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ARQR6 -- 136] A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Southern Research Institute Study ID: 9921.5 April 22, 2003 001599 Final Report on A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey To: 3M Corporation P.O. Box 33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 By: P.E. Noker and G.S. Gorman Southern Research Institute 2000 Ninth Avenue South 35205 P.O. Box 55305 Birmingham, Alabama 35255-5305 001600 ABSTRACT `The pharmacokinetics and urinary excretionofperfluorohexanesulfonate were investigated in male `andfemalecynomolgusmonkeys.Threemaleandthreefemalemonkeyswereadministered asingle iv bolus dose of 10 mg/kg of perfluorohexanesulfonate, potassium salt (T-7504). At various times afterdosing,serumand urine(24-hourcollections)sampleswere obaandiannalyezeddby 'HPLC/MS/MSforlevofeinltasct perfluorohexanesulfonate. The lowerlimitofdetectoiftohne `analyticalmethod was 5ng/mLforserumsamplesand 1 ngforu/ rinem sampL les. Peakserum concentrationsofperfluorohexanesulfonate were similar in male and female monkeys and ranged. from 104,600to 140,900 ng/mL in male monkeys and from 116,400 to 174,000ng/mL. in female `monkeys. Serum concentrationsofperfluorohexanesulfonate in each monkey decreased reltively rapidly during the first 8-24 hours after dosing. At 24 hours, serum concentrations of perfluorohexanesulfonate ranged from 24,870 to 40,405 ng/mL in male monkeys and from 27,115 to 54300 ng/ml in female monkeys. Subsequently, serum concentrations of perfluorohexancsulfonate decreased at a slower rate between 24 hours and the end of sample collection (171 days). On Day 171, serum concentrationsofperfluorohexanesulfonate ranged from 134151021,725ng/mLinmalemonkeysandfrom3,249t020,220ng/mLinfemalemonkeys. The serum concentration versus time data were subjected to non-compartmental pharmacokinetic analysis. The serum elimination half-lifeofperfluorohexanesulfonate ranged from 100 to 200 days (mean: 141 days) in male monkeys andfrom 49 to 140 days (mean: 87 days) infemalemonkeys. `The total body clearanceofperfluorohexanesulfonate was 1.1 to 1.5 mL/day/kg in male monkeys ad 12 to 26 mldaykg in female monkeys. The volume of distribution of perfluorohexanesulfonate ranged from 223 to 391 mL/kg and from 160 to 255 mL/kg in male and female monkeys, respectively. Apparent differences among individual monkeys in the estimated serum half-life and clearanceofthe compound were likely attributable to an overestimationofthe extrapolated AUCau Values for twoofthe female monkeys. Only very low levels (<0.01 t0 0.11% ofthe administered dose)ofperfluorohexanesulfonate were measured in urine during any given 24hour periodof sample collection between Day 1 and Day 70 after dosing. The resultsofthis study suggested that the pharmacokineticsofperfluorohexanesulfonate were similar in male and female monkeys. Perfluorohexanesulfonate was eliminated in urine by both male and female monkeys at low levels for a prolonged periodoftime (270 days) after iv administration. 001601 i . TABLE OF CONTENTS Page SIGNATURE PAGE iii GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL v 10 INTRODUCTION 1 20 MATERIALS AND METHODS 1 21 Test System 1 22 Test Article and Vehicle 2 Test Article: 2 Vehicle 3 Dose Formulation Preparation 3 Dose Formulation Analyses 3 23 Experimental Design 3 Group Assignment and Dose Procedure 3 Clinical Observations 3 Body Weights 3 Urine and Feces Collection 4 Serum Levelsof Perfluorohexanesulfonate 4 BDiaotaanaAlnyatliycsaelsMethod Development and Sample Analysis 44 30 RESULTS 5 3.1 Mortality 5 32 Clinical Observations 5 33 Body Weights 5 34 Serum and Urine Concentrations of Perfluorohexanesulfonate 5 40 DISCUSSION 7 50 CONCLUSIONS 7 60 RECORD ARCHIVES 8 70 REFERENCES 8 001602 " TABLE OF CONTENTS (Continued) LIST OF TABLES Page Table 1: Body Weights 9 Table 2: Serum ConcentrationsofPerfluorohexanesulfonate 13 Table 3: o`fPhPaortmaascsoikiunmetPiecrfPlauroarmoehteexrasneCsaullcfuolnaatteed from Serum Concentrations 1 Table 4: Urinary Excretionof Perfluorohexanesulfonate 15 LIST OF FIGURES Figure1: SerumConcentrationProfileofPerfluorohexanesulfonate 18 LIST OF APPENDICES Appendix A: Study Protocol Al AppendiBx: iAnnaMloytnikcealyMSeetrhuomdafnodrUDreitneerminationof Perfluorohexanesulfonate B1 001603 Signature Page `A Pharmacokinetic Studyof Potassium Perfluorohexanesulfonate in theCynomolgus Monkey Zier Tad) Patricia E. Noker, Ph.D, D.AB.T. `Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: yesfor Date Add Zp Charles D. Hbert, Ph.D., D.AB.T. Director, Safety Assessment pa Date `We,the underwseiregresnpoensdib,lefortheconductof the workandreportingofthe resultsinthe listedsections. We cown ithc theu vier wsrelativetoourbody ofworkasexpressedinthediscussion and conclusions. . an, Ph.D. `Manager, Bioanalytical Chemistry Group 2/0/43 Date 001604 iv Good Laboratory Practices Disclaimer "ThisstudydescribedinthisfinalreportwasnotconductedintrictcompliancewiththeUSS. Food nd DrugAdministration(FDA)GoodLaboratoryPractice (GLP)Regulations(21 CFRPart 58),and neitherthisreportnortherawdatawerereviewedby theSouthernResearchQualityAssuranceUnit. However,thestudy wasconductedaccordingtotheprotocolandamendmentsandtheapplicable. standardoperating procedures,andallstudyprocedures,datarecording,andreportingwereperformed in 8mannerconsistentwiththestandardof GLPs.Thefinalreportaccuratelyreflectstherawdata obtainedduringtheperformanceofthestudy.Therewerenoadversecircumstancesthataffected the qualityorinteogftrheisttudyy. Fotiar &. 20k) PatriciaE. Noker, Ph.D, D.ABT. StudyDirector 4/22 fos Date 001605 v Study Dates: Study Schedule and Personnel Study Initiation: DayofDosing: Last Dayof Sample Collection: Study Completion: February 7, 2001 February 9, 2001 July 29,2001 April 22,2003 Study Personnel: Patricia E. Noker, Ph.D, DAB.T. Study Director Charles D. Hbert, Ph.D, DAB.T. Director, Safety Assessment Norman D. Jefferson, B.A. Associate Director, Safety Assessment Gregory S. Gorman, PhD. Manager, Bioanalytical Chemistry Group Darrell E. Hoskins, D.V.M,, Ph.D,, A.C.L.AM. (Dipl.) Veterinarian LaJuanAa.Durbin, B.S. SuperLvarigesAonirma,l Laboratory D. Wayne May, LATG Supervisor, Animal Care: Carolyn R. Oliver, BS. Supervisor, Study Coordination 001606 1 . 1.0 Introduction The objectives of this study were to determine the concentration of potassium perfluorohexanesulfonate in serum and to estimate urinary clearance at various times following `administorfastiingolne intravenousdoseto monkeys. Acopyof theprotocolandanyapplicable `amendments can be found in Appendix A. 2.0 Materials and Methods 2.1 Test System Thethreemaleandthreefemale cynomolgusmonkeysdesignatedforuseinthisstudywere selected from an in-house colony of monkeys that were housed at Souther Research Institute (SouthernResearch)priortouseon thisstudy. Thesemonwekreepurychassed from CharlesRiver BRF, Inc. (Houston, TX)and werean estim3-a4tyeeardsofagewhen placed on study. Individual animal identification was by chest tattoo. The cynomolgus `monkey isanacceptedspeciestosupportclinicalstudiesofdrugsusedorintendedforuse in humans. Duringthe quarantineperiod,acompletephysicalexaminationincluding afecalexamination forintemalparasites,completebloodcount (CBC),bodyweight,andrectal temperatwuares performed on eachofthe monkeys. The following procedures were performed on the. monkeysduringquarantine:(1)Threetuberculintests wereadministeredto achanimalat 2-week intervals. ~All tuberculin tests were administered intrapalpebrally. The three tuberculin tests were negative for all monkeys. (2) Blood was drawn for CBC and B virus. titer. (3) Fecal cultures (screening for SalmonellaandShigella) were obtained, and fecal flotation tests were performed. (4) In general, primates were examined at least once weekly by an approved veterinarian and were observed (cage-side observations recorded by exception only) twice daily for abnormal clinical signs and mortality/moribundity. Housing, feed, water, and socialization procedures remained the same during the quarantine, holding, and study periods. 001607 2 Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed tothemon2-3ktime eseaychdsay.The quantityofthedailyrationwassufficienttomeet nutritional requirements. In addition, the diet was supplemented with fresh fruithreats sevtimeeserachaweelk. Tapwater(Birminghampublicwatersupply)wasavailabletothe `monkeysad libitumduringthe quarantineandstudy periods.The monwkereehoyussed individuallyinstainlesssteelcagesduringthe quarantine andthestudyperiods. FromDay 010theendof thestudy,themonkeyswerehousedin aroom thatwas maintainedat a temperature of 67.7-72.8 F and a relative humidityof29-74%. The humidity was within therequiredrange(30-70%)over90% ofthetimeduringthestudy;excursionsbelowthe recommendedhumidityrange wereofshort durationandhadnoimpactonanimalhealthor theoutocftohesmtudey. Roomlightswerecontrolledbyanautomatictimersettoprovide 12hooflu ightr (060s 0to 1800hours, CST)and 12hoofduarkr perdsay.Cagesizeand anciarecmonfaormled to the guidelinesoftheGuidefortheCareand UseofLaboratory Animals, 7th edition and the US. Departmentof Agriculture through the Animal Welfare: Act (Public Law 99-198) and to the applicable StandardOperatingProcedures (SOPs) of `SouthernResearch.ThestudydesignwasapprovedbySouther Research'sInstituteAnimal Care and Use Committee. Southem Research is fully accredited by the American Association for AccreditationofLaboratory Animal Care Intemational. `Withthe exceptionofanimal 2053, allofthe monkeysusedonthis studywerepreviously givena single iv bolus doseofperfluorobutanesulfonate (10 mg/kg) on 4/10/00 (Souther Research Study No. 9921.1); a single iv bolus dose of potassium perfluorobutanoate (10 mg/kg) on 6/13/00 (Souther Research Study No. 9921.2); a single iv bolus dose of `potassium perfluorohexanoate (10 mg/kg) on 7/31/00 (Southern Research Study No. 921.3); and a single iv bolus doseofpotassium perfluorooctanoate (10 mg/kg) on 1019/00 (Souther Research Study No. 9921.4). Monkey 2053 was naive. 2.2 Test Article and Vehicle `Test Article: One bottle containing grams ofpotassium perfluorohexanesulfonate (T-7504; expiration date not supplied; SRI EO6/L-1) was supplied by 3M (St. Paul, MN) and received 001608 3 onMay15,2000. Thetestarticlewasstoredatroomtemperatureuntilused.Stabilityofthe test article was the responsibilityofthe Sponsor. Vehicle: The vehicle used for the preparation of the dose formulation of potassium `perfluorohexanesulfonate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 0081050, expiration date August 2003). The vehicle was stored at room temperatureandwasconsideredtobestable whe storedaccordingtotheseconditions. Dose Formulation Preparation: For the single dose formulation of potassium perfluorohexanespurelpfaroendatemg/mL,the requiredamountof testarticle was weighedoutin avolumetflraiskc.Sterilesaline wasaddedandtheformulationwasstirred unintsoliutilon.The formulationwasstoredrefrigeratedandusedfordosingwithin 1 day afterpreparation; it was considered stable during this period. Dose Formulation Analyses: Dose concentration and homogeneity analyses were not required to be performed. 23 Experimental Design Group Assignment and Dose Procedure: Asonly one treatment group wasusedin this study, no formal randomization was required.On Day 0,eachofthethreemaleand three female monkeys received a single intravenous (iv) doseofperfluorohexanesulfonate at 10 mg/kgby injientcoatsupierfoicinalarmor legvein.DoseswerebasedupontheDay-1 individual body weights. Doses were administered at a volume of2mL/kg. Clinical Observations: ~All animals were observed twice daily for signs of `mortality/moribundity. Each primate was examined shortly after dose administration for clinical signsoftoxicity. Additional clinical observations were performed on days ofblood collection. Body Weights: Each primate was weighed on Days -1, 4,7, 14, 21, 28, 35, 42, 49, 56, 63, 70,77, 84,91, 98, 105, 112, and 119. 001609 . Urine and Feces Collection: Urineandfeceswerecollectedfor24-hourintervals on the following days: priorto dose administration (Day -1; baseline), on Day 1 (0-24 hours `postdose), onDay 2 (24-48 hourspostdose), andonDay7s,14,21,28, 42, 56, and 70. The `volumeofeachurine samplewas measuredupon collection.Urine andfeces samplweerse stored frozen (approximately -20 C or below). Fecal samples will not be analyzed unless specifically requested by the Sponsor. `Serum LevelsofPerfluorohexanesulfonate: Blood samples (approximately 3 mL) were collected from each primateatapproximatel0y (predose) minutes; 0.5, 2, 4, 8, 24 and 48 `hours; andon Days 4, 7, 14,21, 28, 42, 56, 70, and 171 postdose. Sampleswere collected intotubeswithoutanticoagulantandwereallotwo eclodtat roomtemperature. Theblood sampleswere then centrifuged, and the serum separated and stored frozen (approximately 20 Corbelow) until analyzed. `BioanalyticalMethod Development and Sample Analysis:Serumandurinesamplweerse analyzedforperfluorohexanesulfonate using a previously validated HPLC/MS/MS method (Appendix B). Data Analyses: The serum concentration data for unchanged perfluorohexanesulfonate were subjected to non-compartmental pharmacokinetic analysis using WinNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc.; Cary, NC). Mean values and standard deviations for each parameter were calculated using Microsoft Excel Software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorohexanesulfonate at each collection interval was calculated and expressed as a percentofthe administered dose. No other statistical analysesofthedatawere performed. 001610 5 3.0 Results 3.1 Mortality All ofthe monkeys in this study survived to the endofthe study. 3.2 Clinical Observations Noadversedrug-relatedclinicalsigaswerenotedforanymonkeyduringthecourse ofthis study. 3.3 Body Weights BodyweightsarepreseinntTaeblde 1. Eachmonkeyeithergainedweightormaintained essentially aconstantweightbetween Day ~1 and Day 119 (last day during thestudythat `body weights were obtained). 3.4 Serum and Urine ConcentrationsofPerfluorohexanesulfonate `Serum concentrationsofperfluorohexanesulfonate in three male and three female monkeys at various times through Day 171 ater administration of a single iv doseof 10 mg/kg are: presented in Table 2 and Figure 1. No sex-related differences were apparent in serum concentrationsofperfluorohexanesulfonate at any timeofsample collection. Peak serum concentrationsofperfluorohexanesulfonate were observed in 1/3 male monkeys and 3/3 female monkeys at 0.5 hours (carlest time point) after dosing. Serum concentrations of perfluorohexanaetsthuisltifmoernaangteed fiom 116,400 to 140,900ng/mLamongthese four monkeys. For the other 2 male monkeys (2053 and 2211), peak serum concentrations ofperfluorohexanesulfonate (104,600 and 128,500 ng/mL) were not observed until 2 hours after dosing. The possibility was investigated that the 0.5- and 2-hour serum samples. collected from these two monkeys were switched during collection and/or analysis; however, it could not be determinedifthe switchingofthe samples had occurred. Subsequent to the timeofpeak levels, serum concentrations of perfluorohexanesulfonate in each monkey decreased relatively rapidly during the first 8-24 hours after dosing. At 24 hours, serum `concentrationsofperfluorohexanesulfonate ranged fom 24,870 to 40,405 ng/mL inthethree. `male monkeys and from 27,115 to 54,300 ng/mL in the three female monkeys. Serum 001611 6 . concentrationsofperfluorodehcreeasxedaatansleowrsatuebeltwfeeon2n4haourts aend the endofsample collection (171 days). During this period, fluctuations were observed in theserum concentrationsofperfluorohexaamonngeisnduivlidfuaolmnonaketyse. Inthat thefluctuationsappearedtobe randomamongthemonkeys,theymayhavebeenattributable to analyticalerrorintroduced as aconseofqtheulaergendilcutieonofeachsamtphaltwaes required prior to HPLCMS/MS analysis. On Day 171, serum concentrations of perfluorohexanesulfonate ranged from 13,415 t0 21,725 ng/mL in the male monkeys and from 3,249 020,220 ng/mL in the female monkeys. Pharmacokinetic parameters calculated from serum concentrations of `perfluorohexanesulfonate in individual monkeys are presentedin Table 3. The values were derivedfromnon-compaanraltysmiseofnthtedaatla. Nodistinctdiffewrereenapcpaeresnt between maleandfemalemonkeysintheestimatedparameters; however,fortwoofthethree female monkeys,theserum half-life andclearanceofperfluorohexaneswuelrfeoshnoartteer and faster, respectively, than observed for the three male monkeys and the other female monkey. AUCyugue, Values ranged from 6456 to 8745 ugeday/m in male monkeys and from 3849 to 8501 ugeday/mL in female monkeys. The terminal halflife of pecfluorohexanesulfonate in serum ranged from 100 to 200 days (mean: 141 days) in the threemalemonankdferomy49so 140days (mean: 87days)inthethreefemale monkeys. `The total body clearance of perfluorohexanesulfonate was 1.1 to 1.5 mLday/kg in male monkeys and 1.2 to 2.6 mLiday/kg in female monkeys. The volume of distribution of `perfluorohexanesulfroannagteedfrom 223 to 391ml/kginmalemonkeaynsd from 160to 255mL/kgin female monkeys. `The amount of perfluorohexanesulfonate eliminated in urine by individual monkeys at various times after dosing is presented in Table 4. Only very low levels (<0.01 to 0.11% of the administered dose)ofperfluorohexanesulfonate were measured in urine during any given 24-hour period ofsample collection between Day 1 and Day 70afterdosing. There was no clear indication of a sex-related difference in the urinary excretion of perfluorohexanesulfonate, The urinary excretion of perfluorohexanesulfonate was 001612 7 prolonged;detectablelevelsofthecompound werepresentinurineonDay70(lastdayof urine collection)afterdosing. 4.0 Discussion `Theresultsofthisstudyindicatedthat perfluorohewaxssalonwleyeslimuinlatfedboynmaaletaned femalemonkeysgiven asingle iv doseof 10 mg/kg. Throughouttheperiodofsample collection, serumconcentrationsofperfluorowerheseimixlaraatcnaceh sasmpuletlimfeinobonthmaalte aend femalemonkeys.Inaddition,therate ofurinaryexcretionofperfluorohexanesualpfpeoanreadteo besimilarformaleandfemalemonkeys.Thus,therewas 0clearindicationfrom thedatathatthere `was asex-relateddifferenceintheeliminationofthecompound. Fortwoofthethreefemale monkeysonthecurrentstudy,theestimatedserum half-lifeandtotal `body clearanceofperfluorohexanesulfonate appeared to be shorter and faster, respectively, than observedforthe thirdfemalemoneyandforthethreemalemonkeys.Theseapparentdifferences may have been artifactual in that both parameters (half-life and clearance) were calculated from AUCogueValues; theselattervalues were obtained from extrapolationofthe serum concentration time curve and may have contained considerable error. That the AUCugs values for two ofthe three female monkeys may have been overestimated is supported by the observation that estimated AUCqiy,values [0tothelas timepoint(Day 171)]forallthreemaleandallthreefemalemonkeys were similar. 5.0 Conclusions No sex differences were apparent in serum concentrations and the urinary excretion of perfluorohexanesulfonate among male and female monkeys given an iv dose. The mean terminal serum half-lifeofperfluorohexenesulfonate was 141 days in male monkeys and 87 days in female monkeys. Perfluorohexanesulfonate was eliminated in urine by both male and female monkeys at Tow levels for a prolonged periodoftime (270 days) after iv administration. 001613 3 6.0 Record Archives `Data, specimens,and acopyof thefinalreportfrom thisstudywillbestoredintheArchivesat SouthernResearchforup to 1yearafteracceptanceofthefinalreportbytheSponsor. Aft1yeearr and withthepermissionofthe Sponsor's Monitor,thedataandanysamples/specimenswilbe shippedtotheSponsorortotheSponsor'sdesignatedarchivalfacility. Ifmatearertoibearetlainsed inthearchivesbeyondthisdate,suchcontinuedstoragewillbefor aspecificfeedeterminedwith theSponsor. AcopyofthefinalreportwillberetainedinthecentralarchivesatSouthernResearch. 7.0 References 1. InstofiLatbourattoery Animal Resources, CommisosniLoifne Sciences, National Research `Council; NationalAcademyPress;Washington D.C; 1996. 001614  ~ svidda; i paid 2 = suiddg 5 i i i af$s igeinst J 535 kg 17 Le oF]; Femi o-osmiig : .ozznisyi i = 23I3]3 - HET HH] = 3 001615 wo 5 3 aeison d PRT ! gs sszfddd pia i:z os ozenisa : = S38 ELE, ain BE Ea EF| CM 7 os aif 2 8 37[a 31 ii ] 5 amjson id - i lg Is 5 2 2 001616 i + aii i + zasiidd 2 = 22380 i fos oumzifi S i : a Ads {ida} 2 2 Lamia iI | : | = 231 | aH Bis ; : 001617 15 3a 3en3ni88.3 )| f #2 afi i 3Eg a33u3n1i3B0f40| i : 3 AAdiacad 25 EE Las & : %z j 2 Anci#m3enn] Foe oaiilg : e 2n7i%3 4 g eo 333i3il ii | HE ix i 001618 o Tabl2e APharmacokinetic Studyof Potassium Perfluorohexanesulfonaitne the CynomolgusMonkey Serum Concentrationsof Perfluorohexanesulfonate = i [MaTTlFe emales s| [[[o2s0sm| [[1121583508000[0]]B545O,003L7000[[]17B00Q5420L00[[]1'o5Bm5.Q51L0000|] TB5.7Q30L0000]||sBTiaOc4L0|) [[eottss[[570677500] 543609960[1sa8a6s0o] 5655951700] s52a.6o00 ||sssuoa]] [[oCatsitess||2206.70050[2as030[22.4935700[5504005005a7n1o0s0[| 7351555] [oDuasy[[35570890%|ssuss4500][1216600[s9a0s0||a37s2o50| 822000]] [[aDayy1216[2e255255 [a5s701750 |225a,a0s050[[3o9n0e1s5 ||s3.o0o0[[a0v0o5] [[bbauyy 225[[51a1100T5270752328285||3s2n.9c900 ||301.,13005[033882700]] [bDuayy 670 [[1is6.506200 [[224251900[1983060 [1116801500][7222900 ] a2n2s0n0o] [Beytat[1.725 [015[905| 325[02m BQL = Below the quantitation limit (<5 ng/mL) * Data repraensaveernagteoftwo samples. . nets 001619 tereer] go||r[Heelsskl s i {1 hse ILN E Be; er LY Frbehes 1 ii: t fz sRlLE 22 73 cif 1 4 peedi to) IE E585 4 . BEER EEREEEE 001620 15 Table 4 APharmacokinetic StudyofPotassium PerfluorohexanientshuelCyfnomnoalgtues Monkey Urinary ExcretionofPerfluorohexanesulfonte Urine me AniDmal Concentraation VoIlu)me| T2ou)l Poca of Do Dwoes)e [20s [MT BOLT Moo| ese [of [ZoTTM Bor a0Toso [ss FT Bol[a0| seo | -- Fos TF Bol | m0 -- 1 wmow [Te [205 TMT 15 T7560 | 6 1doo[oar (2 TMT 7% Te| sr Tso [oo Peer = Ts T77| 7oo6 r (ZoTF eT a0ToTow| oor | Cor| (205IM] 6 1 60 | 40 ] 6000 | 007 | Mm Te Te e w oor [ZiTM 0Ts1s sso | oor | [208TFT"60 1To| 70 |36000 | 085 | [TFT ms Te1 se ons PeTF 70 1 50 1 35 1 410| om | FoomTM w m eT Lamee[wT Tesnowe| norr ]| [2ELE0C [N MT S ve | 2N 00 |N = 5J8 1SiJ c |NCo:or | EelsTw saToo B=OBeLlowthe quantitation limi (<I ng/mL) Pagetofs 001621 16 > Table 4 (Continued) A Pharmacokinetic StudyofPotassium Perfluorohexanesulfonate in the Cynomolgus Monkey `Urinary ExcretionofPerfluorobexanesulfonate: sim Vos| Tw | Doe | mie Mr Urine | Urine -- `Percent of Dose. D Gym) | @L) [7 2) 8) I EE -- [205 [M8 |70| 6 |6000 [001 | [20[M7 | 0 [ 5 |se00 | oor | 2058 TFT v7 | mo | 28 |36000 | 008 | [205 TF] 7 | 200 [15 |37000 | oo | bw | (2055TMT 10 T 40 T 4 T e000 [ oof 1] [2054[M1 27 | 30 | 5 |eo| oor | (200[M[ & ] 0| 2 1s400 [| <oor | [ ose TFT | wo| 5 |36000 | oor | [ms TFT 27[Tio [26 |37000| 007 | (eCTFT wTs 14 a0 | 00) | sa TM] 8 | 0| 7 |eo | oor | [znIM] to |s | 5 1 s4o00 | oor |} fos TF] 16 170| 4 ] 3680 | oor | [oo F1 0 |= 5 2 7]smoe0 | 1 oor} ow -- (seIMT 55 Taw 1 a ]eo |] oe | [20[M1 2a"| #0| 2 |sso| ood | [2058 TF] 7a | 20 | 16 | 36000 | 00s |} [25TFT 28 [vo| 4 | 37000 | oor | BQL = Below the quantitation limit (<I ng/mL) Page20r3 001622 n `Table 4 (Continued) A Pharmacokinetic StudyofPotassium Perfluorohexanesulfonate in the Cynomolgus Monkey Urinary ExcretionofPerfluorohexanesulfonate: si |Conem=ytn Trine = Tl | be | mum Percenotf Dos | D_| (og/mL) @) | we) we _) { 206[MT 3% #0| |20TM 77750| 4 |om [oo | sa00| oor | | | 205% TF[78[50[3 [20% [FIT 110| 2 |36000 | | 37000 | oor | <oor | [ 205 TMT [2s TMT TT 30 [ 5TooToor a0| 16 |em[oo | (20IMI os TF] 5776 2 0 "m0[4 | sam| <oor | sew[oom | | [209TFs 1 0 2 |500 | oo | BQL = Below the quantitation limit (<1 ng/mL) Pagedof3 001623 0 aos --_-- t tt tt t F CC T/T rr 1r 1 1 rrrrrrr E tL[TTT [] r e --r--e +F + FP Ft b Fb] 5 pb e--b m--e ooy Tr r + T rT TT 1 LT | T YT ee ww wm ww om om Tie Days) o IZw. aos | A A 6 t rr t r ? rrrrr g [FT S==--=--=-== I = es Es es | HMHF--T--1 1111 [ elT eTop T T- 1.1 ae Yom ww wm om we mw Time Oay3) Figure1 A Pharmacokinetic Studyof Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum ConcPreofilneoftPerrfluoarohtexanicsuolfonnate 001624 1 waz --e-- --r-- rrr rr sO -- CC CT>T> T[| iCL [| --rrrrrr JIT TT EEE Tow oomw)o mw ww rss , -- r F--t--+ r ----t+-- r +--1r so LL 1[TT[TT|] So Co TL [.] m= Ems -- rr LTTTT] vx 4 wm--wm mw ww Fi1g(Countirnueed) A Pharmacokinetic Studyof Potassium PerfluorohexanesulfonateintheCynomolgus Monkey Serum ConcPreofilneoftPerrfluoarohtexanicsuolfonnate 001625  rion ------ fj. T CCe CT] T] | LO PT 1 CLL arat--t-- 1 [TTTo|] =ommw 5 rt--t--+r--++++ --r-- rrrTr r--T1T Lr . r =rr wmT we T ww Tora omy rom rr rrr 1 fC] | i ==.TTTTT 1 CTT= [| Osenve. --m= r rrr rr rr Tr Tr TTT T] I. ECC Figure 1 (Continued) A Pharmacokinetic StudyofPotassium Perfluorohexanesulfonateinthe CynomolgusMonkey Serum Concentration Profile ofPerfluorohexanesulfonate 00126 Appendix A Study Protocol 001627 -- SwdyPoweoh A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey | Southern Research Study ID: 9921.5 I I February 7, 2001 IH ap 0D SEHD SOUTHERN RESEARCH INSTITUTE 001628 po STUDY NO.: 9921.5 Febru7,a2r00y1 E EEE EE -- , Ti}} 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: Sponsor: 3M Center, 220-2E-02 P.O. Box 33220 St. Paul, Minnesota 55133-3220 `Sponsor's Representative & Study Monitor: John L. Butenhoff, Ph.D., D.AB.T. 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-3220 (651) 733-1962; FAX: (651) 733-1773 Protocol Approval: (initiallastpage also) on 2.Z7Loitictl > reso John L. Butenhoff Date `Test Article: Ship UnusedTestArticle to: Perfluorohexanesulfonate, potassium salt D. Hakes Building B236 3M Center P.O. Box 33327 55133-3327 St. Paul, Minnesota 55144-1000 001629 a3 STUDY NO.: 9921.5 _-- 20 TITLE: Febrar7y, 2001 wan AMonPhkaerymacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus 3.0 OBJECTIVE: `Theobjectiofvtehiss study are todeterminetheconceofn pert flur oroa hext anei sulo fonn ate inserumand urine at various timesfollowingadministration ofa single intravenous dose of potassium perfluorohexanesulfonate to monkeys. 40 TESTING LABORATORY: Southern Research Institute 2000 Ninth Avenue South + 35205 P.O. Box 55305 Birmingham, AL 35255-5305 (205) 581-2335; FAX: (205) 581-2044 50 KEY STUDY DATES: Urs sad Feces Collcons--| Fea ReporDue 1Bisel red 2 20001 anim 71 2 2sp2rr3001e1 225 23530011 wen st nsiovonr 2i7 2pmrme pir at1 parmitn 22 56 335011 wan awn nsoumo |1 daysaferFolSponsor omnes receved[=| 001630 a4 STUDY NO.: 9921.5 --_-- 60 STUDY PERSONNEL: Feber7y, 2000 het `Thefollowingare theprimary contributorsandsupervisorypersonnelparticipatinginthis study. `Study Director: "Patricia E. Noker PhD,DABT. Alternate StudyDirector: JamesD. Johnson MS,MBA. Director, Safety Assessment: `Ward R. Richter DVM, MS,DACVP. Associate Director: Norman D. Jefferson Manager, Bioanalytical Chemistry: James D. Johnson BA. MS. MBA. Supervisor, In-Life Laboratories: LaJuana A. Durbin AAS. Ve eterinarian: e D DarreBll E.IHoskins e PD.YV.MM,AACCLLAAM.N(DGipDl) 7.0 TEST & CONTROL ARTICLES: `The testarticlewillbesupplied by the Sponsor, whowillbe responsiblefor documentation thofestsatbuidlyi,tyr,easisdwuealllbuaslkmtee starttiochflesyo wnitlhdlebseissr,eftuanebdrtoithcoeraSdpteorniisvooatrni.o,n.`Uponcompletionof 71 IDENTITYOFTHETEST ARTICLE: Name: Identification: Supplier: Lot Number(s): Special Handling: Perfluorohexanesulfonate, potassium salt T-7504 3M To be documented in the study data. None Characterization: Documentation of the characterization of the testarticle, including identity, purity, strength, and composition, as well a`srem spone sibt ilioth yoffsto yhnted heSsps iosns,orf. Caopbier sofi ocrhcadrearacitvt eartiiizoant,oiiosnntdah,tae. havebeenprovided tothetesting laboratory. Stabilit&y Storage: The bulk test article will be stored at roomtemperature. Stabilityofthe bulk test article is the responsibilityofthe Sponsor. 001631 As :` STUDY NO.: 9921.5 `Februa7rSy,o2r001 72 IDENTITYOFTHE VemicLE: Name: Sterile Saline Supplier: Commercial supplier LotNumber(s): Tobedocumintehenstutdyedadta. Special Handling: ~~ None Characterization: Documentationofthe characterizationofthe vehiclemaybe attained. by recording all pertinent information from the containerlabels,orbyretainingthecontainerlabels,orcopies theinrtheestoudyfda,ta.The vehicleis acommercially available product. `Stability & Storage: Sterile saline is considered stable through the date(s) of expiration provided by the manufacturer when stored appropriately.Thebulkvehiclewillbestoredinaccordance with the manufacturer's instructions. 73 FORMULATION: : Preparation:Thetestarticlewillbeformulatedinsterilesalineat aconcentration of 5 mg/mL for intravenous administration; briefly, the required amount of test articlewillbemixedwiththe requiredamountofsterilesaline,andthemixturewill bestireduntilthetestarticlei visiblyinsolution. Formulwailtlbiesotonresd refrigerated untilusedfordosing;formulationsofthe testarticleinsterilesalinearc: expectedto bestableforweeks whensostored. Dose Formulation Concentration and Homogeneity Analyses: No analysis of dose formulation concentration and homogeneity will be conducted. 80 TESTSYSTEM: Species & Strain: Supplier: AgeonDay 1: `Weight at randomization: `Numbeonr Study: Cynomolgus monkeys (Macacafascicularis) Charles River BRF, Inc.(Houston,TX) 3-4yearsofage (estimated) 3Ma7lkegs 3 Females -3 Animals were previously dosed with potassium perfluorobutanesulfonate in study 9921.1, potassium perfluorobutanoate in study 9921.2, potassium perfluorohexanoate in study 9921.3, and potassium perfluorooctanoate in study 9921.4. 061632 As ;v, ~ STUDYNO.99215 81 JusTFiCATION: Febu7a,2r00y0 eee ti 01} `Pervialmuaatteisonasorefccoommpmoounnldysuusseeddoirninprteecnldienidcfaolruphsaerimnachoulmoagnisc,aolratnodwhtioxcihcholuomgaicnasl `mbieegxphosetd. 82 Housma: sDtauirnilnegsssqtueaerlan,tsilnea/tac-cbliomcatattgioeonsm.aAnldlsatnuidmya,lasnwiimlallbsehwiolulsbeediinndivriodoumaltlhyathporuosveiddeisn `faom0inmaitmuamtoefm1p0 eirreaoxftc6u4hr-8ae4pnergFhaoenudrs.arCeolnattriovleshwuimlildbietsyeofto3m0a-i7nt0a%i.n Athe1a2n-ihmoaulr iingthhte/s12a-mheourrodoamrkusceycdlfeorwsiltlubdey.routinelymaintained. Animalswillbe acclimated 83 BEoomG: eNxocnreemreenqtuiarbesdofroprtciaongpianngse,cqoumipmpeerdciwailthheaftl-utsrheabalteedphaanrs.dwFooordccahgiepsbeedqduiinpgpweidlwlibteh } ubseerdevoireewxecdrbeymtehnteaDbespoarrpttimoenn.tAonfaVleytseersionfatrhyeMbeeddidciinnge,saunpdplBieidorbeysotuhrecveesn(dDorV,MwBi)ll inotfSerofuetrheewrintRheosreaafrfcehctttohaessouurtectohmaetnofotkhneostwundcyo.ntaminantsarepresent that could 84 por: DMiOe)t.willTbheecpormimmeartceisawlCielrtbifeieodfPfreriemdatfeeCedlitowwic#e50d4ai8ly(,PMwIitFheeadpsp,rIonxci.maSttelLyouithse, wrielclobmemseunpdpeldedmaeinltyerdaiwiotnh favraeislhafbrlueiattoeffaecrhedfdeaeidliynagnidntterrevaatls. oIffneardedditsieovne,ratlhetidmiecst rceaqcuhirweemeekn.ts.ThAneaqluyasneisiotyfotfhetfheeedda,isluyppraltiieodnbywitlhlebveensduoffri,cwiienltlbteormeeveitewnuetdrbityitonhael oDuVlMdBafoffectSotuhtehhereRaesolefattrhcheh atoniamsaslusr. thatnoknowncontaminantsarepreseatthat 85 Waren: q`Wuaatrearnt(iBniearnmidnsgthuadmy ppuebrliiocdswvaitaeransuapuptloym)atwiicllwabteersiunpgplsiyesdteamd.liSbaimtpulmedsourfinwgattehre refrvoimewtehdebayntihmaelDfVacMilBitoyfwSiolultbheerpneRreisodeiacracllhytoaansasluyrzeetdh,aatnndokthneoawnnacloynsteasmwiinlalnbtes arepresentthatcouldaffectthe healthofthe animals. 001633 a7 oy STUDY NO.: 9921.5 --_-- 86 QUARANTINE: `February7,200 ee hela Aolflp3r5imdaatyesswueproenserleceecitpetdfartoSmosuttohcekranRiemsaelasrcthh.atwNeoreqpruoaprhaynltaicnteidcfoorr atmheirnapiemutuimc treatmentswereadministeredduringthequarantineperiod. Standardprocedures conducted during the quarantine period were as follows: 4p)arAasciotemsp,lectoempphlyestiecablleoxoadmicnoautnitoni(nCcBlCu)d,inbgodafyecwaeligehtx, aamndibnofdoarytin(itreecortnanall) te(mapdmeirnaitsutreerewdainstrpaeparlfpoerbmrealdl;yb,)ustihnrgeealtutbeerractueleiynetleisdtssafotrc2a-cwehetkeisntt)erwvearles `rpeelfrroeomfnqucaoaarcarshntpmirenieme.atd;e.) tPhreibmlaotoedstseasmtpeldendergaatwinvfeotroCalBltChwreaestaelsstosuprsieodrftoor measurement of Salmonella and SBhviigerlulsat)itwera;sdo)btaafienceadlsaandmpsluebfmoirttceudlttuorean(scirnodeenpienngdfeonrt bly aavbetoerirfnaoarriatannaaolnyrsdioysb;saernvded)a(lcalgper-ismiadteeosbwseerrveateixoanmsi)tnweidcaetdlaeialsytfoonrcaebwneoerkmlayl clinical observations and mortality/moribundity. ) `Throughoutthesubsequent holdingandstudyperiods,monkeyswil bemaintained t`oubndeerpecornfdoirtimoendsosinmielaacrhtomtohonskeefyorwiqlualriannctilnued.etIunbeadrdciutliionn,teqsutairtnegr,lbyoedvyalwueaitigohnts d10 ateutbeeratrcensdutmbwloidliilybtneenmepueatrhaatntuirzeeimdeiamosmuerdneimaet,enlty..Prithatmresapontdposeitivsely 87 PSYCHOLOGICAL WELL-BEIANNGDSOCIALIZATION: er`Niocnhhmuemnatnsprsidmiarteecstweidllbbye parvoevtiedreidnaarpisayncahonldoagpicparlowveeldlb-byeitnhgeprIoAgrCaUmCfaornsdociianl afeedcingcrewogiitmhrenthedmaopdpiarfoipcrnaitaitocensSOedPa.ilNyonfohrumthaenirprpismyachtoelsowgiilclablepwreolvli-bdeeidncga.geaTnhed `modifications include,butare not limited to:swings,perches, Kong toys, clean 2plietaenrustosf,t adnridnrkabwottfrlueist,. puWzhzelreefpeeodsesrisb,len,utprriitmiaontaelslywislolubnedhporuismeatdeptrroexatism,autneshtoelolneed another for visual and vocal contact. 88 ANIMAL IDENTIFICATION: `The primates `combination. will be Positive individually identification identified by chest tattoo will be required afte every number or letter cage change and prior to blood sampling, dose administration, and observation. 001634 As , STUDY NO.: 9921.5 _-- 9.0 EXPERIMENTAL DESIGN: ee February 7, 2001 he8an Asonlyone `required. treatmentgroupwillbeused in this study, no formal randomization willbe Doseswillbe administered by intravenousinjectionto determine thepharmacokinetics of thpoettaessstiaurmtipcelref.lEuoarcohhepxrainmeastuelf(otnharteeebmyaliensje,ctthiroen eifnteomaalseusp)erwfiiclilarleacemiveor alseignvgeline.doseof Blood samplesforserumdruglevel determinations willbecollectedfromeachprimateat selectedtimepoints duringthestudy. Urineandfeceswillalsobecollectedatpre- A synopsisofthe study design is presented in the following table. peso Pepe lalLT [PComlbeeyagwtOfebgseehrssvati[Tons5]x||x" Txxi|TxxTP]axT[xxx[T(xxx[[Txxx[llxxs[]xT[oxxT]lsF[xCx a Tx" lTaxlxsals fcrrDerr eves| TT[xTxIx x[x[eT- Tx| [x TT 91 RANDOMIZATION & GROUP ASSIGNMENT: Asonlyone treatmentgroupwillbeusedinthisstudy,noformalrandomizationwill `be required. 92 Dose PRocEDURE: Eachprimate ofpotassium (threemales, threefemales) willreceive perfluorohexanesulfonate (10 mg/kg) by a single intravenous (IV) injection into a superficial dose arm or leg vein. Doses will be based upon the most recent individual `body weights. Doses will be admiant aivoslut meoef2rmLe/kd g. "Thedayofdosingwillbe Day 0 of the study. 93 CLINICAL OBSERVATIONS: Daily Observations: All monkeys will be observed once daily during the holding period and twice daily, morning and aftemoon, at least 4 hours apart, during the 001635 A , STUDY NO.: 9921.5 `February 7, 2001 E--r ----------------------------------------------L --L -- studyforsigns ofmortality/moarndiobveurtntodxiictityy.Animalsfoundin extremiswillbe humanelysacrificedbyanoverdoseof barbituratefollowedby exsanguination with appropriate approval. DetailedObservations: Eachprimatewillbeexaminedshortlyafterdose `administrationfordetailed clisignnsiofctoxaicilty. All findingswillbe recorded. `Additionalclinicalobservations willbeperformed and recorded ondaysofblood collection. 94 BobyWeiGHTs: EachprimatewillbeweighedonDays 1, 4, 7,andweeldyintervalsthereafter through Day 91. 95 URINE AND FECES COLLECTIONS: Urineandfeceswillbe collectedforapproximate24hourintervalspriortodosing andonDays 1 (0-24hourspostdose), 2(24-48hourspostdose), 7, 14,282,421,5,6, 70,and91. Thevolumeofeachurinesamplewillbemeasureduponcollection. All ) samplescollectedwillbestoredfrozenat -20 Corbelowpriortoanalysis (urine) oruntilfurthernoticebythe Sponsor (feces). 9.6 SERUM DRUG LEVELS: Blood samples (approximately 3 mL) will be collected from each primate at approximately 0(predose)minutes;0.5,2,4,8, and24hours;and2,4,7,14,21,28, 42, 56,70, and 91 daysafterdosing. Sampleswillbecolilnteotucbestwiethodut anticoagulantandwillbeallowedtoclotat room temperature.Thebloodsamples willthenbe centrifuged,andtheserumwillbeseparatedandstoredfrozenat 20 C or below until analyzed. 97 BIOANALYTICALMETHOD DEVELOPMENT: Bioanalytical method(s) will be developed for the determination of perfluorohexanesulfonate in serum and urine matrices. The method(s) will be validatedforaccuracyandideallywillbesensitive othe |ppmor leslevel.Iffeces and/or other tissues require analyses, these analyses will be negotiated with the `Sponsor. 98 BIOANALYTICAL SAMPLE ANALYSIS: The serum and urine samples from all monkeys will be analyzed for concentrations ofperfluorohexanesulfonate using the previously validated method. The data will be: 001636 a0 ++, STUDYNO:99215 Feira 7,2001 _--_-- lla expressed as equivalents of potassium perfluorohexanesulfonate. Feces will be `anonalyilfreqyuesztedebytdhe Sponsor. 99 ANIMALDISPOSITION: Attheendofthestudy,monkeys willbemaintainedinthestockcolony.Inthe eveat `untowardreactionsorotherconditionswarranttheeuthanasiaof amonkeyatany timeduringthesamplecollectionperiod, aserumsample (5-10mL)wilbe obtained from theanimalpriortoeuthanasia. Aftercuthanasia,theanimalwilbenecropsied andtheliverandbile (asmuchaspossible)willberemovedandstoredat approximat70elCy. 100 DATAANALYSIS: Pharmacokineticparameters (.g., AUC,half-life,clearance)willbe estimatedfromserum concentrationsofunchangedperfluoroheaxs aappnroperisatue alndffeoasinblae,utsieng,a standard pharmacokineticprogram. `The totalamountofperfluoroihnuerinxe wailnlbeecaslcuulatledafndoexnpraesstedien ) termsofpercoefdaoste. Meanvalues andstandarddeviationswillbecalculatedforeachtimepointandsampletype, asappropriate.Nootherstatisticalanalysesofthedatawillbeperformed. 110 RECORDS: Allrawdatapertaining totheconductof thisstudy,andallsamples/specimenscollectedin thisstudy,wil bestoredintheArchivesatSouthernResearchInstituteforupto 1yearafter acceptanceofthefinalreportby the Sponsor. Afte1ryearandwiththepermissoiftohne Sponsor's Monitor,thedataand any samples/specimeas wil be shipped to the Sponsor or totheSponsor's designated archival facility. Ifmaterials retoberetaiinntehde archives beyondthisdate,suchcontinuedstoragewillbefor aspecificfeedeterminedwiththe Sponsor. A copyofthe final report will be retained in the central archives at Souther Research. : 120 FINAL REPORT: Abrief letterreport summarizingtheserumdruglevelresultswillbeissucdas soon asthe information is available. A draft final report will be issued within 60 calendar days after completionofthe in-life aspectsof thestudy. The final report(electronicandhardcopies) willbe issued within 15 working daysaftr receiptofthe Sponsor's final review comments onthedraftreport. The finalreportforthepresetstudywillinclude,butnotnecessarilybe: limittotehde following: 001637 an STUDY NO.: 9921.5 --- Fey 7,200 ------ hella . DCloisneicfaolrombusleartviaotnipornesparation PShearrummacdorkuigneltevieclpdaartaameters Body weight data Urine excretion data. 13.0 REGULATORY REFERENCES: Thisstudywillbeconductedinaccordancewiththeprotocoland the Standard Operating Procedures (SOPs) of Southern Research,and inaccordancewiththeapplicableregulatory `Tequirements, as addressed below. 13.1 PROTOCOL AMENDMENATNSD DEVIATIONS: follow. `Amendments: Allchangesin orrevisions of the approvedprotocol and the reasons. thereofwillbedocumented,signed,anddatedbytheStudy Director, andthe `Sponsor's Monitor. Amendments will be maintained with the protocol. Written `approval (afaxsignatureorelectroniccommunication,suchasemailf)orchanges in theprotocolmaybegranted by theSponsor's Monitor,but awrittenamendmentwill 1 Deviations: Alloperations pertainingtothisstudy,unlessspecificallydefinedinthis protocol,willbeperformedaccordingtotheStandardOperating Procedures (SOPs) of Southern Researchand/or the protocol, and any deviations from protocol or SOP `will be documented. 132 REGULATORY COMPLIANCE: Good Laboratory Practices: This nonclinical laboratory study will be conducted inthespirit of, butwillnot require strict complwiitha,tnheUc.Se. FoodandDrug Administration's (FDA) Good Laboratory Practice (GLP) regulations (21CFRPart 58).`DatafromthisstudymaybesubmittedtotheFDAinsupport ofan IND/NDA application. Quality AssuranceReview:Asthis studywill`notbeconductedinstrictcompliance `with FDA's GLP regulations, neither the in-life activities nor the final report will be `audited by the Quality Assurance Unit at Southern Research. . 001638 An STUDY NO.: 9921.5 ---_-- ee 133 FACILITIESMANAANGDANEIMM ALHE USBN ANDT RY: Febru7,a2r00y1 hla AGnuiidmealilnceasrfeowritlhlebCeairnecaonmdplUisaenocfeLwaibtorhatthoerySOAnPismalosf,S7o*uEtdhietmiRoens(elansrtcihtu,ttehoef NAantiimoanlalRAescoaudrecemsy, PCroesmsm;isWsasihoinngotnonL,ifDeCS;ci1e9n9c6e)s,, aNnadtitohnealU.RSe.seDaerpcahrtCmoeunnctilo;f ReAgsreiacruclthuIrnestitthurtoeuigsfhutlhleyaAccnriemdailteWdbelyftahree AAmcetri(cPaunblAiscsoLcaiawti9o9nf-1o9r8A)c.creSdoiuttahteironn of LabAonimralaCarte(AoAArLACy). 134 ANIMALWELACFTCOAMPLRIANE CE: aBlytesmiagtniivnegstthoitshpreoutsoecoolf,atnhiemaSlpso,nasnodrtshiagtntifhieessttuhdayttdheesrcerairbeednboygtehniesrparloltyoaccocledpoteesd `notunnecessarilyduplicatepreviously conductedorreportedexperiments. tPoromciendiumriezseuosreadvionitdhicsapursoitnogcpoalianr,edidsetsriesgsn,eodtrdoicsocnofmofromrttionatchceepantiemdparlasc.tIicnestahonsde ` c`imrocmuemnsttaarnycoerssilnigwhhtpiacihnroerqudiisrterdessst,utdhyeparnoicmeadlusrweislalrreeceliivkeelayptporocparuisateemaonarlegteshiacns tohreanSetsutdhyeDtiicrseucntloersastndh/eowritShphoonlsdoirnagnodfatphpersoevageednbtysthhaesIbAeeCnUCju.stifiedinwriting by ``nTuhembneurmnbeecresosfaarnyitmoalmseseeltescciteendtfiofricusaenidrnetghuilssattuodryygiusicdoenlsiindeesrfeodrttohbisettyhpeemoifnsitumduym. `wTahisssatupdydpesoirgnn0o7w/av2s6r/ee2v0i0de0w;iedtwbyasthaesIsiAgCneUdCIaAtCSUoCutthreacrkiRnegsneuamrbcehIrn0st0i-t0u7t-e0a3n4d. 001639 a STUDY NO.: 9921.5 Feu, 2000 ------------------------------------------------L --L -- 14.0 PROTOCOL APPROVALS: `This protocol has been reviewed and approved. Study Director: Kear E Tied PSatturdiyciDaiEr.ecNtookrer, Ph.D, DAB.T. 23a Date Sponsor's Monitor: FL ---------------------------- INITIALS ONLY (See page 2) Date 0) ManagementApproval: Aloo 477 22/07/49 Ward R. Ritter, M.S\B\V.M/b.A CVF. Date Director, 1 sesshotnt DpaSourthetr RmeseearcnhIntstit,ute 001640 Appendix B `Analytical Method ifnorMDoentkeermyiSnaetriuomnoafndPeUrrfilnueorohexanesulfonate 001641 Bi . Page Lof14 ANALYTICAL METHOD Method No: BACG-3606 Tite: Determinationof Perfluorohexanesulfonate in Monkey Serum and Urine: Sample: PreparationandAnalysisby HPLCMassSpectrometSrpyec/trMomaestrsy (HPLC/MS/MS) ----e -------- ee 10 PRINCIPLE SPeerrfulumoororheuxranienseulsfaomnaptlee(sPaFrHeS)o.btTahiensedferoromruucryimnnoem(oel.gg,u0s.m5 omLn)kecyosnttrezatie(ndPwiFiHntSgh) issamfporlteisfieadrewitthheannmiinxteedrnwailtshtaanndajornd-p(1aSi)r,inPgerrfelaugoernoto,cbtuafnfcecrarabnodxawlaatteer,(PfFoOlCl)o.wedThbey erxetcrowiatinhcnet9sth5y%ltiamceeoittahtnaet.noTulhceotentthaeyinlaidcnegt1a.te5la%yfeorirsmriecmaocviedd,,5ev%apoSrmatMedatmomdroynniesusm, Sapceecttatreo,meftryi/MilaastnsSdpetercartnrsofemerertderdyt,(oHPaLutCo/sMaSm/pMlSe)r.viTahlse,raanndgeaofnrealialbblyeryezsHulPetLsdeCxtMeandsss fSraommpalbesoucton5ttaoin2in0g,0P0F0nHgS/amtLcoonfcPenFtHrSatiinonssegrruemataerndthfarno2m01,000t0on5g0/0mLnmga/ymLbienudriilnutee.d `rweiltihacbleoresnubltltsanprrkimoorattroliaxnsaloystish.attheconcentrationof PFHSwillbewithintherangeof `TThheeimoanssspsrapyseocurtcrevoolmotfeagPteFrHisSysaenadt -P20O00iCvosltas cwhcichoismlopiwnlentiohuesngehhgtaoetgirvdeeatiloynrmdoudcee. the formationofother potentially interfering ions extracted from the matrix. inCcAluUdTiIngObNl:ooSdi,npcleapsrmiamaantdessemrauym,caarrerty oabneucmobnesriodfezreodoansobsieosh,aazlalrudnsparnedshearnveddletdiswsiuetsh, punriovceerdsuarlesptroecbaeuutsieodnsw.heRnefhearndtloiSngOuPnpnruemsbeervreSdRpIri2m-a5t-e5tifsosruea.description of safety 20 REAGENTS AND SOLUTIONS `The listed reagents o their equivalents may be used. 21 Neat Reagents 211 Water, deionized and organic free (from in-house purification system; e.g, Ingalls 210N) 212 Methanol, HPLC grade 001642 B2 Page20f14 ANALYTICAL METHOD Method No: BACG-3606 Title: DPreetpearrmaitniaotnioanondfPeArnfalluyosrisohebxyaneHsPulLfConaMtaesisn MSopnekcetryoSmeerturymMaansdsUrSipneec:trSoammeptlrey (HPLC/MS/MS) _--_--nmmm--m-- 213 Perfluoroh(eanxalyaten),aespsrouvildefdboytnheacltienet 214 Perfluorooctanecarboxalate (internal standard), 97% 215 Ammonium acetate, HPLC grade 216 Blank control monkey serum 217 SodiumCarbonate,Certified ACSGradeorequivalent 218 Sodium Bicarbonate, Certified ACS Grade or equivalent 219 Ethyl Acetate, HPLC grade 2110 Tetrabutylammonium Hydrogen Sulfate, Aldrich 97% 2111 Sodium Hydroxi5d0%e solution, Cergtradieorfeqiuivealednt 2.112 Blank controlmonkeyurine 2113 Formic Acid, 88% 22 Prepared Solutions Appropriate changes in the solutions may be made at the discretionofthe analyst 221 5 mM Ammonium acetateinorganic free water 2211 Finororegxaanmipcl-ef,reteowparteeparr(ee4.gl,ite4rs,L)m.eaMsuirxe woeultlaamnmdonfiiltuemr tahcertoautgeh(eH.Pg.L1C.5m4o2bgi)leanpdhaadsde filtration apparatus. 222 TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydroxide) 001643 B3 Method No: Title: Page3of 14 ANALYTICAL METHOD BACG-3606 Determinationof Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2221 223 223.1 224 22.41 30 31 32 33 34 3s 36 For example, to prepare 25 mL, dissolve approximat4e.l24y goftetrabutylammonium hydrogensulfateindeionizedwaterandadjustthe pHto 10with 50% NaOHsolution. Note: a more dilute solution of NaOH in water may be usedtoeffect smaller pH `adjustments. Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) Foaprperxoaxmipmlaet,elto2yp.r1e0pgaorefs1o0d0imuLm,bdiicsasroblovneaatpeprionx1i0m0amteLloy2f.d6e5iognoifzsedowdaituemrc.aMrbioxnwateellatnod ensure complete dissolution. Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) Forexamtpoplreepa,re 100mL,dissolveapproximately 10.6 g ofsodiumcarbonateand approximately 8.4 gofsodium bicarbonate in 100 mLofdeionized water. Mix well to ensure complete dissolution. INSTRUMENTS, MATERIALS, AND APPARATUS `The following or their equivalents may be used. HPLC pump(s), autosampler, and triple quadrupole mass spectrometer Autosampler vials with inserts Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with sourceofnitrogen) HPLC mobile phase filration apparatus Filters for HPLC mobile phase filtration apparatus (c.g, Nylon-66, 0.20 um) 001644 B4 ANALYTICAL METHOD Pagedof14 Method No. BACG-3606 Title: DPreetpearrmaitniaotnioanonfdPeArnfalluyosrioshebxyancHsPulLfConaMtaesisn MSopnekcetryoSmeerturymMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) _--mmmm------ 37 Asalytical balance 38 Volumetric flasks (e.g. 10 and25mL) 39 Disposable Pasteurpipettes 310 Micropipettor(s) with tips 311 Culture bes with teflon-lined caps 312 Centrifuge 313 Assorted glassware and syringes 314 Culture tubes (vials) or use withsolvent.concentration apparatus 315 1mLPlasticsyringeswith 0.2 jumPVDFsyringe filters 3.16 Variable speed horizontal platform shaker 317 pHumeter 40 PREPARATION OF STOCKS AND WORKING STOCKS a1 oAfptphroeparniaaltyestc.haAncgteusalindtihleutcioonncsewnitlrlatbieondsoofctumheenstoeldutoinontshmeapryepbaermataidoen asthetehtesd,iscretion Main Stock Solution ofPFHS ~1000 pg/mL 411 aPcrceupraarteelaynwe~i1g0h00abuogu/tm1l0. msogluPtiFoHnSoifntPoFaHS10-inmLdevioolnuimzeetdriocrgfalnaiskc)-.freAeddwadteeiron(icz.egd, organic-free water o dissolve. Dilute to the mark. Altematively, weigh the compound 001645 Bs Page Sof 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: DPreetpearrmaitniaotnioanonfd PeArnfalluycsrioshebxyaneHsPulLfConaMtaesisn MSopnekcetryoSmeerturymMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) _--mm winattoera.naMpiprxopwerlila.teTVreasnssefler(ct.hge,scoulluttuiroen ttuobae)calneadnavdedss1el0imfLdeosifredde.ionized organic-free 42 Stock SolutionofInternal Standard (PFOC), ~200 pg/mL 421 Pr`weepiagrheaabnou~t20100umgg/imnLt.soolau5ti0o-nmoLfvPoFlOumCeitnridceifolnaikz)e.doArdgadnidce-ifornizweadtoerrga(neigc..afrceceurwaatteelry ttohdeiscsoomlpveoaunndddiilnuttoetaoatnheampaprrkopwriitathedevieosnsiezled(co.rg.g,anciucl-tfurreeewtautbeer),AalntedrnaadtdiveSlOy,mweLigohf deionized desired. organic-free water. Mix well. Transfer the solution to a clean vessel if 43 Spiking solutionofInternal Standard (FOC), ~ S0ug/mL 431 2PmreLpaorfetahne~20500pugg//mml.LssoolluuttiioonnoinftPoFaOcCultiunrdeetiuobneiazenddoardgadni6cm-Lfroeefwdaetieornibzyepdiwpaettetirn.g Mix well. 44 `Working Stock Solutions of PHS 44.1 Ttaoblep.rePpraerpewaoreriknin1g0s-tmocLkvsoolluumteiotnrsi,cmfalkaseksthoerportohpeerradpiplruotpironisatsesgslhaosswwnarien.thIfdfeoslilroweidn3g modified dilution scheme can be used and documented in thestudyrecords. 001646 Bs n PageGof14 ANALYTICAL METHOD Method No: BACG-3606 Title: _-- PrDeeptaerramtiniaotniaonnodfAPnearlflyusoirsohbexyanHePsuLlfCoMnaatesisnSMopnekecytSreroumm eantd rSUrpyienc/et:MrSoaammesptlrsey (HPLC/MS/MS) FEE Approximate Concentration| Volume of PFHS solution| Final Volumein (ng/ml) I)TTT TR dferieoeniwzaetderor(gmanLi)c: Lomeoo ssmsomrecommoonmmm| | 1] 0 | [oso ie]m soroma oomarn || 0n ]| wwoo msmwaesoom||we0 0 mmee] ]st on entomm|m0ns]a [sol smionmmmmox| 0 | [oof sotormmmoes| 10 001647 7 ) Page7of 14 ANALYTICALMETHOD Method No. BACG-3606 Tile: PDreetpearrmaitniaotnioannodfPAenrafllyusoirsohebxyaneHsPulLfConaMtaesisn MSopnekcetryoSmeertruymMaansdsUrSipneec:trSoammeptlrey HPLCMSIMS) ---------------------- 442 Summaryofconcenotfrsearutmsitaondanrdss: Standard VolndSupikmeCoene.|_CoAnpcperntorxaitmiaotneof Level PHinseSrum aa (ng/ml) 204of 00000g/L. [2[10m orsoomomgm pee [Timm [= [oowonsosogm|so| WuLorsomognt |aw| [5| ouorsoomgm. [1 | 0| r a ie I a 01648 Bs Page 8 of 14 Method No: BACG-3606 ANALYTICAL METHOD ------------ Title: Determination ofPerfluorohexanesulfonate in Monkey Serum and Urine:Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 50 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriatechanges intheconcentrations ofthe analyst. of the solutions maybemadeatthe discretion 5.1 Multiple (e.g.,aboutthree)sets ofmatrixstandardsand amatrixblank(blank +IS) arc analyzed with cachsetofunknownsamples. Amatrixdouble blank(blank-IS)mayalso `be analyzed ifdesired. 52 `Ianptporoipnrdiiavtiedvuoallu~m2e0a-smdLesccurltiubreeditunbtesh,etpaipbelte ablbaonvkefmoatrrciaxch(es.tga,n0d.a5rdm.LFo).rtPhipebetlainnktsh,e pipet 10 uL oforganiwcat-erfirnsteeaedofthe workingstocksolution. Addthe 10ul, ofinternalstandard stock (~50 pg/mLt)o each tubeexcepthteblank-IS(pipet 10 uLof organic-free water instead) and vortexfor ~5 seconds. 53 For scrum samples add the following to each tube: 500 pLofthe TBA ion-pairing solution, 1 mLof 0.25M/0.25Mcarbonate/bicarbonate buffer, and 1 mL ofdeionized organicfreewater. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. 54 Asedttdi2n.g.5mLofethylacetateandextracton horizontalmixerfor 1hourat alowspeed. 55 Re`mminouvtees,thetube from the shakerandplacein acentrifuge (e.g., 2500 rpm)forabout5 56 Remothevtoepethylacelayteraandtpuetitinto acleantube and evaporate todryness (e.g.,~ 50 minutes inthe Turbo-Vap ) with a gentle stor fniterogaen amnd moderate heat (e.g., 50C). VU164Y