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a ore Contains No Cpy Ivo Viesk Oroerln-R3asn0gaecreicndiinngTTaotxsicity Study g E=R 2 Exportaent to.: Dosing seared: Recovery Intervals Completed: study Direotor: zesting Factitey: vromsoz october 3, 1979 November 15, 1979 6. n. stetfen, 35 S on J] = 43 w>l HsaoteesthyauitEa,veaclWauiuamntmritaoasnso:rLabTooreastory sponsors & EPA-OTS vTf.onric.eecWieaegdcyomSiocrkvices St. Paul, Minnesota 0008117964 S Spantoot r rno, xiycotbiat bz/g1o02 A Mainnevr,esFa,thologGy-Tooxicolo2syis/es0e ts hvatancion Laboratory 03695 TAOFBCOL NTEE NTS Page INLROBUCEION + + + shee e a eee.2 Methods . . + oui i ieee.2 RESULES + + vv ttt. 3 DASCUSSAON + 4 4 hh hh eeeaa h a.22 Appendices LL... i... 23 03696 1 Summary Tthweo oparratls.rangIenfPianrdterItotxheicaictuytestuordayloAfLDTS-O25w0a9sCcCdefininerdatsinwmasalecomCpDlreattesd. in Smiixnigsrtoeurpesd shianvginlge sdoisxesratrsa/nggrionugp,fro5m1-52950d-a1y0s,00o0f magg/ekgw.henIndoPsaedr,t wIeT,reTa-d2-509CoC Twavselvaedmirnaitss/tseerxe/dgrboyupd,ail5y1 dgaaysstroifc iagnetuobnatdioosnetdoamyal1e, waenrdefdeomsaelde CaDt r2a0t0s.or 7a5ndmgf/okrg/ndianye.dayDsosaitngthceontliownueldeveflor (f7i5vmeo/dkagy/sdaayt).theALhLighsurlveivevling(2h0i0ghmg/dkogs/eday) afnoilnlaolwsinagnddoshianlgf.ofSetlheectleodw dlooswedogsreoupanihmadalsa w1e4r-e15eudatyhanraetciozveedryonintdearyva1l0. All isnutrevrivvailn.g anBrianianl,s sintobmaocthh,grliovueprs,weardereneaulthaanndatkiizdendeyattithsesueesndweorfetheevalrueactoevdery microscopically. Tfhiedeanccueteliomritasl oAfLDS5O58o-f13T9-925mg0/9kCgo.C iGnasmtarloeinrtaetsstiwnaasl87i8rrmigt/aktgiowniwtahs95t%hecmoons-t cococmumrornedefifnectsomceausdoesdebygrohuipgsh a2c-7utdeaydsosefso.lloHwainndgldionsgi-npgr.eciTphietatleodwecsotndvouslesigonrsoup (250 mg/kg) was free Of treatment-related effects. aDnadilysexg-arsetrliactedintefufbeacttiso.n OfTheraptrsimwairtyh eTf-f2e5c0t3sCcwCerreesgualstterdicinirtrrietaattmieonnt,-,hadnodslaigneg-ptrreicbiuptietdatteoddocsoangveu-lsrieolnastedanddeamtihcsr.oscoSpeiccondlairvyerefcfheacntgess.at bTohtehseleevfeflesctwserceon-a tthueckdeods-iunpgapppereiaorda,nceh,eadanturnekmoermsptanadppheyarpaenrcaec,tivsiotfyt. feTcehse,hbiogdhydwoesieghgrtoulpo,ssbudturing wneortetnheo mliocwrdoossceopgircoucph,angheasd ihnisttohelogbircaienvisdeecntcieonsoffgraosmtreiictheirrrilteavteilont.o coTrherreelate wnietchrostihse aCNnSd effaftetcytsc.hangTehse. trTehaetmmeanlte-sregleanteerdallliyvehradeffeeacrtlsiercooncsciusrtreidnogfdefaotchasl, more convulsive episodes and more animals with liver changes. CRNeSveresfaflectosf dtihde tnootxioccceufrfecdtusrinogccurrecroevderayf.terThdeosgiansgtrsitcoppleeds.ionsSevaendreotdheelrayed sSaeicno.ndaTrhyeerfefceocvtesrysubasniidmeadlsdduirdinhgavreecmoivelrdy.fatAtlyllirveecrovecrhyangaensimbaults hnaodt wleiivsehrt necrosis. 03697 2 Introduction The two poarratls.rangPearftindIeervatlouxaitceidtythsetuadcyutoef Tor-a2l509ALCDoSCO ininrmaatlse warsatsc.ompPlaerttedIIin aevnadlufaetmealdetrhaetstouxsiicngeffdeocsetslaenvdelsrecsoevleecrtyedfoflrloowmintghedraeisluyltosralindoPsairntgI.in male TThoexicsotluodgyywaSservciocnedsu,cte3dM bCoymptahney,SafSte.tyPaEuvla,luMaitninoensLoatbaowraatsortyh,e study Riker sponsor. Laboratories, rIencp.e,atSdt.osPeauplo,rtiMoinnnwesaostac.ompAlceutteed donosNionvgemsbtearrte1d5,on197O9c.tobeTrhe3,pro1t9o7c9olanwditthhe amendments and a list found in Appendices I of principal participants and II respectively. and supervisory personnel are LSaibnocreattohreysPtruadcytiwcaes a(GLrPa)ngerfeigunldaetrioenxsp,erihmoewnetv,er,ittwhaessteuxdcyluwdaesd cfornodmuGcotoedd in arecqcuoirrdeamnecnetsw.ithThoeurstSotraangdeardloOcpaetrioantinfgorPrsopecceidmuerness,whriacwhdaitnacorapndoraftienalGLPreport is maintained in the record archives for the Safety Evaluation Laboratory. Methods Part from I: The Charles RaipvperroxiLmaabtoreatoorrailesA,LDSIOnc.wasThdeeterratmsinweedreincomnadlietCiDonerdatsin-ohbotuasienedfor approximately with food and woanteerweaevkaiplraibolre toaddolsiibnigt.um. ThAenyimwaelreihdoeunsteidficsaitxi/ownirwea-stopbycaignedivi- dual numbers and humidity caonndtraolcloeldorancdodhiandg asys1t2emh.ourThleigahnti/mdaalrkhcoyucslien.g room was temperature wSeirxera5t1-s5/9dosdeaysgrooludp waenrdewesieglheecdte1d79f-r2o6m2thgeramcsonwdhietniodnoesdeda.nimAalsc.onstThaentandiosmeals vthoelumteesotfar10timcll/ekgtowatsheusaepdproatprieaatceh cloenvecle.ntraDtiisotnisl.led water was used to dilute T14hetaoni2m0aldsaysw.ereBoobdysewreviegdht3s-6wehroeursobtfaoilnleodwinjgustdopsriinograntdo ddaoisliyngtahnedreoanftetrhefdoary Of necropsy. died or were Gross killed natectrhoepseyndabnoofrmtahelitoibesserwvearteionrepceorridoedd for but atlilssuaensimwaelrse tnhoatt saved. Part Inc. II: and cMoanldeitiaonndedfemianl-ehouCsDeraftosr wtewroeweoebktsaipnreidorfrtoom Charles dosing. River Laboratories, The animals were ainnddiwvaitdeuralwleyrehoauvsaeidlaibnlehaangdinlgibictaugne.s hAanviimnaglwiirdeenmteisfhicaftrioonntswaasndbybotitnodmisv.iduaFlood ear had tags a 12 ahonudr ca1gieghtc/adrdasr.k cyTchlee.room was temperature and humidity controlled and 03698 3 Tawseslivgenedrattso/stehxe/l2e0v0elorwe7r5emgs/eklge/cdtaeyd fgrroounp.theOncondadyiti1ontehed aanniimmaallss waenrde 51144d-a1y8s5 gorladmsa.nd Tthhee mtaelsetsawretiigchleedso1l5u4t-i2o1n1sgrwaenrse parnedpatrheedfdeamialleysuwseiingghed duissetdilfloerdeawcahterdoasse tlehveeld.ilueDnots.ingAcocnotnisntuaendt dfoosrefviovelumdeaysofat10tmhle/hkigghwas d(o7s5emgl/evkegl/d(a2y0).0 mgT/hkeg/ldoawy)doasnedgrfoourpnhiande dsaiyxsaantimtahles/sleoxw rdoesteainleedvelfor areycov20e.ry aALfLtersudrovsievindgayan9imaanldsthien rtehemahiingihngdoasneimaglrsouwperweerecutrheatnaaitniezdedfoorn rleocwovdoesrey agfrtoeurp danodsin15g dsatyosppefdo.r tThehehriegchodvoesrey girnotuepr.val was 14 days for the Tohbetaianniemdaplrsedwoesree,obbsiewreveekdlydadiulryingfodrossiignngsanodf toonxcieciotry.twicBeodya wweeieskhtdsuriwenrge rgercoosvsernye.croAplsly aanndimatlisssutehsatwedrieedsaovrewderfeoreuhtihsatnoaptaitzheodlowgeircealsuebvajleucatteidont.o a TThhee d7o5smea/lkeav/edlasyfolrevePlartforIItwheererepseelaetctdeodseatporttheioncoomfplethteionstuodfyPwaarst sI.elected Tthoede2l0i0vmegr/kag/cduamyullaetvievlewa1s4 dsaeylecdtoesde atpopbreoxicmlaotseelytoeqtuhealactuotethenoA-LeDfSfOectvalue. ltheavneltahnedALtDoSOdelvialvueer.a cummulative 14 day dose at least two times greater Results P35a%rtcoI:nfidTehneceacultiemitosralofAL5D5S0Otoof1T3-9925m0g3/CkcgC.in male rats was 878 mg/kg having CDloisnaigcea-lresliagtnesd ignacslturdoeidnteasttiuncakledt-ouxpicaiptpyearvaanscet,hesporfitmafreyce7s-/2d5i0a9rCrohCeaefafnedcth.yporoevaecrtniivgihtty/onhyptohaectdiavyitoyf d(oTsaibnlge 1()f.ounMdosdteaddeaotnhsstuadtytdhaeyhi2)ghwehrilleevetlhsoseoccaturrtehde ldoewaedrhaldeveglrsoswsereeviddeenlcaeyedofthrreodudgehnedstuodryhedmaoyrr8ha(gTiacblgeas2)t.roiMnotsetstiannailmalmsucofsoaund (rable 3). Dtheelay5e00d0,CNS200t0oxaicnidty10w0a0smgev/ikdgenltevaetlssohmaed dcolsoangiec/tloevneilcs.conOvnuelsiaonnismalonatdayesach6,of 2 or were f7 oruensdpedcetaidvewliyt.hin Ea2c4h hoeupriss.ode Hwyapsereparcetciivpiittyateodcobuyrrheadndtlhirnege tanodnitnhee daanyismals cfoovlelroewdi.ng dThoesinobgseartvatthieon20p0e0,rio1d00w0asandext5e0n0dmegd/ktgo l20evedlasysbuatt tthheese10a0n0imaanlds5r00e- mg/kg levels because of the delayed deaths but no additional effects occurred. Tthheeseloawneismtaldsoseshogwreodupno(r2m5a0lmwg/ekigg)htwagsainf.reeAlolf tanriemaatlmsentat-reealcahteddoseeffelcetvselantdhat sofurvobivvieodusfogrrotshse dluersaiotnisonatofnetchreopsstyu.dy had acceptable weight gain and were free 03699 | -- servation Appearednormal Salivaion Lcriaation poresctive Tabte 1 oAfcu1t-e2o50r3a0CToxiincMiatlyeshtautdhy Nunbe1r0,o0f00Amn3i/mka3sGrAofufpe.cted SBiamTooui asdw we meee 733 3 aa Se eTyebiesowwn mmmm mm 2 3 2 ow. o- oo o-oo ooo oo 32 ow ow - - - - oo o.oo W2 o-2 ooooo1 o. o Wozoe ow o-oo. oo oo Chrossdacryorzhea WE Ne 1 We = =. =. o-oo_ . o_o. posctive mows ao o-o ooo. Tucked-upsbdosen NE Ne We 1 = =. o-oo o_o_ D-- eachs rer-- e ereec ctt-- e0stta1t 0S -- overmig-- n te eraser e Ne =votevident 23a - 3S3 Ta1 b(conltinueed) AocfuTt-e2o5r0a9d0uTcoxiinciMtaylostRuadtys Number50of00Anmimaalgs Affected O servato ion e-- s ------eTe T-- e -- ais 3a ET ew nm nw | peedemal 4 4 1 3 2 2 2 2 1 ow = - - oo Satsvation 30 wm a mow om oweow ow oo - o-oo Bloody arescoun 2 Ww Nw 1 wowowow owow - - - - Hyporeaceive Zo ss mom ow ow ow ow - - = oo Dyspnea Poe ow ow owomow owow ow - - oo Soft. feces mass mowow ow owow - - - - oo prosia moo 1 mom oweow owow - - o-oo Tokedwp bios MEN 2 3 Wow Wow ow ow - - - - - . Clonic/tontcconmlston NE WE NE Ne wow ow we 2 we = - = o-oo - Death 0 0 oo 3 1 0 0 1 1 2 =vot evident 2 precipitated by handling 2$a - 3S8s Re1 (continuo AfWrcp toa Sr4aotoSTwhoexlntAesteeoutddeys | --[T--TTTFE it =Se =mTrSSe eme eewmat -- ----S e ---------- ------ --S ------ l -- wreoreemaecatoinet wow6 m2I : owowm ow ow - - - oDronpty ares =mooww oma now hme wm ww Fae mow om ow ow ow LL LLL sowow naaom oo LLL oCtlotnueess conmtsio1=n8 0mow1a rents ow ow Lemooww owow owow wowowow oLoLo L LLLLL mow ow oy owowow LLLL L Hnpoenrsceive pesctive wooew ooww ww ow ommoowoowmowroowaowoew L LL L LLLLLL wow oy owow owoo LLLLL se--ns_e ---- ooo Se nea 0a 1 = vo vigon, reciente by anion 23 . g32 Ta1(beonltinueea) AofcutTe-213o0r0a%0lc00cTnoaxi/ntveNatattyestautasy Wmber of Animals Affected BObseE rvation F=SoECamnm=Su=SM T BOBT Oe SESe STeOST RU eSeASwSwe n CTwm0 Aopeaced norma Clonic/tonicconvlsion NE 6 XE 66 WW 65 wow 6 ow 5s wm 4 ow 3 mom om ow 3 ow 3 ow a ow 3 om 3 we in pereactive mow ow om mow ow ow momomowow low mom om owowe ow 2 ow 3 ow ow wom ow ow ow ow ow ow enors momom om mow ow ow ow ow 1 om ow ow ow ow De_a--ths 0 0 0 0 0 1 10 10 0ro r o o o 2 =votevident Bh 2 precipitated by hanating 22 - 33 8 -- observation Mpewedtorml Hrpereaceive De_at--hs NE =Not vident Ta1 b(conlcludeed) Aofcu1te-2O5r0a5%l000cTnoaxiinycsHiatlyosRtautdsy Mbox of Anisala Affected FBlFTzaai EwRs FF5 I We dse ee vs 5vwwonnn n nhmwmm 66 mow 66 Ew 66 wow 665 mow ow 5 wow 5s 5 5 5 ES 5s ow ow ow ow om 0 0 0 0 0 0 10 0 0r0 r o r o o o Member of25A0nmimaalgs Affocted ` O-- bserv-- ation mes FELTiSH= a=nes opened normal 6 6 De_at--hs o oo seem SE53BO aWSSeSySU SOw SSnSw en Cw=m 6665656 5 & 5 o 0 0 0 0 0 0 0 0ro r o o o 2833 . Taste 2 hoefut1e-23o0r8aCl0CToxiincMiattyeStRuadcyh Death Pattern BLlT oeeverl 3 TE 3 aeme a6wm7 e T4 rSeemm 5 omhrembpioemmeasams 10000 Bet 15 em em am en en en so0mat 0 3 1 0 0 1 1 ae em es oe 2000ng/kg 6 3 0 1 0 1 0 1 os soma 1000 mg/kg. 0o 0o 00 oo o1 11 0o 1o ooo 13/66 200=58708 nak . 250 mg/kg Jooo0J00 0 0/6 95% Confidence Limits = 558 to 1399 mg/kg~ 2 CfboamtepauetfartoimontshisbylbeuvleulthnoPtraubsietd AfnoarlyAsLiSsO, Bcavliczuolmamteinotnasl Research Laboratory 03705 ante 3 AofutToN-e2c5orr0oa5pl0s0yTcoxiRniecsiWatttysesatutdsy usher of Andes Aefected Gross Ghueevati"on Tow 0Tow To05 7 Toon Em Ei No. Dead/to. bossa No Visible Lesions ws os ws re we oe BFuotuhnadnaDteiszaed oo o2 o1 15 s . Roddaned Stomach wicosa Hesorehagic Stomach Mucosa 2 2 2 2 o = 1 o o ' [a Heamocrozrhnagic small Intestinal o o 2 o o 0 [R-- o o o ) o o 2with possible utcer 22 .5 83g n Part II: prominant Terfefaetcmtencta-usaenddbydodsaagiel-yreorlaalteaddmgiasntirsitcratiirornitoaftioTn-2w5a0s9CotCh.e most The hgiagshtridcoseirgrriotuapt,ionb.ut Tnohtisthierriltoawtdioosnewagsrouppr,esehnatd hiinst11oloofgic12emvaildeesnceandof 8ulcofers12wefreemalfeosund(.TabCllein4i).calInsigsnosmeasisnosctiaantceeds weiatrhlyghaesmtorrirchaigrircitgaatsitornic waeprpeearparnecsee,nt doinsabgoet-hredloasteedgrsoofutpsfeacneds infocrludtehde maaldeosoangley-raelnadteadn tucked-up unkempt a(pTpeebalreasnc5eamnodre6).commBoentteirn tshuervilvoawldoastethgeroulpowtdhoansethleevehlighnododsoeubtgraolulpowed Agbrdeoamtienralexpparienssi(otnucokfed-aupseacpopnedaarraynceef)feccto,ntsiuncuhedasforanthuenkefmiprtstapfpeewardaanycse. aopfperaerceovderuyn.kemTphtethsruoruvgihvoiuntg htihgehrdeocsoevearnyimianltservaanld.some low dose animals Defofseacgtes-rweelraetedexpCNrSesseefdfecatsscooncvcuulrsrieodnsat anbdothisotlraetaetdmenitnstlaenvceless. ofThheesaed tfroeumndorsinanadnimhaylpseraacttieviitthye.r lHeiveslt.opatThheolborgaiicnalsbercatiionnslewseiroensowbetraeinendotat othcecurforreed-,inmitdwicaendthhiendnubmrbearinoflemvaellse.s coTmonpiacredortoclofenmiacl/etso:nicscioxnhviuglhsiodnosse mTahleesl,essthrseeevehrieghCNSdoseeffefcetmsa,lesh,eadtwotrelmoowrsdoosre mhaylpeesracatnidviotnye, loocwcdurorseedfeimnale. only one animal signs. One low dionseeacfhemdaolseecognrotuipnueedxcetpotbethhaytpenroachtiigvhedodsuerinmagletshehraedcotvheersye interval. Tihnecrecaosnevulisnivfereeqpuiesnocdyesafwteerredgoesnienrgawlalsy sptroepcpiepdi.tatHeadndbylinhga-npdrleicngipaintdatdeiddconno-t vulsions started on dose day 5 at the high dose level and each animal that convulsed was dead within 24 hours. The low dose female convulsed on study day 9 and survived. The low dose males convulsed during handling just prior ttohiprldadnanyed (ndeacyro8)psiaefsteornddoasyisng8waasndst10o.ppeOdnebuthigthhedofsreeqmuaelnecycoofnvcuolnsveudlstihvee episodes were not increasing compared to the rats dosed acutely in Part I. All animals were handled (picked up) as much during recovery as they were while being dosed. Treatment- and dosage-related deaths resulted from the treatment-related toxicities. Deaths occurred earlier in the study for the hich dose males wtehraen fdoeradtbhye hdiagyh6doasned bfyemadlaeys.8 alSlevetnheofmal1e2smaalnedsseavndentwoof o1f2 1f2emafleemsalweesre weiatdh.twoThesurhvigihvindgosethrfoeumgahleosutcothnetinrueecdovetroydiientetrhvraolu.gh Tdhaey o1n2ly(rterceoavtemreyntd-ay 7) related death at the low dose level was a female on day 9. Tforlelaotwmienngt-rtehleatreedcovmeircyrosicnotperivcall.iveTrhetoxliicvietryefsfheocwtesd evidence of reversal consisted of fatty changes and focal areas of necrosis. Minimal to moderately severe fatty 03707 12 changes were present in five of 12 males and three of 12 females at the hairgehasdoosfenelcervoeslisandocctuhrrereedofinfitvweo males of 12 at the low dose level. Focal males and one of 12 females at the high dose level and three of five males and two of five females at the low dose level. These low dose effects reflect only those rats killed for the the interim necropsy. The recovery interval (died on last days three 9, 11 horigh12)dosheadfneomahliesstotphaatthodlioegdicdaulring acnhiamnagels.witThheonltywofaftetmyalecshanignestheathingehcrodpossye. groThuep tlhoawtdossuervirveecdovheardy oanneimals did not have focal liver necrosis but they still had minimal to mild fatty changes in five of six males and one of six females. The greater number of bleotwtedorsesurrvatisvalcomraptaeredattothehiglhowdodsoese raltesvewlitahnd ltihveersucbhcanhgreosnicrefnlaetcutreedofthethe liver response. gTarienatmoecnctu-rrreedlatdeudribnogdythweeirgehcotvelroyssinotcecruvrarle.d dTuhreinwgeidgohstinglosasndwbasodysiwmeiilgahrtat bAontihmaldsosein lbeovtehlsdobsuet tghreoupfsemaallseos alpopsteamroerdetwheini.ghtThtehansurthveivmianlgehsig(hTadbolsee 7). females and many of the low dose recovery animals continued to appear thin during the recovery interval even though they were gaining weight. Food consumption for all animals appeared normal throughout the study. The only other treatment-related histopathological change was congested and hemorrhagic adrenals in one of 12 males and two of 12 females at the high dose level. The adrenal effect was probably due to stress caused by one or mroerleateodf ctlheinioctahleretfrfeeacttmweanst-ruerliantaerdy tionxciocnittiineesn.ce Ainn ifonufrrehqiugehntdotsreeamtamleenst.The incontinence occurred for several days prior to death but was not associated with microscopic kidney changes. One low dose male was euthanatized fonoradtaeyd8eshoapvhianggusa. soft subcutaneous lump caused by an intubation-induced per- 03708 My ITE: KPiodtnleo oyusi."ng c5om4pleCittaetnigteroIsitas mEiTmEi, OaW 200 220 water apmrierayes stom shoe sunshye Stomen ascents psye 0 stomach anaarasye 7 [--. smpirreayen adeanaie [SR Liver axaraseer 0 Scone re mh ir o --. mi irass frome Liver miireo Fo. 1 te + ntnooerGctrrMooTapnietsnyet.0de,dhi2s7K0t0corslsiooLgssnnciLiocffpiLtecosxuoannFmtiisnchaahstaaeinnoggnceebrrvsievawreovrrdaeeotfnisFoonuosonundt,sbdesbeieno,tnoibLyyiavieeso,mrmsetaonimtatcihns,eaaedrrseeeTnaitisLseeeasn?.d or ecy weat-- ion M[erroe--d--w--s Smaellehnemorrhagic cess Yo signiticant anges Semmerttivaisaeckatayreus Duck rmorzhagicn Lahrsebemorhagtc acess tage blaocknyspore Dark amorstagien Yo signicicant changes Dark Linus Oremcrchuge?) 0 stgnitican chamses S[ma=ack=spucks nad ttnmn Yo significant anges Leoenegrbaiannetitirnas aTeArraottcmihseectraoetuaaetaevrrieTtsshhuesfoiaosmouynrsshaesosastfmermetasnhteesoitfscosetnFeenotpsscoirsctmeoiaocornononsmaaonttssvhare Paricutar rectosty of the Sebcrie manos MrCmorpdErEecorraiutaiaemtlmyiaclssyaaetvrars-ehvivaahmfrsaieive,athsy.a.codhfao+ntgehceshLevreonersetoscpTsroeescoehnittnr,siefsaonome ination: rScatatazetd oect obf caonspeateion aanldpbeimsortehearsedvoereil Ratheeer asuckTed cTonTgestioBnevas presen, sexticutacly SFrceastatnesreidnfoocmk oeftecornseamteisononand hemozhace vere eDFCSeoPcecLaistttahtneehtIraieridinen'fvtoCasoantemvosoaouftmsoeccproemianengetecmsmoetaftiteoasn4ssasatntditMehroeensoopnbreeevrsheooaressresimmeavnnestrce Yo signtttcans changes taste ot processed tor exanination --KBAoLiA--staotFtyhcahahnegpeacoveeprieesinhdaiscapteedomatsananstosroxhtomstels FFBorcsatelantariTenesstohoeefiTpciouenrsgeeiwssiteionmnuseeoaemnsed-nheecmTrooocnoethosase8tivoenroee s f SaeteaLs epeitna)tim (hvelopmant oF an sort EKingsaayl faR tty che anges vfers peresei nt as ssppreoximnstiely 20 stsnicicans changes PForceaslentaceTsnstohfe Scoenvgeersiteioonnasnd.emorrhace vere Km ToMshtan otFtytchehahnagepsatoveeryeinsprmeasdenFoaen aSppvroseisaately fFocralyaceess of pcornogunagtion and esorshase vere : Table 4 (continued) Grons an Microscopie Tissue Gervations u f A r ED Grows chesrvations Wieroscopic coeervations 20 ma/ig mates (con's smsaay stomach BLaercgoenbaigeeckstsypots hf ers vep re focal axess of congestion and hemoesha Liver Mo signiticunt changes lInaotneiaocens mtheecrreaewiensre several smail fost of mssay 0 -- No significant changes RTheemrteenvagee ifnocatlheagreassstriocf mcoonrgens.tion and Liver iTcaarabanTdoon dete TaIdhoienlstaiiccoeunsd,tcoba"tlahaerarvreeevdsero--cefomscisonlaiypmvaislspcpifeaostnrteydneccthroousnbitese.sa asIn rSrparexpinaaealyfat0vaocfwotihees:hapatooyies had Fine Spleen Aepears pate Hasse not processed for exemtnation. mdaanaer seomacn Leaorgceenpiangcehesypots eEForrcaeatleLntearneioantsttaohtfeecavuoirmttghreinceticmorncooraienssd oahfnedsotahrteehssaeegvveeevreaielereareas I Taine memmrmasie Sastric vice 200 msg vemsies mTeahenaedey stoma Laargee dark ares Gemor ADehceosorrsehoafstcheulscaerstvraise pSprietsmeenittiwmi:th localised aacenats sack rmorzhagter) RReamtohteirnaegxetovnenrisveprceosnegnets.tion and some areas of amehay seomach Dsemmcaeebnhaegcekstspots Foctentofpceonrgepsotritonionandofhetmhecrgrhaasrterivcemruecopurse.sent Liver Mo significant changes arSecastetnPetec.iesdanifsonsgwtiiltoehcfianccioossnosmteoaftoifmomntohmonasecceiroocnsrtisocrvoelertesfoct. mwrses smn saver Dark xed Linas Wo significant chnges 0 tmiicant changes KLLA fatty changes vere present as approximately Ion of "a hepetaceien hat eviopiamiis Fat sales medsadsay 1) (none) miarlaear stomacn Dark rea streaks Fock ofecorngesrteigoionnsanotfhetmheorsrhuasgierivcesruechperne:sent mresye stomach Mo signiticant charges A anal, shallow bemorzhasic ulcer vas present. pheea ay -- LCamrsgremmblaapceketsypots Cmaeiprfeosceanltaicenssheofoceosntigieestmieotnonasn.a hemorshase mersseo 0 stomach hBienmodrutsnkagdiennes wo sismiticant canes 03710 EEkEe orn 200 mig remstes (eons dmoanyrs,csomtgaoruninnda5ted Liver ayo) mtera's 51 stomach arena aver sm{aonmacsoympound 5 Carminatad day 200 smtenas'eday 12) 25 mapag nates dmoanveu,coSmeproiunna9dted uve ay dmTyonns,ceotmhpeomuinnasated iver ayn smoinracsosmpound Liver dave,iorSerminated smdoianrmea,cooomtpeoruminnda5ted Liver aio arenas smdoanrv'se,coomvpeoruend 5 Arenate + niver alta 100 saovmne:coomspeoruminnda3ted Laver + wor able 4 (continued) 15 Gross 4nd Kicroscoptc Tissue Corervations rons comervations Wo significant changes BOlraecmkoczthianzeesr?) EOnrloamrcgrerdhagaincd) duck Mo significant changes tone) MeNircrwoswclopeichcmoesamrvtatiioonns oFaFtttyhechheapnagteoscyvteerse hpardesecnyttopalsasampipcroxfiatastveacluyol3e0s-.40 Yo atanieicant changes Atoss of congestion and hemorzhage vere present. KhLeidhefpaatttoycyctheasnawhsadvecryesapprleasmemnitc aastabvoauctuol1e0s8.of [== RE Mo Hunificart charges Mo significant changes Pale serous and foct [ep-- mtargea? Fae streak Mo significant changes a10c-u30r0a"o:f the hepatacyses has -- cyeopiionic Far K1i0l8eoffatthtey hcehpaantgoecsytweesrehpardesceynttoplasasmaippcrofxaitmavtaecloyrle psNlpoipdacesorixacitnefalatyteslveyavce3ur0oe%leofsfa.tttyheIcnhheaspndageetisocicoywnee,erseihnpraesnseceynttsoec.atnion ochroaaotpneiulcCalaterinoernscnueeviecrlrasolsicslsenvaaiinlti,hngiftonpcfaistlhtersaetcaiesorntuoofiafrSnlduiemcbekrriosu.s To0hnemsuuLlbeaastciiuootnesn ncIehncafrulorasvmiemsda.togrryAosrsMeliysmpdovtneesrseesvfaaosctalpreaosrfeensstscstoef adjacent to and vichin some Of hese Tock. Anreaes ofcorcvoenrgestion ant hesorehage vars resent Mo signiticant chases htLhieoaniatnraeedcsrsaomsociuhsne,tervofefrdeaqgcurueotnsetslly0ysvmceuobrosemcpuastntei.eemdsinobffylacmomnastuolrayvATpaiptcrtyoexsicmh:aantgeelsy a1s0-i2n0d8iocfatecdhebhyepEayttoocpyitaessaiehadfot re oan 25 meg ates (conte) (moni%c0o3mpound "Liver adayys,aterminated sm{iornsc0o4sgoua + ver aayye,acarmimated dm oanys,cos mtpoocumin, nda3ted Lgver aay a amTo7yu3to0ha6natisea Becphagus Adrenals a{yons,conCgooruminnda9ted aay 5s) sm(ioznzc0o8mpound 3 dayLso,ver + terainited day 10) 25 mg remsten smC(oiornzmicsnosamtpoodundday3 d1a01ys, . s(amtasday 100 Aacenals + m{oan scosmgound 3 Liver + days, carminated day 10) m(onsceompoun9d Mer 0 anys. Carminaced aay 10) sda{aoynns;scoSnogromiwnaasted ain) 16 Table 4 (continued) Gross and Kicroscepic Tissue hrervations Gross cbeervacions fe -- Mo significant changes M1i-n2im0a0l tfhaettyhecphaatnogceystesverheadprceysteonptlasamsicappartoxvisaactuelo Mo significant changes hWienpiamtaolcytfeasttyhadchasnygeeosplwaemrmeicprfeatsevnatcsaosle1r-21 of the Mo significant changes EApEprvoaxciumoalteeslyin1d-i24catoifngthmeilhaepfaatiocrvyiecshahnageds.cyeoplas apnutnecrtiuorre thoolethojruasctic Sets leads to large in- No tiasve saved einctloattiecaxsiulblcauta-neionutsubaarteison Deanrlkarg(erdemorzhagte), Congestion and hesorshage ere present. (nena) "Mo aigniticant changes 1K0i014offtahtetyhcehpaantgoecsytveesrehadindciyceaotpeiadsmaiscapfourrovxaicnuaetteelsy (none) endacgear Soasible pale ares pale stress (sone) iConngTehesticoonrtaexndanfaocmaledhiensao.rehages were present No significant charges h`ceougluelsaitoinosn OF in acute ctnboescersrouvsbeisdscutagecrcoiosmsnplfayinuivmeeardteobryfyociaenlxfuidlaatrteresastioonf 3 Tobe 4 (comciotess rons nd Micrncepc Tansn comrvasions Ei ow emai mene 25 maa vemos com T mfionrsseooE on 3 saver ear 11 RA. S my aceeveree focat eaT as of ch oupleteseucerote adeannie dP oonreoT 3 Novas aniarges Ro Hioniticunt chugs Yo ianiticans ahargn TThaozervase mpinieasltfaattyachaTnrgees asTapprosimetely a mn moinknoseanscsverunrdh9sass,. [-- fra mayL o tam Ce . aS mroayrseeL ener -- SonIcomEgenna sw+f [-- SmonnsIomonTna 3 one 03713 ste vo WeeoRkkepaOeartasbroonasng%1Eeeatf0iamncaiktntsga Study imate teceessouy -- Ce SI em teet B e ---- 1 O e ----a --n ----e ----a--CR--aT--Rn `-- ScEkedeu-- pespeacance wE 2w23iE o4lmrmE dioma e HUiormininearyfosamcaotnatiionnetnce Ek wwo eeeddmeSee5w3wom1 Unkenoe. appearance Deaths W w0w0e0w16331 2 200 my/xq/eny vemos ANJ ipcpnkeeaar-er udpnsoprpmea-- nlcance TiBonlnkokeocsspySrnoorseveseansinaodmnmteouth Tupersctive suathe 25sapa/enywares feoSFreo-- cnkediue-- epesppencance Bevoravseont tem mWm12o1me1m2 moo1l1wE6 6 wNwE rNE2 Nm 6E6 N4lE5 m1NE4 omO h 2om3N o3eE o11 m mC owCm1oTCN ww1Nm oWE wEEewWNC E TWm wN E TNwEE YwWE A WmEEE EE mwmo1Wwom2 fwmedm e WwdmNmomweema owwn mEoi Emma hea WW mw ew 0000 2411 13 mm a Ne ee 01100000000 mW 2w2smw 9w39 27lw 4sism4]31 1 l 31e1 Twme1sm1 eece1 we1 eEw Se1 e ee rr 5 Table 5 (concluded) v0 MeaRk 0Sex1hpbomoraeenEnoieanfnieonctmitannsrgs sy i Rerertearey -- Retest T1 S 7 34 5E6T7T8 ST5 1Rm0 Nm11 1P2 T13 W14 E15 R16 E17 E16 1E9 E30 TFEB ES w[in ---- E mw ssm w 223 5e 3 53 3d0wme 0w 3i3a 3w 2323232e 212220: iTTSnoiokcsnoeissmcytpcroenAnmopvousurselsssatinadonnmcsoeuth impon Tortside NE E WEE mNEE EE NEEENwEmENA EweENEeNEeE TeWE1oNwE1E R 1lENdmEm NwwEoNmmE E NREERMwNREMwNE NbEe wNbA EeA NmE NE aNE NE seats 0 000 00 Bo Ro 00000000o0o 25 ma/kg/day Females Aeepkaneairea naoprpmeeadrence Eerpicatory congestion mE WB oEwEmoEmwmmmr oEe3 7w45w45a416 e w 4552o 14 1w 3 i 23ww 3 mm 2w 2To 2T w 2s m E 2aw Tm aa! ClTnrokEneoicrT/etosornnpisecncrodanenvncuslesnion M[EW E NEEEmEEWEEENmEEEmNEEENE mEEwNEENmEEwI1 NH wE NwEE NwE NEA1 NEH AN1 E NE1E NE N1 AEH hNE1 wNEoN1 E NE -- Deaths 00000000 6r0r0r000000000 BS 3e cE=omvmmoie tsmeivvie.daenpsisodes geerally cccureed ducing banding (dosing, weighing, ote.) ie IEpive outhanatized, one found dead " 20 Table & Two Week Oral Rangefinding Study of 7-2509C0C in Rats Summary of Clinical Observations Number Affected/Group Csliignnical 204 No signs through study onz Treatment-related deaths 12/12 Soft feces 8/12 Tucked-up appearance 1/12 hin 6/12 unkempt appearance 4/12 Tonic or clonic/tonic convulsions 6/12 Hyperactive 0/12 Head tremors 0/12 Urinary incontinence 112 Respiratory congestion 0/12 Bloody nose and mouth 0/12 Lump on left side on2 Hair loss on abdomen 0/12 D2o0s0eG row(mg/k7g5/4day TE 0/12 112 112 10/12 0/12 112 0/12 2/12 oz 12/12 6/12 6/12 1/12 e/12 8/12 312 8/12 6/12 312 2/12 112 112 o/12 112 0/12 112 0/12 312 0/12 0/12 0/12 712 1/12 112 1/12 0/12 0/12 112 0/12 o/12 012 1/12 03716 able 7 No okof O7r-a2R5an0go5ifni0nRd0aitnsg stucy Group Mean Body Weisnts Gar.ow e = T m5y mm = To T LaeomSs noEot 20mm S1sn aamwen m1eMe ae en an - 20mAg/eys 1304 169.208 m1o4s2 aMomoes Hidsde wsoeosn oy an Teeny 1f1m3 awame 1w0e9n smeRm uHseam aaaram ahessae os an T-- Smafosday Se17356 1d6o3e.7s Quneo 1n2s9.e5-- % heeass rdemear s10s.7i oa ae |: 2 vDoosisntg ddiissccoonnttiinnuueedd ffoollltaodtnngg adaayy 5 2 8 Sg 2 Discussion Ddoasialgye-gasatndricsexi-nrteulbaatteidonefoffecrtast.s with The Tp-ri2m5a0r9yCoCeffreecstusltweedreingatsrteratimcenitr-r,itation, handling-induced responses to the convulsions and gastric effects mwiecrreosacotpuicckedl-iuvperapcpheanagreasn.ce, Doasnaguen-kreemlpatted raepsppeoanrsaenscetaondthseofgtastfreciecs;effoencltystwheermealtehsinhneasdssaonfdt bfoedcyes.weiTgrhetatlmoesnst-druerliantged dosing. stomach iTrhreitafteimaolneswhilcohstimnocrleudweedigahtfewtheaanrltyhehmeamloersr.hagGircosgsastarnidcmiuclrcoerssc,opwiacs porcecsuernrtediinntmhoerehigahnimdaolsse girnoutpheonhliyg.h Convulsive episodes (CNS stimulation) dose group than the low dose group and meofrfeectmsalweesrethahneafdemtarleemsorshaadncdonhvyuplesriaocntsi.vityL.essDofsraegqeu-entandtresaetxm-ernetla-treedlatleidveCrNS cthoaxnigceistyooccccuurrrreeddininmotrhee mfaolremsofthafnocaflemanleecsr.osis and fatty changes. The liver Dosage-related deaths were caused by T-2509CoC treatment. More high dose males died study. than high dose females and the males started dying earlier in the The recovery interval resulted in reversal or cessation of the toxic effects. Gastric lesions were not present in the two surviving high dose females. Gastric pain (tucked-up appearance) continued for the first few days of raneicmoavlesr,y haonwdevaerf,ewhaadnibmoadlys wceoingthitnugeadintotahprpoeuagrhouutnketmhpetreancdovetrhiyn.intAelrvlalr.ecoTvheery later-occurring deaths in the high dose female group were probably due to a generalized debilitated condition because none of these animals had microscopic cLhiavnegresn.ecroSseivserweasdenlaoytepdrecsoennvtulsiinontshedirdeconvoteroycacnuirmadlusr.ingTrheecolvoewryd.oseForceaclovery group had more males than females with mild fatty changes in the liver. 03718 23 Appendix I Two Week Oroafl7-R2a5n0g9eCfoiCndiinngRTaotsxicity Study Protocol TITLE: P(rSottuodcyolNumfborera1T7w9oRR0W2e3e)k Oral Rangefinding Toxicity Study in Rats TEST ARTICLE: A clear, chemical pale will byeellsouwpplsioeldutbiyonToixdiecnotliofgiyedSeasrviTc-e2s5,09C3o4C.CompTahniys, st. Paul, Minnesota. OBJECTIVE: Tihneraptrsimadurryinogbjetcwtoiwveeewkisllofbedaitloyigdaesnttriifcy ainnytumbaajtoirontoofxiTc-2e5f0f9eCcotCs. hBoewfeovreer,thethedosaecsutweilolrableALsDeSlOecotfedT-t2o50c9oCmopCletien the above rats will objective need to be dseutpeprlmiiern,ed.straTihnisanadcuatgeeporratnigoenowfilrlatsusaesmadleesscroinbleyd from the same below for the rEveapleuaattidoonseMepotrhtoidon6.05ATwhiellgenbeeraulsedprofcoerdutrheesacfuotreadoRsikienrg Safety except that susiex mtahleesa/cluetveeldawtialltobeseldeoscetd.theThdeosasgpeonsloervelasndfosrtutdhyedtiwroecwteoerkwill repeat dose study. Atthethsetudcyondcilruescitoonrofhavtehethtewoowpetieokndoosficngontiinnteurivnagl,thetheobsspeornvsaotrioannd pexetreinoddedfoorbsaebrovuattoinoen-phearlifodthweilmlalebse and two wfeeemkaslesinatleenagcthh level. The and dosing will not occur during that interval. CONTROL ARTICLE: None SPONSOR: Toxicology Services, 3 Company, St. Paul, Minnesota TESTING FACILITY: Ssta.fePtayulE,valMiunanteisoontaL.aboratory, Riker Laboratories, Inc., DOSING INTERVAL: September-October, 1979 DOSAGE LEVELSb,ecaRuOsUTeE,itGRaOpUpPe,arsSIZtEo,beEICa.n:appOrraolprigaatsetrriocutientfuobrateivoanluwialtlinbgetuhseed systnic toxicity in rats. The rats will be dosed daily for two weeks (1) Alevceoln.staDnitstdoislelevdowlaumteerowfil1l0 bmel/kugsewdilflorbeallusnedecfeosrsaeraychdilduotsiagoens of the test article. 03719 Appendix I (continued) 2 Dose Group. High Low Dosage Levels (2) a(fttoevre tsheeleacctuetde pcoormtplieotnedh)as been Group size 124,129 124,128 ANALYTICAL CHARACTERIZATIONS: The sponsor has on file one or more of the following or other pertinent characterizations of the test article: identity, synthesis, strength, purity, stability. Ptrheepatreasttioanrtiacnldemweaislulrbeemenctonsriecdoerrdesd afdoerqutahteevavreiroiufsicdaitliuotnioofns of croanntgeenftindainndgcsotnucdeyn.tration of the dosing solutions for this TEST SYSTEM:01dTwoenntdyo-sfeoudramyal1,e wainldl2b4efuesmaeldefCohrartlheesreRpievaetr CdDosreatsst,udy4.5-55Thedays (3) acangiemsalswiwtihllwibree mheosushedfloionrdsiviadndualflryontisn.haTnhgeinrgosotmawiinlllesbsestteeemlperature and humidity controlled with the lights on a 12 hour light/dark cycle. JUSTIFICATION FOR SELECTION OF TEST SYSTEM: Charles River CD rats will be used because of historical in-house data on the strain. TEST SYSTEM IDENTIFICATION: Each animal will be assigned a number which will be indicated on the outside of the cage and on an individual ear tag. RANDOMIZATION OF TEST SYSTEM: The animals will be indiscriminately removed from otfhecasgheisppifnrgombolxeefst btyo ArniigmhatlsCtaarretipnegrsaotnntehle atnodp palnadcweodrkiinngthedowrna.ck The study director will assign dose groups by vertical rows. DIET SPECIFICATIONS: Purina Laboratory Chow and water will be available ad libitun throughout the study. BIOAVAILABILITY OF TEST ARTICLE: During previous acute toxicity studies in rats, systemic absorption of the test article appears to have occurred in the form of delayed CNS effects. CLINICAL OBSERVATIONS: The animals will be observed daily throughout the dosing interval for evidence of treatment-related toxicity. Body weights will be recorded approximately one week prior to dose day 1, on dose day 1 and biweekly thereafter. Food consumption will be eresttaiimnaetdedfwoerektlwyo atdhdriotuigohnoault wteheeksdosfionlglowiinntegrvdaols.ing,Ifthaenyimwaillslarbee observed and weighed as indicated above (4). 03720 Appendix I (continued) 25 TISSUE PATHOLOGY: during Gtrhoessstundeyc.ropsIineasniwmialllsbweitchonaduutcotleydsiosn, alals rats which a minimum, die bseamfpliexsedofinlfuongr,malliinv.er,Mikicdrnoesyc,opibcraiexnamaindnatainoyngwriolsls dleetseiromninweillif itnheatmiosrsiuebsundareconsdaittiisofnactsohroyuldfobrehitsetromliongaitcedditoagnionssiusr.e sAantyisfraacttory tthiesssueelescatmpeldess.urvAivpoprrsoxiwimlaltebley k2i4llheodursforfoglrlooswsinngectrhoepslyastexdaomsien,ation. The any aarneimasleslecsteelde,ctweidllforbeadkdiiltlieodnaflorobgrsoesrsvatnieocnrowpisythoeuxtamidnoasitnigo,n if at tfhoermeanldinofarethaltisitendtebrevallo.w. Samples to be fixed in 108 buffered 12.. EMyaemsmar(y2) Gland (female) 3. 4. TThhyymruosid/Parathyroid/Trachea/Esophagus 65.. HLeuanrgt 7. Liver 6. 9. AKiddrneenyasls (2()2) 1101.. UTreisntaersy/EpBildaidcdyemris or Ovaries 12. 130 Uterus or Prostate spleen . 1154.. sPtaonmcraecahs 16. 17. SLmaarlgle IInntteessttiinnee (at least two areas) 11980. MsecsieanttiecriNcervLeymph Node 22100. SBrpaiinnal Cord/Bone Marrow (cervical and thoracic) 22. 23. pAintyuiGrtoasrsy Lesion Tsheeletcitsesduebsy tthoebepaptrhoocleogsissetd aantdtheexamcionnecdlushiiosntoolfogithceallgryoswsillnecbreopsy. pTrhoecetsissisnuge s(5e)l.ections will be reviewed with the sponsor prior to DATA ANALYSISwiAlNlDbFeINsAeLleRcEtPeOdRTa:t tThheecoanpcplruospiroinatoefsttahetissttuidcya.l aTnhaelysperso,posiefdadnya,te f(oarpptrhoeximfaitnaellyre2p-o5rtmonitshs1-a2fmtoenrthnsecraofptseyr).slide reading is completed 03721 2 Appendix I (concluded) AMENDMENTS: 1. 2. 3. 4. 5. The was last day of dosing day 5 and the last for the high dose day for dose group (200 mg/kg/day) the low dose group w(e7r5emgo/bksge/rdvaeyd) fwoars adpapyrox9.imatAelllysutwroviwveienkgshifgohlldoowsiengantihmeallsast dtohsee.apprFooplrlioawtiengnucmobmpelreotfiomnaloefsdoasndingfemataletshewelroew dkoisleledletveol reach a total of 6/sex dead. served for approximately two moTrhee wreeemkasi.ning 6/sex were ob- The selected selected low high dose dose level was 200 mg/kg/day level was 75 mg/kg/day. and the Age day range for the of dosing. acute rats extended to 59 days old on the Body weights obtained rather than biweekly. once or twice a week during recovery The tissues processed for liver, adrenals, stomach, hkiidsnteoypsatahonldogbiracianl. evaluation were 03722 2 Appendix IT To Week Oroafl7-Ra3n5g0e5f0i0ndiinngFaTtoexicity Study List of Principal Participating Personnel Nene Marvin 7. Case ames D. Henderson, Ir. Cathy 2. Ludemann Gary c. Pecore Tnara Porietis Jan H. Skeons G. Ray Steffen Function Veterinary Pathologist Stat veterinarian Coordinator - Clinical Pathology Coordinator - Anisal Care Histopathology Technician Toxicology Technician Study pizector 03723 DISTRIBUTION LIST M. 7. Case X. L. Ebbens F. Db. Griffith W. C. McCormick R. A. Nelson G. R. Steffen (2) 03724