Document g21eVaY2Bnbk7LaKNvqkV9awN

AR2IG -- 1340 8-2 Telomer B Alcohol Repeated-Dose Oral Toxicity Range-Finding Study in Rats `Work Request Number 13690 Service Code 1583 LT0E3E8 = == = &S - Data Summary 12 Summary "The data in this document are from a range-finder study that was performed in order 10 set dose levels fora 90-day subchronic toxicity study with 8-2 Telomer B Alcohol. The data from the 90day subchronic toxicity study are located in Haskell Laboratory Report 9478. Theprotocol and protocol amendments for the range-finder study are located in appendix 1 of thisdocument. In the 8-2 Telomer B Alcohol range finder study, male (10/group) and female (S/group) rats were dosed nil the attainment of steady state blood fluorine levels, and allowed to recover for 75. dayspost-dosing (5/group, males and females). Serial bleeding for analysis of total fluorine and parenumetabolites was conducted during the dosing and recovery phases of the study. Blood fluorine levels reached steady sate between test days 60-84. At steady state blood fluorine levels, the liver and kidneys ofS males/group were weighed; females were not evaluated. At necropsy, liver kidney, and fat were saved from males and females for analysis of parent compound. PFOA, and PENA. In males (on test day 81), mean body weightsweresignificantly decreased and relative liver weights were significantly increased i rats administered 125 me/kg/day 8-2 Telomer B Alcohol; kidney weights were not affected. After 75 days of recovery (test day 159), relative kidney weights were significantly increased in male rats administered 125 mg/kg/day 8-2 Telomer B Alcohol; final body, liver, and testes weights were not affected. In female rats (est day 159), kidney (absolute and relative) and relative ver weights were significantly increased in rats administered 125 mg/kg/day 8-2 TelomeBr Alcohol; final body weights were not affected. In addition, pooled plasma samples obtained from males and females over the duration of the inlife phase of the study were analyzed for parent compound, the 8-2 acid, PFOA, and PENA. At steady stat (test day 84) pooled plasma were analyzed for PFOA, parent compound, and total fluorine levels. At steady state, the level of parent compound detected did not exceed 0.074 ppm in cither males or females. In males, PFOA represented approximately 85% of the total plasma fluorine detected at test day84. In females, PFOA represented approximately 5% of the total plasma fluorine detected at test day 84. The 8-2 acid of 8-2 Telomer B alcohol and PFNA were also found in both males and females over the duration of the study. % Study Parameters "Test Species Dose Adminisation Body Wight Clinical Observations Male & female Crl:CD(SD)IGS BR rats eAlsltanDiamyal0s-1verstadiosnemdenutnoftsheeaadsy sdoisee egrsotudpasyi89n,d steady state for total fluorine in the blood. Tpowricweenpkersweeekdaot 3s4wdaeypimervlas uring dosing one: avery weighing Serial beeing or fluorine a{nApdppraorne.nt2/mheetsaaboflritdeosanianlgy)sis senpeythrstte Palogy) Sferloemctmtisausaitlsaimepsaldeycsoaltleecbtiloonod fluorine levels Predosing bleed. Day (4) DPuorsiindgodionsgiDnga-y D3a. y141, &3w1ee.kl6y40,5&.7,5 Bsloouopdafonr psaerpeanrta/tmeedtaibooliptleaasnmaalyosnisd pRoBoCl.ed per dose mSemkagledsaydhoes0e5,ro2u5p,s asnadc1r25ifoincesdt wdalygh81ed(lifvoerr,ukuidrneay,ratndyfaotftipsasrueencmolelteacbteodliasnd). fSreolleolcotmitnsgrscssiiemnsiadlnmtpaoltoeomnacsosolflebcltoioond lone evel ddAolanlyea7tg5sr(oneutprh,se K0e,e5e,ys2.5,aoannnddpFo1tr2tt5dimosnleskgolmday colcted and weighedfor futuranaloyfsuoiinse levels). ig Results 16 ATRTA T SAY T| = wig ui |{ . Ee _ TouSood ineAisi Ferae tsSadwi Teor52 cn . | | = 17 | - = Sg Bo: 82z ofl 8Oe 05z9g0 Sn 7o 82 8| 3z |g I E1595 338 7 |Z " i= | zs 8 8 2IN I|5 EIFE2 38iA 3 5% = FE " E3 li= e 5 l E a <3 gsg3 8 | = EE I Sg iit szg 22 [3c3e3 z 2 o E|3% = si a g[Ef =5 si s0: os5 fzfiiss 52 & 3 Bi Sl, Le ase Hz EE82SE5EREcs 9:S 271|s= $233 8 2 EE es: ets E g 372$ 3R 23E2% N8gEE ES$R33E38 2 |E 5P 2E $|2E =7l E:Pz: s 2Esf 8Rw2Eg5 fzT =e55d 8d& } z 13 g 52aN El e 3B 33 2E;8 z = 2 2 a1:$$38Ff8g si: Eig5 233z2 g , 2% IEEE 2 3 = f 3 2 oo 88 8s od 8 f&0 LiE fatEH BEE 2 ~lg5%s & elis pg ogs CE3 Ee5i8 d EE Zs w 8= =5z2 s0&z8 38 2 gr fi 3: 3 2 a = I]s 2 2 5 3o = of of 8g Se FE = El = a" " < 2z 19 2 = 3 3 &i E2F g lw:f v5F oggow fsF oflgsfL Io 2 |5 5 &SPEE =z 8 = sligPyeg zg fsTS 522 53 3I gfFEFs == | eg ~ 8 = S|2<%f i i 5 =8 2 Ee 2: o3 %3 25 z =|28 F - Z(8% 2= _ ~8 25 2a S5732 Z= 3ys 5 33 85 z2l5i%z 5 38 & 3 )2 | fz lls. il3 w L30s% 2 18932 5 |2 AEIE8 318%:=3 x=3 |Z fg:: ERATE z = 3 |3 S35 = 2 23 85 g g 3 ocfie Z |= 5 52 | 2 22 9 S |g 4 :Eo ES 2 = 8 les 2 g | WE 8 Ss LEE8 =8 Fs Sgll33222% Sos & J i3: Flees aSHllEziacHde 2 %|is S lang Je: . HE $ HE 3i 5 : BE S 29wl2 zg |% 2 8.2 2 EE |e i|g- IEEE 8 oS |= SE | ~ zgz= |* 28= i 3 | (S 2 gg 3 es o| E i :zio:3|3 3 &| 2 g cl gl H3IE:3 5| & : &lez Analysis of 8-2 Telomer B Alcohol, PFOA, and PFNA in Serum and Selected Tissues Pooled plasma samples obtained from males and females at steady state (test day 84) were analyzed for PFOA, parent compound, and total fluorine levels. The level of parent detected did not exceed 0.074 ppm in either males or females. The following plasma levels of PFOA were. detected in males and females dosed with 0, 5, 25, and 125 mg/kg/day 8-2 TelomeBr Alcohol. Male | Male PFOA| Male total| Female| Female| Female PFOA total Dose Group| (mgkgday) | PFOA | converted to | (ppm) | ppmF | plasma | PFOA| converted| plasma F F(ppm* | (ppm)| toppmF| (ppm) 0 007 005 NM 00 00 NM s |2 219 151 sar | ss NM 008 | 00s NM si [ose | om | sss 125 6560% | 45.16 24 fuss| 1 2.20 "Determined using Wickbold Torch; *mean of two separate runs; NM- not measured Based on the data presented above, the PFOA in males accounts for a large portion of the. fluorine present in the plasma, however, this is not the case for females. While what is in the blood in males is predominately PFOA, it is not possible to determine what percentage of the 8-2 `Telomer B Alcohol dose is converted to PFOA (c.g. information is lacking on mass balance and biodistribution). Further, it i loss certain as to what the metabolites may be in females as both parent and PFOA were present in low levels and did not account for the total fluorine levels in the blood. 23 "Tissue samples (Fat, iver, and kidney) obtained at necropsy from males and females at steady state (est day 81)were analyzed for the concentration of parent compound, PFOA, and PFNA. "The data are summarized below. `Tissue Analyses from Range-Finder Study (Test Day 81) hE een aan ies 182Telomer BAlcohol(mgkgiday) | [Cem[fT a T |_ s oor@on|TT To2m5en||] iwmsos|| Fe TwieeeTTTw omom|ocnorooo|| oowmeen]] Twa Teiom| sa@io] ia] T[Kw idney | TT0o0s0i0e0m0|| oo1vrt0oemn|iosmsaeme]| Serum samples were collected throughout the in-life phase of the study and pooled within cach dose group (per sex) for analysisofparentcompound,PFOA, PENA, and the 8-2 acid (CuoHhOFy). Since there is no standard available for the 8-2 acid, we are unable to quantitate or positively idenify this specics in the plasma (fat, liver and kidney were not analyzed). However, there is an unknown at mass/charge ratio of 477, which is consistent with the 8-2 acid. This 477 species appears o have a dose response relationship, futher supporting the premise that this species is related to the metabolism ofthe 8-2 alcohol. Since ti not possible to quantitate, we have presented the data as area counts rather than absolute concentrations. Although we cannot quantitate,ifwe assume that the response factor for this unknown metabolite is approximately equal to the response factor for PENA, then the concentrations would be in the low ppm range (i. mor similatro PFNA than PFOA).Thedata are presented below. ab Pram 0A Concanatonin eleFs - 1 [I | | gal | $f2 .= [~z ie--rOmram nga |||| | 84) [Se esmoro | l eT ee r e mrm re ee s Tea | =- lz iEd PlasmaPFOAConcentrationin Female Rats. | aro|ra|| amon |Smoo/day | o EAE f y er. | oh eee -- _ a5 Plasma8:2 Telomer BAlcoholConcentration InMaleFats. =- | Zoo] | Sod |Lnsefe eTTr | 8 | I ] [Smo|| {a zsmonote | [e125moray | ----r---- 2 HiE$=0 fe tr J -- a rr , ay [[g5e]l | &,. |y P E=rErr || . === Son re | [-- | wo, } Pp 7 | LI | | eH e Cree hS neon ea od = | 12r5otor ||| 217 IB Pisa Mote77Concentration n Mal Pats yi | 20000000 1JT8r0ro0c0co0e0n0dn|+ | 12000000 pr] Sone rere melS 0 e 7 m e omm kor mm mog ew Tondo || Te [Series seesol -- [ | on | otoeocoonom0 | 2 10000000 | | 2000000 | rememe [= 4000000 SN | CT Hm me ee ew Testday | T_R ---- | --a--25mphkg/day | 125moody | | 28 Appendix| 29 -_-- DeuPomnt63s57 8:2 TelGoamveaBrgeAlRcaonhgoel-FiRnedpienagteSdt-uDdoysienORraatlsToxicity `Work Request Number 13690 Service Code 1583 Protocol `DuPont Animal Welfare Committee Number: CBT-075-P ' `2GovaTge ReingmBeAFelincdoishnoglSRuedpyetineR-aDtosse Oral Toiciy Duponi6357 TAOFBCONLTENETS Page INTRODUCTION tse 3 OBJECTIVE... SPONSORANDTEST FACILITY ccd STUDY DESIGN sss d MAATTESUESUABSRNIEDNIMCEETAc HOLDSSes c sesn oer 6 CB. ATEMSUASPEHCUISEDSAc NMIY co os o et 6 6 FEE AE SSEMEN.10 GIOUPS..c E ooo eeesereosoes F. G. DAodsmiinnigsStorlauttiioononfPrDeOpSaIrNatgioSnUaSPnEdNSSIAONPSIN.g..............o.o.r.s.es.o.e.s.o.s...r..e-e..r..orr 8 1H. BCOliAnYicaWlEHOBAbISservaanc tdMiorotas nlisty s ...._...__________.......mm 88 KJ.C. ABNlGoIoOdMCIoClAlIecPtAioNnOfIorOBToYtav l Fluando oParrenUMietanbc oleite Evaluae tion ..................9 SAFETY AND HOUSEKEEPING vn 10 RECORDS AND SAMPLERETENTION... ssn PROTOCOLAPPENDIX snes 11 _-- 2 31 G8a2vaTgeelRoamenrgeBFAilncdoihnoglSRteupdeysitneRda-tDsose Oral Toxicity DuPont357 INTRODUCTION `iTmhteertmeestdisautbestianntchee, p8r-o2dTucetlioonmoefBrnuAmlceorhoouls,oirsgaannoofrlguaonroifnleuoprriondeucctso.mpTohuend5,u2s5e,daansdan s1u2b5chmrgo/nkigc/dstauydideossewiltehveslismifloarrthliusosrtoutdeylowmeerre-bsaelseecdteadlcboahsoelds oanndthpeertfolxiucoirtoyocotbasneorivceadciind a(dPmFiOnAi)s.terSeidgn1if0i0camngt/lkygl/odwaeyromregarneatbeordyofwseiimgihltasr fcloumopraorteeldotmoerc-obnatsroeld awlecroehoolbss.erLvievderinanradts lkeivdenlesywofeifglhutosrowteelroemearl-sboassiegdniafliccoahnotllsy iasnclroewasaesd 2c5ommpga/rkeg/ddtaoy.conFturrotlheinrmroatrse,addmaitnaissutgegreesdtdotshea.t rtheepePatFdOoAsemesttuadbioclsi,tePFmOayAaactcduomsuelsaotfe 1in0amngi/mkagl/sdadoysoerdlwoiwtehrtphreo8du2ceTdetlhoemfeoBlrloAwlicnoghoeflf.ectIsn: hinocrrmeoasneedlleivveelrs.we`iTghhetsh,igihn-cdroesaeseldevheelpaotfic12c5llmgp/roklgi/fdearaytifoonr tahnids rbeatnageo-xfiidnadtiinogn,staunddyawlatesresdelseecrteudm sbiamsieldarolnutohreotabeilloimtyero-fbraastesdtoaltcoloehroaltseaand10i0s emxgp/ekcgt/eddaytodopsreodleuvceltoixniscuitbychwriotnhiocutsteuxdcieesssiwviet.h w`mhoirltaelitthye. 2T5hmeg/lkogw/ddoasyedoosfe5 imsegx/pkge/cdtaeyditsoeixnpdeuccteedmitnoibmealthteoxnioc-itoyb.served-adverse-effect level, OBJECTIVE, 9`T0h-edoabyjoercatlivseubocfhtrhoinsiocrasltugdayv.agTehreaonrgael-foiuntdeinogfsatdumdiyniisttroadtieotnerwmaisnesetlheecdteodsebelceaveulssefiotrisusae ina potential routeof human exposure. SPONSOR AND TEST FACILITY "tThheisdastteudtyheisspsopnosnosrorseudbmbiytttehedTtehelowmoerrkRoensetahrecWhoPrrkogAruatmh.oriTzhaetisopnonFsoorrm'.s aTphperosvtauldywawsilefbfeective. Ec.on1d. udcutePdonattdHeasNkeelmlouLrabsoarnatdoCryomfporanToyx,iNceowloagryk,anDdelIanwdausrter.ial Medicine, 3 32 i: 3 i I: so: i Fp ot = 3g 2 E 22 223 oF 3 cw a 8 tH Tk | 38.538 2 3 sz gift ff] i "e. 238 ss. es. s. zii |3 i4f | 3 FR | |: 3 z 23 |2 gx 2 333 |2 3% z - WE8.28,.28,. 2872 ,i5r5s% &: `Ls FESEEzEESRESyf1iil3g5i5s #235 23 3s Loe = = [feMiiEieEs Bos: 2 FC 7 Cong H& I8oz 3 x =- o= s F= [I|zFEz EiFRiaNiSz | 2 id SBs E3 Fds = _oso -5 |UdLEFLEEE: 3% t TT . 33 [ibkaei iEy i [731 HEH zt 2z 5B5i35 a3 3:f: | HH HE || 55| BF sist 1 af | 3 min th Tex | 5r fdEis g3Eglilz|Ede | | EE sri 93]58 Bip28ysd 188582323 iol:ii : LE 8 noi If ERNE I li 4 BL HSgE L FfAfRsT: i[[1TFEh2 3k 8:2 TelomerB Alcohol: Repeated-DoseOral Toxicity OnogeRangeFindingSudyinkas~~ pwemo MATERIALS AND METHODS A. Test Substance `LTahbeorteasttosruybNstuamnbceesrw2i4l69b1e.supplicd by the sponsor. The test substances) were assigned Haskell B. Test Species rIMneacp.loret".TahnTedhafededmCrarelsles:CCoDrf*lt(:hSCeDDs)*uIp(pGSlSDi)eBrIRG(ScriaBttyR/hsatrstatebs)ewewiinllllsebbleeecodtboetdcauoimnneetndhtfeerdboamisneCsthhaoerflseetxsutdReynisvrieevcreoLreadxbspoerarantidoernfiicneeasl,with stehnissisttirvaiitny,atanHdaslkoewlliLnacibdoernacteoroyfanspdointtsansueiotuasbidliisteyasweist.h respect to hardiness, longevity, C. Animal Husbandry rAlolomastswwiilllbebemahionutsaeidneindsattaaintleemsspesrtaeetlu,rweiorfe-2m2e+sh3caCgaensdsuasrpeelnatdievde ahbuomviedictaygoefb0oar+ds2,0A%nimal lAingihmtadlarrkocoymclse.willbeartificially illuminated (fluorescent light) on an approximate 12hour All Inc. zCaetrtwiifliledbeRopdreonvtidLeadbtDaipetw 5a0a02dtalidebiltirubimt.umA.ll rats wilbe fed PMI Nutrition Internationl, fAosllsopweciinfgiepdroicnetdhuereHsasakreelpleLrafboorrmaetdorpyerainoidmicaalllhyeatlotahsasnudreetnhveitrcoonnmteanmtianlamnotnlietvoerlisnagreprboeglroawm, the those that would be expected to impact the scientific integrity of the study: aWnadtoetrhesramcopnlteasamirneaanntsa.lyzed for total bacterial counts, and the presence of coliforms, lead, SFaemepdlesasmpflreosmafrreesahnlaylwyazesdhefdorctotaal bgaancdteecraisgale, srpaockrseaarnedafnuanlgyazledcotuontesn.sure adequate sanitation by the cagewashers rCeerqtuiifrieemdeanntismaanldfneoetdtios eusxecde,egdutaartaendtemeadxbiymtuhemmcaonnucfeancttruarteiorntsoomfekeetyscpoencitfaimeidnanunttrsi,tiionncalluding pspreecsiefniceed ohfetahveysemectoanltsa,maifnlaantotxsinb,elcholwortihneamteadxhiymdruomcacrobnocnesn,traantdioonrgstaantoepdhobsyphtahteemsa.nuTfahceturer Would not be expected to impact the integity of the study. --- O00 0s6 35 8G:a2vaTgeelRoamnegBreFAilncdoihnogl:SRteupdeyaitneRdaDtsose Oral Toxicity Dupon357 "lTahbeoraantiomraylanheiamlatlhvaentedriennavriirano.nmDeanttaalarmeonmiationrtianignepdrsoegpraarmatieslaydfmrionmisstteurdeydrbeycotrhdesaattnedndmianyg be: included in the final report at the discretion of the study director. D. Pretest Period `quUaproanntairrniev.alTahteHarastkselwlillLabbe:oratory, all rats will be housed 1percage, sexes separate, in + quarantined for a minimum of5 days. identified temporarilybycage identification. + weighed at least 3 times during quarantine. + observed with respecttoweight gain and any gross signs of disease or injury. "The rats bases of wbioldlybweerieglhetasseadndfrcolminqicuaalrasnitginnsoefbayllthreatlsa.boratory animal veterinarian or designe on the. dRiastcsatrhdaetdarweiatchcoiudtennetcarlolpysyk.illRedaotsr trheamtoavreedfofurnodmdsetauddyordusarcirnigfitcheedpirneteexsttrpeemriisodduwriilnlgbtehe pretest ddpiiessreciarosedet.iwoinDleopfbeetnhsdeeensntttutdouyPpdaoitnrhetochlteoorsg,eyafaipnnaddtihgnogilsvo,egnifsuatr,tghroeorrstsdhieeaxglanaombsiotnriaacttoiprorynocatenodicumhraeelcsvkmetfaeoyrrintbahereieapmnr.pelsoeTynhceeedroaeftsutlhtes will not be reportedi the final report unless considered significant to the evaluationof the study. E. Assignment to Groups cRlaitnsicoafl csaigcnhssoefx,disseelaescteeodroinnjtuhreybwaislelsboefdaisdterqiubautteedbboydycowmepiutgehrigaziend,anstdraftrifeieeddormanfdroommizaantyion diinftofesrteundcyesgraomuopnsgasgrdoeuspigbnoadteydwienigthhet SmteuadnysDweistihginn, asosext.hatTthheerweeairgehntovasrtaitaitsitoincaolflysseilgencitfeidcarnatts will not exceed 20% of the mean weight for cach sex. wAifltlerinacslsuidgentmheentuntioqgureouHpass,keclalchanriamtawlilnlubmebheoruasnedd tihnediuvniiduqaulelyi.ndiIvnifdouramlatiideonntoifnictahteiocnagneumlabbeerls aosnstihgenetdailtoofeaecahchratr.at.The last3 or 4 digits of the unique Haskell animal number will be tattooed abAnUoddsyrteuwpdelyiagschteatdrstwt(ihetashttardartaesynho0at)vtwihinteghrbianosd=ywi2wl0le%ibgeohf7stwhweeietmkheisanonltdh.waittOrhaninngtaeests(esdxua,byjwei0cl,tltwobhteehrneepmsooasvsmieebdlsefe,rleorcmatsisotwnuidtyh criteria as the original ras). _-- a2 36 G82aTveagleoRmaenrge8FAilncdoihonlg:StRaepyeisnteRda-tDsose Oral Toxicity Dupont6357 dRaayts0,thfaotrhoauvte-onfo-trabnegeenbaosdsyigwneeidghtt0.,awtiesltbgeroruepleoarsewdhifocrhohtahevrelbsebeonrarteormyovpuerdpofsreosm,sotrudbey: on test sacrificed by discretionof tchaerbsotnuddyidoixriedcteora.sphyxiation and discarded without pathological evaluation, at the F. Dosing Solution Preparation and Sampling w"iTlhle tbeestprseupbasrteandcdeaiwliyl.l be suspended in methylcellulose. Dosing solutions of the test substance. G. AdminiofsDotsirngaSutspeinsoionns `iTnhteubtaetsitosnuubnsttialnscteewaidlylsbteatseuisspoebntdaeidneidnamtetahvylocleulmlueloosfe5. mTLh/ekgraotfs bwioldybweeidgohste.d bAyniimntarlasgawsitlrlicbe `dwoesiegdht7s cdoalylsecatweedetkw.icTehaewaemeokuanntdotfhteesstussupbesntsainocneccoanccehntrraattrieocne.iveCsonwtirlollbeatbsasweldlboendthoesebdodwyith `methylcellulose at a volume of 5 mL/kg of body weight. H. Body Weights "The rats willbe weighed twice per week at 310 4 day intervals during the study. I Clinical Observations and Mortality bDeuhraivnigotrhaentde/sotrpaepripoeda,racangcee-saimtoe negxarmaitnsawtiilolnbsetcoodnedtueccttemdoartibleuanstd towridceeaddairlaytstahnroduagbhnooutrmtahel. estaucdhy.ratMwoirlilbbuendinrdaitvsidwuiallllbyehsaancdrliefdicaenddanedxadmiisncaerddefdorwaibthnoourtmanlecbreohpasyv.iorAatnedvearppyewaeraingchei.ng, bCleirnieccaolrdseidgnassn"ontoedabwinlolrmbaelirtecyodredteedc.teAdn"i(mNalAsD)thiant dthoensottuedxyhriebciotrdcsl.inical signs oftoxicity will aPrnidolrortostsaucdryifdiicreecotform,obrlioboudnsdarmaptsi,eispfoosrshiebmlaetaonldogayt athneddcilsicnriectailocnhoefmitshtercylimneicaaslupraetmheonltogsismtay be collected for clinical pathology evaluations. 5 `G2avaTgeelRoamnegBreFAilncdoihonlg: StRodyein pRaDisoeseaOratlToexicidty Dupon6357 J. Blood Collection for Total Fluorine and Parent/Metabolite Evaluation Prior to dosing (est day 4), day 0, and7 days ater study tart, and weekly thereafter until steady osrtbaitteaflosritnoutsalofflbuootrihnmeailnebsloaondd ifseamtatlaeisnewdh,iblleouondd(earpplirgohxticmaartbeolny d|imoxLi)dewialnlebsethceoslilaefcotredtoftarlom the. fluorine and parent/metabolite analysis approximately 2 hours (+ 30 minutes) after dosing. For `males, the frst 5 animals in Groups I, I, V. and VII are designated for blood collection. The blood will be collected in plastic tubes containing EDTA while on ice. Approximately 0.5 ml of blood from each animal willbe removed and stored frozen until analyzed for total fluorine levels. "The remaining blood (approximately 0.5 mi) from each animal will then be pooled by dose group. and separated into plasma and RBC.Theseparated plasma/RBC willbestored frozen until a qualitative analysis for parent and metabolite is performed. For each bleeding, blood should be claosltledcotseedgartoaupppraottxaiimnastsetleyatdhyestsaatmeefotritmoetoafldfalyuo.rinAellinanbliomoadl.s will continue to be dosed uniil the Blood willalsobe collected for otal fluorine analysis at 3,7, 14, 30, 45, 60, and 75 days postdosing for each dose group. The blood samples collected postdosing may be analyzed as needed to answer questions regarding distribution and metabolism of the test substances. K. Anatomical Pathology Lo Pretest `See Materials and Methods, Section D. Pretest Period. 2. Dosing Phase Moribund rats and rats scheduled for sacrifice will be sent to Pathology for sacrifice. Rats will bneeccruoptshya.natRiaztesdfboyucnadrdbeoanddwioixlibdeeadniessctahredseida waintdhoeuxtsannegcurionpastyi.on and willbediscarded without @ Select Tissue/Sample Collection following attainment of steady state blood fluorine levelsofall dose groups. `The second group of five male rats in the control, low,intermediate, and high dose groups wil be sent to Pathology for sacrifice. Rats will be euthanatized by carbon dioxide anesthesia and exsangauination. The following tissues will be collected and weighed: Digesltiivevre System Miscefaltlaneous Excretory System Kidneys "These tissues will be placed in plastic freezer bags and stored in the freezer until a qualitative analysis of parent and metabolites can be performed. 5 5G:2aTvagee RlanogBemAFlicneodhiornlg:SuRedpyesintReadt-sD.ose Oral Toxicity _ Dupont6357 3. Posidosing Phase # fSleuloercitnTeilsesveules/.Sample Collection following elimination of blood Al remaining rats in the control, low, middle, and high dose groups will be sent to Pathology for sacrifice and a gross evaluation and collectionoftissues. Rats will be euthanatized by carbon dioxide ancsthesia and exsanguination. "The following tissues will be collected and weighed from rats sacrificed by design: Digestive System iver Reproductive System Male testes Miscellaneous fat "The tissues will be placed in plastic freezer bags and sored in th freezer until analysis of total fluorine levels. SAFETY AND HOUSEKEEPING Good housekeeping procedures will be practiced to avoid contamination of dosing solution preparation facilities and potential health hazards. To avoid skin contac, gloves will be wom whahnednlehdanidnlaincghetmhiectaelsthsouobds.taDncoesionrgdsoosliuntgiosnosluwtiilolnsb.e Ipnraedpdaitrieiondn,ptrhoepteersltysvuebnsttialnacteedwcilabse Animal carcasses and feces willbe incinerated. RECORDS AND SAMPLE RETENTION AClolmporaingiyn,alNreewcaorrkd,s DweillabwearreetoarinaetdIartonHaMsokuenltlaLianboRreactoorrdy,s EM.an1.adguemPeonntt,de20N0eTmoodudrssLaannde, `eWqiulimpimnegnttonr,ecDoerldaswawriel. beLarbeotraaitnoerdy-astptechiffiacciolirtysiwthe-esrpeectifhiecwroerckorwdasssudcohnea.s personnel files and 0 5Go2vTageeRaongBmeAFliecnodhironlg:SoRd ein RpaDosseeOraltToec oo _ DuPoneis? PROTOCOL APPENDIX Study Function StudyPersonnel Study Director: Anatomical Pathologist Study Dates Prebleed InitiationofTest Substance Administration Scheduled Sacrifice SGerneigoroRrSey.seLaardcihcsS,ciPehn.tiDs.t G.SeTnriaocryRMeaskeoarvcehe,ScDi.eVn.isMt.. April 16,2001 April 20, 2001 Deleipmeinndaetinotnurpaotenotfhefluorine dforsoemgtrhoeubplsood ofthe various u v2aTg oRongmBAFleicoihrnolg:StRyeRpaDiaosiOdnlToi Doms? SIGNATURES Approvedby: piFTnaoinredBrTita eonaBmS.m 7 Geman GDTirpalyoSmaaktoevAecC,VPD.VM Senor Research Sie 1gpalTM ol regen Date ci JMoehmniyCnoVnaniollrancourt ChSaureiCernaeveSnmith Janes Mstanka GVPaiaraudylaHJnieinprdseCoranlptkear NiSthaawBnakGearnnon iCcok Sadr,CP,hD. DABT. GenerDairTeocwteorology ebamo'. 5ds A 0, DAB. SeerResear Scents `Study Director BAPDaRe-2001 (2 fBparct-doi u 41 `5:G2ivTaegeloRmienegB:FAilncdoihnogl SRuedpeyatinedR-aDtosse Oa Toxicity `Work Request Number 13650 Service Code 1583 + Protocol AmendmenNot. 1 Durene357 `The protocol is amended as follows 1. Page 8, Materials and Methods, F. Dosing Solution Preparation and Sampling, add as second paragraph: "Homogencity and concentration verification and stability for 8-2 Telomes B Alcohol will be conducted during the dose range-finding study." Rationale: Troefersitgaebrlaitsehdmailxoinnggwiutnhif-ohrmoiutry ranodomsttaebmilpietrya(t5u-rheo)urforrotohem9t0e-mdpaeyrarteupreeatandods7e-sdtauydy. Ait: boaTaSd i70d.eDAB. artiteam SirStRayeDcihceSrlr co JMoehmnalOy'nCoVnaniollrancourt TJruadcyyStMaadlkeorve SJauneinCeraBvreitnton JCahnaertleMnaeslSamniktah GPaaurlyHJienpdseorlniter SVihaadwanmiGranCanpolnea Nita Baker 8G:2avaTgeelRomaenrgBeFAilncdoihnogl SRteupdyesitndR-aDtosse Oral Toxicty Duont6357 Work Request Number 13690 Service Code 1583 Protocol Amendment No. 2 "The protocol is amended as follows (changes are bolded): 1. Page 4, Study Design, Study Design table, footnote d, change to read "The second group of5 rats in Groups I, Tl,V,andVIIare designated for select tissue csotlelaedcytisotnatfeoilslaotwtianigneadttataianllmednotsoeflesvteelasd,ytshteastee abnliomoadlsflwuiolrlinbeelseavcerlisfaitceadlladnodsleivleervse,lsk.idWnehyesn. and fat will be collected and weighed, then placed in plastic freezer bags and stored in the freezer untila qualitative analysis of parent andmetabolitescan be performed." 2. aPtaagienm5,enSttoufdsyteDaedsyigsna,teSbtluodoydPfalruoarmienteelrevtealbslaet,aSlelldeocsteTilesvseules,/ScahmapnlgeeC(o0lrleeactdion following "The second group of males fromth 0, 5, 25, and 125 mg/kg/day dose groups willbe sacrificed, banadgsliavenr,dKisdtnoeryesd, ianntdhfeafrtiesesuzeewriulntbielcaolqcucaleidtaatnidvweeaingahleyds,itshoefnppalraecnetdainndplmaesttaicboflrieteezsercan be performed." Rationale: To complete the collection and storage methods for the specified tissues. _-- Yr -- fo soicer -- 1y 94d, o ~200) Sry Director ce MJuedrylSyandeVraillancourt TJroahcnyOM'aCoknorveere CJhaanirnleenBeriStmointh SJuoeneCrMaavselnanka GViuardyaJmeiprsConapa PSahualwHniGnadnenrloenr Nita Baker `2GavTageelRoamnegBreFAilncdoihnogl SRteupdeysiinedRa-tDso:se Oral Toicity Dupont6357 `Work Request Number 13690 Service Code 1583 Protocol Amendment No. 3 "The protocol is amended as follows (changes are bolded): Page 8, H. Body Weights, change to read "The rats will be weighed twice per weck at 3 to 4 day intervals during dosing, then once per week after dosing is stopped. Rationale: To accurately describe the frequency of body weight collection. Appvd: /, . AdD.yDABT. StdDistr cc: JMuedmyaSluynndeVarillancourt TJroahcnyOM'aCkoonvneoer CJhaanrileenBeriStmointh JSauneetCrMaavselnank GVairsydaJmeiprsCoanpka SPahualwHninGdaenrnlotenr Nia Baker 19dD-u3ye4;doo) `6G:a2vTaegleRoamnergBe-FAilcnoihnoglRSetupdeyatienRdaDosseOral Toxicity DuPont6357 `Work Request Number 13690 Service Code 1583 Protocol Amendment No.4 `The protocol is amended as follows (changes are bolded): 1. dPaaygefsr5o,mS3t0u1d0y 3D1e.sign, parameter for serial bleeding, change post dosing blood collection 2. pPaargaegr9a,pJh.,BflrosotdseCnotlelneccet,iocnhfaonrgTeobtlaoloFdlucoolrlienceteionnddPaayre3n0yMtoet3a1b.olite Evaluation, second Ratiforoitenmsa1alnde2: The blood collection schedule was changed. 3. Page, K. Anatomical Pathology2,. Dosing Phase, fist paragraph is changed to read: Moribund rats will be euthanatized by carbon dioxide anesthesia and exsanguination and will be discarded without necropsy. Rats that are found dead will be discarded without necropsy. Rationale: To accurately describe the dispositionofmoribund rats. J-- Aug 3 a `SLtaddysD,iicto.r ABT. ce: JMuedmylSyindeVrallacoun TJroahcnyOM'aCkoonvneoer CJhaanrilneenBeriStmointh JSauneetCrMaavsetnnka VGirsydaJmeiprsConapa SPhaauwHniGanndneonr Nita Baker Aydt 2GrvTagoeomReanrgBeFAilncdoihnogl SReeaeyaitneRda-tDsoscOral Toxicity Work Request Number 13690 Service Code 1583 Protocol Amendment No. 5 Duponeas? The protocol is amended as follows: bPlaogoed1f0l,uo3r.inPeosltedveolssi,nagcPdhatshee,fao.lSelloecwtt oiTintshsguel/sSaomfptlsesuCcosl.lection following elimination of Excretory System kidneys Rationale: The kidneys were inadvertently omitted from this listoftissues in the protocol. Approvedby: \ S-Ladi, PED, DAB. `Study Director JMuedyrSlaydnleVrailancout TJooabcayOM'Caoknonor CJaanrneeBnriStmon SoTienMCraavnenia GVaerdaoaConpka SPhaaulwHniCnodnenroiner Nis Baker b-Seft-doo]