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ST-300 ToxDocs 10-170 TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY 10-170: ORAL STUDY OF MTDID 208 AND MTDID 1004 IN MALE RATS FINAL REPORT ToxDocs Study Number 10-170 Test Material MTDID 208 MTDID 1004 Lot Number(s) 217 (MTDID 208) 332 (MTDID 1004) Strategic Tox Lab Study Number | ST-300 Other Identification Number(s) |K'PFOS (MTDID 208) NH;"PFOA (MTDID 1004) 19F/TH-NMR Analytical Report | See Appendices 1 & 2 for Certificate GID Number of Analysis Study Director Jill Hart, AAS, LATE Laboratory Administrator Principle Investigator: (Study Monitor) John Butenhoff, Ph.D., DABT, CIF Corporate Scientist Testing Facility Tn-Life Start Date 3M Strategic Toxicology Laboratory 3M Center, Bldg 270, room SB314 Saint Paul, MN 55144 Tune 21,2010 Tn-Life Termination Date Tune 25,2010 Tof14 ST-300 ToxDocs 10-170 TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY 10-170: 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: de Ft JillHart, AAS, LATg Laboratory Administrator Study Director Goma. CotsZdl John Butenhoff, Ph.D., DABT, CIH. Corporate Scientist Principle Investigator 21 Tw @oil Date 9 ga 20s Date 20f 14 ST-300 ToxDocs 10-170 1 Summary The objectiveofthis pilot study was to determine whether administrationofezetimibe (Zetia) to male rats reduced the absorptionoflow-dose PFOS and PFOA. The hypothesisofthis study was that perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA), when present in the gastrointestinal system from oral 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 (NPC111) protein. Because ezetimibe (Zetia)is a drug used to block cholesterol absorption by binding to and inactivating the NPCI11 protein, absorption of PFOS and PFOA in ezetimibe-treated (5 mg/kg/day, days) and vehicle-treated (com oil) male rats was evaluated by measuring serum PFOS and PFOA, as well as serum cholesterol concentrations, before and after oral KPFOS (MTDID 218, 20 pg/kg) and NH; "PFOA (MTDID 1004, 20 pg/kg) administration. All ats appeared normal during the study and survived to scheduled study termination. Interim serum samples (via tail vein bleeding) were obtained prior to administrationsof $ mg/kg ezetimibe on day 1 and also prior to the administration of 20 ug/kg K'PFOS/NH,"PFOA on day 5. At necropsy (4 hours post K'PFOS/NH;PFOA dose), serum and liver were harvested. Serum samples were analyzed for cholesterol concentrations and both serum and liver samples analyzed for PFOS and PFOA concentrations by LC-MS/MS. "The mean serum cholesterol levels for rats prior to first and after last ezetimibe treatments were 135.5 + 5.1 mg/dL and 135.8 + 14.4 mg/dL, respectively. Four hours after K'PFOS/NH;PFOA administration, mean serum cholesterol was 116.5 + 6.5 mg/dL. Mean serum PFOS and PFOA concentrations were 3.5 + 2.8 ng/mL and 34.7 + 14.2 ng/mL, respectively. Mean liver PFOS and PFOA concentrations were 209.5 + 74.4 nglg and 142.5 = 33.6 ng/g, respectively. "The mean serum cholesterol levels for rats prior to first and after lat vehicle (corn oil) treatments were 174.0 = 76.4 mg/dL and 119.5 + 13.2 mg/dL, respectively. Four hours after K'PFOS/NH;"PFOA administration, mean serum cholesterol was 108.8 16.5 mg/dL. Mean serum PFOS and PFOA concentrations were 2.3 + 1.1 ng/mL and 26.6 + 8.0 ng/mL, respectively. Mean liver PFOS and PFOA concentrations were 171.2. 92.8 ng/gand 1103 + 50.3 ng/g, respectively. `When administering to rats at 20 pg/kg, the respective PFOS and PFOA concentrations in liver were 59 ~ 75-fold and 4-fold of that in serum. 2 Study Objective The objectiveofthis pilot study was to determine whether administrationof ezetimibe (Zetia adrug used to block cholesterol absorption by binding to and inactivating the NPCII1 protein) to male rats reduced the absorptionoflow-dose PFOS and PFOA. The 3of14 ST-300 ToxDocs 10-170 hypothesisofthis study was that perfluorooctanesulfonate (PFOS) and. perfluorooctanoate (PFOA), when present in the gastrointestinal system from oral aexnpdoasbusroerobredbialliaornygewxictrhetcihoonleasttelroowl,dovsiae,thmeatyrabnesppoarrttiparlolcyefsosrmmeeddiaintteodmbiyxetdhemNiiceelmleesn Pick C1 like 1 (NPC111) protein. Absorption of PFOS and PFOA in ezetimibe-treated (50 mg/kg/day, 5 days) and vehicle-treated (corn oil) male rats were evaluated, as well as Serum cholesterol concentrations, before and after oral K'PFOS (MTDID 218, 20 pg/kg) and NH; PFOA (MTDID 1004, 20 pg/kg) administration. 3 "Test Material Information 31 Test Substance MIDID208 I Chemical Name Other Identifiers Appearance Purit Potassium Perfluorooctanesulfonate K'PFOS. White solid 86.9% |stability| Stable under normal conditions |ExpiraDatteion| une 2019 Characterization The test material isassignedas MTDID 208 and its characterization is attached as Appendix 1 Storage aT The test article will be stored tightly sealed in its original Handling `Standard protective equipment will be used. Lab coats, Precautions gloves and eye protection will be worn while handling the test article. Disposition Any remaininn g unuses d test article will be kept in the Study [Cendy [rom 004 I Chomont Nore Other Identifiers NH,PFOA White solid Lrwiy |o799% 4of 14 ST-300 ToxDocs 10-170 Densit Characterization Storage PrHeacnadultiinogns Disposition NA Stable under normal conditions June 2019 | The test materiali assigned as MTDID 1004 and its characterization is attached as Appendix 2. "The test article will be stored tightly sealed in ts original container at ambient room temperature. Standard protectiveequipmentwill be used. Lab coats, gloves and test article. eye protection wil be worn while handling the AFanciylitryemuanitniilnegxpuinruasteidontedsattea.rticle will be kept in the Study 32 Dose Preparation Dose Preparation Physical State of administered material Route ofAdministration "The test materials were prepared and mixed in the same test tube. They were dissolved in water as 20 pg/mL. solution. Liquid [Oral | 4 TestSystem 41 Test System Information [SR pecies a] [Strain "Tsp | [S"oAgueracteInitiation of Treatment Harlan Laboratories Weight at Initiation of "Approximately 300 360 & Treatment [Numberandsex T8males | [TIACdUeC nAntimialfUsiagceation| U20n0i8q-u0e4t5a9il mark in indelible ink Application Number 42 Animal Husbandry Sof 4 ST-300 ToxDocs 10-170 Housing DictWater Housing Environment `All animals were group-housed in solid-bottom cages throughout the study except during urine and feces collection in which rats were single housed in wired bottom metabolism caging for urine and feces collection for 24 hours prior to designated study termination time. | HarlanTeklad RatMouse 2018 Diet (HarlanTeklad, Madison, WI) and tap water were available ad libitum throughout the study. Temperature Range 72 = 3F | Humidity Range 30 -- 70% Minimum of 10 exchangesofroom air per hour 12hour light/darkcycle 5 Study Design [om TS oo Tow [os[oe[wrens] SOmghe Zea" | hous | [ee meme me|S20 uEghg CEoNmH/RoPlFOA_| NHK',P7PF0FOSO/A 2 | 4| Comoil | Comoil | Comail| Comoil | 2020uugggksNKH',PPrFOOSA dose * Fach Zetia tablet contains 10mgezetimibe as the active ingredient, the entire: tablet weighs 100 me. In order to achieve 5 mg/kgofezetimibe dose, 50 mg/kg AofdpmuilnivsetrriazteidoZne/tDioas"etaPbrleeptarpaotwidoenrbewillolwbfeoradmmaitneriisatlerperdeptaorraattsi.onSdeeteaiMlse.thod of All ats were dosed on per table above on designated days. Interim bleeding (via tail vein) were performed on Day 1 and Day S (prior to Zetia/Comn oil administration). All rats were euthanized via CO; asphyxiation at designated times followed by gross necropsy with blood (forserum) and livers collected. 6 Parameters Evaluated 6.1 Clinical Observations Each animal was observed immediately following dosing and throughout the study for `mortality and morbidity. Any notable findings were recorded. 62 Body Weights All animals were weighed daily prior to treatment for the purposeof calculating dose volumes andpriorto necropsy. 6of14 ST-300 ToxDocs 10-170 63 Serum Collection Interim bleeding (via ail vein) was performed on all ats on Day 1 and Day 5 (prior to Zetia/Com oil administration). At euthanasia, blood was collected via the abdominal aorta and transferred to labeled collection tubes without anticoagulant. The blood samples were allowed to clot for 15 to 30 minutes at room temperature and then centrifuged at 2000 x g for 15 minutes. The serum was transferred to 1.7-mL polypropylene microcentrifuge tubes labeled witha tube code and stored frozen at -70C pending analysis. 64 Gross Necropsy, Organ Weights and Tissue Collection Following euthanasia, a gross necropsy was performed on all animals. Liver samples were harvested and the corresponding weights were recorded. All samples were stored in a freezer set to maintain 70 C for future analysis. No other tissues were collected. 65 Analysis Cholesterol levels were determined (by Marshfield Laboratories) in all serum samples collected. PFOS and PFOA concentrations were also determined in all serum and liver samples by LC-MS/MS. 7 Statistical Analysis `There were no statistical analyses performed on data collected in this study. 8 RawData `The raw data for body weights, organ weights and dosing volumes are recorded in 3M Noteboo#k154823,pages 13 - 16. 9 Results 9.1 Clinical Observations Al ats survived until scheduled necropsies. Clinical observations were normal throughout the study. 92 Body Weights and Organ Weights Body weight data are presented in Table 1. All animals gained weight during the study. Liver weight data are also presented in Table 1. Tof14 ST-300 ToxDocs 10-170 9.3 Gross Necropsy `There were no gross lesions or other abnormalities noted in any rats during necropsy. 94 Analytical Results Cholesterol concentration (serum), PFOS concentrations (serum and liver), and PFOA concentrations (serum and liver) are presented in Table 2. 10 Discussion & Conclusion `The mean serum cholesterol levels for rats prior to first and after last ezetimibe treatments were 135.5 5.1 mg/dL and 135.8 + 14.4 mg/dL, respectively. Four hours after K'PFOS/NH;"PFOA administration, mean serum cholesterol was 116.5 + 6.5 mg/dL. Mean serum PFOS and PFOA concentrations were 3.5 + 2.8 ng/mL and 34.7 + 14.2 ng/mL, respectively. Mean liver PFOS and PFOA concentrations were 209.5 + 74.4 ng/g and 142.5 33.6 ng/g, respectively. `The mean (+ SD) serum cholesterol levels for rats prior tofirstand after last vehicle (cor oil) treatments were 174.0+ 76.4 mg/dL and 119.5 + 13.2 mg/dL, respectively. Four hours after K"PFOS/NH,PFOA administration, mean serum cholesterol was 108.8 + 16.5 mg/dL. Mean serum PFOS and PFOA concentrations were 23 1.1 ng/mL and 26.6% 8.0 ng/mL, respectively. Mean liver PFOS and PFOA concentrations were 171.2 +928 ng/g and 110.3 + 50.3 ng/g, respectively. `When administered to rats at 20 pg/kg, the respective PFOS and PFOA concentrations in liver were 59 ~ 75-fold and 4-foldof that in serum. Sof 14 ST-300 "ToxDocs 10-170 Table 1: Body weight (BW) and liver weight (LW) data ere c Dom secre oup)| lg) B | gh B LB G B a) BW |DaAyminCiomeptoauonnd|| Da5yLV [R346|Semoilmkgbiieay[i mo oa[or[me | [[oTRORE||(spurvepearrzemdszreotma[ [5|E 02 || %07 3| |s%e6s 7 || w3o8]] [ORS [=e| oT134| 33| B71 | [[Ro3R=1z]] [[s3e6[s31i6[|s2u1[[s2i3[| 5m3]| (RE ] | 300 [300 134|37| 308 | @Kokuegrrkoss) * muro Ougkg) [113| `Table 2: Mean cholesterol (in serum), PFOS (in serum and liver), and PFOA (in serum and liver) data DoseGroup moTowPe oar) | (Days ahs posrryiosprdosn [chal]| [chal] | [000] | eog mg | mgaL | mga || gg | nog [ORG]exst5migikegiGdraaypes d13o4 | t520 1 | t2h6 | |1258||m353|| tqeos||t14u1 ]| [OR346 |frompuverizedZetia 126 | 21 | 713 | 17 |22 | iw | i | [0R3%8| tabletincomWole) n w1es| |vtsea | | 1e15s|| 3765| |83167 [|031s8 |[r1e0s0 || LT & | 744 | 65| 26 | 742 | 744 | 336| _ 2E 2 | BT | 31 |32O |300| ie0|75| Rs Moon 1NW4AT0 || Ta mas | | 6ai 6s[|T 3"125 | | Pmaea|| i8m r428||e8r3ie6s| C77 7 | m2 | 5| 71| 80|ws| 503| NEZA:: ENzoetsiemriubmesamples were obtained. 9of14 ST-300 ToxDocs 10-170 Appendix 1: MTDID 208 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS REVISION 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023- FT m s] 018A 3M Product: PFOS,Lot217 Reference #: SD-018 Purity: 86.9% [__TestName[Specific| atiReo suln t s| NR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium' 5. Nickel 6. lon 7. Manganese mMR m Lgacos sTM |] |] 1. 0.005 wt/wt% 2. 0.001 wi/wt% 3. 1439 wnt 45.. 6<.08.40901wt/wt% 6. 0W.0w05 %Win% 7. <0.001 mTeEw WWL% Bolweewr| (GC/MS) POAA (TGA) [PuInroirtgyanbiyc DAnSioCns (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate* enw 0.33 wt/wt% | TT NotApplicable" | 1. <0015 WU 2 059wm% 3. <0.040 WU 4. <0.009 w/w 100f14 ST-300 OrganicAcids* (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5._Fluorine ToxDocs 10-170 5. <0.006 WLW 6. <0.007 WLW 7. BT6wW% 1. <0.0 wtt% 2. <0. wtiwt% 34..002180 wwiiwmt%% 1. TheorVealtuei=c17a.8l% 11248wi/wm% 2. Theoretical Value =0% 2.0244 win 3. Theoretical Val=u0e% 3174 wm% 4. Theoretical Value =5.95% | 4. 884 wi/wi% 5. Theoretical Value =60% 5. 540 wid of 14 1-300 ToxDocs 10-170 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"lPuuroirtiyde=, 01.005%9%-+(NsuMmRofimmepturailtiism,pu1r.i9ti3e%so,r1g.a4n5i%c+aLciCd/iMmSpuriimtpiuersi,ti0cs.,388%+.PO4+AInA1o,rg9ani%c. 033%) Total impurity from alltests = 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 materialsoflow purity. No endotherm was i"Snuolrfguarniicn atnhieosnammepltehoadppceoanrdsittioonbse. coTnhveeratneidontoreSsOul;t aangdreheesnwceelldewtietchtetdheussiunlgfutrhe determination on the results, itnhethSeOc4leismennottaclonansaildyesriesd, laennidmipnugrictoyn.fidence to this interpretation. Based "HTFFABA NFPA PFPA TrHiefpltuaofrlouaocreotbiuctyarciicdacid Nonofluoropentanoic acid Pentafluoropropanoic acid "Theoretical value calculations based on the empirical formula, CsF1;SOyK" (MW=538) `This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160) 120614 ST-300 ToxDocs 10-170 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023018A LC/MS Purity Profile: [1 [ew Tp a ---- zai 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 130f 14 ST-300 Appendix 2: MTDID 1004 Certificate ofAnalysis ToxDocs 10-170 March 2, 2000 FC-143, Lot 332 Richard M. Payfer `"TThhiesrseasumlptlsoefwtahsesaenatleystzsedshuosiwngthGeCs/aAmpElDe,toLCco/nMtSa,in"tHhe-NfMolRl,owainndg GweCi/gMhtSpteercchennitques. composition: [CC oRcomm-- T oow emcomu, em | [CC eRconm TT omw | CC oRcomm 7---- oroeeg | [oFsc TTom eeewm" CC okgoM 1 ome tAdedcihtniioqnuaelslya,ndthfeoiusnodmteorcdoinsttariibnuttihoenfoofltlhoewisnagmmpolleewpaesrcdeenttercommipnoesdiutsiionng: maClFc(haCinF,-whCeOre0xiNsLmCai)nly ) CF(int(eCrnFalCCmFoHnoF CmEe)th2 ,yC! Ob)rCan)chN, HL) wher(e CxFy )isCmaFin-l(y CdC,Fa0),n,xN#d0L,C)20) tsopropy branch, where xis mainly 4) (bu(tCyFl)b,rCa-n(chC,Fw2h)e-rCeOx()isNmaHinl)y 3) "F-NMR ED PosCsiPbleC)C"C"F)(CwFh)er,eCO)i NundHef)ined oT (temal gemm-adinmyeth3.y3lanbdraxn2c0h,)w--here xy is v f vOy F 01% possiblewhere Ris undefine'd P(oASSlTpIhEaCbrFasnc-hC, Fw(hCerFex:)is-mCaiOnlyNI5L) of 14