136: MIN 2520004312 APPENDI6X (Additional comments from functional observational bat--tconetinrueyd) Week1 Group 4 Sex F 2 (1) Slight brown nasal staining 22 (22) In the arena: Scratching 23 (23) ISnligthhte barreonwan: naCslailmbesdtaionninfgront edge of arena 25 (25) LIonwetrheteareetnha:palCelimbed on front edge of arena [REN MIN 2520004312 APPENDIX 6 (Additional comments from functional observational bat--tconetirnueyd) Week2 Group 1 Sex M 18 (8) Slight brown nasal staining D138: APPENDI6X MIN 252004312 (Additional comments from functional observational bat~tconetirnueyd) Week2 Group 2 Sex M CINCI Slight brown nasal staining 9 5) Slight brown nasal staining 10 (10) In the arena: Climbed on front edge of arena D139: MIN 2521004312 APPENDIX 6 (Additional comments from functional observational battery --continued) Week2 Group 3 Sex M 1 ai) Slight brown nasal staining 12 uz) In the arena: Climbed on front edge of arena 1 ae) Slight brown nasal staining 1140 MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetinrueyd) Week2 Group Sex M 2 2) Slight brown nasal staining 3 G6) Slight brown staining head and neck 4a SInligthhte barroenwan: naCslailmbsetdaionninfgronatndedsgleighotf barroewnna staining head and neck 5s (5) Slight brown nasal staining and slight brown staining head and neck ca: MIN 2520004312 APPENDIX 6 (Additional comments from functional observational batter--y continued) Week2 Group 1 Sex F n @) In the arena: Climbed on front edge of arena D142: MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week2 Group 2 Sex F 36 (3s) In the arena: Climbed on front edge of arena nn a1) In the arena: Climbed on front edge of arena 38 (38) In the arena: Climbed on front edge of arena 39 (39) TIinpthoef atraeinla:misCsliinmgbed and walked on front edge of arena 0 (0) In the arena: Climbed on front edge of arena 143 MIN252004312 APPENDI6X (Additions! comments from functional observational battery~ continued) Week2 Group 3 Sex 21 (@1) SIlnigthhte haareinra:losCslihmebaedd on front edge of arena 28 (28) In the arena: Climbed on front edge of arena 29 (29) MIondtehreatearehnaai:r lSocsrsatchheiandganadndnecclkimbed on front edge of arena 30 (30) Scabs right ear and slight hair loss neck : 144 APPENDI6X MIN 252/004312 (Additional comments fromfunctional observational bat-tconetirnueyd) Week2 Group 4 Sex F 2 (1) SInligthhte barreonwan: naSscaraltcshtianign,ingeyes front edge of arena slight to closed on occasions and climbed on 2 (22) In the arena: Climbed on front edge of arena 23 (23) In the arena: Climbed and walked on front edge of arena 26 (24) SInligthhte alraecnka:ofClgirmoboemdingonrufmrpont edge of arena 25 (25) BIynestheslairgehnta:toCl1imcbleodsedononfroonctcaesdigoensofandarlenoawearndtejetuhmpepadleout of arena 14s: MIN 252/004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week3 Group 1 Sex M 1 oan Slight lack of grooming rump 20 (20) Slight lack of grooming rump 146 : MIN 252/004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week3 Group 2 Sex M 6 (6) In the arena: Climbed on front edge of arena 70 In the arena: Climbed on front edge of arena 8 (8) MSloidgehrtatebrobwrnownstnaaisnailngshteaaidn,inngeck and muzzle ss) SSlliigghhtt bbrroowwnn nsatsaailninsgtahienaidngand neck 10 (0) SSlLiigghhtt blraocwknofnasgarloomsitnaginirnugmp In the arena: Climbed on front edge of arena P47: MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week3 Group 3 Sex M 12 az) In the arena: Climbed on front edge of arena 13 a3) In the arena: Climbed on front edge of arena 1 ae) SInligthhte barreonwan: naCslailmbsetdaionninfgront edge of arena 15 as) In the arena: Climbed on front edge of arena 14: MIN 2520004312 APPENDI6X (Additional comments from functional observational bat-ctonetinrueyd) Week3 Group 4 Sex M 1a) SInligthhte abrreonwan: sHtuanicnhiendg noneckoccasions 2 2) SInligthhte barreonwan: sEtyaeisninslgignhetckly closed on occasions 3a) Slight brown staining head and neck iw MSloidgehrtatberbowrnownstnaaisnailngstnaecikning Slight lack of grooming rump 5 5) SSlliigghhtt bbrroowwnn nsatsaailninsgtaihneiandgand neck D149: eo re -------- econ MIN252/004312 APPENDIX 6 (Additional comments from functional observational bat-tconetirnueyd) Week3 Group 1 Sex an @) In the arena: Climbed on front edge of arena 3 (33) ISnligthhte abrreonwan: naCslailmbsetdaionninfgront edge of arena 34 (34) In the arena: Clinbed on front edge of arena 150: MIN 2520004312 APPENDIX 6 (Additional comments from functional observational bat--tconetirnueyd) Week3 Grow 2 Sex F 36 (36) MSLoidgehrtatelabcrkowonf gsrtoaionmiinngg hreuamdp, neck and ears In the arena: Climbed and walked on front edge of arena 37 37) SSlliigghhtt bhariorwnlosstsainneicnkg head and neck In the arena: Climbed and walked on front edge of arena 38 (38) SIlnigthhte abrreonwan: nCalsailmbesdtaainndinwgalked on front edge of arena 39 (39) TSilpighotf btraiolwnmisstsaiinnging head and neck In the arena: Climbed on front edge of arena 40 (0) In the arena: Climbed on front edge of arena D151: MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week3 Growp 3 Sex 26 (26) In the arena: Climbed on front edge of arena 2 @) In the arena: Climbed on front edge of arena 2 (2) SSlliigghhtt hbariorwnlsostsainneicnkg ears In the arena: Climbed and walked on front edge of arena 2 29) In the arena: Climbed on front edge of arena 0 (30) SSlliigghhtt bhraoiwrnlonsassalandstbarioniwnngstaining neck SInnaitlhesacraebnar:ighCtlimebaerd on front edge of arena ss: . MIN 252004312 APPENDIX 6 (Additional comments from functional observational battery continued) Week3 Group 4 Sex aa Slight lack of grooming rump 2 G2) SSlliigghhtt blraocwknofnagsarloomsitnaginirnugmp In the arena: Climbed on front edge of arena 3 @) SSlliigghhtt hbariorwnlosstsaidnoirnsgalhead In the arena: Climbed and walked on front edge of arena 2 Gi) SSmlailglhtnilcakckriofghtgroeoamring rump In the arena: Climbed on front edge of arena 3 Gs) LSolviegrhtteleatchk opfalgerooming rump SEIylneisgthhteslabirrgeohnwtanl:ystCcallioinsmeibdnegdoohnneoacdfcraosntionesdge of arena 1183: MIN 252004312 APPENDIX (Additional comments from functional observational bat--tconetirnueyd) Week4 Group 1 Sax M 16 (16) SIlnigthhte barreonwan: naCslailmbesdtaoinninfgront edge of arena oar) Approach response: Bit probe 18 as) Slight brown staining head 19 as) In the arena: Climbed on front edge of arena D154: MIN 2521004312 APPENDI6X (Additional comments from functional observational bat-tconetirnueyd) Week4 Group 2 Sex M 6 (6) ISnligthhte barroenwan: nCalsailmbesdtaionninfgront edge of arena 700) ISnligthhte barroenwan:naCslailmbesdtaionninfgront edge of arena 8 (8) SSlliigghhtt bbrroowwnn nsatsaailninsgtahienaidngand neck In the arena: Climbed on front edge of arena 3 9) Slight brown staining head and neck Lass: MIN 2521004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week4 Group 3 Sex M u oan) SIlnigthhte barreonwan: naCslailmbesdtaionninfgront edge of arena 12 az) SInligthhte barreonwan: sCtlaiimnibnegd ohneadfraonntd enedcgke of arena 13 a3) In the arena: Climbed on front edge of arena uo) In the arena: Climbed on front edge of arena 15 as) MSoldigehrtatberobwrnownnassatlainsitnaginihnegad and neck In the arena: Climbed and sat on front edge of arena 1156 : MIN 252004312 APPENDI6X (Additional comments from functional observational bat-tconetirnueyd) Week4 Group 4 Sex M 1a) Slight brown staining neck SIlnigthhte barreonwan:nCaslailmbesdtaionninfgront edge of arena 2 2) MIondetrheatearebnrao:wnScsrtaaticnihnignghead and neck Touch response: Rears 3 a) Approach response: Bit probe aw In the arena: Climbed on front edge of arena and scratching 5 (5) Moderate brown staining head and neck Slight lack of grooming rump 1157 MIN 2521004312 APPENDIX 6 (Additional comments from functional observational bat--ctonetirnueyd) Week4 Group 1 Sex F ae) ISnligthhte barreonwan: sCtlaiimnbiendg eoanrsfront edge of arena During manipulations: Moderatsly vocalising throughout 32 (32) Slight brown staining ears and head 33 (33) In the arena: Climbed on front edge of arena EER SSlliigghhtt blraocwknofstgarionoimnigngearrsumapnd head In the arena: Climbed on front edge of arena 1158 MIN 2521004312 APPENDI6X (Additional comments from functional observational battery --continued) Week4 Group 2 Sex 36 (36) MIondetrheatearebnrao:wnClsitmabiendingandhewaadlkaendd onneckfront edge of arena Dhuarnidnlge manipulations: Loud/aggressively vocalising throughout and awkward to nar) MIondtehreataerebnrao:wnClsitmabiendinagnhdewadalaknedd noneckfront edge of arena ATopupcrhoarcehsproensspeo:nseR:earAsttempted to bite probe 38 (38) SInligthhte barreonwan: stCaliinmibnegd hanedadwaalnkdendeocnk front edge of arena Temperature: Moderately vocalising 39 (39) STliipghotf ltaaiclk omifsgsrionogming rump SIlnigthhte barreonwan: stCaliinmibnegd haenaddwaalnkdednecokn front edge of arena Touch response: Rears 40 (0) ISnligthhte alraecnka:ofClgirmoboemdingandruwmaplked on front edge of arena During manipulations: Moderately vocalising throughout 1159 : MIN252/004312 APPENDI6X (Additional comments from functional observational bat--tconetinrueyd) Week4 Group 3 Sex 26 (26) In the arena: Climbed on front edge of arena 21 (21) Slight hair loss right hindlimb SInligthhte barreonwan: stCaliinmibnegd ahnedadwaalndkednecokn front edge of arena 28 (28) SSlliigghhtt bhraoirwnlnoasssalheasdtaining IPnupitlhereafrleenxa:: CRilgihmtbedeyeanpdupwiallkdeidlaotnedfrounntderedgleighotf aatrenlast attempt 29 (29) TInoucthhereasrepnoan:se:CliRemabresd on front edge of arena 30 (30) SSlliigghhtt blraocwknofstgarionoimnigngheardump Small nick out of Right ear In the arena: Climbed on front edge of arena 1160: MIN 252004312 APPENDI6X (Additional comments from functional observational bat--tconetirnueyd) Week4 Group 4 Sex F 2 (21) In the arena: Eyes slight to 4 closed on occasions and climbed on front edge of arena : 2 (2) SSlliigghhtt bbrroowwnn snatsaailninsgtaihneiandg In the arena: Climbed and walked on front edge of arena 23 (23) Slight brown staining head and neck SIlnigthhte alraecnka:ofCglriomobmeidngandruwmaplked on front edge of arena Pupil reflex: Left pupil dilated under light on first attempt, on 2nd attempt constricted as normal 20 (24) MSomdaelrlatneicklaocuktooffgrroioghmtingearrump 25 (25) MLoodweerratteeetlhackpaloef grooming rump AIpnprtohaecharerneas:ponCslei:mbBeidtonProfbreont edge of arena Touch response: Rears 16: APPENDIXT Haematology -ndivdusl values Tome mm am om om we me wr om oe wo wa mess vm we a meen Son 5 - 5 sews 8 Bom um Pe i > am 5 SOT aE .E3 ; TT Jr. (Haemat-coonltionugedy) EEE ELL LT 5 ~ 1 BEE LIUBZLR ( CID Clottedsample. | | ) ) 5 g APPENDIX? Enca-ncontoneot) www our ONL OED z > 5 ::::: am Clotetsmpe 5 Armmoney emmy -contane) wwe men vm ve a en Ba & pot om OME OD Momo oa | - z APPENDIXT (Haematology continued) J aShmaoEsa oBana The wen mewn me me Eom W mma ow - Booamoomoe nou nom 2 | all | ;i APPENDIXS Bloodchem- idivsidutalvraluyes Ten on Sih mn wen mn ma man | g = 3 "83 Pod om mam am : | meteors go. FEL oEEEEG Pom oH BH om OF oF 3 ; : | Svtarint : LE EE : | wy | Pomom HOE OH 5 : APPENDIXS (Bloodchemistry continued) wos wn aa wr Mr MM mw cw Ge Gm om A 3 - 3 moon 8 Poms i moe i | arrmonss [I mneeex San E emDoEmeN om OaB a EE Blood che-mcionsane) u a 5 | tewors t | TE EE OEE OB PE . i : APPENDIXS Absoluteorgan weghts dividual values | : : APPENDIX 10 Individual pathological findings MIN 252/004312 "tT0hea irnoiutitailneexpaemeirnarteivoinewwabsy uandseerctoankdenpabthyoltohgeisstt.udyTphaethdoiloagginsots,esthreerpeosrutletds ohferwehircehprewseernet tthheencsounbsjeencstuesd opinions of both pathologists. Study pathologist: Peer review: Sheila E. Begg, MA., Vet MB, MRCV.S, FR.CPath., CDoenpsaurlttmaennttoPaftPhaoltohgoilsot,gy. David J. Lewis, Ph.D., FRC Path., CDoenpsaurlttmaennttPoafthPoaltohgoilsto,gy. S17: Framers 5 | oi pAPPENi DfIXo10s g nin g tt riertescn Arrmorxio (Uniden ptholgn fa-consan) 8 [Sirmmwrremn i | : tetpops cin i Btw i Td Tm :i | Amon [TP -- i Ee = deed i rs | A aaa oy i | 3 Ed r-- (ndividua pathfoindlingo-cgontiinuced) ded pt re 3 i tp J--tst no : | | Arroin0 adi publ tdi cots g Bed = ded a B sri ect ce 5 { APPENDIX 10 (ndividul pathofaldinogsg~iconctianueld) } EEE IEa Tm, | rbgatnts cc i Bend de bedeed es : APPENDIX 10 (individu pathological fading -continued) arrose Gndid fogs tan | :: | ArvmorXio [rE -- : [ 3 pr-- [T--R -- T_T hopes re orem eoot a ew... | | Armorxio [Sr -- Ee rem | mem AprENDIX 0 ndidun piston nin conned) ER ES Eeme gomsnsJse--ngenege; 2 g | ssmanoosseses- EEE EELem 2 EB | ArpmOIXI0 J -- Emme titiismeerer---------- TE | sone ess gong | | 5 CasArrtENDIXaI0 no YE ETwe BedR SEER | | teppei APPENDIX 10 Ia Arrmoie (individualpathofilndionggs - icocntianueld) [pte 2 tisric cei i Been ob ded Tm 2 mane (Individualpathofilndionggs-iconctianueld) PB de ded seeemrr : | 3 ArpmNOIXI0 dividualpathBoilnsocgontiinnccd) ere em APPENDIX 10 (Individual paihologial fdiogs continued) 8 | | ossaoema | 5 ombusdoimsin 2 [-- J---- smsmr---------- : i | | Se | : Arresoixio lndbitat publitndins cota) 5 || 2 | | eos nopgsns nts : metreamssn MIN 2520004312 ADMINISTRATION OF T-7499 BY INHALATION TO RATS Author Stuart Cracknell 1219: CONTENTS MIN 2520004312 Page TEST SUBSTANCEANDADMINISTRATION... 21 EXPOIRETYEIEM ccsmmsmtessssmmATO--riiEE ZROCTDNRE conned TARGET CONCENTRATIONS cera 225 EXPOSURE CHAMBER CONDITIONS vss 225 CALCULATIONS commons26 CVHAAPMOBUERRCT ONCE ENTM RATP IONAE N.D.RR .EcLAA cTrIVcTEiHnUUMsIsDRIsTsYE sssrvsscrnorsroseooronrones 22218 DISCUSSION .sccnammmmmmisssssmmmmmmmmnsmsmsmmmn 20 FIGURES J B. Schematic ofthe XPOSUIE SYSIE USCA0 EXPOSETAS ...rrrorv-- rovrn-- rrrov-- rororn 231 C. Schematic ofthe 3u10Mated SAMPIG SYSIEM......rororrnrnrorosnr orn 232 TABLES AB.. OCphearmabteinrgccoonncdeinttiroantsioonfstohfeTE-X7P49O9S--EdSaYilSyTMEME..V.AIIES.....rroverrornrnrorneoror 223334 F D. Chamber tempL erature and relativeJRUmidit--y CXPOSUTC TEAR VAIIES....v.vrn--rorr--ror--nroror 236 APPENDICES AB.. CMehtahmobdesrocfonscaemnptlreatcioolnlseoctfiTon-7a4n9d9an~aliynsdiisvifdouralT-S7A49I9Cv Vales .s .....s ...r.-c..-ror22-341.6 1220: `TEST SUBSTANCE AND ADMINISTRATION MIN 252/004312 TEST SUBSTANCE `sTuhlefotneyslt fsluubosrtiadnece(,PBwShFi)chanisd r2ef--er4r%edpteorftlhuroroousguhlofuotlatnhei.s IrtewpaorstsausppTl-i7e4d9a9s,ailsi9qu6id--in9a8s%maplelrfclyuloirndoebrut(anneet contents 35 kg/cylinder) and was received at these laboratories on 26 April 2000. Information from the `Sponsor indicated that T-7499 was suficietly stable for use in this study. The T-7499 was administered to theratsas a vapourdilutedwithair. ADMINISTRATION ``TThhee cT-h7a4m9b9ervaaptomuorspwhaesreasdmwienriestperroeddutcoedthebyramtsetbeyriwnhgotlhee-bloiqduyidextpesotsusruebsitnanccheamibnteorsstdaeisalcersisbesdtebeellcoowi.l `Vgaepnoeurartigoennesryasttoermswtahsrofuugrhthewrhidiclhutaedswtirtehamaiorftodrgiievde tahire fwiansal pcahsasemdb.erTcohneceanttmroastipohnesreoftpersotduaecreodsobl.y the `tTyhpee igna-slimneetaeirrfdluorwitnog tthhee vparpeoliumrigneanreyrpathiaosneoafpptahreatsutsudayn.dDtuhreidnigltuehnetsatiurdsyutphpley awierfrleowvetroiftiheed autsmionsgpahedrrey `generation system was monitored throughout cachofthe exposures using calibrated in-line tapered tube gas flowmeters. The settingsofthe test substance metering system required to obtain the target chamber concentrations wcherroematdoegtrearpmhiinced(GdCu)rianngalytshiesofpartemliomsipnhaerrye gseanmeprlaetsi.onMitnrioarlsadwjiutshtomuetntsawniemraelsm,adbeatsoedtheotnesttmhaetergiaasl delivery rates in order to maintain chamber concentration close o target. Animals assigned to the control group received an exposure to compressed air only, from the same source as used for the generationofthe test atmospheres. `The duration of administration was a single 6-hour exposure on consecutive daysofeach week for 4 weeks. The combined usageofT-7499 was determined, for each dayoftreatment, for th three test groups. 11: EXPOSURE SYSTEM MIN 2520004312 tEaapcehredexnpeoesdulree fslyuisdtefmlocwomcopnrtirsoeldvaalvwe,hodliel-uebnotdyairinchoanltartoilonvaelxvpeso,suarecocmhparmebsesre,d aairvaspuoppulrygaennedractoonrt,rola vsaylsvteesm ttohaetawcehregenceormatmoornantod ailnl-lgirnoeupasirfwleorwe mtohneitteosrtinmgatefrlioawlmectyelrisn.der,Coamlpooandencetlsl aonfdthites gaesnseorcaitaitoend display for determining the `material supply pipework. test material usage (weight loss from the cylinder) and the stainless steel test Schematic FigureAs adnidagB.ramTsheofcothmepovnaepnoturpagretnseorfatthioesnyssytsetmesmarancddaesncerixbpeodsuirnefucrhtahemrbdeertasiylsbteelmowa:re presented in Vapour generation `cTohme mvoapnourersegrevnoeirratoifonliqsuyisdtTe-m74fo9r9,Grwohuipcsh 2, 3 was maanidnt4ai(nLeodw,at Iantperremsesduiraeteofa4n0d pHsiiguhnddeorseN)itcroomgepnr.iseTdhea pressurised Valves) to vessel supplied each of the test the test substance to chambers. The liquid separate delivery lmienteetrointghevmaeltveersin(gNuvparlovesS Series Needle was fitted with (tsotgagilnelevsaslsvteesel,to1a5ljluomw piosroleatsiizoen")o.f tFhleuitdesptasssuibnsgtatnhcreousguhpptlhye amnedtewriansgavlaslovefsittweadswidtelhivaepraerdtidciurleacttelyfiilntteor ttheempaeirratiunlreeot ftoapsptraoinxliemssatsetleeyl6v0apCo.risTahteiotnestcosiulbsstthaantcewevrapeouirmamnedrseaidr miinxtaurweatwearsbsautbhsemqauiennttaliynecdarartiead tcohasmebceornsd,artyhrdoiluugthiPoonlvyeTsesterlasFclounosrtorEucttheydleonfegl(aPsTsFaEn)d tsutbaiinnlgeswsisttheela,naedxjtaceemnatltdoitahmeetienrhaolafti6onmmex.posTuhree `pTr-o7d4u9c9evtahpeoeuxrpoinsuarier maitxmtousprheefrreso.m the generators was further diluted with air in the secondary vessels to `The air residual psaurptpilciueldatteoantdhewavsapdoruiredg(edneerwatpoorisnta~n2dCs)e.condary dilution vessels was filtered to remove any `tTohaed tceesltlmaatnedritahlerweesiergvhotirof(tthheeorreisgeirnvaolircyalninddecronstuepnptlsiewderbeydtihseplSapyoendsocro)ntwianusoumsoluy.ntTehdeocnonatnenetlsecotfrotnhiec cylinder were maintained at constant pressure (40 psi) under dried nitrogen. Lstieqeul,id7wuams tproarnespsoizret)e"dwferroemitnhceorbpaosreoatfetdhientroeesearcvhoilriqtuoidthdeelmievteerryinlignveablveetsw.eePnartthiecudliastterifbiulttieorsn (msatnaiinfloelsds maentdetrheedvablyvetshteovparlovteescwttahsemdelfivreormedbltoocktahgeevabpyorainsyatpiaornticcouillastetthhraotumgihgPhtTFhEavteubbeienng porfesaepnptr.oxTihmeatleilqyui2d mm intemal diameter Aline drawingofthe generation system is given in Figure A. ** NHuupnrtloeiCgoh, IWnidlulsotruigahlbyC,onOthriools44L0i9d4,,LUoSadA Cell Division, Portman Moor Industrial Estate, East Moors, Cardiff, South Glamorgan, CF22 2HB p22: MIN 252004312 iFonroatlhlegrbaosuepsofetxhpeosseedctoondTa-r7y49d9i,luttihoenvvaepsosuerl/aaincdmiwxatsurmeixperdodwuictehdainfutthehevraspuopuprlgyenoefrcalteoarns wanads dprayssaeidt spuafsfsiecdientthrtoouegnhsufrleexiablteotadluccthianmgbteoraaitrafnlgoewntoifalapipnlreotximmoautnetleyd1a5t0 tUhmeina.pexThoef etshet aaptpmroospprhieartee wexapsostuherne. chamber "The control group received clean sr only at a ateofapproximately 150 Vin. Inkalation chamber "fTihteedexwpiotshurae cphyarmabmiedraslwebraeseofanstdaitnolpe.ss sTteheel ainndtegrlnaalssvcoolnustmreuctoifoncaacnhd ccohnasmibsteerdowfasa acupbporiodxailmabtoedlyy b0.e7l5owm't.hisAwtatsheaappeerxfoorfattehde cuapnpiestrerp,yrwahmiicdhalenfsiugurreedwaeqsutahleditsatnrgiebnuttiiaolnloyftmhouentteesdt aaitrmoduscpth.erIemmwietdhiiantetlhye chamber. AStcacienlsessstosttehle cfrhaammeb.erThweadsootrhrwouagshstehaelefdrounstionfgthmoeublodxedsercutbiboenrvsieaalainhginsgteipd.door with a glass panel and EExapcohsulerveelciageasblceontsotrhuocltded4ofcasgteasi,nlweistshstceaeclhmecasghewcearpeabsluespoefndheodusoinnag 4frraamsewinodrikviadruralalnyg.edTohnis4 glaevveelsa peovteelnt2iaalnadniaimralsaemxpploessurweercaepwaciitthyodrfa6wn4 faotr.anaIlnysthiissfirnovemsthiigsatlieovenl,. 1N0oanciamgaelscwoemrpearptrmeesennttsownerleevuelssed1 oonr 4 Athewgeltasasn-dpadnreyllaeldcdoohoolr baunldbwtahseumsmeodhytgormoomneitteorrcwhaasmsbuesrpteenmdpeedraitnutrheeanchdamrbeelart.ivTehhiusmiwdaistyv.isible through "dTrhaeinpaygreamsiydsatlembavsiea oafbcaalcvhaclveh. amwabs ieterd with a2-nch drain. Thedrain connected with acommon SAitusaqtueadrie ttuhbeulpayrraemxihdaaulstbapsle.enTuhmi,s3coinnncehcetsedintodtihaememtaeirneaxntdrapcetrfsoyrsatteed. along the ventral surface, was "aiTrheftlootwalwcahsammbeearsauirrefdlouwswinagsa1t5a0pelrietdretsumbineutfel.owAmireteensittutahteeedcrhataemthbeedrfrotnhtroofugah pthuerpnolseet-bduuicltt. stDaiilnlleesrs SSitmeillatrrofllloeywmoentewrhmiocuhnttheedsoenctohnedavraypoduirlugteionnervaetsisoenltrwoallseym.ounted. Generation aif was measured on a `"AThMisagwaaeshemloiucnptreedssuorne tghaeugseec(o0n-d2a5rymmdiwlautteiorngaveusgsee)lwtarsollceoynnaencdtewdawsituhseedacthocmhoanmibteorrbthaye antymloosnpthuebre.e pressure inside the chamber, relative to the exposure room. Ebuxitlrdaicntgionwiotfthhdreawcihnagmbierrstwhraosugahcc&ommpalniisfhoelddbtyomwehaincshofaall csihnagmlbeefrasn mwoeurnetceodnnoencttehed.ouTthseidechwaalmlboefrtahier extract was ventedtoatmosphere viaan exhaust stack. 23: MIN 2520004312 Efixlterrasc.t Tfhleowinwtaesmaaldjpuresstseudreuswiintghignateeacvhalcvheasmmboeurnwtaesdmianintthaeienxetdraicntthdeucrtainnggeb-e2twtoe--en4 tmhemcwhaatmebrebrelaonwd ambient pressure when operational. `The control only. animals were exposed using a similar systemtothat usedforthe test groups, but received air PROCEDURE Aidesnteipcaarlateexpeoxspuorseucrehacmhbaemrbteorthwaatsusuesdedforfotrheatecsht group. groups. The The icnohnatlraotlioannsiymsatlesmwwearseseetxpuopsaesdduessicnrgibaend above. c`Tohmepraarttsmweenrtesotrfatnshfeererexdpofsruormetchaegehso.ldiTnhgecaagneismaalnsdwpelraecedloicnataedproendelteevremli2noedftsehqeuecnhcaembienrt.heIninodirvdiedruatlo cahvaomidbearn,ythvearpioastiitoinosn ionftthhee danoismealrsecweiitvheidndtuhee ctohatmhbeerspwataisalcahrarnagnegdemaetn7t-sdaoyf itnhteeravnalismaalcscowridtihnignttohae previously assigned sequence. The `The diluent and generator chamber Magnehelic gaaiurgfleowwsaswecrheecskwietdcthoedenosnuraendthattheopcehraamtbioenr odfootrhse wcehraembcelrosteodoaknpdlasceecurweidt.h internal pressure below thatofthe room. a`Tnhdettheestesxupbosstuarnecetasrutptpliymetodgogcluemevanltveeds.between the pressure vessel and the metering valves were opened m`TohveedmeelnivteorfyoefnttreasitnemdatvearpioalurtboutbhbelevsapinoutrhegPenTeFraEtdoerlsicveoruyldtubbiengm.onitored during the exposures by the cDhuarmibnegr.theTfhirestsnaimnpeleesxpwoesrureesc,olslaecmtpeldesintwoer2e0 cmoll,legcatsedtiagthta,ppporloxyipmraotpeyllyenheousrylriyngienste.rvSalasmpflroemcoelalcehcttieosnt wwaalsl paenrdfofrlmusehdinbgytihnesesrytriinnggethweistyhriangmeinneiemdulmeotfhrtohurgeheavsoelputmeusm soefalchlaomcabteerd aiinrtbheefoirnehadlartaiwoinncghuapmbtehre seqaumiplilberaftoer, aSnamllysoifs.theAfctehraamdbeleaairyr owfasapdpirsopxeinmsaetdelbyac5ksienctoondtsh,e etoxpaolsluorwe tchheamprbeesrsuarneditnhethseyrsiynrgiengtehetno sealed. The samples were injected directly into sample loopofthe gas chromatograph. tIhnethsetapretrtiiomdefrfoormtehxepoesxuproesu1r0e,anadstehqruoeungchionugt tdheevirceemaainnddesraomfptlhiengstpuudym,pimtmhaetdicaotnetlroylalfetdertdheocauumteonmtaitnigc csoalmlpelcetisofnorfoamtemaocshpchhearmebsearmpinletsuwra(sGsrwoituc4phe~d Gornoaunpdt1h)teottihmeerGrCesesty.stTehmi,sadte1vi5c-emidneultieveirnetderavtamlsosdpuhreirneg the 6-hour exposure period. m`TihneuCtehaimntbeervralasirtfhlroowu,gtheomuptertahetuerxepoasnudrreeplaetriivoed.humidity were monitored and recorded at approximately 30p24 MIN 252/004312 oAftttehset emantdeorfiatlhepreexspsourseurveestsheeltweastsrseucbosrtdanecde.sTuhppelvyatpoogugrlienvathlevetseswtecrheamsbweirtcshweadosfaflalnowdedthteocflienaalrbweefiogrhet. the animals were removed. At the endofthis time, the rats were unloaded from cages.The chambers werewashedwithhotwater. the chambers and retumed to their respective holding. A summaryofthe operating conditions used is presented in Table A. TARGET CONCENTRATIONS `The target concentrationsof T-7499 were as follows: Group Designation 23 ((moewrd.odsoese)) 1 (Highdose) Concentration (o5p0m) 415500 a`Tvhaeilatbalregedtatca.oncentrations were selected in consultation with the Sponsor, following the review of EXPOSURE CHAMBER CONDITIONS Analysisofchamber concentrationsofT-7499 dFiorrectthley ifinrtsot tnhienesaemxpploesulroeosp, osfamtphleegsasofchcrhoammabteorgraaiprh.werTehecroelalfeicetredmoinnitgoarsintgighwtassypreinrgfeosrmaendduisnijnegctaend automated system for on-line sampling and injection. The air samples collected by bothofthe sample caonlalleycstiisoanremedteshcordisbewdeirneAptapkeenndiixn As.equence from Groups 4 to 2. Methods of sample collection and The method of analysis was adapted from a method supplied by the Sponsor to accommodate the IanrheagliavteinoninToAxpipceonldoigxy AD.epartment equipment and procedures. Detailsofthe analytical procedures used Airflow and pressure Taphperoxaiirmfaltoewlyin3t0o-meiancuhte cihnatmerbvearls wtahrsoumgohnoiuttoreeadch uesxipnogsutraep.ereTdhetubcehamrobtearmenteegrastiavendprcehsesucrkeedwaast edinsspulraeytehdecdoinstpilnauyoeudsvlaylaunedwaasdioncuthmeentatregdetcrhaencgke.was performed at approximately 30 minute intervals to 125: MIN 2520004312 `Temperature and relative humidity `The temperature of the generation approximately 30-minute intervals wduartienrgbeaatchhaenxdpoaslusroet.he air in each exposure chamber was recorded at a`Tphperowxeitmaatnedlyd3r0y-mbuilnbutteeminpteerravtaulrsetshorfougahotuhteremaochhyegxrpoomseurtee.r pRlealcaetidveinhuemaicdhitcyhawmabserfowuenrduesirnecgoradleodoka-t up table supplied with the instrument. CALCULATIONS dIantoardienrtthoismirenpiomritsehatshebeceunmulcaaltciuvleateerdrocrosntthiantuoreussullyt fusrionmgruepneraotuendderdounnduimnbgerosf naunmdbeornsl,y mruoucnhdeodf tfhoer printing. Consequently, these rounded numbers will include rounding errors in the last significant figure and recalculation may lead to small apparent discrepancies with other data in the report Chamber concentration Mean chamber concentrations were calculated where equal sampling intervals were employed. When aadvjeursatgmeecnotnscetnottrhaetigoennewraasticoanlcuslyastteedm. were necessary and sample intervals were varied, a time weighted 226: RESULTS MIN 252/004312 "VAPOUR CONCENTRATION Analysed concentrationofT-7499 `rTehsepeectxipvoesluy.re mean and individual sample concentration values are presented in Table B and Appendix B, `The exposure mean concentrations for each group exposed to T-7499 are presented below and were in `800d agreement with target concentrations. Group 32(I(nLteorw. ddoossee)) "4 GHigh dose) TargeCthamber concentrationA(npaplyms)ed 50 M4an 69d0) V19%3) 415500 49 22016 B4557 `cTohnecehnitgrahteirontsharnefelexcpteecdtveadricaoteifofnisciiennttesotfflvuairdiadteiloinvesreyetno tfhoer vbaoptohurthgeenLeroawtorasnodnIenatcehrmoecdciaastieonextphoatsuthree. sbuybsbtleemswwaisthsinwitthcehetedstonf.luidThdieslipvehreynpoimpeensoanndwawsasfoeuvniddentto tbheroautgtrhiobuuttatbhlee sttoudthyefofroralmlattieostngorfouvpaspobuurt h`iwgahseslte.ss mMairnkoerdaadtjutshtemHeingths adnodsereecxopnofsiugrueractoinocnesnwtrearteiomnawdheetroetthheegdeenleirvaetriyonrastyeosftetmhsedtuersitnsgubWseteankce1 waansd 2 of exposure to minimise nucleation of vapour bubbles and associated interruptions to the liquid test substance flow. Nominal concentration of T-7499 The data are presented in Table C and are summarised below: Group Study mean nominal concentration AN ratio 2,3and 4 (Low,Inter. 2p1m) 104)6 and High dose combined) arom) `Nominal concentration. The nominal concentration for cach exposure period was calculated, for the three treated groups. catommobsipnheedr,icfrpormestsuhreeomafs7s6o0fmTm-74H9g9adnedluisveerdetdheintmoetahne cgheanemrbaetrortse.mpTehraetucralefcoulratthieotnharseseumtreedataedcgornosutpasn.t It should be noted that useofthese values may have artificially depressed the nominal concentration and increased the analysed to nominal ratio. 1227: MIN 252/004312 S`Tyhsetecmalwcualsatpeedrfvoarlumeinogfatshdeeasniaglnyesd.ed to nominal ratio was sufficiently close to 100% to confirm that the CHAMBER TEMPERATURE AND RELATIVE HUMIDITY `The daily mean chamber temperatures and relative humidities are presented in Table D. `mTehaesucrheadmbreerlattievmepehruamtiudrietsieasndrehcuomriddeidtiedsurwienrgetshiemisltaurdyforwearlle groups lower tohnaneacthhe dtaayrogeftthreangsetud(y4.0%Thteo 6fr0o%m RaHc)omrpefrleescstoinrgstyhsetegmeneirnactoirpoonraatnidngdialurteiforniogfertahnet dcrihear.mbeTrhiastmdoesvipahteiroensfwriotmhatihe tdheastigwnascosnudpiptliioends shtauddyn.o discernible effect upon the animals and is not considered to have affected the outcome of the 128: DISCUSSION MIN 252/004312 Cgroenattreorl voafritahteioTn-7b4e9t9weveanpooucrcadseiloinvseroyftgoetnheeraetxipoonsuwraes cehvaimdbeentrsfowrasthgeenLeorwallayndsatIinstfearcmteodryiaatletheoxupgohsutrhee gmreoaunpscotnhcaenntwraatsiosnesen(1a9t.t3h%e, High dose. 13.7% and This 4.5% rweaspsecrteifvleelcytefdorinGrthoeupcsoe2f,fi3ciaenndts4)o.f variation for the daily `The study mean for all groups. concentrationsofT-7499 in each chamber were within 8%ofthe target concentrations cGoonocedntargatrieoenmse.ntTwhaesdectalecrumliantaetdiobneotfweaennotmheinaavlercaognecenatnraaltyisoend tahnatd wtahes csloimgbhitlnyedbenloomwintahle acnhaalmybseedr `chamber concentration was attributed to a combination ofactors as follows: ~ theuseof a single barometric pressure (760 mm Hg) inthecalculations; ~ the calculation ofan average nominal concentration for all groups; = useofthe average chamber temperature foalrthree est groups in the calculations; ~ trehseultteinndgeinnctyheodfeltiheverteysotfmaatmeriial xtooftvvaapupooriursreaenidn the delivery liquid. pipe-swork to the vaporisers i229: FIGURE A Schematic diagramofvapour generation system =) VE MIN 252004312 i i od o] eee oSem] 0] TET Y Li 1] Front view Key a Air supply b Rotameterand control valve. d PAriershseuarteigngaucgoeilivapour generator CSohnuttrooflfvvaallvvee(generation trolley air supply) Bh NTietsrtomgaetner(iparlestsaunrke) inet Ji TPeTsFtEsutbessttamnacteeriinalprdeeslsiuvreirsyedpirpeeservoir k Toggle valve Sie view 115umfilter m7 pm filter n MTeisctrosumbesttearnicnegfvraolmvepressurised reservoir qp TVeasptomura/taeirriamlixfteuerdein(ttoo acihrasmubpeprl)y line: s WTeamtpeerrbaattuhre probe and digital display u TLohaedmeoc-elsltimer unit Vv Load cell digital display 1230: -- `Schematicofthe exposure system used to expose rats MIN 252/004312 {/D @ w I| CaHEEENn m= mm -- - (ETNTOTN EfA annn ---- A Key: B Air flow control and chamber monitoring CD DEixsppoesrusrieonchdeavmibceer (0.75 m') EF SaAmnipmlailngexppoorsture cages A H Drain J1 GPraetfeilvtaelrve LK MPoawienreexdhaexutsrtact filter 1231: Schhee FImmGtURdaCEsatplinig c gmoanlgSusLpts LSellouLt - Fume Extract sss sii =f ; fg. wie 2 Bypassflow 7 [Cow] x To i TABLE A Operating conditionsof the exposure system MIN 2520004312 I T 2 7 "Target concentration (ppm) (Control) 0 (Low dose) 50 (Inter dose)_ (High dose) 150 0 Atmosphere generation Test material feed NA At40 psi direct from the Sponsor supplied Test material flow control NA Tapered gas cylinder Tapered Tapered cnoenetdrolle vfallovwe_ cnoenetdrolle vfallovwe cnoenetdrolle vfallovew. Test material usage measurement NA Weightloss from coyaldincdeelrl measured using a Chamber airflows (Vin) Generator NA 50 50 50 Diluent `Chamberextract 150 150 100 150 115000 110500 Chamberpressure (mmwater)| _2to-4 210-4 210-4 204 1233: TABLEB MIN 252004312 `Chamberconcentrations of T-7499 -- daily mean values pNoor. e Group? Grou Group 21 5 52 (Low dose) S =761 (Inter. dose) (Hip4g6rh6 dose) as 535 i5s6 f1t8 17 iisst a :7 i5s 1261 at7 5 1s 14s 9 1ui0n" pid3s5 11164648 4ai7s03s 1i4 paH 15 5s riiss 17 5i1s0 57 1Is a i1m54 1s 5 150 1pr9e in 219 33 2140s6 iwsrs FVela 750 6221 2006 A oSvanedna ET 193 17 4s C+ V FCoornExfpiosturoefsVa1r0i1a0ti2o0n, =co(nscde=ntmreataino)n sa1m0pl0es were collected by an automated sampling system 124s MIN 2520004312 i = TABLEC Combined nominal concentraoftTi-o74n9s9 and analysed to nominal concentration ratio NoT |7.74wG9agwr d| Temp7eCrsure. [1-257ap4o9 20 5 Ts 23 P| aosst 2a1 om 2 32n0ss4 221008228 22500 110244 19 ie 5 | o03a 3 | om 22 2 535223 mbsd 528 m nms n0m2 06 102 58 0 | | aosus ow 22 2 336502 352 mwm w mm pi) 15052 101 nnoo|| 5o| oowm oss a2 2 288 384 m0 zm 22119s 20 1h0y6 57 iuso || ooam | os 2a 3 535228 382 mm a E20d 24 11095 10 iuo| | wo | oookas aa 2 32834 528 Fmd wm patn) Fr 0572 ns | o5is 00s Ena03 330829 208 ImT 127 amn1t07 55 0AN ASmaaidyastcidnoemvaidnconcentaionaioexpresseda perccaiage * Calfroem thue foallowtingeequatdion: vaYWxiDTIRxRR, 000 Where WTEZZ= WAGevigeshhctgoeomcfahtenasmt(bm0aet0res8rm0iap3lersaAeuurdmeimt(ohKle)e(xKpe'o)saunret(e5m)p(C) +273) Nw = Vioweilgh oafPsBSF (302.09g/mol) Calfrcomuhe lfollaowitngeqeuatidon: Nominalconcenaton = 1v100V5a0(rp2vpm)) Where Va = Chamber ison ()frthe exposure (162000lies): Nmoemainnoaesconeacaadien ovinatiinod cnlsuedaer drevedn though adiscrepancyinweightlos was ned. Nochangeinthe os: TABLED MIN 252/004312 Chamber temperature and relative humidity exposure mean values Eo GCoonwro]) | _(Goowpdo?s) |GniSemoediwstse dose)| _ctSigoh awoe) 2 T 2 s lz =El os [[2 2xxoza|a onx ||bn 2= c is s2lz=0 [[[22 2%N 0% n||nn o%n % ||BB |B aun OB sss la|lz2 oxw zm [22 [2 o3o x | [na oxx0 |hanm |B 0&% 4 wnewiozzlmoox2a | |m|zz omom |(ax |a xax au ||[BB haiA W3w 2lz 5 lz xn ow |[|z2z 3ow ow [([aaa om om |[h2n (A oR om vB 6o zzl2 onwwun ([z2m |z oown ow |[aa [a oomm x |[a [a 0a% V2evm 2l|2Z om xwn m[[2 an3w% |a|a @x ||na hu maa i ovaenS|a|nd ovsa devon 62s0 || 0t2o aloa || ovss 4i5s || 9o5s aasa cv Coefficientof Variation = (sd +mean) x 100 26: APPENDIX A Methodsof sample collection and analysis for T-7499 MIN 2520004312 SAMPLE COLLECTION Chamber concentration eAxllpossaumrpelseswewreerematnaukeanllfyrosmamaplseamdplbiyngwiptohrdtrlaowciantgedcohnamlbeeverl a2tomfotshpeheerxepofsruorme achsaammbpelri.nTghpeorftirsotnnitnhee eexxppoossuurrees,chaanmbaeurtoinmtaotaedposlaympprloipnyglesnyesstyerminwgeasfitutsededwittoh tarasnesafleirngsvaamlpvle.esFovirathPeTtFeEnthtuabnidngsutboseaqueganst chromatograph. Sampling lines were purged withthe chamber atmosphere for 12 minutes prioor sample collection. Aline drawingofthe automatedsamplingsystem is given in Figure A. cTohnetroalutcohmaamtbeedrssaomnplain1g5 smyisntuetme csywciltec.hesDusreiqnugenctaiaclhlycycbleet,wteheen stahmeplHiinggh,liInnet(er4m"edTieaftleo,n)LaonwdaGnCd gAaisr S`asmupplpilnegmelnotoparwyertiemperursgteadrtweditthhethGeCchaanmdbesro aitnmjoecstpehderteh.e cAotntaepnptrsooxfitmhaeteslaym1p2limnignultooeps. inTtoheeascahmcpylcilneg, bflyopwasvsafllvoesw mweetreer wahdijulsetoendlsyo20thmati/mmoisnpta(s6s0e0dtmhlr/omuignh)otfhtehGeC cihnjaemcbtieorn vaatlmvoesp(vhiear1e/8p"asTseefdlotnhrtuobuignhg).the `gaWshebnatghetoatuhteomaaptperodprsiaamtpelipnorgt.syTshteempwuamspiwnausset,hiennjescwtiitoncohefdgaosn baangdsiwmamsedcioantdeulcytaefdtebr,ytahtetagcahsibngageatcahp cwoansceonptreanteidont,o atlhleowptuhmepteswtacsomspwoiutnchdedto obne dfroarwnaalpeornigotdheosfam3p0lesleicneosn.dsFobrefionrjeectsitoanrstoifngthtehesagmaes chromatograph and for dissimilar concentration, 60 seconds was used to purge the sampling lines. METHOD OF ANALYSIS Cdehtaaimlbede,rtaotgemtohseprhewriethsaamspulmesmawreyroeftanhaelymseetdhboyd gvaaslidcahtrioomna,tiongtrhaephIyn.haTlahteiomneAtnhaoldyotifcsalamPprolceedaunraleaystitshies endofthis appendix. 127: MIN 2520004312 APPENDIX A (Methodsofsample collection and analysis for T-7499 continued) CALCULATIONS GC analysis `VTahpeosuarmsptlaensdoafrdschparmebpaerreadtimnosgpashebraegsw.eTrheeinmjeecttheoddinftoor acaglacsulcahtrinogmatthoegcroanpche,ntwrhaitciohnowafsTc-a7l4ib9r9atferdoumsitnhge, `mass used to prepare cach vapour standard is given below in equations 1 and 2. Corcent=r=T artex ioxn10001,000 00 ppm my WxRxT 760mmHg v TMM Am @ where. VWo == M= R T= = AVim == gmaassesooufs vT-o7l4u9m9eo(fmgT)-7499 (mi) 0mo0l8e2c0u5lamrlweaimg/htmomfoPlBKSF (302.09 ghmole) temperature (K) avtomlousmpehoefriacirpr(emsls)ure (mmHg) dIantoardinertthoismirenpiomritsehatshebeceunmulcaaltciuvleateerdrocrosntthiantuoreussullyt ufsrionmgruenpreaotuendderdounnudimnbgerosf annumdbeornsl,y mrouucnhdoefdtfhoer parnidntriengc.alcCuloantsieoqnuemnatylyl,eatdhetsoesrmaolulndaepdpanruemntbedrisscwrielplanicnicelsudweitrhouontdhienrgdaetraroirnstihnetrheepolrats.t significant figure 238: MIN 252/004312 APPENDIX A (Methodsofsample collection and analysis for T-7499 -- continued) COMPOUND SPECIFIC INHALATION ANALYTICAL PROCEDURE FOR T-7499 `The analysis of T-7499 in air sample substrate T`mhoenitmoertihnogdtesotuatltimnoesdphienretshisindaoncIunmhealnattihoansTobxeiecnolvoaglyidsattueddy.and is considered fit for the purpose of This document details the basic procedures for the analysis of T-7499 sampled by syringe from test ee iswis opm Birt. NprOesTenEt,TbhurtowuiglhobuetitnhcilsuddeodcuimneantS,tutdhyesspyecmibfoilc supipnldeimceatnet.that the relevant information is not available at Test substance T-7499 is mainly perfluorobutyl sulfonyl fluoride (PBSF), which has the formula CiF110;S. Equipment Balance Syringes Sartorius H`aHammiillttoonn Gas sample bags Syringe valve Vacuum pump Flow meter General laboratory glassware Consumables Syringes SKCINC Mininert AEG J& W Scientific Sigma Aldrich BP4100 1500000sseerriieessggasa-tisgh-t ((tS100i00magln)hd2t5 mi) Tedlar 232-series (1 and 3 dm' capacity) Push button valve ADEB 56(orequivalent) ADM1000 (acoustic displacement) 20 ml polypropylene 1239: MIN 252/004312 APPENDIX A (Methods ofsample collection and analysis for T-7499 -- continued) Preparationofsamples for analysis `ssyySrraiimnnpggleee.snToehfedetlhveealtisvesetiniasstecmrltooessdpehdienartnoedtathrheeecssoaylmrlpienlcgitenedgreupmsoiorntvgeadandf2r01o0mmItmhlseysroiafnmgtpeelsitfniagttepmdoowsripfthoherraien"jMiesicntwiiiontnehrodtnr"taovwatnlhveienG,tCo.Tthhee pTohretogfatshseamvpallvien.g vTahleveovafltvheoefGtCheissysretintgoetihseo"pleonaed"d paonsdittihoen caonndtetnhtessoyfritnhgeesiysrpilnagceeadreinptoastsheediinnjteocttihoen sampling valve. activated. The valve is then switched to the "inject" position and simultaneously the run stat is Preparation ofcalibration standards dSettaanidlaerddisnatrheepsrteupdayrespdecuisfiincgstuhpeplfeomlelnotw.ing method. The actual standard concentration ranges used are as oAfvTo-l7u4m9e9of(4T0-0u7l4)99anids diinsjpeectnsiendtofraomgatshebacgy,limndiexr tihntooroaugschilnytilalnadtiomnavkieal.upWetioghsetapvporloxuimmeatweiltyh6a7ir0tgo provide standard S1. The T-7499 is vaporised by gentle warming using a hot air blower. oEfvaaicru. atUesainsgergieassoftigghatssysraimnpgle(esb)afgistotefd awpiptrhopariseaatleicnagpvaaclivtey,aancdcuirnattreoldyudciesbpyenssyerimnegaesmueraesduvroeldumveo(lsu)meosf cthoenceTn-t7r4a9t9ionvarpaonugre dientsocrtihbeedgianstshaemsptluedybsapgescivfiicastuhpeplienmjeecntti.on port to produce standards covering the Storageofstandards and samples `The maximum storage periods for the various sample types are detailed below Sample type Storage conditions Storage period GSyarsinsgtaensdaarmdpsles RRoooomm tteemmpp., alimgbhitent 515d0aymsinutes $240: MIN 252004312 APPENDIX A (Methodsofsample collection and analysis for T-7499 -- continued) Calibration and quantification Calibrate by injecting duplicoafctaecsh calibration standard,a detailed in the study specific supplement, bebeamino g a bgarae es estsore med. FSoarmpelaecuhsiinnjgectthieoneoqfuatthieonsabemlpolwe:measure the peak area responseand determine the amount present in the Amountppm)= A=0 Where A = Penk rearesponse of Perfluorobutyl sulfonyl fluoride (PBSF) in the sample Sofon e deckedom cationdata Chromatographic conditions. Analytical column Carrier gas Split vent Septumpurge Split ratio `Make up CP SIL SCB (100% dimethyl polysloxanc), 30m x 0.53 mm i.d. Spm film `Helium (4.25 m/min, head pressure 18kPa, 3psig) Helium (20 mVmin) Helium(1 ml/min) 1:47 Helium (31 mVmin) Oxidant Fuel Injection volume Gasvalvetemperature Injectortemperature Detectortemperature Column temperature Retention time Air (450 mVmin) Hydrogen (45 ml/min) 250 pl via gas valve injection loop arc src 1o0rc asc PBSF approximately 2. minutes un MIN 252006312 APPENDIX A (Methodsofsample collction and analysis for T-749--9 continued) Quality assurance measures When the method s established on achromatographic system sx injections ofa standard will be used 10 `verify performanceofthe system. TaPlilnPgaaorauomnent((eUrUsS)P) Theparametersand acceptance criteria Typi1cS0aala0sv%ane aresetout below; Accep2rta3b0rle5limits RQepcesOiiCeabtniayei(nCVs,ioaq6) = Spreed SPEhreeo "iTnhdeepheingdheensttlyc.aliTbhreataiotnisotfanrdeasrpdonwsielfabcetocrosmwpialrlebd aacgaeienpsatbalestwanidairhdofhesirmainleasr ocosncernptration prepared A quality check standard must follow every 6 concentration samples for the analysis to be regarded as valid. The resultsofthe quality check standards must lie within the QC tolerance limits. AumquwailliltybechreecgakrsdteadnadarhdoefcolnocwecnoTntcienotnraotfitohnewlilolwebset raucncetpotvaebrliefyStUheRLOeQfkortmhoe druin. The LOQ for the Summary of method validation `The raw data for the method validation is located in study MIN/244. Comparisonoftest blanks, standards and test samples showed tha he analyte was well esoved from any potential interfering peak. 1P0r,e0c0is0i1o0n d5a0t0apsphmowaenddceosssfitchiaennt2s.o0f%vtaorsiiaatniodnarfdosrtPoB|S0F0opfpmless than 1.4% with standards in the range of traUocancnwe1gep0eit,ga01bh00l0te0ep0dhp0elmec)taoks"tprs1soq0adu0naudrcaeerpsdpdm,r.aehgcTroohewresersevilLeoairntm,iiaonttnahlecoyofseLifQOsfuiaoGcnfitpeainenftadiockfaLa0tiir.moe9na:9r9(oe9fLs9OpD1oGan)esaencfdhaorgoraeniTlna7s(t4tiLv9cOeo5Dne)crweraoinrrtlseralbtteeisosesntroshfsabtntya2niy.dh1ae%r1dLin(e1t0rh0e 5so9l.u4t5ioanondf c1o7n.c8e3npiaptmiornes1p0e0ctpipveml.y (calculated `statistically using the standard deviation obtained for a sSutbasnedqaurednstloyfaTn-al7y4s9e9d aignaitnhset rfraensghest1a0n0dartods 1s0h0o0w0edpcpomncesnotraesdonats wrioboimn Ste5mpoefrarteurseefoosri5n.days and maSgabamiptnls,etsforcefsohaldysoitnnajnsedcatr(eddo(sotacnmdaardst1,e0p00esrphpiomew)eedofnCoTen-cr7e4n9tnr9oartsmitaoolnrsedlwgihAninhtgh5,e 5iCnojoencttdihoonens)yPriaonngdewsfooru1b5p0smeeinauntens yundcer 22 MIN2520004312 APPENDIAX (Methodsofsample collection and analysis for T-7~co4nti9nue9d) GAS CHROMATOGRAPHS IN INHALATION TOXICOLOGY AT 25 SEPTEMBER 1997 wet Packard 5 heartoedmagoasgasapmhpwliilngCvaaplvae, ECID aend FID. HeHowwlleettttPPaacckkaarrdd HowletPackad 11835599368CX GISDAX }} 7673 Autosampler ) TThheermmooQQuueesstt* Pye Unicam SPPCai500000 POTD AIntDegiranttieornfascoetvare Chomatograph with gs Valve ad FID TBhyeerUmnoiQcuaemst ThemoQuest SPPUaAsT0O00 PCI) AIIAnuDtteogisrnaattmeiproifneascroeftware SShimmaaddzu oACOC'1400 AutSorsmaamiopglrearph wit FID TShheimmoaQduzeust ThermoQuest ASpaCs0A0 Pein AAuDtoiinntjeerfcaocre integration sotvare PPyye e UUnniiccaamm ThermoQuest PU03TO SPa400 ACuhrgosmaamtpolgerraphwith FID negrator PThyeerUmUnoniiQccuaaemmst PU04T00 SP4400 `Autoomsaatmopgisearph wil FID ntgrator ShSihimmaaddzzuu Shimada MCGRiS-A oC `Aiuetgormaattoerd gs valve hromaiograph wit FID SShhiimmaaddzzuu MCRGiS-A4 Hiewler Packard HR n`tAeugrtaotmoatgeids valve ormatographwillGaplry Tle, heated somatic gassampvallveiandnFgD. HHeowwlleetttPPsacckkaarrdd HewlPackad G1I5S9I6ICAK GISDAX }) 6890 Series Autosampler 5 TThhPeeemrmokoiQQEnuui eesstt PSPCaiso0n0n tory AIDnteignrtaetirofnascoeftware Smatographwil programmapblaey Te, x heatedautomaticga samplingvalveand FID. *formerly Spectra-Physics 23: MIN 2520004312 APPENDIX A (Methodsofsample collection and analysis for T-7499 -- continued) STUDY SPECIFIC PBSF (T-7499) SUPPLEMENT TO THE INHALATION ANALYTICAL PROCEDURE FOR ne Ho tn ditions sod amendmen othe `procedure to be used forthe GC assay of PBSF The assay, incorporating the additions and amendments, `concentrations within the range of 25 to 1000ppm. is suitable for the analysis of PBSF, in air, at Details given in this supplement supersede those in the `compound specific AP. Analytical standard Chromatographs `The analysis is performed using chromatograph 8. Caries gas Helium (275 mUinin) Split vent Helium (24.5 mUmin) Split ratio Detector temperature DetectorRange Retention time 1:89 150C 0 PSFapproxim2.a7tmienleys Summary ofmethod validation Manusl injection Precision data showed costicentsof variation for PBSF ofles than 0.8% with standards in the range of 1000to 25ppm.Thisdata is locatedwiththe MIN/252 study data. 204: MIN 252/004312 APPENDIX A (Methodsofsample collection and analysis for T-7499 -- continued) aLgeaaisntstsqucaornecsenrtergarteisosnioonfansatlaynsdias,rdwi(t2h5 atnoun1w0e0i0ghptpemd)lipneraordurcegerdessaiocno,mrfeolratpieoank caoreefafirceiesnptonsoef 0L.i9mi9t99o8f4aQnuadnrtifeicaltieoarnro(trLsOilQes)vstfhoearnPB1.S3F%iwnitllhebreansegteb1y00t0hetolo5w0esptpamccaenpdta4b.l3e%cahte2c5k psptman.daTrhde, however, the LOQ and LimitofDetection (LOD) are potentially as low as 1.69 and 0.51 ppm. respectively. Calibration and Quantification Calibrate using standards with nominal concentrations S00 and 50 ppm. Preparationofstandards Preparestandardsinthenominal ange2510 600ppm. EFFECTIVE DATE: Calibration and Quantification Calibrate at 4 concentrations across the standard range. Summary ofmethod validation Automated injections rParnegciesoifon60da0ttaos2h5owpepdm.cTohefifsidcaietnatsoslfocvaatrieadtwiiotnhftohrePMBISNF/o2f5l2essstutdhyadnat4a..3% with standards in the Lareeaastrseqsupaornessereaggraeisnssitoncoanncaelnytsrias,tiwointhof1s/tcaonndcaerndtra(t2i5ont'o we6i0g0htpepdm)linpearrodruecgreedssiaon,corfroerlapteiaokn. `coofeQffuiacniteinftiocfati0o.n99(9L9O4Q0)anfodrrPelBaStiFveweirlrobresleseststbhyatnhe1l.o3w%esitnatchceeprtaanbglee6c0h0ectok 2s5tanpdpamr.d,ThhoeweLviemri,t the LOQ and Limit of Detection (LOD) arc potentially as low as 12.28 and 3.68 ppm respectively. 25: MIN 252006312 APPENDIXB Chamber concentrations of T74ind9ivid9ual sample values Yor | mGoeowaT)l| onodTe = nGrrTormdose) GiGgherdoose 35132 a"" pr} a 111863s6 167 oiiso iad CWo5rAs a "3 m 5557 ] EiE wo 12 225555 204 s0:B5 23 a&& 13p:6 PP.eo . ofos) 55 5@a i18s 1 ii 0 [Wora o -Tr mT5r ii`s] 12 ":" 1I5%8 a:ps 2o53s3 555 16166 aaiitss TWA [Time Wweig5AhsedT verge"S si5m of aged ConcTesoisoirons ime) calfoeaclhsatmplding ovsiosn Follows: pI Smeaeois----w]eed for Wei}ghtedconcnraio}n o(popmm)) = CConceEntratRiEoxnpgo0Esppumr))exEuxraTTtiiiOmmoneew(emeRiilng)ghiOgn"g rnnini)) hWheemriedtphoeit"sibmetwweeiegnhcionngiecsuihevsreSsapmepcltiivneg ionvtcearviaolnbseienenaajusme entwsis othdesexposesystem o ss: MIN 2527004312 APPENDIX B (Chamber concentrationsofT-7499 ~individual sample values-continued) 7 intervlael ou ous) | Lowdose) intrro, udose) idpose 12 52z7 23 awwTn : 23 "- 125097 20 n:: 34 a"- 221175 fr] !: 61 46s : 5 11530 17 46:s is :: . 11113 s 301 ::: 12 7" f2r% 23 1 144 a"21 3 & +s @ 1154i00 ie 456 ss 56 146 pi] 212 3s3 i11am a50 6 p3s4 7& 7 56 57 172s 11664 4549 a5937 y : aot WA timTiems) cWaelicguhlatteeddfoarvecraacghesaSimmp,linofg o"cWceiagshiedaon oaleioswsa:ons (Wp CoReEnaaons whied for `Weightedconcentration (ppm) = CNonCcHeTn2tEr0aoNn(pm(gpm)) xx""TTiimmeewweeiigghhttiinngg""((mmiinn)) Exposure duration (min) `tWhheemriedhpoeintsimbeewtewiegehnticonngs!eicstuthievreesspsemcptliivneginotceeravsiaolbnsetwneoensadajumstemenntws atrthneecxposuresystem or 127: -------------------- Ee -------- MIN 252004312 APPENDIX B (Chamber concentrations ofT-7-in4div9idu9al sample val--countienuesd) Gow)| owdose) sir Js igh dose) 2132 2%@9 1122259 sa4ss 3o544 aa"@ 111275 apPipd oT] 2 17 2"6 ; 2312 565i3 pe] " 11155>57 1% aip-ini i 54 00 15> p5it0] 2132 5aal I1r5>% i40 Hn 3i 5a5s 3a 1# 11552 a> paiod 33i5z 33a 11155s00 ipiro 5p=8; 7i 1550 i4 WA TSi[emeaYrweeidgehrevd|oanverage: um of |[SW] "weighted concentrations' sample concentrations weighted for in leafocti sampling ocesionas lows: Weighedcen pny ECE) x Time eight') h Exposuredustion (min) WEhemriedtpnhee tm1b03ew0eiwegehrtceinmogop!etissotrheedresbsipynepcslttiiovnemgaionictecedarsvsaielonnpbsleitfnwgeaensadujussimeentwsas0mhaedeexpos syst of cs APPENDIX B MIN 252/004312 (Chamber concentrationsof T-7i4ndi9vid9ual sample val--countienuesd) pNoore|| inStaemprlveal | Cour) | (Lorwotdose) Chamberconrcoeumraion(pm) Gnir dose) igh oudose) 2132 3i 54 38 11540 7 157 w36s3 61 o5s6 35s 131567 1166s5 2132 3is3 Ss [ar 2132 o3s4 Se 555 " 113379 15s w398 is i"s ji151 a61 TsTe i2s & 1114ss 1a "0 11476 2132 38 33 3s i1s51t 151 0si 8 osse aas 111590 aan Sandra doviaron 3u3o TWA Time time) weighted calculated average: sum of `weighted concentrations' for each sampling occasion as follows: (sample concentrations weighted for `Wei.ghtedconcentration(ppm) = --`Conocentnratcion(e(ppppmmn))xxi""TTizimmeeawweeiitgghhttiiinngg""o((mmininn)) i Exposureduration (min) WhEhxeepromseiudrtpheosi"n1ts0i1mb0eew2tewiwegeehnrtceiomnnogsneiictsuotthrieevredebsspayemscpetlitivonemgaiOnsEtecedarssviamlopbnlseitInfwngeonaaddjujskumsentwtsamtsoemtanhdteexposuresystemor Fir samopflcaech grwaosmuanupal 1249 ee MIN 252004312 APPENDIX B (Chamber concentrationsofT-7--in4 div9 idu9al sample value--s continued) E Sample Go0ow1w)| _(ow5dose (tTe,isdose) ighGados) 2132 5a 11522 SS2010 i4s 51 55 @ 119904 1m aas6 50 woTa 12 ss m Temr er &581 2334 is 21553 mn 15 iss To[ t56 Wa153 ar a7w 2132 4is T5V6A ToTMewan io 5% wo 2132 a2r 4 i 115575 15s p-r pr] i5s6 TVA 52 115553 o8>ss PMeYan f: a1832 TWA TiSmaerwdeidgehvtieadtioanverage: sum of ime) calculatedfo ach sampling o"cwceaisgihotnedascoanlcleonwtsr:ations" (sample concentrations weighted for Weighted concenraon (gpm)= SCoSnceEntEraotinon (0ppmm))xx"*TTiimmeewweeilgghhtiinngg""((niinn)) hte o> `Exposureduration (min) Wh{heermeitdhpeo`initmebewtewiegehnticnognsiescuhtieveressapmepcltiivnegionctcearsvialonbseifwenoenadajdujsuemsetnmtewnatsosmtahedeexposure stem or Exposures 1010 20 were monitored by"a2u5to0mated sampling. MIN 252004312 APPENDIX B (Chamber concentrationsof T74ind9ivid9ual sample va-lcountienuesd) ExNpoorse|| iSnatmerpvlael oT Group roi (hoou1rr2s) 23 (L3ow9dose) (int1e15r3.5s4dose) a08 (High dose) as i5s4 5 23 5 115566 154 i0s pets 5 o12r 23 171 1a7s3e waon 2534 aa is 3 11673 16 wsaas pi 5s5s 2: 12 i Sard Msedan deviation 2a576 TWA tTiimmee) cwaelicgulhaiteedd faovrercaagceh:ssamupmlionfg o"wevigehtneds5coionlcoelnatnr:ations" (sample concentrations weighted or Weig} htedconcentration(pm)= C ConcentratiE2 oxnp(op9sppummr))edrs*0 TTtiiimoene(wwmeeiiing)ghh0 tiinngg""m(miinn)) * syEWsxhteperomesoetrhseth1`e0tm1ii0mde2p0owwieneitgrshebtimenotgnw'ieoeisrncetohdenbsyreecssuputeticovteimvaseatmeipndtlseiranvmaglloicbcgeattwieoenns fadojusstmtenutsptao the exposure 251: ---------------- ee