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AR &26 -- 1356 A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey `Southern Research Institute Study ID: 9921.6 April 22, 2003 : 000378 Final Report on A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey To: 3M Corporation P.O.3BMoxCe3n3te3r2,72245-5I1N3-30-43327 St. Paul,Minnesota 55144-1000 By: P.E. Noker and G.S. Gorman Souther Research Institute 2000 NintPh.0A.vBeonxue5S5o3u0t5h 35205 Birmingham, Alabama 35255-5305 000376 ABSTRACT `Thepharmacokinetics andurinaryexcretionofperfluorooctwaenreeinsveustligfatoedninamtalee andfemalecynomolgusmonkeys. Threemaleandthree femalemonkeyswereadministered a single iv bolus dose of2 mg/kg of perfluorooctanesulfonate,potassiumsalt (T6295). Atvarious timesaferdosing,serumandurine(24-hourcollections)samples wereobtzinedandanalyzedby HPLC/MS/MS for levels of intact perfluorooctanesulfonate. The lower limit of quantitation of theanalyticalmethodwas 10ng/mLforserumsamplesand 5ng/mLfor urinesamples. At0.5 `hours after dosing (eartlimiepeoisntt), serum concentrations of perfluoroocianesulfonate appeared 10beslightlyhigherinmalemonkeysthaninfemalemonkeysandrangedfrom 13,790to 16,250 ng/ml in male monkeys and from 7,871 to 11,790 ng/mL in female monkeys. Serum concentrations of perfluorooctanesulfonate then decreased relatively rapidly in each monkey through the first 24 hours afer dosing and, at 24 hours, ranged from 7,039 to 9,538 ng/mL in `malemonandk5,0e420y7,s294ng/mLinfemalemonkeys. Withtheexceofpontemialoeannd one female monkey, serum concentrations of perfluorooctanesulfonate remained essentially constant between Days 7and70. Thereafter, serum concentrationsofperfluorooctanesulfonate decreasedinallanimalsandrangedfrom 3,803 t0 3,840ng/mLinthemalemonkeaynsd from 2,019102,781ng/mLinthefemalemonkeysonDay 161. Theserumconcentvrearstusitoimne. data were subjected to non-compartmental pharmacokinetic analysis. The serum terminal half-life ofperfluorooctanesulfonate ranged from 122 to 146 days (mean: 132 days) in male monkeys and from 88 to 138 days (mean: 110 days) in female monkeys. The total body clearance of perfluorooctanesulfonate was 1.0 to 1.2 mL/day/kg in male monkeys and 1.6 to 2.1 mL/day/kg in female monkeys. The volumeofdistributionofperfluorooctanesulfonate ranged from 178 to 220 ml/kg and from 231 to 327 mL/kg in male and female monkeys, respectively. Only very low levels (<0.1%ofthe administered dose) of perfluorooctanesulfonate were measured in urine: during any given 24-hour period of sample collection between Day 1 and Day 91 after dosing. The results of this study provided no clear indication that the pharmacokinetics of perfluorooctanesulfonate were different in male and female monkeys. Perfluorooctanesulfonate was eliminated in urine bybothmale and female monkeys at low levels for a prolonged period of time (291 days) after iv administration. 000377 i TABLE OF CONTENTS Page SIGNATURE PAGE ii GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL v 10 INTRODUCTION 1 20 MATERIALS AND METHODS 1 21 TestSystem 22 Test Article and Vehicle 1 3 Test Article 3 Vehicle 3 Dose Formulation Preparation Dose Formulation Analyses 33 23 Experimental Design Group Assignment and Dose Procedure: 3 3 Clinical Observations Body Weights 3 4 Urine and Feces Collection 4 Serum Levelsof Perfluorooctancsulfonate Bioanalytical Method Developmentand Sample Analysis 4 4 Data Analyses 4 30 RESULTS 5 3.1 Morality 32 Clinical Observations 5 5 33 Body Weights 3.4 SerumandUrine Concentrations of 5 Perfluorooctanesulfonate 5 40 DISCUSSION 7 50 CONCLUSIONS 7 60 RECORD ARCHIVES 7 70 REFERENCES 8 000378 i TABLE OF CONTENTS (Continued) LIST OF TABLES Table 1: `Table 2: `Table 3: Table 4: Table 5: Figure 1: Appendix A: Appendix B: Appendix C: Rage Individual Body Weights 9 Serum Concentrationsof Perfluorooctanesulfonate 10 Replicate Analysis Comparisons for Potassium Perfluorooctanesulfonate Ii in Monkey Serum Pharmacokinetic Parameters Calculated from Serum Concentrations 12 `of Potassium Perfluorooctanesulfonate Urinary ExcretionofPerfluorooctanesulfonate 13 LIST OF FIGURES Serum Concentration Profile of Perfluorooctanesulfonate 16 LIST OF APPENDICES StudyProtocolandComments onStudyData. Al Analytical Method for Determination of Perfluorooctanesulfonate Bl in Monkey Serum and Urine SummaryofPharmacokinetic Parameters Calculated Concentrations of PFOs and Urinary Excretion Data from Serum for PFOs C1 000379 iv Good Laboratory Practices Disclaimer `Thisstudy describedin this finalreportwas not conductedin strictcompliancewith the U.S. Food and Drug Administration(FDA)GoodLaboratoryPractice (GLP)Regulations(21CFRPart58),and `neither this reportnor the rawdatawere reviewed by the SouthernResearchQuality AssuranceUnit. However,thestudywasconductedaccordingtothe protocolandamendmentsandtheapplicable standardoperatingprocedures,andallstudyprocedures,datarecording,andreportingwere performed in amannerconsistentwith thestandardof GLPs. `Thefinalreportaccuratelyreflectstherawdata `obtainedduringthe perforofmtahenstcudey.There wereno adversecircumtshattaaffnecctedethse `qualityorinteogftrheisttudyy. Lari E ade) PatrEi.Ncokiera, Ph.D, D.AB.T. StudyDirector ufeafos Date 000380 Signature Page `A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in theCynomolgus Monkey Poiaeir EPA) Patricia E. Noker, Ph.D., D.AB.T. Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: Hazfos Date Chet 0 2por Charles D. Hbert, Ph.D., D.AB.T. Director, Safety Assessment ozs Date `We,theundersigned,were responsible for theconductofthework and repooftrhertesuiltsnin tghe listedsections. `Weconcur with the views relative to our body ofworkasexpressed in the discussion and conclusions. Lo Gorman, Ph.D. `Manager, Bioanalytical Chemistry Group Date 000381 v Study Dates: Study Schedule and Personnel Study Initiation: DayofDosing: Last Dayof Sample Collection: Study Completion: Stady Personnel: July 26, 2001 July 30, 2001 January 7, 2002 April 22,2003 Patricia E. Noker, Ph.D, D.AB.T. Study Director Charles D. Hbert, Ph.D, DABT. Director, Safety Assessment `Norman D. Jefferson, B.A. Associate Director, Safety Assessment Gregory S. Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Darrell E. Hoskins, D.V.M,, Ph.D., A.C.L.AM. (Dipl.) Veterinarian LaJuana A. Durbin, B.S. Supervisor,LargeAnimal Laboratory D. Wayne May, LATG `Supervisor, Animal Care Carolyn R. Oliver, BS. Supervisor, Study Coordination 000382 1 1.0 Introduction The objectives of this study were to determine the concentration of potassium `perfluorooctanesulfonate in serum and to estimate urinary clearance at various times following administration ofa single intravenousdoseto monkeys. Acopyofthe protocol can be found in Appendix A. 2.0 Materials and Methods 2.1 Test System `Thethreemaleandthreefemalecynomolgusmonkeysdesignatedforuseinthisstudy were: selected from an in-house colony of monkeys that were housed at Southern Research Institute (Southern Research)priorto use on this study. These monkeyswere purchased from CharlesRiver BREF, Inc. (Houston, TX) and were an estimated 3.54.5 yearsofage when placed on study. Individual animal identification was by chest tattoo. The cynomolgus monkey is an accepted species to support clinical studies of drugs used or intended for use in humans. "Duringthe quarantineperiod,acompletephysicalexaminationincluding afecalexamination for intemal parasites, complete blood count (CBC), body weight, and rectal temperature was performed on eachofthe monkeys. The following procedures were performed on the `monkeysduring quarantine:(1)Threetubercutlesitns wereadministered toeachanimal at 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. 000383 2 Certified, commercial,dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed tothemon2-3ktimeeseaychdsay.Thequantity ofthedailyrationwassufficienttomeet nutritional requirements. In addition, the diet was supplemented with fresh frittreats severaltimeseachweek. Tapwater(Birmpuiblincwagtehrsuapplmy) was availatobtlhe `monakd elibyitsum during the quarantine andstudyperiods. The monkeys were housed individuallyinstainlesssteel cagesduring thequarantineandthestudy periods. FromDay 0't0theendofthestudy,themonkeyswerehousedin aroom thatwasmaintainedat a temperature of66.4-71.5 F and a relative humidityof25-74%. The humidity was within the required range (30-70%)over90% ofthetimeduringthe study; excursions below the recommendedhumidityrangewereofshort durationandhadnoimpactonanimalhealthor theoutocftohesmtudey. Room lightswerecontroledbyanautomatictimersettoprovide 12 hoursoflight (0600to 1800houCSrT)san,d 12 hoursofdarkperday. Cagesize and `animal care conformed to the guidelinesofthe Guidefor the Care and UseofLaboratory Animals, 7th editionTM and the U.S. DepartmentofAgriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPs) of `SouthernResearch.ThestudydesignwasapprovedbySouther Research'sInstituteAnimal Care and Use Committee. Souther Research is fully accredited by the American Association for AccreditationofLaboratory Animal Care Intemational. `With the exceptionofanimal 2053,allofthe monkeysusedonthis study were previously given a single iv bolus doseofperfluorobutanesulfonate (10 mg/kg) on 4/10/00 (Southem Research Study No. 9921.1); a single iv bolus doseofpotassium 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 ResearchStudy No. 9921.3); aansindgle ivbolusdoseofpotassiumperfluorooctanoate(10mg/kgo)n 10/9/00 (Souther Research StNou.99d21.y4).Inaddition, all monkeysusedonthsstudywere giavsinegle iv bolus dose of potassium perfluorohexanesulfonate (10 mg/kg) on 2/9/01 (Southern Research Study No. 9921.5). 000384 3 2.2 Test Article and Vehicle `Test Article: One bottle containing 6.65 gramsofpotassium perfluorooctanesulfonate (T6295; expirationdatenotsupplied;SRIEOS/L-w1a)s supplied by 3M (St. Paul, MN) and received on May 15, 2000. The test article was stored at room temperature until used. Stabilityofthe test article was the responsibilityofthe Sponsor. Vehicle: The vehicle used for the preparation of the dose formulation of potassium perfluorooctanesulfonate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 102049F, expirationdate February2004).Thevehwasistorcedalt roeom temperatureandwasconsideredtobestable whenstoredaccording totheseconditions. Dose Formulation Preparation: For the single dose formulation of potassium perfluorooctanesulfonate prepared at 1 mg/mL, the required amountoftest article was `weighed outin avolumetfrlaikc.Sterilesalinewasaddedandtheformulationwasstirred until in solution. The formulation was stored refrigerated and used for dosing on the same dayofpreparation; it was considered stable during this period. Dose Formulation Analyses: Dose concentration and homogeneity analyses were not required to be performed. 2.3 Experimental Design Group Assignment and Dose Procedure: As only one treatment group was used in this study, no formal randomization was required. On Day 0, cachofthethreemale and three. female monkeys received a single intravenous (iv) doseofperfluorooctanesulfonate at 2 `mg/kg by injection into a superficial arm or leg vein. Doses were based upon the individual body weights obtained on the day of dosing. Doses were administered at a volume of 2. ml/kg. Clinical Observations: All animals were observed twice daily for sigas of `mortality/moribundity. Each primate was examined shortlyafterdose administration for 000385 Te ) clinical signsoftoxicity. Additional clinical observations were performed on daysof blood collection,withtheexceptionofDay21whenclinical observationswerenotperformeddue to technician error. Body Weights: Each primate was weighed on Days 0,4,7, 14, 21, 28, 42, 56,70, 85, 105, 126, and 151. Urine and Feces Collection: Urine andfeceswere collected for24-hourintervalsonthe following days: prior to dose administration (Day -3; baseline), on Day 1 (0-24 hours postdose), on D2a(24y-48hourspostdose),andon Days 7,15, 21, 28, 42,56, 70,and91. The volumeofeach urine sample was measured upon collection. Urine and feces samples were stored frozen (approximately 20 Corbelow). Fecal samples will not be analyzed unless specifically requested by the Sponsor. Serum LevelsofPerfluorooctanesulfonate: Blood samples (spproximately 3 mL) were collected fromeachprimateatapproximately0 (predose) mites; 05, 2, 4, 8, 24 and 48 hours; and on Days 4, 7, 14, 21, 28, 42, 56, 70, 105, and 161 postdose. Samples were collected into tubes without anticoagulant and were allowed to clot at room temperature. The blood samples were then centrifuged, and the serum separated and stored frozen (approximately -20 C or below) until analyzed. Bioanalytical Method DevelandoSapmpmleeAnanlystis: Serumandurinesamplweerse: analyzed for perlucrooctanesulfonate using a previously validated HPLC/MS/MS method (Appendix B). The lower limit of quantitation of the method was 10 ng/mL for serum samples and $ ng/mL. for urine samples. Data Analyses: The serum concentration data for unchanged perfluorooctanesulfonate were subjected to noncompartmental pharmacokinetic analysis sing WinNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc; Cary, NC). Mean values and standard deviations fo cach parameter were calculated using Microsoft Excel Software (Microsoft 000386 5 Corporation; Irvine, CA). The urinary excretion of perfluorooctanesulfonate at each collectioninterval wascalculatedandexpressedas apercentoftheadministereddose. No other statistical analysesofthe data were performed. 3.0 Results 3.1 Mortality Allofthemonkeysin thisstudysurvivedtotheendofthestudy. 3.2 Clinical Observations No adverse drug-related clinical signs were noted for any monkey during the courseofthis study. 3.3 Body Weights Body weightsarepreseinnTtaeblde 1. Eachmonkeyeithergained weightormaintained essentially aconstantweightbetweenDay 0and Day 151(lastdayduringthestudythat `body weights were obtained). 3.4 Serum and Urine ConcentrationsofPerfluorooctanesulfonate Serum concentrationsofperfluorooctanesulfonate in three male and three female monkeys at various times through Day 161 after administrationof a single iv dose of2mg/kg are presented in Table 2 and in Figure 1. At 0.5 hours aftr dosing (earliest time point), serum `concentrationsofperfluorooctanesulfonate appeared to be slightly higher in male monkeys thaninfemalemonkeysandrangedfrom 13,790to 16,250ng/mLinmalemonkeysandfrom 7871 to 1179 nghl in female monkeys. Serum concentrations of `perfluorooctanesulfonate then decreased relatively rapidly in each monkey through the first 24 hoursaterdosing and ranged from 7,039to9,538 ng/mL inthethreemalemonkeaynsd from 5,042 to 7,294 ng/mL in twoofthe female monkeys at 24 hours; datafortheother female monkey was not obtained at this time point. A trough in serum concentrations of perfluorooctanesulfonate was observed in individual monkeys between Days 2 and 7. This trough was followed by an increase and subsequent plateau in serum concentrations of 000387 6 perfluorooctanesulfonate; for all except one male and one female monkey (M2054 and F2058), serum concentrations of perfluorooctanesulfonate remained essentially constant between Days 7 and 70. Thereafter, serum concentrations of perfluorooctancsulfonate: decreasedinall monkeysandrangedfrom3,803t03,840ng/mLinthemalemonkeysand from 2,019 t0 2,781 ng/mLinthefemale monkoenyDasy 161. To confirm the reproducibilityofthe analytical method, selected serum samples collected fromindividualmonkeysat various timesduringthestudywerere-analyzed approximately 3moanfterttheihrinistial analysis.Theresultsofthesedeterminationsarepreseatiedn `Table3. Theresultsofthefirstandsecondanalysesofeachserumsamplewereingood agreement (within 20%ofthe initially determined concentration) with each other. Pharmacokinetic parameters calculated from serum concentrations of `perfluorooctanesulfonate in individual monkeys are presentedin Tabl4e. AUCo.uu values ranged from 1666to 1877 geday/mL in male monkeys and from 932 to 1259 pgeday/mL infemalemonkeys.Theterminalhalflifeofperflucrooctainnseesruumlrafnogendafrtoem 121462day1 s(me0an: 132days)inthethreemale monkeysandfrom 88to 138days (mean: 110days)inthethreefemalemonkeys. Thetotalbodyclearanceofperfluorooctanesulfonate was 1.0to 1.2 mL/day/kignmalemonkeys and 1.6 to 2.1 mL/daykg in female monkeys. `The volumeofdistributionofperfluorooctanesulfonate ranged from 178 to 220 mL/kg in `male monakndferoym 2s31to 327mL/kginfemalemonkeys. `The amount of perfluorooctanesulfonate eliminated in urine by individual monkeys at various times after dosing is presented in Table 5. Only very low levels (50.1%ofthe administered dose)ofperfluorooctanesulfonate were measured in urine during any given 24`hourperiodofsamplecollebectwteein Doany 1andDay91aftrdosing. Thwaesnrocleear indication ofa sex-related difference in the urinary excretionof perfluorooctancsulfonate. `The urinary excretionofperfluorooctanesulfonate was prolonged; detectable levelsofthe: compound were present in urine on Day 91 (last day ofurine collection) after dosing. 000388 7 4.0 Discussion `The resultsofthisstudyindicatedthatperfluoroocwatsdaetnecteabsleuinlmafleoanndfaemtaele `monkeysforaproloperniodgofetidmeafteradministofraastinigloeinvdoseof 2 mg/kg. Among individual male and female monkeys, the apparent terminal elimination halflife of perfluorooctancsulfonate was estimated to be 88-146 days. In addition, it was found that only low levelsofperfluorooctancsulfonate were excreted in urine byeithermale or female monkeys during any given 24-hour period of sample collection. For either sex, the urinary excretion of perfluorooctancsulfonatewasprolonged;thecompouwnads still presentinurine at 91 days after dosing(longesttimepointthaturinesamples were collected). From theserum concentravteirsouns timedataandtheurinaryexcretiondata, therewas10clearindicationthe pharmacokineticsof perfluorooctanesulfonate were different in male and female monkeys. Asummaryofestimatedpharmacokineticparameters andurinaryexcretiondataobtainedduringthis andpreviousinvestigationswith variousperfluoro-compoundsispresentedinAppendix C.Forthe C6 and C8 carboxylated derivatives, and possibly the C4 carboxylated compound, the serum pharmacokineticsappearedtobedifferentinmaleandfemalemonkeys. Sexdifferenciens pharmacokinetic parameters were less apparent for the C4, C6, and C8 sulfonated derivatives. 5.0 Conclusions `There was no clear indication that the serum kineticsofperfluorooctanesulfonate were different in male and female monkeys. Perfluorooctanesulfonate was eliminated in urine by both male and female monkeys at low levels for a prolonged periodof time (291 days) aftr iv administration. 6.0 Record Archives Data, specimens,and acopyofthefinalreportfromthisstudywillbestoredintheArchivesat Souther Research forup to 1yearafteracceptanceofthefinalreportbythe Sponsor. Af1tyeearr and with the permissionof the Sponsor's Monitor, the data and any samples/specimens will be shippedtothe Sponsoror tothe Sponsor'sdesignatedarchivalfacility. Ifmataerertoibearetlainsed 000389 8 inthearchivesbeyondthisdate,suchcontinuedstoragewillbefor aspecificfe determinedwith theSponsor. Acopyof thefinalreportwillberetainedinthecentralarchivesat Souther Research. 7.0 References 1. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington D.C.; 1996. 000330 CE TEE b2 ok = HY ls |[E] Fel Fe 8 [ep |WaElE aEe L1E8BF h SC [EFL I $ EPiE2 FTs20ReRte] Es Fr oF52g] ] =H F| E 2 3 33 z SER] 5 LE g i S55 000391 10 `Table 2 `A Pharmacokinetic Study ofPotassium PerfluorooctaninetshueClyfnoomnoalgtues Monkey Serum Concentrations ofPerfluorooctanesulfonate -- Serum Concentration (ng/mL)_ [0[BOL |BL| BoC| 58 | BOL |BAL| 16250|15,120| 13,790 11,79 [2s [113% 11280|7.991| 9,500|9,207| [ns [10275 [11,508| 9.585 [6080|9.450 | 9.200| [sin | 0.578 [11.475[10015[735% [10525 sur | [2bs[asa [053|7009[5 |a0 |72 .004| 7.998|7,636|7.501| 5.165 |7,860 |7.436| Dera[rss|oas7 [7m[as|o6cas0| 7.25 | Day?[7.585 | 10090 [7550 | 516 |sa |Gas ms| [Daria [as30 rooio|osts[ss [50s [ n136 | [Day21|8547|7205"|8.380|6972|s75 | 003| [Day[ 21868"| 11,746 [Da|oyssz| 10,580| 8358 |6.403|5730|6.14| | Day ss| 7.880 | 8.695 |7,|4a06562| 6.265 [Day70| 5.038[0885|6624|aas7|5.735 |6925| [Day105[4,596 |4425| 5.750[om|aio |3m | B=Q BeL low the quantitation limit(<10 ng/mL) * Initial analysis ofthe sawm as pp robll emate ic; therewas an insufficient volume ofsafm orap real nalye sis. Data represent an averageoftwo analysesofthe sample. Pagetont 000392 u Table 3 A Pharmacokinetic Studyof Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey Replicate Analysis Comparisions for Potassium Perfluorooctanesulfonate in Monkey Serum | |_sample Results fro|m Results from | 10/15/01 19/02 Percent (ag/mL) (og/mL) | Difference | [F0F61oD2easym1sa|| rsm.55s | nsaenw ie | [P2F06o1Dsys7e| 6m 6s7ai7 | eas0]| "These samples were randomly selected from a group ofsamples containing a sufficient volumeofserum for analysis. Pagelofl 000393 lL Ef ; Fr I2 ifeel 3 Eleaf g {||UO Fe -2OF SPA]IEE= EEEHEE] ER 2 i z DLR] Eg = alle i 2: Hie] 1 , :1DbITTeglEz|8Rel8 ElLd]a5 i2 EEA LalRRgNElRidsTE H[E2E5 TBE eiisisEe BEEeld) seadzs 5 Table 5 A Pharmacokinetic Study ofPotassium Perfluorooctanesulfonate in the Cynomolgus Monkey Urinary Excretion of Perfluorooctanesulfonate Animal mo _| ConceUnritnreation UVnoliunmce| Tout gm) | 0) w| heme Dose PercUenrt iofneDo: ww -- ----0--)-- (oo [mM Bor | 0| - | mew|] [20[MT Bor | 3%| -- [sea |} oases ------] [205 [FT BOL | m0| moo|| mow Tso159 1mow Too [2s MT 3% 1310 | 69 | sew| oo | ([2m0s0s[TMF[]2 2 [ "T770 55% [ |7a55[|264a0w [ | oomo |}| [2059 [TF 5 |m0| 15 | am| oo | mime Moms [Mar iw | 6s |mao | om | [20 M72 [m0| as | asa | oor | J IE om| E I C 7 EE A FS I -- | [(2z0n[[mMM5 >[|500|| 5d5iz ||mwaa| | ooms || [O2E E05% [Fa | iw | S 7a |maw | oo | BQL = Below the quantitation limit (<5 ng/mL) * One ofthese samples was mislabeled. during collection; due to uncertainty regarding correct identification, the results are not reported. Page 1of3 000395 1 `Table 5 (Continued) A Pharmacokinetic StudyofPotassium Perfluorooctanesulfonte in the Cynomolgus Monkey Urinary ExcretionofPerfluorooctanesulfonate: sim | CoTemee n VTor ie | Tol | Dos Peroent of Dose] Lo (ng/mL) my _| wg we) ) | Dwi] [sa wa se erase | oh | [zoIM] 51 7so 50 1 2640 | ood | [2058 |F357 | Te0 | 67 | ad | 005 | [2059 TFT 40 1 0 [ 36 | 1&0| 005 | [o aw Tss e meno [om | [20IM] 21 | 560 | 18 | 2640 | ood | [208 |F157 m0[48 iaa0 | 00 | [209|F1 75 | 180 [ 15 1 14000 | 010 | [2053 TMJ 31 [ 410 T T7 T 26000 T [2osa TM" 20 Tsi0 [doz[sew | [2M] 2"Tse wa [sew| [ossTFT sr m0[71[aw | [2059 TFT 5% 1 180 [ 68 |100| 005 ood | oo) 005 | 005 | _______ owe [ 205 TMT 6 [80 T a9 1 6000 | [zn[wv] 0Tw] wa Toa 1 am ww [os FzToo[54 |maw | [209[F358 1 Wo | 44 T4000 | | 002 J wr | wm] ow | 003 | 'BQL = Below the quantitation limit (<5 ng/mL) * Oneof these samples was mislabeled during collection; due to uncertainty regarding correct identification, the results are not reported. Page2of3 000336 1s `Table 5 (Continued) `A Pharmacokinetic StudyofPotassium Perfluorooctanesulfonate in the Cynomolgus Monkey niDal | Urinary Excretion of Perfluorooctanesulfonate | ConcTentmration| VoTlueme| Toa Dose Ggml) | wb) | ww wg Fewer inUrine 0 [250TMT 12 [20[MT 77 Tom| 1 | seo | om | [77560 [67 | 26400 | 003 | [[220598T[FFTT |1 3500[|15- | aaoa0| | oor || 7-- [ 2058[MT 15 300 | 74 F (2sETM F mT0T [s 5e 9 | ase0 | | waao | | 0; ooom | || [zs TF | 0 aa |woo[om | Mose Tw 0[0| so ssa | oo | [F20e[M ow[5 s 36 h |1 204e 0 |0 0a 01 | 2][F120 T7708 | a0 | oor | BQL = Below the quantitation limi (5 ng/ml) * One ofthese samples was mislabeled during collection; due to uncertainty regarding correct identification, the results are not reported. Pagedof3 ~ 000397 e es= L eemeL sT r -essL s | FN A i rr rT 3 TTT] 3 rrr rr ! " 100 2 "0 100 10 `Time (Days) == = TTT rrr $C rrT { == L= L. 3 rr TTT= [TT1711 = ! x 100 2 "0 10 0 Figure 1 A Pharmacokinetic Study ofPotassium Perfluorooctanesulfonate in the Cynomolgus Monkey Serum Concentration ProfileofPerfluorooctanesulfonate 000398 n wan reer | e LLI[TJ [| tt i ttt srr E ee r J s r s ir isr ms sna LTT ! " Tim (ay1s0)0 1" " 100 0 Fass o eoe i r r r rr r tr p J I | E P eeer 1 CT rTTT ! " Tino (os0)0 2" wo 0 0 Figure 1 (Continued) A Pharmacokinetic StudyofPotassium Perfluorooctanesulfonate in the Cynomolgus Monkey Serum Concentration Profileof Perfluorooctanesulfonate 000399 8 F208 -rree--s---------------- | IOB rrr | i-- TTS s [T1 T TT T Pt F A i i ee= I is I i rr fe T--=--1 1 Ih Tm ew ww wm ww ow "Timo(Pays) F081 0 gr rrr t r--1T--1 Po rr P+ 5 r r rr ----r ee LT] t ew m ww wm wm we ww Time Os) Fi1g(Cuontrinueed) APharmacokinetic StudyofPotassium PerfluorooctanesulfonaitnetheCynomolgus Monkey Serum Concentration Profile of Perfluorooctanesulfonate 060400 Appendix A Study Protocol and Comments on Study Data. 000401 at I suapowcr | | A Pharmacokinetic Study of Potassium ! Perfluorooctanesulfonate in the Cynomolgus Monkey I I Southern ResearchStudyID: 9921.6 i| | I I | July26,2001 -- | | I || Sd<HDpBDS SOUTHERN RESEARCH INSTITUTE 000402 " STUDY NO.: 9921.6 -- eee 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: July26,2001 Sponsor: 3M Center, 220-2E-02 P.O. Box 33220 St. Paul, Minnesota 55133-3220 `Sponsor's Representative & Study Monitor: PGriotaocwol Apppreoyval 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 fr Za] John L. Butenhoff Apalz, 200% Date `Test Article: Perfluorooctanesulfonate, potassium salt Ship Unused Test Article to: D. Hakes Building B236 3M Center P.O. Box 33327 55133-3327 St. Paul, Minnesota 55144-1000 000403 a3 STUDY NO.: 9921.6 ty26,2000 -- hed} 20 TITLE: A Pharmacokinetic Study of Potassium Perfluorooctanesulfonate in the Cynomolgus Monkey 3.0 OBJECTIVE: The objectivesofthis study are to determine the concentrationofperfluorooctanesulfonate in serumandurineat various timesfollowingadministration of asingleintravednosoeuosf potassium perfluorooctanesulfonate 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: One soFeces [PT Collections -- | 1B0o.2e4 hoGure)do) 27 2448 how) p1t 3@ 5i] st 21: 24 hours postdose i7 211 Ba5 1r0si [eBee eeore 30000 3wo0r z20o0n1 ssrirooor s10a0o1 1029701 3i1o0r1 wweoll p20r0e1 sartoo sToa0o1 11/21120011 I 000404 a4 STUDY NO.: 9921.6 --_-- July 26,2000 dau 6.0 STUDY PERSONNEL: The following are the primary contributors and supervisory personnel participating in this study. SwdyDirector: Paul. Noker Altemate Study Director: James D. Johnson Director, Safety Assessment: `Ward R. Richter Associate Director: NommD. Jefferson Manager, Bioanalytical Chemistry: James D. Johnson `Supervisor, In-Life Laboratories: ~~ Laluana A. Durbin Veterinarian: Darrell E. Hoskins FAD.DABT. -- MS, MBA. DVM, MS, DACVE. BA. MS, MBA. AAS. DVM, ACLAM Dipl) 70 TEST & CONTROL ARTICLES: The test article will be supplied by the Sponsor, who will be responsible for documentation ofstability, as well as methodsofsynthesis, fabrication, or derivation. Upon completion of the study, residual bulk test article will be retuned to the Sponsor. 71 IDENTITY OF THE TEST ARTICLE: Name: Perfluorooctanesulfonate, potassium salt Identification T-6295 Supplier: 3M Lot Number(s): To be documented in the study data. Special Handling: ~~ None Characterization: Documentation of the characterization of the test article, including identity, purity, strength, and composition,aswell as methods of synthesis, fabrication, or derivation, is the responsibilityofthe Sponsor. Copiesofcharacterization data have been provided to the testing laboratory. Stability & Storage: The bulk test article will be stored at room temperature. Stability of the bulk test article is the responsibility ofthe Sponsor. 000405 As STUDY NO.: 9921.6 -_-- 72 IDENTITY OF THE VEHICLE: July 26,2001 heSau Name: Sterile Saline Supplier: Commercial supplier LotNumber(s): To be documented in the study data. Special Handling: ~~ None Characterization: Documentationofthe characterizationofthe vehicle may be attainedbyrecordingallpertinentinformation fromthe containerlabels,orbyretainingthecontainerlabels,o copies theinrtheestoudyfda,ta.The veihsaicomcmerlciaelly 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: The test article will be formulated in sterile saline at a concentration of 1 mg/mL for intravenous administration; briefly, the required amountoftest articlewillbemixedwiththerequiredamountofsterilesaline, andthemixturewill be stirred until the test article is visibly in solution. Formulations will be stored refrigerateduntilused for dosing; formulationsofthetestarticle insterilesalineare expected to be stable for weeks when so stored. Dose Formulation Concentration and Homogeneity Analyses: No analysis of dose formulation concentration and homogeneity will be conducted. 80 TEST SYSTEM: Species & Strain: SuApgpelioenr:Day I: Weight at randomization: `Number on Study: Cynomolgus monkeys (Macacafascicularis) Charles River BRF, Inc. (Houston, TX) 3-4yeaofragse (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 000406 As STUDY NO.: 9921.6 -_ 9921.3, potassium perfluorooctanoate in study 9921.4, and potassium perflourohexanesulfonate in 9921.5. iy 26,2000 umf 81 JUSTIFICATION: Primates are commonly used in preclinical pharmacological and toxicological `evaluationsofcompuo seduorn intd ends edforusein humans,orto whichhumans `might be exposed. 82 HousiG: During quarantine/acclimation and study, animals will be individually housed in stainlesssteel,slat-boctatgeosm. Allanimalswillbehousedin aroom thatprovides `a minimumof10airexchapnergheousr.Controlswillbe settomaintaintheanimal room at a temperatureof64-84 Fand arelative humidityof 30-70%. A 12-hour light/12-hour dark cycle will be routinely maintained. Animals will be acclimated in the same room used for study. 83 BEDDING: None required for caging equipped with flushable pans. For cages equipped with excrementabsorption pans, commercial heat-treated hardwood chip bedding will be `usedforexcrementabsorption. Analysesof the bedding,suppliedby thevendor, will be reviewed by the Departmentof Veterinary Medicine and Bioresources (DVMB) of Southern Research to assure that no known contaminants are present that could affect the healthofthe animals. 84 Dm Diet will be commercial Certified Primate Chow #5048 (PMI Feeds, Inc.; St. Louis, rMOe)c.ommTehndeepdrdiamialtyesratwiiolnl abveaiolfafbelereadt efaecedh tfeweidciengdaiinltye,rvawli.thInapapdrdiotxiiomna,ttehleyditehte `will be supplemented with fresh fruit offered daily and treats offered several times `reeaqcuhirweemeekn.ts.ThAenaqluyasnetsiotyf tohfethfeeedda,isluyprpaltiieodnbwyiltlhebevesnudfofri,ciweinltl tboemreeevtienwuetdribtyiotnhael 'DVMBofSouther Research to assure that no known contaminants are present that could affect the healthofthe animals. 000407 a7 STUDY NO.: 9921.6 Jy 26,2000 --ehTaf} 85 WATER: Water (Birmingham public water supply) will be supplied ad libitum during the `quarantine and study periodsviaan automatic watering system. Samplesof water from the animalfacilitywill be periodically analyzed,and theanalyseswilbe reviewedbythe DVMB ofSouthernResearchtoassurethat no knowncontaminants arepresentthat could affect the healthofthe animals. 86 QUARANTINE: Allprimateswereselectedfromstockanimalsthatwerequarantinedfor aminimum of 35 days upon receipt at Southern Research. No prophylactic or therapeutic treatments were administered during the quarantine period. Standard procedures conducted during the quarantineperiodwere as follows: 4) A complete physical examination includinga fecal examinationforintemal parasites, complete blood count (CBC), body weight, and body (rectal) temperaturewas performed;b)threetuberculintestsat2-weekintervals (administered intrapalpebrally, using alternate eyelids for cach test) were perfonoearchmpriematde. Primatestestednegativetoalltreetestsprior to reflroemqauarasntiene. ;c)thebloodsampledrawnforCBCwasalsousedfor `measuremoefnBt virus titer;d) afecalsampleforculture(screening for Salmonella and Shigella) was obtained and submitted to an independent laboratory for analysis; and ) all primates were examined at least once weekly. by aveterinarianandobserved(cage-sideobservations)twicedailyforabnormal clinical observations and mortality/moribundity. `Throughout the subsequent holding and study periods, monkeys wil be maintained `under conditions similar to those for quarantine. In addition, quarterly evaluations 10 be performed on each monkey will include tuberculin testing, body weight determination, andbodytemperature measurement. Primates that respond positively toa tuberculin test will be euthanized immediately. 87 PSYCHOLOGICAL WELL-BEING AND SOCIALIZATION: Nonhuman primates will be provided a psychological well-being program for social enrichment as directed by a veterinarian and approved by the IACUC and in accordance with the appropriate SOP. Nonhuman primates wil be provided cage and feeding regimen modifications daily for their psychological well-being. The modifications include, but are not limitedto; swings, perches, Kong toys, clean 2liter soft drink bottles, puzzle feeders, nutritionally sound primate treats, unshelled 000408 As STUDY NO.: 9921.6 July26,2001 _-- ee heli `peaanndruatwfsrui,t. Wherepossible,primateswillbehousedproximatetoone another for visual and vocal contact. 88 ANIMAL IDENTIFICATION: The primates will be individually identified by chest tattoo number or letter combination. Positive identification will be required afte every cage change and prior to blood sampling, dose administration, and observation. 90 EXPERIMENTAL DESIGN: Asonly one treatmentgroup willbe usedinthisstudy,noformalrandomization wil be required. Doseswillbe administered by intravenous injection to determinethe pharmacokinetics of thetestarticle. Eachprimate (threemales,threefemales)willreceive asingledoseof potassium perfluorooctanesulfonate by injection into a superficial arm or leg vein. Bloodsamplesforserumdrugleveldeterminationswillbe collectedfromcachprimateat selectedtimepoints duringthestudy. Urineandfeces wilalsobecollectedatpredetermined intervals. Asynopsisofthe study design is presentiend the following table. IS---- fotudy Procecures [TTT0TTT2 [417[1472128[35[42[5670[84[51 [Tos[126 147]161} % i HE ERR RBA n ClinicalObservations] [x[x[x[x[x [xfx[xl JxTxl[x][x][ I [ } [rned@Feces TTTxTx|Txlxlxlx|Tell]Tx] "TT BenmbrogCevels|TxTx xTx xlx]x]x] [xJxlx]Txlx] 1 Tx] * Denotes week, 91 RANDOMIZATION & GROUP ASSIGNMENT: Asonlyonetreatmentgroupwillbeusedin thisstudy,noformalrandomizationwill be required. 000409 Ao STUDY NO.: 9921.6 July26, 2001 I -------- SE 92 Dose PROCEDURE: oEafcphotparsismiautme p(ethrrfeleumoarloeosct,tahnerseuelffeomnaatlees()2wmigl/kregc)ebivyeiansjienctgiloeninitntroavaesnuopuesrf(iIcVia)ldoasrem or legvein.Doseswillbebaseduponthemostrecentindividualbodyweights. Doses willbe adminataivsolutmeeofr2meL/kdg.Thedayofdosing willbe Day 0ofthe study. 93 CLINICAL OBSERVATIONS: `DpaeriiloydaObnsdetrwviactieodnasi:ly,Almlommoinnkgeaysndwialfltebrenoobosne,arvtedleoanstce4dhaoiulrysdauprairntg,tdhuerihnolgdtihneg esxtturdeymifsorwislilgabseohfummoarntealliytys/amcroirfiibcuenddibtyy aanndovoevredrotsetooxfibciatryb.ituArnaitmealfsollfoowuendd biyn exsanguination with appropriate approval. Detailed Observations: Each primate will be examined shortly after dose `administration for detailed clinical signs oftoxicity. All findings will be recorded. `Additional clinical observations wil be performed and recorded on daysofblood collection. 94 Bopy WEIGHTS: Each primate will be weighed on Days 1, 4, 7, 14, 21, and 28 and at bi-weekly (Days 42, 56, 70, and 84) or tri-weekly (105, 126, 147) intervals thereafter through the endofthe study. 95 URINE AND FECES COLLECTIONS: Urine and feces will be collected for approximate 24 hour intervalspriorto dosing andonDays 1 (0-24 hours postdose), 2 (24-48 hours post dose), 7, 14, 21, 28, 42, 56, 70and 91.Thevolumeof cachurinesamplewillbemeasuredupon collection. All samples collected will be stored frozen at ~20 C or below prior to analysis (urine) or until further notice by the Sponsor (feces). 9.6 SERUM DRUG LEVELS: Balpporoodxismaamtpelleys0 ((parpepdroosxei)mmaitneultyes3; 0m.L5,)2,w4i,ll8,baendco2l4lehcotuerds;farnodm2,ea4,c7h,p1r4i,m2a1t,e28a,t 42,56, 70, 105, and 161 days after dosing. Additional blood samples may be taken at subsequent times ifequested by the Sponsor. Samples will be collected into tubes 000410 a0 STUDY NO.: 9921.6 July26,2001 helt s`awmitphloeustwainltlitchoaegnubleantceanntdriwfiulgledb,eaanldltohweesdteroumclwotilaltbreosoempatreamtpeedraatnudrset.orTehdefrbloozoedn. at-20 C or below until analyzed. 9.7 BIOANALYTICAL METHOD DEVELOPMENT: Bioanalytical method(s) will be developed for the determination of perfluoroocitnasenruemasnudulrifnoemnataritcees. The methowdi(llsb)e Validatedforaccuracyand ideallywil besensitivetothe 1ppmorless level. Iffeces andorothertissuesrequireanalyses, these analyseswillbenegotiawtietdh the `Sponsor. 938 BIOANALYTICAL SAMPLEANALYSIS: `Theserumandurinesamplesfromall monkeyswillbeanalyzedforconcentrations of perfluomusiongtcheptrevaiounslyevalisdatued mlethfodo.Thnedaatawtillebe expressed as equivalents of potassium perfluorooctanesulfonate. Feces will be `analyzed onlyifrequested by the Sponsor. 99 AMMALDISPOSITION: Attheend ofthestudy, monkeys willbe maintainedinthestock colony. Intheevent `untowardreactionsorotherconditionswarranttheeuthanasiaof amonkeyatany timeduringthesamplecollection period, aserumsample (5-10mL)willbeobtained. Sromtheanimalpriortoeuthanasia. Afereuthanasia,theanimalwillbenecropsied andtheliverandbile(asmuchaspossible)willberemovedand storedat approximately -70 C. 100 DATA ANALYSIS: Pharmacokinetic parameters (.g., AUC, half-life, clearance) will be estimated from serum concentrationsofunchanged perfluorooctanesulfonate, as appropriate and feasible, using a standard pharmacokinetic program. "The total amountofperfluorooctanesulfonate in urine will be calculated and expressed in termsofpercentofdose. Mean values and standard deviations will be calculated for each time point and sample type, as appropriate. No other statistical analysesofthe data will be performed. 000411 Ad STUDY NO.: 9921.6 110 RECORDS: July26,2001 Mellen Allrawdatapertainingtotheconduct ofthisstudy,andallsamples/specimenscollectedin thisstudy,willbestoredintheArchivesat SouthernResearchInstituteforup to 1yearater acceptanceofthefinalreportbytheSponsor. After 1yearandwiththepermissoiftohne. Sponsor's Monitor, the data and any samples/specimens will be shipped to the Sponsor or totheSponsor'sdesiagrcnhivaaltfaceilidty.Ifmaterialsaretobe retainedinthearchives beyondthisdate,suchcontinuedstoragewillbefor aspecificfee determinedwiththe Sponsor. AcoopftyhefinalreportwillberetainedinthecentralarchivesatSouthern Research. 120 FINAL REPORT: Abriefletterreportsummarizingthe serumdrug levelresultswillbe issuedas soon asthe. informisaavtaiilaoblne. Adraftfinalreportwillbeissuedwithin60calendardaysafier `completionofthe in-lfe aspectsofthe study. The final report electronic and hard copies) willbeissuedwithin 15 workingdaysafte recofteheiSpopnsotr finalreviewcomments onthedraftreport. Thefinalreportforthepresentstudy willinclude,butnotnecessarilybe: limited to the following: Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data 130 REGULATORY REFERENCES: `This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs)ofSouther Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 PROTOCOL AMENDMENTS AND DEVIATIONS: Amendments: All changes in or revisionsofthe approved protocol and the reasons thereof will be documented, signed, and dated by the Study Director, and the Sponsor's Monitor. Amendments will be maintained with the protocol. Written approval (afax signoaretlecutrroneic communication, suasecmahi forchanges in the protocol may be granted by the Sponsor's Monitor, butawrittenamendmentwill follow. 000412 an STUDY NO.: 9921.6 July 26,2000 -------------- 1A Deviations: Alloperationspertaining to tis study, unlessspecificallydefined in this protocol, will be performed according to the Standard Operating Procedures (SOPs) ofSouthern Research and/or te protocol, and any deviations rom protocolor SOP will be documented. 13.2 REGULATORY COMPLIANCE: Good Laboratory Practices: This nonclinical laboratory study will be conducted in the spirit of, but will not require strict compliance with, the U.S. Food and Drug `Administration's(FDA)GoodLaboratoryPractice (GLP)regulatio(n2s1 CFRPart 58). Data fromthisstudymaybesubmittedtothe FDA insupport ofan INDINDA application. Quality AssuranceReview: As thisstudywilnotbeconductedinstrictcompliance: with FDA's GLP regulations, nither the in-lfe activities nor the final report will be audited by the Quality Assurance Unit at Southern Research. 133 FACILITIES MANAGEMENT AND ANTVAL HUSBANDRY: Animalcarewillbe incompliance withthe SOPsofSouthernResearch,the Guidelinesfor the Care and UseofLaboratory Animals, 7* Edition (Institute of Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, DC; 1996), and the USS. Department of Agriculture through the Animal Welfare Act (Public Law 99-198). Southem Research Institute is fully accredited by the American Association for Accreditation ofLaboratory Animal Care (AAALAC). 134 ANIMAL WELFARE ACTCOMPLIANCE: By sigaing this protocol, the Sponsor signifies that there are no generally accepted altematives to the use ofanimals, and that the study described by this protocol does not unnecessarily duplicate previously conducted or reported experiments. Procedures used in this protocol are designed to confortom accepted practices and tominimizeoravoidcausingpain,distress,ordiscomfortintheanimals.In those circumstances in which required study procedures are likely to cause more than momentary or slight pain or distress, the animals will receive appropriate analgesics or anesthetics unless the withholdingofthese agentsbasbeenjustifiedinwriting by the Study Director and/or Sponsor and approved by the IACUC. 000413 ass STUDY NO.: 9921.6 July26,2001 --eeerenoororo helo la `Thenumberofanimalsselectedforuseinthisstudyisconsidered tobetheminimum `numbernecessaryto meetscientificandregulatory guidelines forthistype ofstudy. `This study designwasrevi bytehewIAeCUdC atSouthernResearch Institute and `was approved on 07/26/2000; it was assigned IACUC tracking number 00-07-034. 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. Paliicar . Ith) swaybirector: OnisEd Patricia E. Noker, Ph.D, D.AB.T. Study Director S/4foz 3/acfu Date. somrorimion; _T _Pe2 AAL pil7zoos INITIALS ONLY (See page 2) Date Management Approval: __Jka} Gohl ZsJoy `Ward R. Richter, M.S., D.V.M,, D.A.C.V.P. Date Director, Safety Assessment Department, Southern Research Institute The Study Monitor did not rebum orginal probocol. A copy of +he oviginal was suned aga bythe Study dvechor + Sovwarded to the mondor for signahare, Hos 000414 Au Comments on Study Data o`tThheerfwoillsoewniontgeids, atlhiesSt toufdpryoDtiorceocltaonrddeotreSrOmiPndeedvtihaetisoendsetvhiaattoicocnustrroedhdauvreinnogtahdivsesrtsuediym.paUcntleosns theoutocftohesmtudey. `aThcecporomtmoocodlatrtehqeuilraebdortahtaotryursicnheedcuollel,ectthieocnoslbleecctoionndsuscpteecidfioendfDoaryDa1y4.14Iwneorremdaedtroe onDay15. Dthuee"tSoeranumoDverrusgighLteavteplrso"tsoeccotliodneovfeSleocptmieonnt,5t.h0.epDroatyoc1o6l1 lwisatss Dacatyua1ll6y1 oans11/27//3012/,0a1nidn sewarscoullecmted onthatday insteadof 12/31/01. Dueto technician roacrlini,cal observawtaisnoont recoorndDeady 21 (8/20/01). ItnhtehaeppdlaitcaafbolrethSiOsPsst.udy, recording errorsoccurredbutwerecomet inaccordancewith 000415 Appendix B Analytical Method for Determination ofPerfluorooctanesulfonate in Monkey Serum and Urine 000416 B1 Method No: BACG-3607 ANALYTICAL METHOD Page 1of13 Title: Determinationof Preparation and Perfluorooctanesulfonate in Analysis by HPLC Mass Monkey Serum and SpectrometryMass Urine: Sample Spectrometry (HPLCMS/MS) _--_--mm---- 10 PRINCIPLE SPeerrfulumorooroctuarnicnseulsfoanmaptlee(sPFaOrSc).obTthaeinseedrufmroormurciynneo(meo.lgg.u0s.5 mmoLn)kceoynstaitnrienagte(dPFwOiSt)h siasmfpolretisfaierdewtihtehnamnixinetderwnialthsatannidoanr-dpa(1iSr)i,nPgerrefalgueornoto,cbtuafnfeecraarbnodxwaalatteer,(fPoFlOlCo)w.edTbhye erecxonsttitrutweiadthicent9hty5l%iacmeoteattnhea.nTohl ecoentthayilnaicnegta1t.e5la%yefriosrmriecmoacviedd,,5ev%apoSrmatMedtaomdmroynneisusm, Sacpeetcattre,omfeitltreyr/edM,asasnSdpetcratnrsofmeertrredy (toHPaLuCt/osMaSm/pMlSe)r.viTahlse,raanndgeoanfarleylziesdblebyresHuPltLsCextMeansdss fr`oSammaplebs oco2nu0tattioni1n0g,P0F00OnSg/atmcLonocfePnFtrOaStiionnssgererautmearnthdafnro1m01,000t0ong5/0m0Lnmagyb/einmduirliuLnteed. r`weiltihabcloentrresoullbtlsapnrkiomrattoriaxnalsyositsh.at the concentrationof PFOS will be within the range of ``TThhee imoansssprspaeycstoruormceetvroylotfagPeFiOssSetaantd -P2F0O0C0voisltasccwohmipclhiisshleodwienntohuegnhetgaotgirveeatiloynrmeodduece. the formationofother potentially interfering ions extracted from the matrix. iCnAclUuTdiInOgNbl:ooSdi,npcelapsrmiamaatnedssmearuym,caarreytao nbuemcboensriodferzeodoansosbeiso,haazllarudnspraensdehravneddlteidsswuietsh, purnoicveedrusraelsptroecbaeuutsieodnsw.heRnefhearndtloiSngOuPnpnruemsbeervreSdRpIri2m-a5t-e5tifsosruea. description of safety 20 REAGENTS AND SOLUTIONS `The listed reagents or their equivalents may be used. 21 Neat Reagents 211 Water, deionized and organic ree (fiomin-housepurification system; .g., Ingalls 210N) 212 Methanol, HPLC grade 000417 B2 Page20f13 ANALYTICAL METHOD Method No: BACG-3607 Title: PDreetpearrmaitniaotnioanondfPeArnfalluyosriosocbtyaneHsuPlLfConaMteasisn MSopnekcetryomSeetrruym/Maansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) --_--_--m-- 213 Perfluorooctanesulfonate (analyte), as provided by the client 214 Perfluorooctanecarboxalate (intemal standard), 97% 215 Ammonium acetate, HPLC grade 216 Blank control monkeyserum 217 Sodium Carbonate, Certified ACSGroraequdivaleent 218 Sodium Bicarbonate, Certified ACS Grade or equivalent 219 Ethyl Acetate, HPLC grade 2110 Tetrabutylammonium Hydrogen Sulfate, 97% 2111 Sodium Hydroxide 50% solution, Certified grade or equivalent 2112 Blankcontrol monkeyurine 2113 Formic Acid, 88% 22 Prepared Solutions Appropriate changes in the solutions may be made at the discretion ofthe analyst 221 mMAmmaceo tatein norgi anicfu reewm ater 22.1.1 Fionroerxgaamnipcl-ef,rteeopwraetpearre(e4.lgi,te4s,L)m.easMuirxeowuetllamamndonfiiluemratcehtraotueg(heH.gP, L1m.obgi5)laen4dpaha2dsde filtration apparatus 222 TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydroxide) GCo4LE B3 ' Page30f13 ANALYTICAL METHOD Method No: BACG-3607 Title: PDreetpearrmaitniaotnioanondfPAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetyroSmeerturmyMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) --_--_ s Mm ---ms 2221 Finordeexiaonmipzleed,twoatperreapnadr2ad5ejmusLt,tdhiespsoHlvteo41.024wigtohf5t0e%tNraOaHbsuoltutyionl.ahymdrmogoennsuilfuatme aNdojtues:tamemnotrs.e dilute solutionof NaOH in water may be used to effect smaller pH 223 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) 223.1 Faoprpreoxximaatmeltypo2pl.r1e0epga,orefs1o0d0miuLm,dbiiscasroblovneaatpeprionx1i0m0atmeLloy2f.d6e5iognoifzseoddwiautemrc.arMbionxawteelalntdo ensure complete dissolution. 224 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) 224.1 Faoprpreoxxaimmpaltee,ltyo8p.r4egpoafreso1d00iummL,bidciasrsboolnveataeppinro1x0i0mamteLloyf1d0.e6igonoifzsedodwaituemr.caMriboxnawteellantdo ensure complete dissolution. 30 INSTRUMENTS, MATERIALS, AND APPARATUS `The following or their equivalents may be used. 31 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 32 Autosampler vials with inserts 33 Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) 34 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with sourceofnitrogen) 35 HPLC mobile phase filtration apparatus 36 Filters for HPLC mobile phase filtration apparatus (c.g., Nylon-66, 0.20 jm) 000419 B4 Method No: Title: Page dof 13 ANALYTICAL METHOD BACG-3607 DeterminationofPerfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 37 Analytical balance 38 Volumetric flasks (e.g. 10 and 25 mL) 39 Disposable Pasteur pipettes 310 Micropipettor(s) with tips 301 Culturetubeswithteflon-lined caps 312 Centrifuge 313 Assorted glassware and syringes 3.14 Culture tubes (vials) for use with solvent-concentration apparatus. 315 1m Plasticsyringeswith 0.2 pum PVDF syringefilters 316 Variablespeedhoripzlaotfonrmtshaakler 317 pHmeter 40 PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrationsofthe solutionsmaybe made at the discretion ofthe analyst. Actual dilutions will be documented on the preparation sheets. 41 Main Stock Solutionof PFOS ~1000 pg/mL 411 Prepare an ~1000 pg/mL solution of PFOS in deionized organic-free water (c.g, accurately weigh about 10 mg PFOS into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound 000420 Bs Page Sof 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: DPreetpearrmaitniaotnioanondf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetyroSmeerturmyMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) ---- winattoera.naMpiprxopwerlila.teTversseal n(t.hgse.s,ofcluulettiurornetutboeac)laenadnvaedsdse1l0imfLdoesfirdeedi.onized organic-free 42 Stock Solutionof Internal Standard (PFOC), ~200 g/mL 421 P`wreeipgahreabaonu~t21000mgg/minLtosoalu5t0io-nmoLfvPoFluOmCeitnridcefiloansikz).edoArdgadnidce-ifornoiezweadtoerrgaeni.cg.,faocecurwaatteelry t1h0deicsosomlpvoeuanndddiinltuote atoatnheapmparrokpwriiatthedveieosnsiezle(de.ogr.g,acnuilct-ufrreeetwuabtee)r.aAnldrmaadtidve5l0y,mwLeiogfh deionized desired. organic-free water. Mix well. Transfer the solution to a clean vessel if 43 Spiking solutionofInternal Standard (PFOC), ~ SOpg/mL 43. 2PrmeLpaoreftahne~25000pgg//mmLLssoolluuttiioonnionftPoF&OcCultiunredetiuobneiazenddoaddr6mgfLroeeafwdaetineornbiiyzepdiwcpaettet-ring Mix well. 44 Working Stock Solutions ofPFOS 44.1 tTaoblper.epParreepawroerkiinn1g0s-omcLkvsoolluumteiotnrsi,cmfalakseksthoerportohpeerradpiplruotpironisataesgslhasoswwnarien.tIhfe fdoelsliorweidnag `modified dilution scheme can be used and documented in the study records. WAoprpkrionxigmSattoeckCoLnecveenltr(aWtSioLn)| (g/mL) Volumeof PFOS solution| Final Volume in mSorm mon || 0] ] dferieoeniwzaetderor(gmaLn)ic 000421 5s Page of 13 ANALYTICAL METHOD Method No.: BACG-3607 Title: DPreetpearrmaitniaotnioannodf PAenrafllyusriosocbtyaneHsuPlLfConaMteasisn MSopnekcetyroSmeerruym/MaansdsUrSipneec:trSoammepnlrey EPLCMSMS) _--_- Emr 31250] smiofezsoomgmi | 10 | -- a] ty [som smtusoommimwn| Limo solomon 10 | [2 [eo snopes 442 Summaryofconcentrationsofserum standards: = Standard Volume and Spike Cone.|_CoAnpcperaotxriamtaotneof Level PROS in serum (ng/mL) [5 [omorsooomm|so00| = =-- STuet-- remra:----| 000422 B71 Method No: Title: Page7of13 ANALYTICAL METHOD BACG-3607 Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) [= Temommwa|m0] [x [ooworsooongm|aw| [ooc [iocmoorrsnomoommgpam [0| [+ [owornommger | m0 | oopoarsogomgaml ||510]| 50 PREPARATION OF SPIKED STANDARDS AND BLANKS oApfptrhoeprainaaltyescth.anges inthe concentrationsofthesolutionsmay be madeat thediscretion 51 Maunlatliypzleed(wei.gt.h,acbacohutstehtorefeu)nskentsoowfnmastarmpilxesst.anAdamradtsriaxnddoaumbalterbilxanbkla(nbkl(abnkl-a1nSk) m+IaSy)aalrseo be analyzedifdesired. 52 I`anptporoipnrdiiavtieduvaoll~u2me0-amsLdecsuclrtiubreedtiunbetsh,eptiapbeltebalbaonvkemfaotrriexac(he.sgt,an0d.a5rdm.LF)o.rPtihpeetbliannktsh,e opfipientte1r0naulLsotfanodragradnisct-ofcrke(e~w5a0tperg/imnsLt)eatdooefatchhetuwboerkeixncegspttotchkesbollauntki-oInS. (Apdipdetth1e0 1p0LuoL.f organic-free water instead) and vortex for ~5 seconds. 53 For serum solution, 1 samples add the following to each tube: 500 pL of the mLof0.25M/0.25M carbonate/bicerbonate buffer, and 1 TBA ion-pairing mL of deionized organic free water. 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/LOM carbonate/bicarbonate buffer and 5 seconds. 1 mLof deionized water. Vortex each tube for about 000423 Bs Page8of13 ANALYTICAL METHOD Method No: BACG-3607 Title: DPreetpearrmaitniaotnioannodf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasins MSopnekcetryoSmeetrruym/MaansdsUrSipneec:trSoammeptlrey (HPLC/MS/MS) _--_--nmm 54 Add2.5 setting. mLofethylacetateandextract onhorizontal mixer for 1hourat a low speed ss Remove thetubefrom `minutes. theshakerandplacein acentrifuge (e.g, 2500 rpm)forabout 5 56 T(ake6o~f5f:0thme8itnoup,teetshiynltahceeTtautrebloa-yVeraapndp)wuittihtiatgeontalcelsetarnteaumbeoafnndietvraopgeonraatnedtmooddreyrnaetses heat (eg, 50 C). 51 cReocnotnasitniitnugt1e.t5h%e froersmidiuceaicnid50a0nduvLorotfex5b%r5 imetMofmalimxym.oFnilituermthaecestaamtep:le9s5t%hrmoeutghhan0o.l2 1m PVDForNylon syringe filters ino autosampler vials, 60 PREPARATION OF SAMPLES 61 Alvilgoorwoeusalcyh. sPeirpuetmsaanmaplilqeuottootfheaawcthosraomopmltee(mep.egr,a0t.u5rme.LV)oirnttoexinedaicvihdsuaalmp~l2e0b-rmiLecfullbtyuur,te `tuebxepse.ctedIfconnecceenstsraartyi,ondoifluttheeatnestalaritqiucolte boefinagnyansalaymzpeldewiwlilthfalbllwaintkhimnatthreixconscoentthraattitohne ratunbgeeaonfdthvoerstteaxndfaorrdacucrouvpel.eoAfsded1co0npdsL.ofintemalstandardstock (~50pg/mL)toeach 62 Fsoolrutsieonr,um1 msaLmpolfes0.a2d5dM/th0e.2f5oMllcoawribnognattoec/baicchartbuobne:ate50b0ufLfero,fatnhde1 TmBLAofiodne-ipoaniirziendg organic free water. following to each Vortex tube 1 cach tube for mL of TBA about 5 seconds. For ion-pairing solution, u1rimneLsaomfpl1e.s0aMd/d1tOhMe c5asrebcoonnadtse./bicarbonate buffer and 1 mLofdeionized water. Vortex each tube for about 63 Add 2.5 setting. mL of ethyl acetate and extract on horizontal mixer for 1hourata low speed 000424 Bs Page9of13 ANALYTICAL METHOD Method No: BACG-3607 Title: DPreetpearrmaitniaotnioannodf PAenrafllyusoirsoocbtyanHesPulLfConaMteasisn MSopnekcetyroSmeetrruym/MaansdsUrSipneec:trSoammeptlrey (HPLC/MS/MS) _--_-- 64 Rmeimnuotvese.thetubefromtheshakerandplacein acentrifuge (c.g.,2500 rpm)forabou5t 65 (Te8a,o~f5fkt0hemteionupteetsihnyltahceeTtautrebola-yVeraapndp)wuittthinagteonatclleseatnretaumboeafnndietvraopgoenraatnedtomdordyernaetses heat (e.g, 50 C). 66 Reccoontnasitniitnugte1t.h5e%rfeosrimdiuceaicnid5a0n0dkvoLrotfex5br%iefSlmytMoammimx.onFiilutmeratcheetsaatme:pl9es5%tmherotuhgahn0o.l2 wm PVDF or Nylon syringe filers into autosampler vials. Cap vials for analysis. 70 ASNPAELCYTSRIOSMBEYTHRIYG/HMAPSESRFSOPREMCATNRCOEMELTIQRUYI(DHCPHLCR/OMMSA/MTSO)GRAPHY MASS 71 Conditions are to be optimizedifnecessary. 711 HPLCConditions Analytical Column: Keystone Scientific Aquasil C18, 150 mm x 2 mm ID, or equivalent `Guard Column: EInljuetcitoinonFlvoolwurmaete:: Mobile phase: KeystoneAquasil C18 400 pL/mi, 10 mm x 2 mm SL BA:: 1S.m5M% faomrmmiocnaciiudmiancemteattheabnuoflfer Gradient Profile: Temperature: 0-3min. 3-Smin. S-8min. 10min. Ambient S0%A:50%B 102%0%AA:: 9800%%BBsltienpegarragdriaednitent 50%A : S0%B step gradient 7.12 SPoEftSwcaireeex::APP!PIE3S0c0ie0xTTruirplbeoQTuurbaoQduarnupole Mass Spectrometer Condit0io0ns0425 B10 Method No: Title: Page 100f13 ANALYTICAL METHOD BACG-3607 Determination of Perfluorooctanesulfonate in Monkey Serum and Urine: Sample Preparation and (HPLCMS/MS) Analysis by HPLC Mass Spectrometry/Mass Spectrometry TNuortbe:oiVoanlSupersalyisStoeduruncdeer "MS/MS Acquisition Conditions" override parameters in this table. Auxiliary Gas: Air (e.g. Grade0.1)at 85popuersnquadreinsch Parameter Value 5 2000 NC 0 TEM 450 OR 20 RNG 120 @ 10 Qt n sT 15 ROI n Q2 20 RO2 50 sT3 60 RO3 52 DF 250 CEM 1800 000426 B11 Page 110f13 ANALYTICAL METHOD Method No: BACG-3607 Tite: DPreetpearrmaitniaotnioannodf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetyroSmeerturmyMaansds UrSipneec:irSoammeptlrey (HPLC/MS/MS) _--m `Parameter NEB Value 15 CUR 6 CAD 5 QE 0 3 POL 1 vem 0 PE 0 MSSanSteA:ccsMRoMnConditions APcoqluairistiyt:onmode: PNreogfaitlieve Pausetime: 5milliseconds. Masses requested: PrOS: Q81M9ass(am) oQs3sM.uss(em) Du2e0llTime(ms) PROCS) Qa1Msass(am) RDOo2 me R1033 Qt sT Q36M8e9ssmi) 3S5at 3S5up 3as7 3 i1s 118 3 5 D2ue0llTie(ma) 000427 B12 Page 120f 13 ANALYTICAL METHOD MethodNo: BACG-3607 Title: PrDeeptaerramtiinaotniaonnodfPAenraflluyosriosocbtaynHesPuLlfConMataesisn MSopnekcetyroSmeerturmyMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) --_--_--nmmm x201 21 21 80 CALCULATIONS 81 A`ptetakhseheanpdeo,fatnhdepaenaalkyhteiicgahltruann,drpeevaikewareeaacdhetcehrrmoimnaattoigornoafmttohetnessutraerttihcelreaetnednttihoenIStiamree, parcocfeiplteasblaet.tThheefodlaltoawimnagymabses-stmoo-cohtahregdearsataiopsp:ropriate. For quantitation, use the ion AnPaFlOySte PFOC I4o9n8P9ro1f0i4l9e89 4129103689 82 PlforotmthaelplesatkaanrdeaarrdesvsepronssuesotfhPeFOcSondcievnitdreatdbioynothfetpheeakatresetaarretsicploensienotfhtehsetIaSnd(aPrFdOsC.) Alitteomafttihveedlayt,at(hee.8p.e,aqkuhaediragthitcs mftaywbeieguhsteeddiwnisttheacdoonfcpeenatkraarteiaosn.ofOthebtetshtaebaretsi itclceurnovrea. cuquravderaatnicd fiit).maNoytbe:eTnheecebsessatrcyutrvoehfaitvmeamyobreedtehpaennodennetsotnantdhaerrdacnugrevoeftfhoersvtaarnidoaursd concentration ranges using the following: y=adsbxic where y= Peak height response ofPROS dividedbypeak height response of the IS (PFOC) in standards. . x = Concentrationofthe PFOS in standards. a,b,c =Constants derived fromtheregression analysis. 000428 Page 13 0f 13 ANALYTICAL METHOD Method No: BACG-3607 Title: mee. Determinationof Preparation and Perfluorooctanesulfonate in Analysis by HPLC Mass `Monkey Serum and Spectrometry/Mass Urine: Sample Spectrometry (HPLC/MS/MS) 83 Using the standard curve, calculate the levelofPFHCineachunknown sample. Correct the resou fsl amptlessforanydilutions. NOTE: Duetounresolvable interferences with thetestarticle and/or intemal standard fromthematrix,externalstandard quantitationmaybeusedatthediscretion ofthe supervising massspectrometrist. 9.0 ACCEPTANCE AND REJECTION CRITERIA 9.1 Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for `standards is 80-120% of theoretical. 10.0 REPORTING 10.1 Reasreultotsboeffaillleadnianlytsheesaaprperotparbiualtaetesdt,uadnydftihlee.rawdata, originalchromatograms,and reports Author(s): hb. ConA Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group sty Sgn > eg Gorman, Ph.D. Manager Bioanalytical Chemistry Group 2/02/. 3 Date sfor Date 000429 Appendix C SummaryofPharmacokine`taincd PUarrianmaertyeErxscCraeltciounlaDtaetdaffroormPSFeOrsum ConcentrationsofPFOs 000430 or 31315 Rivi: sgEil PPLiEE Ei i g I FO Ii H 8 Sip 3z ii E gE t;H IEB] lesIs~ altIs (hese iin B|s2 lzla Aa E:b 5 RB 2 4el. E2] en | shiedA shite 000431 lila RIE | BR EL : : 2a! 8 Hal] : | Fr Iidl : 5 iH Bls|s|le s 2 Eee she i I 1 i IT " i il 5 el 5 FE 8 EEE :2 BIrEEERERREER :sun ? breed .EET 2 "| d : li TE Hi i ial : lee i er dii Ji 000432