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ST311 ToxDocs 10-196 TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY 10-196: ORAL STUDY OF MTDID 208 AND MTDID 1004 IN MALE RATS FINAL REPORT ToxDoes Study Number 10-196 Test Material MTDID 208 MTDID 1004 Lot Number(s) 217 (MTDID 208) 332 (MTDID 1004) Strategic Tox Lab Study Number| ST-311 Other Identification Number(s) | K PFOS (MTDID 208) NH:PFOA (MTDID 1004) T9F/TH-NMR Analytical Report| See Appendices | & 2 for Certificate GID Number ofAnalysis Study Director Jill Hart, AAS, LATg Laboratory Administrator Principle Investigator: (Study Monitor) John Butenhoff, Ph.D., DABT, CIH Corporate Scientist Testing Facility 3M Strategic Toxicology Laboratory 3M Center, Bldg 270, room SB314 Saint Paul, MN 55144 Tn-Life Termination Date fAuguest 13, 2010 Tof14 ST-311 "ToxDocs 10-196 TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY 10-196: ORAL STUDY OF MTDID 208 AND MTDID 1004 IN MALE RATS Compliance Statement and Signature Page `This study was conducted for research and development purposes and does not conform to regulatory guidelines. "This final report has been reviewed and approved by: aYo Tht Jill Hart, AAS, LATg Laboratory Administrator Study Director tom R algal] John Butenhoff, Ph.D., DABT, CIH. CProirnpcoirpalteeInSvcieesnttiigsattor ITN2011 Date G Iw zon Date 20f 14 ST311 ToxDocs 10-196 1 Summary `toThmeaolbejercattisvreeodfuctehids tshteudaybswoarpsttioondeotfelromwi-ndeowsheePtFheOrSadamnidniPsFtOraAt.ionThoef ehzyeptoitmhiebseis(Zoeftithais) sprteusdeynwtaisn tthheatgpaesrtrfoliunotreosotcitnaalncssyusltfeonmaftero(mPForOaSl)exapnodspuerrefloruobrioloicatryaneoxactreet(iPoFnOaAt)l,owwhdeosne, `trmaanysppoarrttipalrloycebsesfmoerdmieadteidntboymithxeeNdimeimceelnlePsicakndC1ablsiokrebe1 d(NalPoCn1g11w)itphroctheoilne.steBreocla,uvsiea the einzaecttiimviabteinigs tahechNePmCicIaIl1upsreodtetion,blaobcskorcphtoiloenstoefroPlFaObSsoarpntdiPonFbOyAbiinndeiznegtitmoibaen-dtreated (5 mg/kg/day, suspended in 0.5% Tween 20, 5 were evaluated, as well as serum cholesterol days) and control (water-treated) concentrations, before and aftr male oral rats K'PFOS (MTDID administration, 218, 20 pg/kg) and NH, PFOA (MTDID 1004, 20 pkg) All rats appeared normal during the study and there was no mortality prior to scheduled sadtmuidnyitsetrrmaitniaotnisoonf.ezIentteirmiimbeseornudmasyam1palnedsa(lvsioa tprailorvetiontbhleeaeddmiinngi)stwrearteioonbtoafined prior to sKa'mPpFlOesS/wNeHre,PanFaOlyAzeodnfodracyho5.lesAtternoelcrcoopnscye,ntsreartiuomnsanadndlibvoerthwesreeruhmaravnesdteldi.verSsearmupmles were analyzed for PFOS and PFOA concentrations by LC-MS/MS. `tTrheeatmmeenatns(w&eSreD)1s1e7r2u+m c6.h8olmesgt/edroLlalnedvel1s14f.o8r +at4s.1prmigo/rdtLo,firresstpaenctdivaefltye.r laFsotuerzchtoiumrisbe aMfetearnKs'ePrFuOmSP/FNOHS; aPnFdOPAFaOdAmicnoinsctreanttiroant,iomnesawnerseer7u.m0%ch6o.l0esntegr/omlLwaasnd993.90.5%5.73.6mg/dL. ng/mL, and 135.0 respectively. Mean liver PFOS + 23.1 ng/g, respectively. and PFOA concentrations were 229.2 + 47.0 ng/g Ttrheeatmmeeantns sweerruem 1c2h8o.2l.este1r1o.l8lmevge/lds Lfoarncdon1t2r2o.l7a+ts10p.ri8omrgt/odfLi,rstraesnpdecatfitveerllya.st Fwoautrerhours amfgt/edrLK.'PMFeOaSn/NseHr;uPmFPOFAOSadamnidniPstFrOatAiocno,ncmeenatnrastieornusmwcehroele4s.t7er+ol1.w0ansg1/0m9L.7a+nd103.89.9 5.4 ng/ml, respectively. Mean liver PFOS ng/g and 141.6 + 20.7 ng/g, respectively. and PFOA concentrations were 235.3 43.0 `liWvheernweardemianpipsrteorxiinmgatteolryat3s2at~2500-pfgo/lkdg,antdhe3r-efoslpdeoctfitvheatPFinOSseraunmd.PFOA concentrations in 2 Study Objective `The objective of this pilot study was to determine whether administrationofezetimibe (a chemical that is used to block cholesterol absorption by binding to and inactivating the hNyPpCoItIheIsipsrootfteihni)stostmuadlyewraaststrheatdupceerdfltuhoeroaobcstoarnpetsiuolnfoofnlaotwe-(dPoFsOeS)PFanOdS and PFOA. The perfluorooctanoate (PFOA), when present in the gastrointestinal system from oral Jofle ST-311 "ToxDocs 10-196 exposure or biliary excretion at low dose, may partially be formed into mixed micelles and absorbed along with cholesterol, via the transport process mediated by the Niemen Pick C1 like 1 (NPCII1) protein. Absorption of PFOS and PFOA in ezetimibe-treated (5 mg/kg/day, 5 days) and control male rats were evaluated, as well as serum cholesterol concentrations, before and after oral K'PFOS (MTDID 208, 20 ng/kg) and NH;"PFOA (MTDID 1004, 20 pg/kg) administration. 3 Test Material Information WM TewSubstanes [Cena [romans i 217 Chemical Name Other Identifiers Potassium Perfluorooctanesulfonate K'PFOS. [puiy seo | [Densy [wa Stability June 2019 Stable under normal conditions Characterization The test material is assigned as MTDID 208 and its characterization is attached as Appendix 1 Handling Precautions `The test article will be stored tightly sealed in its original containaetr ambient room temperature. `Standard protective equipment will be used. Leb coats, Sere gloves and eye protection will be wom while handling the [deni|MB romE sons Te I Disposition Any remaining unused test article will be kept in the Study Chemical Name Other Identifiers Ammonium Perfluorooctanoate NH, PFOA 57.99% 4of14 ST-311 "ToxDocs 10-196 Stability Characterization Storage Handling Precautions Disposition 32 Dose Preparation June 2019 Stable under normal conditions "The test material is assigned as MTDID 1004 and its characterization is attached as Appendix 2. `a The test article will be stored tightly sealed in its original Standard protective equipment will be used. Lab coats, gloves and eye protection will be worn while handling the test article. Any remaining unused test article will be kept in the Study Facility until expiration date. Dose Preparation The test materials were prepared and `mixed in the same test tube. They were dissolved inwater as 20 pg/mL solution. Physical State of Liquid `RaodumtieniosfteArdemdinmiastterraitalion |Ol | 4 Tetsystem 41 Test System Information [[pSetdaiins Trsap | [Source | iaran Laboratories Age at Initiationof Treatment |8-10weeks| Weight at Initiation of "Approximately 258 -- 290 [NTurmeabtemrenatndses Identification [Smal | Unique tail mark in indelible ink TACUC Animal Usage 2008-0459 Application Number Sof 14 sT311 ToxDocs 10-196 42 Animal Husbandry Housing DicvWater Housing Environment c`Aalgleasnitmharlosugwheoruet tghreouspt-uhdoyuesxecdepitn sdoulriidn-gbuortitnoemand `fweicreesdcbooltletcotmiomnetinabwohliicshmrcatasgiwnegrefosriunrgilneehaonudsefdeciens ctoelrlmeicntaitoinonfotri2me4.hours prior to designated study | HMaardliasnoTne,kWlIad) RanadtMtoapuswaete2r01w8erDeieatva(iHlaarbllaenaTedklad, libitum throughout the study. Temperature Range 72 + 3F | MHuimniidmiutymRoafn1g0ee3x0ch-an7g0e%s of room airperhour 12 hour light/dark cycle 5 Study Design |{oom cow come [ ome Snake casimbe | hous 20uugshwgahtNeeHrPrFOAs_| NKHw'CPPoFFOSO/A 2020uugghkgNHKLPPFFOOSA| dose Avlelinr)atwsewreerpeerdfooserdmeodnopnerDataybl1e aanbdovDeaoyn$(dpersiiogrntatoedezdeatysi.mibIen/tweartiemrbolreaeldaidnmgin(ivsitartaatiilon). Anlelcrroaptssywewrietheubtlhoaondi(zfeodrvsiearCumO);aansdphlyixviearsticoonllaetctdeeds.ignated times followed by gross 6 Parameters Evaluated 6.1 Clinical Observations Emoarcthalaintiymaanldwmaosrboibdsietryv.edAnimymneodtiaabtleelyfifnodlilnogwsiwnegrdeosreicnogrdaendd. throughout the study for 62 Body Weights AVlolluamneimsaalnsdwperrieorwetoignhecerdopdsayi.lypriorto treatment for the purposeof calculating dose Gof 14 sT311 ToxDocs 10-196 63 Serum Collection Ienzteetriimmibbel/eweadtienrga(dvmiianiasitlravteiionn))w.asAtpeeurtfhoarnmaseida,onblalolodatwsaosncoDlalyect1eadnvdiaDathye Sab(dporimoirntaol asoarmtpalaensdwterraensaflelrorwededtotolacbleoltefdorco1l5letcoti3o0n mtiunbeustewsitathoruotoamnttiecmoapgeurlaatnutr.e aTnhdetbhleonod cpeonltyrpirfoupgyeldeante2m0i0c0roxcegntfroirf1u5gemitnuubteess.labTehleedsweirtuhma wtausbetrcaondsefearnreddsttoor1e.d7f-rmoLzen at -70C pending analysis. 64 Gross Necropsy, Organ Weights and Tissue Collection wFeorlleohwairnvgesetuetdhaannasdiath,eacgorrorsesspnoencdrionpgsywewiagshtpserwfeorremreedcoorndeadl.l anAilmlalssa.mplLeivsewresraempstloersed in a freezer set to maintain -70 C for future analysis. No other tissues were collected. 65 Analysis cCohlolleecstetde.rolPlFeOveSlsawnedrPeFdOetAercmoinnceendt(rbatyiMonasrswhefrieeladlsLoabdoertaetromriinese)d iinn aallll ssecrruummsaanmdpllievser samples by LC-MS/MS. 7 Statistical Analysis There ware no statistical analyses performed on data collected in this study. 8 RawData `The raw data for body weights, Notebook # 154823, pages 86 - organ 89. weights and dosing volumes are recorded in 3M 9 Results 9.1 Clinical Observations NCloinmiocratlaloibtsieersvaotcicounrsrewderdeurnionrgmtahlistshtruoduyg,haolultratthse survived study. until scheduled necropsies. 92 Body Weights and Organ Weights BLiovdeyr wweeiigghhtt ddaattaa aarree palrseosepnrteesdenitneTdaibnleTa1b. leAl1l animals gained weight during the study. 93 Gross Necropsy `There were no gross lesions or other abnormalities noted in any rats during necropsy. 7of 14 ST-311 ToxDocs 10-196 94 Analytical Results Cholesterol concentration (serum), PFOS concentrations (serum and liver), and PFOA concentrations (serum andliver)are presented in Table 2. 10 Discussion & Conclusion `The mean (& SD) serum cholesterol levels for ats prior to first and after last ezetimibe treatments were 117.2 + 6.8 mg/dL and 114.8 + 4.1 mg/dL, respectively. Four hours after K'PFOS/NH,PFOA administration, mean serum cholesterol was 99.0 % 5.3 mg/dL. Mean serum PFOS and PFOA concentrations were 7.0 6.0 ng/mL and 39.5 + 7.6 ng/mL, respectively. Mean liver PROS and PFOA concentrations were 220.2 + 47.0 ng/g. and 135.0 + 23.1 ng, respectively. The mean serum cholesterol levels for control ats prior to first and after last water treatments were 128.2 11.8 mg/dL and 122.7 + 10.8 mg/dL, respectively. Four hours after K'PFOS/NH,PFOA administration, mean serum cholesterol was 109.7 10.9 m5.g4/dnLg./mMl,earenspseecrtiuvmelPyF.OMSeaanndlPivFeOr APFcOoSnceanntdraPtFioOnAs wceonrcee4n.t7ra+ti1o.n0snwge/rmeL23a5n.d33=8.493.+0 ng/g and 141.6 + 20.7 ng/g, respectively. `When administered to rats at 20 pg/kg, the respective PFOS and PFOA concentrations in liver were approximately 32 ~ 50-fold and 3-foldof that in serum. Sof 14 ST-311 ToxDocs 10-196 Table 1: Body weight (BW) and liver weight (LW) data Dosecroup | EW| Daya) 26W{Day |G3) {Day|G 43 |G |DaAym5CmomepnouDanyd5|g1] RA[20T 288|2030|2080| 203| 20Fao 0 0 0 0 [ORR4T0. O]evriaerdedvtootma[ | 2Z5| ] 22077|82075T19287 6 |720 85| | KePFOS (20ugkg) | Raz] |22 1 2% |21 120|280 | + is] |ORa7a] [Z 2% | oT r2 |255 | Nearron [[227s6T22rs3 TT70782|856||22868 | | Oughg) [28T20 |22 5 |6 21| [260 | 283 | 284| 205 | 204| [10] [[1219]] [720] ProEZcom] 3 hourspostPROSPRdOosAe Table 2: Mean cholesterol (in serum), PFOS (in serum and liver), and PFOA (in serum and liver) data fez] meme yy (Chol =1oso] [Chol | [m2][Chol] | mm]serum om] Lidl Ny CE JC720 XO 0 2 Smghkg/day seuzseptoirmdebdein 16 TT01O75 | | 6 |jo |oes |27| tes | #0] 06 Ti9e1 || 3tasr| | a#4r5| | 200s0|| tvoero|] [OR471|05% Tween20) OR4T2 w112z8 1 | |7 i7u8 2s[| |woieosr ||17 r3@m o6| |a2540| | |22t6e71s [| |T1aa1os0|]| F2 EC | iT 8 Teave 2 0HT 0 57 GL: NE20 CY 70 A | os] Ve 1RE2C | iz Ti |3480|%0| 20J 6 |75| ORATT. IB | 5Tr158 |dae | air | 63 | [14m |1r7 | 1e08r| [aaortr | m6e |z2y5| Temraes]) L 78 | ms |o 09|70| 5s|430 |27] EZ: Ezetimibe 9of14 ST311 ToxDocs 10-196 Appendix 1: MTDID 208 Certificateof Analysis INTERIM CERTIFICATE OF ANALYSIS REVISION 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023- 018A 3M RPerfoedruectn:ceP#:FOSSD,-L0o1t8217 Purity: 86.9% [__TestName [Specifica|tiRo etns| Identification NR Metals (ICP/VES) 21.. CMaalgcnieusmium 3. Sodium 45.. NPioctkaeslsium? 6. Iron 7. Manganese Wp | eee (NMR Total % Impurity LC/MS) Ew Te| (GCMS) Related Compounds -- POAA RTeGsiAd)ual Solvents [PuIrnoirtgyanbiyc DAnSiCon_s_(_IC_)--| 1. Chloride 23.. FBlruoomriiddee 4. Nitrate 65.. NPihtorsiptheate. 7. Sulfate Positive 1. 0005 wiwt% 20.001 wi/w% 3. 1439 WAn% | 4 6849 wm% || 5 <Wo0Um 6. 0.005 wt/wt 7. <0.001 WUWL% SAT WW 0.33 wi/wt% None Detected NotApplicable' | 1. <W001Ms 2 059wm% 3. <0.040 Ww% 4. <0009 wi/wt% 100f14 ST311 Organic Acids 1. TFA (IC) 2. PFPA 3. HFBA 4. NFPA Elemental Analysis 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur S._Fluorine ToxDocs 10-196 5. <0.006 WLW 6. <0.007 WL 7. 876WAn% 1 <01 wim 2. <0.1 wimt% 3.0.10 wm% 4. 028 wt/wt% I. Theoretical Value = 17.8% 1. 1248 wtint% 2. Theoretical Value = 0% 2. 0244 wt/w%t 3. Theoretical Value = 0% 3.174 wm% 4. Theoretical Value =595% | 4. 8.84 wi/wi% 5. Theoretical Val=6u0e% 5. 54.1 wt/wt of14 ST311 ToxDocs 10-196 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023- 018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 F"lPuuorirtiyde=,01.0509%% -N(MsuRmofimmeputrailtieism,pu1r.it9i3e%s+,or1g.a4n5i%c+aLciCd/iMmSpuriimtpiuersit,i0es.,388.%4+1P%O+IAnAo,rganic 033%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% "Potassium is expected in this salt form and is therefore not considered an impurity. "oPbusreirtvyebdyfoDrStChisissagmepnleer.ally not applicable to materials of low purity. No endotherm was "iSnuolrfguarniicn atnhieosnammepltehoadppceoanrdsittioonbse.coTnhveeratneidontoreSsOul;t aangdreheesnwceelldewtietchtetdheussiunlgfutrhe determination on the results, in the elemental analysis, the SO is not considered lending confidence an impurity. to this interpretation. Based TFA HFBA NFPA PEPA TrHiefpltuaofrlouaocreotbiuctyarciicdacid Nonofluoropentanoic acid Pentafluoropropanoic acid "Theoretical value calculations based on the empirical formula, CgF17SOyK" (MW=538) "This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). 120014 ST-311 ToxDocs 10-196 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023- 018A LC/MS Purity Profile: CC mphy wm% | [ Ca ---- Ifo] ee Ta og Tae Cl sai Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The CS value was calculated using the average response factors. from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed By: John Flaherty Date Laboratory Manager, Centre Analytical Laboratories 1Bof14 ST311 Appendix 2: MTDID 1004 Certificate ofAnalysis ToxDocs 10-196 March 2, 2000 FC-143, Lot 332 Richard M. Payfer `This sample was analyzed using GC/AED, LC/MS, "H-NMR, and GC/MS techniques. The results of these tests show the sample to contain the following weight percent composition: CiF;CONH,~ [--ceRC TTomomN w% mT| CF ;CO, NH, TFwmy 1 Coro' Tome | Atdedcihtniioqnuaelslya,ndthfeoiusnodmteorcdoinsttariinbutthieonofofltlhoewisngammpolleewpaesrcdeenttercommipnoesdiutsiionng: "'F-NMR forCmaFl(cChaFn),-wChOer0e)xNisAmCai)nly 6) Te Ch (@nieC( mRalCmFoC n(omCeFF t)hy,lC) OraLnc)h,NH) 26% where xy ismainly 4, and x #0. 2 0) isopropylbranch, whxeisrmaienly 4) buy(CFb)rCan-cChF,2wh)e.rCeOx)sNmaHinIly) 3 oT PosCsiFbleC)C"CF") (C"Fwh2e)re-CO,)isNunHdeLf)ined internal gemm-aiinmlyeth3,ylnbdraxnc2h,0)where x+y is Possiblewhere Ris undefine'd P(OASSlBpIheaCbruanech-,CwFh(eCrFe:x)-isCmOaAinlyNL5C) 140f 14