Document O53ND0rLrrNmr07oXybbGeZp

TC ARARG -- 1362 A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Southern Research Institute Study ID: 9921.4 January 14, 2003 001650 Final Report on A Pharmacokinetic Study of Potassium Perfluorooctanoate 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 001651 ABSTRACT `The pharmacokineticsandurinaryexcretion ofperfluorooctanoatewereinvestigatedinmaleand femalecynomolgusmonkeys.Threemaleandthreefemalemonkeyswereadministered asingleiv bolusdose of10 mg/kgofperfluorooctanoate,potassium salt.Atvarioustimesafterdosisnergu,m and urine (24-hour collections) samples were obtained andanalyzed by HPLC/MS/MS for levels of intact perfluorooctanoate. The lower limitof quantitationofthe analytical method was 20 ng/mL forserumsamplesand 10ngfor/ urinem sampL les. At0.5 hoursafterdosing,serum concentrations ofperfluorocctancwaerte similarinmale andfemalemonkeysandranged from 91,130to96,660 ng/mL in male monkeys and from 88,040 to 96,400 ng/mL in female monkeys. Serum concentrations of perfluorooctanoate subsequently declined but, beyond the first few days after dosing,therateofdecreaseappeared tobefasterinmalemonkeys thaninfemalemonkeys. OnDay 28,theconcenotfpreraflutorioocotannoasteinserumranged from 1863t0.27,140ng/mL.inthemale: `monkeys and from 7,145 to 33,680 ng/mL in the female monkeys. Due to the relatively high serum concentrationsofthe compound on this day, the lengthofserum collection was extended through Day 123. On Day 123, the serum concentrationofperfluorooctanoate was at or only slightly above the limitof quantitation (20 ng/mL) in male monkeys and between 885 and 4701 ng/mL in female monkeys. The serum concentration versus time data were subjected to non-compartmental `pharmacokinetic analysis. The terminal halflife ofperfluorooctanoate in serum was 13.6, 13.7, and 35.3 days inthethmarlemeonke eys and 26.8, 29.3, and 41.7daysinthethreefernalemonkeys. The total body clearance was 4.0, 15.8, and 17.5 mLiday/kg for male monkeys and 3.1, 3.9, and 9.1 `mLdaylkg for female monkeys. These estimated values for half-life and clearance indicated that twoofthe three male monkeys eliminated perfluorooctanoate at a faster rate than did the female 001652 `monkeys. The volumeofdistributionofthecompounadt steadystate(Vd,) wassimilarfor both sexes and ranged from 168 to 192 mL/kgformalemonkeys and 133 to 270 mL/kg for female `monkeys. Perfluorooctanoatewasslowlyexcretedinurinebybothmaleandfemalemonkeys; levels ofperfluorooctanoate,representingas muchas 1%oftheadministereddose,werepresentin rine 28daaftey r doss ing. Theresuolftthsisstudysuggested thatthepharmacokinetiocfs perfluorooctanoate may bedifferentinmaleandfemalemonkeys. 001653 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 2.1 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 3 Serum LevelsofPerfluorooctanoate 4 Bioanalytical Method Development and Sample Analysis 4 Data Analyses 4 30 RESULTS 4 3.1 Morality 4 32 Clinical Observations 5 33 Body Weights 5 3.4 Serum and Urine ConcentrationsofPerfluorooctanoate 5 40 DISCUSSION 7 50 CONCLUSIONS 8 60 RECORD ARCHIVES 3 70 REFERENCES 8 001654 . `TABLE OF CONTENTS (Continued) LIST OFTABLES Table 1: Table 2: Table 3: Table 4: Figure 1: Appendix A: AppendiBx: Appendix C: Page Individual Body Weights 9 `Serum ConcentrationsofPerfluorooctanoate: 10 `Pharmacokinetic Parameters Calculated from Serum Concentrations un ofPerfluorooctancate. Urinary ExcretionofPerfluorooctanoate 12 LIST OF FIGURES Serum Concentration Profilesof Perfluorooctanoate in Monkeys 14 LIST OF APPENDICES Study Protocol and Amendments Al MDeettehromdinVaatliiodnatoifonPeRrefplourotr:oocVtaalniodaatteioinnoMfoAnnkaleyytiScealruMmetahnoddUsrfionre Using B1 HPLC/MS/MS Analytical Method for Determination of Perfluorooctanoate in `Monkey Serum and Urine cl 001655 iii Signature Page A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Foti E. Ztded Patricia E. Noker, Ph.D, D.A.B.T. `Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: 1 Litfos Date ldo 2A Charles D. Hbert, Ph.D, D.AB.T. Director, Safety Assessment Loves Date `We, the undersigned, were responsible for the conductof thework and reportingofthe results in the listedsections. Weconcurwiththeviewsrelativetoour bodyofworkasexpressedinthediscussion and conclusions. y `Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Date 001656 iv Good Laboratory Practices Disclaimer "ThisstudydescribedinthisinalreortwasnotconductedinstrictcompliancewiththeUSS. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations (21 CFR Part 58), and neitherthisreportnortherawdatawere reviewed bytheSouthernResearchQualityAssurance Urit. However,thestudywasconductedaccording totheprotocolandamendmentsandthespplcable standardoperatingprocedures, ndallstudyprocedures,dataecording,andreporting wereperformed in 2mannerconsistentwiththestandardof GLPs. Thefinalreportaccuratelyreflectstherawdata biainedduringtheperforomftah nstcudey.There werenoadversecircumsthtataaffnecctedethse quality orinteogftrheisttudyy. Boron EPs) PSatturdiyciDaiEr.ecNtookrer, PhD, DAB.T. 1/14/05 Date 001657 v Study Dates: Study Schedule and Personnel Study Initiation: DayofDosing: LastDayofSample Collection: Study Completion: October 4, 2000 October 9, 2000 February 9, 2001 January 14,2003 Study Personnel: Patricia E. Noker, Ph.D, DAB. Study Director Charles D. Hbert, Ph.D, DAB.T. Director, Safety Assessment `Norman D. Jefferson, B.S. Gregory S. Gorman, Ph.D. Associate Director, Safety Assessment Manager, Bioanalytical Chemistry Group Tsu-Han Lin, PhD. Pharmacokineticist Darrell E. Hoskins, D.V.M., Ph.D., A.C.L.AM. (Dipl) Veterinarian LaJuana A. Durbin, B.S. Supervisor, Large Animal Laboratory D. Wayne May, LATG Supervisor, Animal Care: Carolyn R. Oliver, B.S. Supervisor, Study Coordination, 001658 1 1.0 Introduction `Theobjeofcthitsstiudyvweeresto detethercomnceintrnatieonofpotassium perfluorooctanoate inserumandtoestuirinamryacleatraneceat various times following administrationof a single intravenous dose to monkeys.A copyofthe protocol and any applicable amendments can be found in Appendix A. 2.0 Materials and Methods 2.1 Test System `The 3maleand 3 femalemonkeysdesignatedforuseinthisstudywereselectedfromaninhouse colony of monkeys that were housed at Souther Research Institute (Southem Research)priorto use on this study. These monkeys were purchased from Charles River `BRF, Inc. (Houston, TX) and were an estimated 3-4 yearsof age when placed on study. Individualanimalidentiwfasibcycahetstitaottono. The cynomomnkoeyilsangacucepsted species to support clinical studiesofdrugs used or intendedforusein humans. During the quarantine period, acomplete physical examination including fecal examination for intemal parasites,completeblood count (CBC), body weight, and rectal temperature was performed on each of the monkeys. The following procedures were performed on the `monkeys duringquarantine:(1)Threetuberculintestswereadministeredtoeachanimalat 2-week intervals. All tuberculin tests were administered intrapalpebrally. The three tuberculin tests were negative for all monkeys. (2) Blood wasdrawn for CBCand B virus titer. (3) Fecal cultures (screening for Salmonella and Shigella) 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 clinica signs and mortality/moribundity. Housing, feed, water, and socialization procedures remained the same during the quarantine, holding, and study periods. Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed to the monkeys 2-3 times each day. The quantityofthe daily ration was sufficietnot meet 001659 2 . nutritional requirements. In addition, the diet was supplemented with fresh frit/treats several times cach week. Tapwater(Birmingham public water supply) was available to the `monkeys ad libitum during the quarantine and study periods. The monkeys were housed individually instainlesssteelcagesduringthe quarantineandthestudyperiods. FromDay 0totheendofthestudy,themonkeyswerehousedin room that wasmaintainedat a temperatureof 68.0-70.3 F and a relative humidity of 22.2-65.7%. The humidity was withinthereqruanige(r30e-70d%)over90% ofthetimeduringthestudy;excursionsbelow therecommended humidityrangewereofshort duration. Room lights werecontrolledby anautomatictimersettoprovide 12hoursoflight (0600to 1800hours,CST)and 12hours of darkperday. Cagesizeandanimalcareconformed to the guidelinesoftheGuifodrtehe Care and UseofLaboratory Animals, Tth edition and the U.S. DepartmentofAgriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPs) of Southern Research. The study design was approved by Souther Research's Institute Animal Care and Use Committee (IACUC). Souther Research is fully accredited by the American Associate for Accreditation of Laboratory Animal Care Intemational (AAALAC). `The monkeys used on this study were previously givena single IV bolus dose of perfluorobutanesulfonate (10 mg/kg) on April 10, 2000 (Souther Research Study No. 9921.1); a single IV bolus dose ofpotassium perfluorobutanoate (10 mg/kg) on June 13, 2000 (Southern Research Study No. 9921.2); and a single IV bolus dose of potassium perfluorohexanoate (10 mg/kg) on July 31, 2000 (Southern Research Study No. 9921.3). 2.2 Test Article and Vehicle Test Article: One bottle containing 5.2 grams of potassium perfluorooctanoate (T-7507; expiration date not supplied; Southem Research Lot No. EO9/L-1) was supplied by 3M (St. Paul, MN) and received on May 15, 2000. The test article was stored at roomtemperature until used. Stabilityofthe test article was the responsibility ofthe Sponsor. 001660 3 Vehicle: The vehicle used for the preparation of the dose formulation of potassium perfluorooctanoate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 8070655, expiration date July 2001). The vehicle was stored at room temperature and was considered to be stable when stored according to these conditions. Dose Formulation Preparation: For the single dose formulation of potassium perfluorooctanoatepreparedat mg/mL,therequiredamountoftestarticlewasweighedout in a volumetric flask. Sterile saline was added and the formulation was stirred until in solution. The formulation was stored refrigeratedandusedfordosingwithin 4 days after preparation; 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: Asonly onetreatment group was used in this study, no formal randomization was required. OnDay 0, eachofthethreemaleand three female monkeys received a single intravenous (IV) doseofperfluorooctanoate at 10 mg/kg. byinjectioninto asuperficairamlor legvein.Doseswere based uptheoDayn-1 individual body weights. Doses were administered at a volume of2 aL/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 daysof blood collection. Body Weights: Each priwamsaweitgheed on Days -1, 4,7, 14, 21,and 28. Urine and Feces Collection: Urine and feces were collected for 24-hour intervalsonthe: following days: prior to dose administration (Day -3; baseline), on Day 1 (0-24 hours 001661 4 postdose),onDay 2(24-48hourspostdose),andon Days 7,14, 21,and28.Thevolumoef eachurinesample wasmeasureduponcollection. Urineandfecessampleswerestored frozen (approximately 20 C or below). Fecal samples will not be analyzed unless specifically requested by the Sponsor. `Serum LevelsofPerfluorooctanoate: Blood samples (spproximately 3 mL)werecollected from each primateatapproximately (predose) minutes; 0.5, 2, 4,8, and 24hours;andon Days2,4,7, 11, 14,21, 28,57,79, 87,and 123 postdose.Sampleswerecolleinctotteubeds `without anticoagulant and were allowed to clot at room temperature. The blood samples werethencentrifuged, andtheserumseparatedandstoredfrozen (approximately 20 C or below) until analyzed. Bioanalytical Method Development and Sample Analysis: A bioanalytical method was developed fortheanalysisofperfluorinoseorucmatndaurnineomaatritcees.The method was validated for accuracy. This method was used for the analysisofserum and urine `samples collected during the study (see Appendices B and C). Data Analyses: The serum concentration data for unchanged perfluorooctanoste were subjected tonon-compartment pharmacokinetic analysis using WiniNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc.; Cary, NC). Mean values and standard deviations for cach parameter were calculated using Microsoft Excel software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorooctanoate at each collection interval was calculated and expressed 2s a percent ofthe administered dose. No other statistical analyses ofthe data were performed. 3.0 Results 3.1 Mortality No test article-related deaths occurred during the study. One of the three male monkeys (2052) was euthanizedonDay 79 becauseofrepeated episodesof self mutilation; there was no indication that the self-mutilation was related to administrationofperfluorooctanoate. 001662 5 32 Clinical Observations No adverse drug-related clinical signsoftoxicity were observed for any monkey. Male `monkey2052wasobservedonDay 57ofthestudytohaveseveralwoundsontheright leg. The attending veterinarian diagnosed these wounds to be self-inflicted lacerations. The `woundswerecleanedandsubsequentlyflushedwith aweakbetadinesolution every3-4days for 4couorftrseatemenst.Thesocialenrichmentofthemonkeywasalsoaugmentedin orderto providethemonkeyadditionalstimulationand tochangehisfocusofattention. On Day 79,the monkeywasexaminedandseveralseverelacerationswereobservedontheleg `and footofthe animal. These lacerations were diagnosed as self-inflicted wounds. Due to the repeated instancoefs self-mutilation and the likelihoodofcontinued episodesofself`mutilation, the monkey was euthanized on Day 79 for humane reasons. 33 Body Weights Body weights for individual monkeys are presentedin Table 1. Thebody weight ofcach monkey remthaesiamenbeetweden Days-1and 28. 3.4 Serum and Urine Concentrations of Perfluorooctanoate During the study, an HPLC/MS/MS method was developed and validatedforthe analysis of perfluorooctanoate in monkey serum and urine. Details of the method validation procedure are provided in Appendix B. Detailsofthe analytical method are provided in AppendiCx. The lowerlimitof quantitationofthemethodwas20ng/mLforserumsamples and 10 ng/mL for urine samples. Serum concentrations of perfluorooctanoate in three male and thre female monkeys at various times through Day 123 after administration of a single iv dose of 10 mg/kg are presented in Table 2 and plotted in Figure 1. At 0.5 hours afte dosing, serum concentrations ofperfluorooctanoate were similar in male and female monkeys and ranged from 91,130 to 96,660 ng/mL in the male monkeys and from 88,940 to 96,400 ng/mL. in the female `monkeys. Serum concentrationsofperluorooctanoate subsequently declines at a slow rate; the rateofdecline appeared to be similar in male and female monkeys through atleast the. 001663 6 firsttwodaysaftrdosing. At 48hours,serumconcentrationsofperfluorooctanwoearte between 48,530and65,810ng/mLinthemalemonkeys and 39,820 and 61,600 ng/mLin the female monkeys. Beyond this time, serum concentrationsofperfluorooctanoate in two ofthethreemalemonkeysdecreasedat fasterratethanthatobservedforthethirdmale monkey and for the three female monkeys. On Day 28, the concentrations of perfluoroinosecrtumaranngoedaftroem 18631027,140ng/mLinthemalemonkeysand from 7,145 to 33,680 ng/mL in the female monkeys; due to these relatively high serum concentrationsofthe compound on this day, the length ofserum collection was extended throughDay 123.OnDay 123,theserumconcentrationofperfluoroocwatsaantooraontley slightly above the limitofquantitation (20 ng/mL) in the two surviving male monkeys and between 885 and 4701 ng/mL in the three female monkeys. Pharmacokinetic parameters calculated from serum concentrationsofperfluorooctanoate in individual monkeysarepresentedinTable 3. Thevalueswerederivedfromnoncompartmental pharmacokinetic analysisofthe data. AUC gs, values ranged from 571 to 2501 ugeday/mL (mean: 1235 ugeday/mL) for male monkeys and from 1094 to 3224 ugeday/mL (mean: 2293 ugday/mL) for female monkeys. The terminal halflife of `perfluorooetanoatein serum was 13.6, 13.7, and 35.3 daysinthethreemale monkeys and 268,29.3,and 41.7 days inthethree femalemonkeys.Thetotalbodyclearanwcase 4.0, 15.8, and 17.5 mi/day/kg for male monkeys and 3.1, 3.9, and 9.1 mL/daykg for female `monkeys. These estimated valuesforhalf-lifeand clearanceindicatedthat twoofthethree male monkeys eliminated perfluorooctanoate ata faster ate than did the female monkeys. `The volumeofdistributionofthe compound at steady state (Vd) was similarforboth sexes and ranged from 168 to 192 mL/kg for the male monkeys and 133 to 270 mL/kg for the female monkeys. 001664 7 . `Theamountofperfluorooctanoate eliminatedinurineby individualmonkeys atvarious timesafterdosingispresentedinTable4. Sexdiffinetherurienarnyexccreteionsof `perfluorooctanoate were notreadilyapparentprior toDay 21;onDay 21andDay28, the femalemonkeysappeared toeliminate higherpercentageofthedoseinurinethandidthe `male monkeys. Theurinary eliminationofperfluorooctanoate was prolongedforboth the `maleandfemalemonkeys; onDay28from0.1101%oftheadmindiossewtaserercoveerded in urine collected from the individual monkeys. 4.0 Discussion Perfluorooctanoatewas slowlyeliminatedbymaleandfemalemonkeysgiven asingleivdose of 10 `mg/kg.Theresultsof thisstudyindicatedthatbeginning onaround Day 7andcontinuingthroughout the remaining periodofsample collection, serum concentrationsofperfluorooctanwoearte lower intwoofthethreemalemonkeysthanin thethreefemale monkeys.Onthe finaldayof serum collection (Day 123), serum concentrationsofperfluorooctanoate were below or only slightly above the quantitation limit (20 ng/mL)inthe two surviving male monkeys, whereas, on the same day, serum concentrationsofperfluorooctanate ranged from 885 to 4701 ng/mL in the three female `monkeys. These differences in the serum concentrationsofperfluorooctanoate were reflected in a shorterserum half-lifeofperfluorooctanoate in these two male monkeys (13.6 or 13.7 days) than observed for the three female monkeys (range: 26.8 to 417 days). The clearance of `perfluorooctanoate also appeared to be higher in twoofthe male monkeys tha in the three female `monkeys. Although the number ofmonkeys was limited, these resultssuggestedthat the kinetics ofperfluorooctanoate may have been different in male and female monkeys. It was observed that the serum concentration data for oneofthe male monkeys (2052) more closely paralleled the serum concentration profile observed for the femalemonkeysthan that observed for the other two male monkeys. It is possible that the study procedures (c.g. handling, short-term restraintin achair)orotherunknownfactorsmayhaveproducedstressinthismonkeyresultingin the release of high levels of cortisol. Cortisol is known to affect various physiological changes including changes in carbohydrate, protein, and/or lipid metabolism, shifts in electrolyte and water balance, and increases in plasma proteins. Such changes could have possibly led to changes in the 001665 8 dispositionofperfluorooctanoate in this male monkey. Itsalsopossiblethat the self-mutilation activitiesofthismonkey,which leadtotheeventualeuthanasiaoftheanimal,were aresponseto stress. Although theserumlevelsofperfluorooctanoateindicated thatat leasttwoofthe three male `monkeysmayhaveeliminatedperfluorooctanoateat afasterratethandidfemalemonkeys, itwas difficulttodiscemn a differenceintheurinaryexcretionofthecompound by maleandfemale monkeys.Thismayhavebeenrelatedtothefactthattherateofurinaryexcretionof thecompound byallthemonkeysonstwuasdsloyw. Lessthan 20% oftheadminisdtoesreweadsexcretedin urine by either male or female monkeys within the fist 48 hoursafterdosing. Subsequently, on Day 28, perfluorooctanoate was present in urine collected frommaleor female monkeys at levels that represented as much as 1.0%ofthe dose. 5.0 Conclusions `The mean terminal serum half-life ofperfluorooctanoate was 22.0 days in male monkeys and 32.6 days in female monkeys. Unchanged perfluorooctanoate was slowly excreted in urine at a slow rate by male and female monkeys. 6.0 Record Archives Data, specimens, and a copyofthe final report from this study will be stored in the Archives at Souther Researchforupto 1yearafteracceptanceofthe finalreport bythe Sponsor. Afte1r year and with the permission of the Sponsor's Monitor, the data and any samples/specimens will be shipped to the SponorstoothreSponsor'sdesignated archival facility. If mataerertoibearetlainsed in the archives beyond this date, such continued storage will be foar specificfeedetermined with. the Sponsor. Acopyofthefinalreportwil beretainedinthecentralarchivesatSouther Research. 7.0 References 1. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington D.C.; 1996. 001666 , li bE er 2 g E gzE[3N n 1 ERE Sith : i Eis E1LH 3 EEFE Lo [REA Ee : El lelel| Ieee :a i 2 001667 0 Tabl2e A Pharmacokinetic Studyof Potassium Perfluorooctanionatthee Cynomolgus Monkey Serum Concentrationsof Perfluorooctanoate -- Serum Concent(rnga/tmli)o_n_ oC me wo [5[a mw [ost [o1130 91370 [2m [67500 [s1.| o6n3o0[ 7azso 88540|94080| [77870 [7600 [a|t780s00 [ 89.160 81,620| 69380 [241s| 7510,221000|5 60,8600 || 6743,13 66500 | 620 890 | 61520 60760|41.940 [4|s 658t 10 | 48,530| 48.930 49,670| 39.82|0 |Day [43370|42.930.94900 | 70.520 4370 [D|a458y 90 | 27.910 [29.100| 59.820 36.760 | 39,450| 19,320 38,980| 53,080| 37,500 37,360| 11.220|13.430| 40.450 |457|2919100| [DDaayy221s[[3237,164000|13.988653||5.sa9a8n|9| 338a7s500[|23473.80500]7.145 | [Fo57y [15010 S10] 16570 [Daayms7|o--s|[os~ |i~ n [ss-es1[s-ess1-[2m1m] [Dwizs| --[mo | 3s | aest[amo] uss| * Sample taken prior to humane sacrifice ofanimal. B=QBeL low the quantitation limit (<20 ng/mL) Pugs Loft 001668 BlelslFsPF i; :3 A 2] R 2 2:| eREEE ER FIRS 53 = E|FFFP i:2 :g eh SERRE i3i . ff 22 2 EE a| -fleakke i 5i30 | EFPPCE fEellaegks O4iE Fa iF see i $3F | BRERRRE ! 2g5282 82 lnfo |= Sz g Leh 2 git LoEll || [[EEe2e88 3fEF s fimolinzeid 552% salaleied TiEcgEaiiis 001669 2 . Table 4 A Pharmacokinetic StudyofPotassium Perfluorooctanoate in the Cynomolgus Monkey I=[=] Urinary Excretion ofPerfluorooctanoate - Urine | Unne -- 1 | Percenotf Dose sex=| gm) |oD (mn) |[7 (2) | ) [Mwosn[|ww|| sBooLL || wmo0| | - [|seoo w | | || [Foosso ||rF|| BBoorr[|wwso| | - || ssaomo| | || -- 1 -- 1} [[o2osms[[wW|2o7om0 || a0w || 3m ose | Goo| |seo | 61 | tse | ose[7| [0% [7| ss toe | m0[tow | 80[ew || sssaow| | 3a17 || my [[2m0s5e2[ ][ WM T 0&] 7 |2m0 o ||7aw6||G eo ww||1 os2|| zn[wos | m0| we |sow | or | [ose[e 7 wm | m0e | ser | sae o | 17 | ---------- wr-------- 1 [zs vom [m0| sos |eno [ai | [[ozmne[M7 omm er[|ooww| | swaer ||ssoooe[ |2e 6 || Coo[Fs | mo| de |mow| 13 | BQL = Below the quantitation limit (<10 ng/mL) *sCtoanncdeanrtdrcautrivoen;otfhpereerffolrueo,ruoroicnteancooantceenitnratthieosnavmapllueegmraeaytlcyonetxacienesdiegdnitfhiecahnitgheersrtorconcentration in the Due to technician error, urine was discarded after the volume was obtained. "Concentrationofperfluorooctanoate in the sample was slightly above the highest concentration in the standard curve; urine concentration value should have no significant error. Page 1012 00160 n `Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorooctanoate in the. Cynomolgus Monkey Urinary Excretion ofPerfluorooctanoate -- D Animal -- Urine Urine a) Concentration| Volume ow = Twoota)l -- |Percent of Do | Dowsoe 6) in Urine: | CFFaoassneeT[[wFmwseopn [[[omoww0 |[m wamw ||s smoooe | | |i oo| se || ooTFs [mo| om | moo| a | Fa asEN[[Fw0eE eas [[owwo| [oapwJO ||eawwJ e| [o0 CsE || Fo [Faw[mo [se | mow [to | zmns twww Tw eo[[ssem ms rw ao| wm [|esww| [0| 2 | |woo | os | ow [Fm[0| se | mow [10 | BQL = Below th quaniiaion limit (<10 nginl) *aCnondcaenndtrcautrivoneohfrpeerffolru,oruooncetancoonacteenitnratthieosnavmlplueegmraeaytlSyoexncaeesdiegdnitfhiecahnitghresot concentration in the Due to technician error, urine was discarded after the volume was obtained. "iCnohnceensttraantdiaorndocfuprevr;fluuoirooccotnacneonattreatiinotnhevaslaumepslheowuladsaslvieghtmloy saibgonviectahnetheigrhe,st concentratPigone2012 001671 EE , me Twp fn wassz _ | we r=, 1 t ------t ------ ] ---- P= + Ce --Prdam w 5ee 3 x T ee> 1 -fr ] )= `Time (day) wanes EE p= ee ee] T oaE LE He | fES EBE EBEBVBVmMm------~1~ m 5P S$ =Emm = e a ---- | fo =~] ree Pe oor . :1 > Time don) 5mT 1 = 1= 001672 Figur1e A Pharmacokinetic StudyofPotassium Perfluorooctanoate in the Cynomolgus Monkey . `Serum Concentration ProfilesofPerfluorooctanoate in Monkeys 1s M2211 --- er -------- oe elele] iP w--e_+ r ss ---- -- l -- ~~ I | VFVFm------------------ w r ==] = wet .r PA wtt ) ws 9 = _ ree F205 om --_-- t EPP t Z we 5 --+ Ei=o e ---- e g er i orl t eeeee 1 t e 1 1 . a wo 3 PY = Time (day) . + ens TM Figure 1 Continued) 001673 A Pharmacokinetic Study of Potassium Perfluorooctancate in the Cynomolgus Monkey `Serum Concentration Profilesof Perfluorooctanoate in Monkeys 1 Fase ---- Eee ee Tw r+1 1 1 Ee -- ------------------ Wo -- Bem ------ --= -- 1~ +---->-- ~[>--_-- --> --|~ Ft 5 a 1 *oum W t FF T t F : Ed 0 i 100 125 < `Time (day) 0 EF Z wep Fave __ | 11| ] Derr tf, pes e s= Wl rr SE Time (day) Figure 1 (Continued) A Pharmacokinetic StudyofPotassium Perfluorooctancate in the Cynomolgus Monkey `Serum Concentration Profiles ofPerfluorooctancate in Monkeys 001674 Appendix A Study Protocol and Amendments 001675 a ~ I ! ---- i Study Protocol: I i A Pharmacokinetic Study of Potassium | (Il Perfluorooctanoate in the Cynomolgus Monkey l| | i i Southern Research Study ID: 9921.4 | I II |i October 4,2000 i| ( | i a -- PDoD SED SOUTHERN RESEARCH INSTITUTE 001676 a2 STUDY NO. 9921.4 : 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: October,200 wan 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.A.B.T. 3M Center `SBt.uiPladuiln,g M2i2n0n-e2sEo-t0a2 55144-3220 (070 (651) 733-1962; FAX: (651) 733-1773 `iPtroatoclaoslt pAapgpersosvoa)l: Test Article: got 2 Bitaldaff John L. Butenhoff 10/5) zo0e Date Perfluorooctanoate, potassium salt , Ship Unused Test Article to: D. Hakes : Building B236 3M P.O. Box 33327 + 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 001677 As I STUDY NO.: 9921.4 Octo4b,2e00r0 20 TITLE: A Pharmacokinetic StudyofPotassium Perfluorooctanoate in the Cynomolgus Monkey 30 OBJECTIVE: Tshereuombjaencdtiuvreisneaofttvhaisrisotuusdtyiamrees tfooldleotweirnmginaedmtihneisctornacteinotnorfataisoinnogflepeirnftlruaovroeoncotuasndooastee oinf potassium perfluorooctanoate to monkeys. 40 TESTING LABORATORY: . Southern Research Institute 2000 Ninth Avenue South + 35205 P.0. Box 55305 `(B2i0r5m)i5n8g1h-a2m3,3A5;LF3A5X2:55-(250350)5581-2044 50 KEY STUDY DATES: [ve 1Sequemce@a) | Date(s Veron | !, [PUaryioneraTnrdeFaetcemseCnotllections [03 -- Tw1o0/w60w0 | 1 2 11001110/10000 7 1 | o1031i6/0000 21 2 103000 11/600 Serum Drug Levels 0: ho0u(rprsepdoossted)o,se0.3, 2, 4, 8 md 24| 109-1070 23 1011/00 1013/00 7 u 1016/00 1020000 211 102300 1030/00 Draft Report Due 260 Calendar days afer completion of| 1S1160010 the in-life p Final Report Due. 5 days ater finalSponsorcomments L received - 001678 a4 STUDY NO.: 9921.4 October 4,2000 6.0 STUDY PERSONNEL: The following are the primary contributorsand supervisory personnel participating in this study. J Study Director: Patricia E. No-- ker ---- PD,-- DABT. Alternate Study Director: Director, Safety Assessment: James D. Johnson `Ward R. Richter MS, MBA. DVM. MS, DACVP. Associate Director: NomanD. Jefferson Manager, Bioanalytical Chemistry: James D. Johnson BA: MS, MBA. Supervisor, In-Life Laboratories: Veterinarian: LaJuana A. Durbin Darrell E. Hoskins AAS. DVM, ACLAM. @il) 70 TEST & CONTROL ARTICLES: oTfhsetatbeisltitaryt,icalsewweillllabsemseutphploidesod fbysytnhteheSspiosn,sofrab,rwichatoiowni,lorbederreisvpaotniosni.blUepfoorndcoocmupmleenttiaotnioonf { the study, residual bulk test article will be returned to the Sponsor. 71 IDENTITY OF THE TEST ARTICLE: Name: Identification: Supplier: Lot Number(s): Special Handling: Perfluorooctanoate, potassium salt T7507 TMo be documented in the study data. None Characterization: Dionccluumdeinntgaitdienotnityo,fpturhietyc,hasrtarcetnegtrhi,zaatnidoncoomfpotshietitoesnt, aasrtwicellel, raesspmoentsihboidlsityoofftshynethSepsoinss,ofra.brCiocpaiteioson,focrhadrearcitveartiizoant,ioinsdtahtea have been provided to the testing laboratory. Stability & Storage: The bulk test article Stability of the bulk tweisltlarbteicslteoirsedthaet rreospoomnsitbeimlpietryaotfutreh.e Sponsor. 001679 As Cosme 72 IDENTITY OFTHE VEHICLE: Sas Name: Supplier: Lot Number(s): Special Handling: Characterization: Sterile Saline TCoombmeerdcoicaulmesnuptpeldieirn the study data. None Datotcauimneedntbaytiorneocofrtdhiengchaalrlacpteerrtiiznaetnitonionfftohremavteihoinclfermoamytbhee ctonthaineeirlnartbehleess,otorubdyyfdraett,aai.nTinhgtehvecoe ntaihinesrli2acbeolmcsm,eorrlccioapelielsy available product. Stability & Storage: Setxepriirlaetisoanlinperoivsidceodnsibdyeretdhestambalneuftahcrtouurgehr thwehednate(sst)oroefd appropriately. The bulk vehiclewillbe stored in accordance with the manufacturer's instructions. 73 FORMULATION: Porfe5pamrgat/imoLn:fTorheintterstavaertnioculse waidlmlinbiestfroartmiuolna;tberdieifnlsyt,ertihlee sraelqiuniereadt aacmoonucnetnotrfatteisont baretisctlierwreidlubnteilmitxheedtewsittahrttihceleriequviirseidbalymoiunnstooluftsitoenr.ileFosarlmiunle,atainodnsthweimllixbteursetowrieldl. refrigerated until used for dosing; formulationsofthe test article in sterile saline are expected to be stable for weeks when so stored. Dose dose fFoorrmumlualtaitoinocnonCcoennctreanttiroantainodn and Homogeneity Analyses: No homogeneity will be conducted. analysis of 80 TESTSYSTEM: Species & Strain: Supplier: AgeonDay 1: `Weight at randomization: Number on Study: CChyanrolmeoslRgiuvesrmBoRnFk,eyInsc.(M(Haocuasctaofna,scTiXcu)laris) 3-4 yearsofage (estimated) 37kg Males 3 Females 3 A99n2i1m.a1l,spwoetraesspiruemvipoeursfllyuodroosbeudtawniotahtepointasstsuiduym9p9e2r1f.l2u,oraonbdutpaonteassuslifuomnate in study - perfluorohexanoate in study 9921.3 001680 as Sid 81 JUSTIFICATION: Pevrailmuaatteisonasroefccoommpmoounnldysuusseedd ionr ipnrteeclnidneidcaflorpuhsaermianchoulmoagnisc,aloratnodwhtioxcihcohluomgiacnasl `might be exposed. 82 HOUSING: Dsuritngaqsuteaierla,ntnsilantebl/oatctecolmimscaatgiseons.aAnldlsatnuidmy,alasnwiimlallbsewhiolulsbeediinndaivriodoumaltlhyathporuosveiddeisn 3rm oom i at ateon mfp1er0aai tiurerxeocm fha6n4g-eu 8s4perFm haonudr.aCroenltartoivleshwiulmlibdietsyetotfo3ma0i-n7t0a%i.ntAhe1a2n-ihmouarl light/12-hour dark cyele will be routinely maintained. Animals wil be acclimated inthe same roomusedforstudy. 83 BEDDING: `eNxocnreemreenqtuiarbesdorfpotricoangpianngs,eqcuoimpmpeerdciwailthhefaltu-sthraebalteedphanasr.dwFooordccahgiepsbeeqdudiipnpgewdilwlibthe . `buseerdfeovrieewxecdrbemyetnhteaDbseopraprttimoenn.tAonfaVleytseersionfatrhyebMeedddiicnign,esaunpdplBiieodrbeystohurecveesn(doDrV,MwiBl)l oinftSerofuetrheewrintRheosreaarffcehctttohaesosuurtectomeoh1f0tkahneostwtundyc.ontaminants are present that could. 84 Dum DMiOe)t.willThbee cpormdimameialrtyceisraawtliiCloelnrtabivefaiieoldfabfPlereriemadtatefeaeecCdhhtfoewweidc#ie5n0gd4aii8nlty(e,PrvMwaIli.tFheIenadpsap,drdIointxcii.omnaS,tt.etlhLeyouditishe,et rw"iealcclhobmwemeseeuknp.dpeldTehmeenqtueadntwiittyhofftrehseh fdrauiiltyofrfaetrioend dwaiilllybaensdutfrfeiactisenotffteormedeestevneurtarlittiiomneasl DreVquMirBeomefntSso.utAhnearlnysReessoeafrtchh tfoeeads,susrueppthlaitednobyknthoewvnencdoonrt,amwiilnlanbtesreaveieprweesdebnyt tthhaet could affect the healthofthe animals. 85 WATER: `qWuaatrearnt(iBnieramnidngsthuadmy ppuebrliiocdswavitaeransuapuptloym)atwiicllwbateersiunpgplsiyesdteamd. liSbaimtpulmedsuoifnwgatteher Sroem tvhebiaynteihmeawlDefVaciMdlityofwiSlolutbheerpeRreiosdeiacracllhytaonaaslsyuzreedt,haatnndokthneowannacloynsetsamwiinlalnbtes : arepresentthatcouldaffectthehealthofthe animals. 001681 a7 . STUDY NO.: 9921.4 October, 2000 eee BRL 86 QUARANTINE: All priwmeraesteleectsed from stock animalsthatwillhavebeen quarantined fora `minimumof 35 days prior to study start. No prophylactic or therapeutic treatments will be administered during the quarantine period. Standard procedures to be `conducted during the quarantine period are as follows: 4) A complete physical examination including a fecal examinationforintemal parasites, complete blood count (CBC), body weight, and body (rectal) temperature will be performed; b) three tuberculin tests at 2-week intervals (administered intrapalpebrally, using altemate eyelids for each test) will be `performed on eachprimate(primatesmusttestnegativetoallthreetests priorto releasefrom quarantine;primatesthatrespondpositivelyto atuberculintestwill be euthanized immediately, while non-responding primates housed in the same room will be held in quarantine for an additional 90 days); the blood sample: drawnfor CBCwill alsobeusedfor measuring oBf virus iter(primates found to have a positive B virutiterwil be euthanized immediately); ) a fecal sample for culture (screening for Salmonella and Shigella) will be obtained and submitted to an independent laboratory for analysis(ifany primates are found ; to be positive for Salmonella, Shigella, or parasites, the Study Director, in `conjunction with thestaffveterinarian, will determine the courseofaction); and ) all primates will be examined at least once weekly by a veterinarian and. observed (cage-side observations) twice daily for abnormal clinical observations and mortality/moribundity. 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 accwoithrthedapparoprniatceSOeP. Nonhumanprimateswilbeprovidedcageand feeding regimen modifications daily for their psychological well-being. The `modifications include, but are not limited to: swings, perches, Kong toys, clean 2liter soft drink bottles, puzzle feeders, nutritionally sound primate treats, unshelled. peanuts, and raw fruit. Where possible, primates will be housed proximatetoone another for visual and vocal contact. 88 ANIMAL IDENTIFICATION: During quaratnetpirinmaete,swillbeindividuallyidentified bychesttattoomumber or leter combination. Positive identification will be required after every cage change and prior to blood sampling, dose administration, and observation. 001652 as STUDY NO.: 9921.4 October 4,2000 -- afl} 9.0 EXPERIMENTAL DESIGN: As only one treatment group will be used in this study, no formal randomization will be required. Doses will be administered by intravenous injection to dette hepr harm maci okin netiecs of the test article. Each primate (three males, three females) will receive a single dose of `potassium perfluorooctanoate by injection into a superficial arm or leg vein. Blood samplesforserum drug level determinations willbecollected fromeachprimate at selected time points during the study. Urine and feces will also be collected at pre- determined intervals. A synopsisofthe study design is presented in the following table. fasuty pProocecduroes [[FrTeO[rTZ[3e[7e56[ 7P8RE9T[RwinE[TuSlnTluEznT] JE ose FT epaiS on | I | T AT] A| t [BColmgcyawlOengshersvas[ionsx|_[x|[x|[x|| [[xx T[ [[=xx[ [| [| [|=x| || [[xxl[x|xola=sl] f[SOernimcb&FregcLeesvel |[[|x[[x=[[xx| 1TxTT Tsx]T 1J[[x| l | J[xxllxslxx]] * Denotes week 9.1 RANDOMIZATION & GROUP ASSIGNMENT: As onlyonetreatmentgroupwillbeusedinthis study, no formal randomizatwiiolnl be required. 9.2 DOSE PROCEDURE: Eachprimate (three males, three females) will receive a single intravenous (TV) dose ofpotassium perfluorooctanoate (10 mg/kg) by injection intoa superficial arm or leg vein. Doseswillbebaseduponthemostrecentindividualbodyweights. Doses will be administered at a volume of2 mL/kg. The dayofdosing will be Day 0ofthe study. 9.3 CLINICAL OBSERVATIONS: - Daily Observations: All monkeys will be observed once daily during quarantine and twice daily, morning and afternoon, at least 4 hours apart, during the study for signsofmortality/moribundity and overt toxicity. Animals found in extremis will 001683 Ao { STUDY NO.: 9921.4 `Oct4o,b20e0r0 _-- hdd `be humanely sacrificed by an overdose of barbiturate followed by exsanguination with appropriate approval. Detailed Observations: Each primate will be examined shortly after dose administration for detailed clinical signsoftoxicity. All findings will be recorded. Additional clinical observations will be performed and recorded on daysof blood collection. 94 BobY WerGHTS: Each primate will be weighed on Days -1, 4, 7, 14, 21, and 28. 9.5 URINE AND FECES COLLECTIONS: UrineandfeceswillbecollectoendDays-3, 1 (0-24 hourspostdose), 2 (24-48 hours post dose), 7, 14, 21, and 28. The volumeofeach urine samplewillbe measured upon collection. All samples collected will be stored frozen at ~20 Corbelow prior to analysis (urine) or until further notice by the Sponsor (feces). 9.6 SERUM DRUGLEVELS: Blood samples (approximately 3 mL) will be collected from each primate at `approximately 0 (predose) minutes; 0.5, 2, 4, 8, and 24 hours; and 2,4, 7, 11, 14,21, and 28 days after dosing. Samples will be collected into tubes without anticoagulant `and will be allowed to clot at room temperature. The blood samples wil then be centrifuged, and the serumwillbe separated and stored frozen at ~20 Corbelow until analyzed. 9.7 BIOANALYTICAL METHOD DEVELOPMENT: Bioanalytical method(s) are to be developed for the determination of perfluorooctanoate in serum and urine matrices. The method will be validated for `accuracyand should be sensitivetothe 1 ppm or less level.Iffecesandothertissue `methods need to be developed, such a decision would result in additional cost. 9.8 BIOANALYTICAL SAMPLE ANALYSIS: The serum and urine samples from all monkeys will be analyzed for the concentrations of perfluorooctanoate using the previously validated method. The data will be expressed as equivalents of potassium perfluorooctanate. The analytical data on samples collected through and including Day 14 should be - available by Day 21 postdose. The Sponsor will use this information to make 001684 Ado i STUDY NO.: 9921.4 `October 4, 2000 -- e ---- ee apelt decisions concerning possible blood samplingofanimals beyond Day 28 postdose. Feces will be analyzed onlyif requested by the Sponsor. 99 ANIMAL DISPOSITION: At the endofthe study, monkeys will be maintained in the stock colony. 100 DATA ANALYSIS: Pharmacokinetic parameters (e.g, AUC, halflife, clearance) will be estimated from serum concentrationsofunchanged perfluorooctanoate, as appropriate and feasible, using a standard pharmacokinetic program. `Thetotal amountofperfluorooctanoateinurinewillbecalculatedandexpressedintermsof percent ofdose. - Meanvaluesandstandarddeviationswillbecalculatedforeach timepointandsampletype, as appropriate. No other statistical analysesofthedatawill be performed. 0 110 RECORDS: All rawdatapertaining to the conductofthis study, and all samples/specimens collected in this study, will be stored in the Archives at Southern Research Institute for upto 1 year ater acceptanceofthe final reportbythe Sponsor. After 1yearand with the permissofitohne `Sponsors Monitor, the data and any samples/specimens will be shipped to the Sponsor or to the Sponsors designated archival facility.Ifmaterials are to be retained in the archives beyond this date, such continued storage will be for a specific fee determined with the Sponsor. A copyofthe final report will be retained in the central archives at Southem Research. 120 FINAL REPORT: Abriefletter report summarizing the serum drug level results will be issued as soon as the information is available. A draft final report will be issued within 60 calendar days after `completionofthe in-life aspectsofthe study. The final report (electronic and hard copies) will be issued within 15 working days after receiptofthe Sponsor's final review comments onthedraftreport.The finalreportforthepresentstudywillinclude, butnotnecessarilybe limited to the following: Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data 001685 an STUDY NO.: 9921.4 Oct, 200 130 REGULATORY REFERENCES: T`Phrioscesdtuurdeysw(iSlOlPbse)ocfonSdoucuttehderinRaecsceoarrdcahn,caenwdiitnhtachceoprrdoatnocceowliatnhdthteheapSptlaincdaabrlde rOepgeurlaattionrgy requirements, as addressed below. 13.1 PROTOCOL AMENDMENTS AND DEVIATIONS: Athmeernedofmewinltlsb:eAdlolccuhmaenngteesd,in osirgrneevdi,siaonnsdofdattheedapbpyrotvheedpSrtoutdoycoDliraenctdotrh,earnedasotnhse `aSpppornosvoarl's(aMofnaixtsoirg.natAumreeonrdemleencttrsonwiiclclobmemumnaiicnattaiionne,dswuicthhatsheemapirolt)ofcoorl.chaWnrgietsteinn {the protocolmaybe granted by the Sponsors Monitor, but a written amendment will follow. Dpreovtioactoilo,nswi:llAblleoppeerrfaotiromnesdpaecrctoairndiinnggttootthhise sSttuadnyd,aurndleOspsersapetciinfgicParlolcyeddeufriensed(SnOtPhSi)s ofSouthern Research and/or the protocol, and any deviations from protocol or SOP will be documented. | 132 REGULATORY COMPLIANCE: Ginotohde sLpairbiotroaft,obruty wPrialctniocterse:quTihriesstnroincctlcionmicpallialnabcoerwaittohr,y tshteudUy.Sw.ilFloboedcaonndduDcrtuegd A'5d8m).inDiasttrafatrioomn'tshi(sFsDtAu)dyGmoaoydbLeabsorautobrymPrtiaoctttihcteeFe(DGAdLP)inrseguulpatpiooonfsra(tn21IGNFDRINPaDrAt application. QuwiatlhiFtyDAA'sssuGrLanPcreeRgeulvaiteiwon:s,Ansetithhsesrttuhdeyinw-illilfenaocttbievictiesononr thdeinfsiutnarliccrtecpotormtplewiilalndcbee audited by the Quality Assurance Unit at Southern Research. 13.3 FACILITIES MANAGEMENT AND ANIMAL HUSBANDRY: Animal care will be in compliance with the SOPs of Southem Research, the GAuniidmeallineRsesfoourrctehse,CaCroemmainsdsiUosneoofnLaLibfoeraStcoireyncAensi,maNlast,ion7a*lEdRietsieoanrc(lhasCtoituuntceilo;f NAgartiicounlatlurAecatdheromuyghPrtehses;AnWaismhailngWteolnf,arDeC;Act199(6P)u,blaincdLtahwe U9.9S-.19D8e)p.artSmoeunttheorf Research Institute is fully accreditbeyd the American Association for Accreditation ofLaboratory Animal Care (AAALAC). 001686 an ( STUDY NO.: 9921.4 --_-- 134 ANIMALWELFAREACT COMPLIANCE: `October4,200 hello By signing this protocol, the Sponsor signifiesthatthearreneo generally accepted altematives totheuseofanimals,andthatthestudydescribedbythisprotocoldoes not unnecessarily duplicate previously conducted or reported experiments. Procedures used in this protareodecsiogneld to conform to accepted practices and to minimizeor avoidcausingpain,distress, ordiscomfortintheanimals. Inthose circumstancesin whichrequiredstudyproceduresarelikelytocausemorethan `momentary or slight pain or distress, the animals will receive appropriate analgesics or anesthetics unless the withholdingofthese ageats hasbeen justified in writing by the Study Director and/or Sponsor and approved by the IACUC. `Thenumber ofanimalsselectedforuseinthisstudyisconsideredtobetheminimum number necessary to meet scientific and regulatory guidelines for this typeofstudy. `This study design was reviewed by the IACUC at Souther Research Institute and was approved on 07/26/2000; it was assigned ACUC tracking number 00-07-034. 001687 as ,- STUDYNO.:99214 140 PROTOCOL APPROVALS: "This protocol has been reviewed and approved. [--ena Study Director: Pde SF daded PatriciEa. Noker, Ph.D., D.AB.T. `Study Director u)ufe Date `Sponsor's Monitor: g2& INITIALS ONLY (See page 2) [a a Date. Management Approval: Modlle `WardR. Richter, M.S, D.V.M, D.ACV.P. og Date Director, Safety Assessment Departmerit, Southern Research Institute 001688 Appendix B Method Validation Report: ValidationofAnalytical Method for Determination of `Perfluorooctanoate in Monkey Serum and Urine Using HPLC/MS/MS 001689 Bi METHOD VALIDATION REPORT VALIDATION OF AN ANALYTICAL METHOD FOR DETERMINATION OF PERFLUOROOCTANECARBOXALATE IN MONKEY SERUM AND URINE USING HPLC/MS/MS STUDY ID: 9921.4.2 Southern Research Institute 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 001690 B2 SUMMARY `SouthemResearchInstitutehassuccessfullyvalidatedfor3Mananalyticalmethod (BACG 3548) entitled "Determinationof Perfluorooctanecarboxylate in Monkey Serum and Urine: `Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS)". Calibration standards were prepared by spiking samplesof eitherblank `monkeyserumor humanurinewithknownamountsoftestarticle,perfluorooctanecarboxalate (PFOC) and intemal standard, perfluorohexanesulfonate (FHS). The concentrationoftest articleinserumcovered acombinedrangefrom 10to100,000ng/mLwhiletheurineextended from 5to500 ng/mL.Quantitationlimitsweresetat 20ng/mLforserumand 10ng/mLfor urine. Compositestandard curvesgeneratedusingintemalstandard quantitationwereusedto determine the correlation coefficients. Fortheserum, threecompositecurves composed ofa total of44individualcalibration standardsextendingoverconcentrationrangesfrom 5000 100,000ng/mL, 10-0 10,000ng/mLand 10 500ng/mLproducedcorrelationcoefficientof 0.9874, 0.9750 and 0.9977 respectively.A single composite curve prepared from urine over a concentration range of5 ~ 500 ng/mL and containing 20 individual calibration standards `produced a correlation coefficient of0.9960. 001691 James D. Johnson, M.S., MBA MBiaonaangaleyrtical Chemistry Group Gregory S. Gorman, Ph.D. StaffChemist `Bioanalytical Chemistry Group Lester Williams, B.S. Associate, Chemist II `Bioanalytical Chemistry Group George Dollar Associate Biologist I Bioanalytical Chemistry Group B3 KEY PERSONNEL 001692 B4 1. OBJECTIVE `The objectiveofthis study was to provide avalidated analytical method for the determination of perfluorooctanecarboxalate in monkey serum and urine. 2. SAFETY All necessary proceduresto ensuresafetyoftheanalystswerebased on informationcontainedin the Material Safety and Data Sheets (MSDS), provided by 3M for the test article used in this study. CAUTION: Since primates may carry a numberofzoonoses, all unpreserved tissues, including. blood,plasmaandserum,aretobeconsidered asbiohazardsandhandledwithuniversal precautions. Referto SOP number SRI 2-5-5 for a descriptionofsafetyprocedures to be used `when handling unpreserved primate tissue. 3. EXPERIMENTAL 3.1 Analytical Procedures `The sample preparation and analysis procedures as described in the analytical method entitled "DeterminationofPerfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLCMS/MS)" were employed for all analyses. For the preparationofthe calibration. standards, a known volume of blank serum or urine (c.g. 500 kL) was spiked with aknown amountoftest article (PFOC) and internal standard (PFHS). To each standard was added 500 L of TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized water. This mixture was vortexed for approximately 5 seconds. Then 2.5 mL of ethyl acetate was added and the mixture was placed ona horizontal platform shaker at a low speed for 1 hour. The mixture was then centrifuged at approximately 2500 rpm for 5 minutes. The ethyl acetate layer was transferred to a second tube and evaporated to dryness at approximately 50C under a streamofdry nitrogen. The residue was reconstituted in 5% SmM ammonium acetate in deionized water : 95% methanol containing 1.5% formic acid and filtered through a0.2 um syringe filter 3.2 Method Validation Validation for BACG 3548 "DeterminationofPerfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS)" consistedofanalyzing standard curves prepared in monkey serem or human 001693 Bs 5`0ur0innego/vemrLvfaorriosuesrcuonmcaenndtra5t--io5n0r0anngge/sm:Lf5,o0r0u0ri--ne1.00,000 ng/mL, 100 -- 10,000 ng/mL and 10 - 51 .00000, ng0 /mL(0 Ser0 um) `One composite calibration curve comprising 3 setsofcalibration standards in a single analytical runin the 5,00--0 100,000ng/mLconcentrarngaegteneiraotend from atotofa2l2 calibration `standards produced a correlation coefficient of0.9874. A statistical `summary for the composite curve is shown below: STANDARD COMPOSITECURVE 1(ng/mL) MorSee [3T2733153] 35] Conc. (ng/mL) 5000 10,000 20,000 25,000 50,000 75,000 100,000 (SidDevistion | 385 |31 | 153| 3917 |7016|304g|13980 100 10,000ng/mL(Serum) `Two composite curves consisting of a total of 12 calibration standards encompassing a concentration range of 100 -- 10,00 ng/mL produced correlation coefficient of 0.9750 The 5,000ng/mLstandards weredroppedduetopreparationerror. composite curve is given below: A statistical summaryforthe STANDARDCOMPOSITECURVE 2(ng/mL) [MCFeoaonncr.S(&nAgc/cmuL[ )r. |7 913005[|22100051|1251040 1| 190230081|28026001 2[110,0]0700| {SiDevision | 18.5 32.0| 06 |147]307 |2| 10-500ng/mL (Serum) A single composite curve consisting of 11 standards takenfroman analyticalrunencompassing acacloinbcraetnitornatsitoanndraarndgsewoefre10dr--op5p0e0dnfgr/ommLtpherordunu.cedA astcaotrirsetliactaliosnucmomefafricyifeonrtothfe0.c9o9m7p7o.siNteocurve is given below 001694 Be STANDARDCOMPOSITECURVE 3(ng/ml) Conc.(ng/mL) 1020 50 100 200 500 [fofSils -- "T"2T2T2172T1T2] [Mean% Accur."| 966 |100|Tol|106 [ 906|101| (Std.Deviation |0ST |0.97|1.04|208|-- 381| 5-500ng/mL(Urine) . pArcoodmupcoesditaeccourrrveleaptrieopnacroeedfifincuirenitneocfon0s.i9s9t6i0n.gofOn2l0ystoannedacradlsiobrvaetrionarsatnagnedaorfd,55-- n5g0/0mnLg/wmaLs dropped ducto given below: lowinternalstandardresponse. Astatisticalsummaryforthe compositecurveis STANDARDCOMPOSITECURVE 4(ng/mL) Conc. (ng/mL) 5 1020 50 100 200 500 [FofSs--"TT2 35 T37 1353 T135 13] [Mean %Accur.| 947 | 107 |107 |827|107|101|995| [SWDeviation |007 |027[34T |420|82 |90[146] 33 Calculations Csaelrcuumlaetxitornasctwse(rneg/pmeLr)fowramsedbuascikngcaTlcuurlbaoteQduaunsin(gVearsciaolnibr1.a0t)i.on Tchureveamgoeuntnrtatoefdafnraolymtea isnettohef tcahleibbersattifoint cstuarnvdear(des..g.Tlhineeacra,liqburaadtriaotniccuertvce.)waansdgaemnoeruantteodfbyweiaghrteignregs.siAonqaunaadlryastiisctfoitdewtietrhm1i/nXe weighting wasdetetrobmetihebneset fdit: yeas br tec where: = Peak height responseofPFOC divibydpeeadkheightresponseofthe 1S (PFHS) in standards. x= Concentrationofthe PFOC in standards. a,b,c = Constants derived from the regression analysis. 00175 B7 4.0Conclusion pAeqrufalnutoirtoaotcitvaenemceatrhbooxdal(aBteAGinC3m5o4n8k)ehyasserbuemenutdileivzeilngopLeCd/aMnSd/vMaSl.idaQtueadntfiotratthieondeftoerrtmhiinsamteitonhoodf i`csobnacsenetdraotnioanrnainngteefmoarl astsaenrduamrmda(tPrFiHxSi)su2s0in-g10a0,10/0x0wenigg/hmtLeadndquad~ra5t0i0c enqgu/amtLifono.ruTrhineetwoittahl limits ofquantitation of 20 and 10ng/mL respectively. 001696 Appendix C Analytical Method for DeterminationofPerfluorooctanoate in Monkey Serum and Urine 001697 ct Page 1of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: DePtreerpamriantaitoinonaonfdPAenarlysfislbuy oHrPLoCoMcastisnaMSponencketercoymSeaetrrruym/bMaanosdsxUrSaipneelc:trSaoamtmeptelrye (HPLC/MS/MS) _--_---- 10 PRINCIPLE SPeerrfulmuoorroocutrainneecasrabmopxalleastaer(ePFoObCt)a.ineTdhfersoermucmynoromuroilnegu(sem.go.n, k0.e5ymsLt)recaotnetdawiinitnhg `(TPheOsCa)mpilsefsoratrifeietdhewnitmhixaendinwtietrhnaalnstiaonn-dpaaridri(InSg),repaegrefnltu,obruofhfeexranaensdulwfaotneart,ef(oPlFlHoSw)e.d dbryyneexstsr,actrieocnonwstiitthuteetdhyilna9ce5ta%te.metThhaenoelthcyolntaacietnaitneg l1a.y5er%is froermmoivceda,cide,va5po%ratSedmtMo `HaPmLmConMiausmsaScpeteactter,omfielttrerye/dM,asasndSptercatnrsofmererterdy t(oHPauLtCo/sMaSmp/lMeSr),viaTlhs,earnda`aonfnalryegzleidaebblye rensgul/tmsLeinxurintef.roemSaanmbpoludets20csotnota1i0n0i,n0g0P0nFgO/CmLatofcoPncFeOnCtriantisoenrsugmraenatdefrrtohman110t00o,500000 wnigl/l mbLe mwiatyhibnethdeilruatendgewiotfhrecloinatbrloelrbelsualntks mpartiroirxtosaonatlhyastist.he concentration of PFOC ``TThheemiaosnsssppreacytrsoomuertcreyovofltPaFgeOCisasnedtPaFt H-S2i00s0acvcoolmtpslwihsihcehdiinstlhoewneenogugahitotonigmrovedaeetl.y Irnedourcdeerthteo fmoarxmiamtiiozne osfenostihtievritpyo,tetnhtiisalmleytihnotderifserbiansgeidoonsnemxitxraecdterdefarctoimonthmeonmiattorriixn.g of the negative fragment ion (M-COOH)' for PFOC. CinAcUluTdIinOgNb:loSoidn,ceplparismmaateasndmasyercuamr,ryaraentuombbeercoofnsziodoenroesdesas, bailohuanzparredsseravneddhtaisnsduleesd, with universal precautions. Refer to SOP number SRI 2-5-5 for a description of safety procedures to be used when handling unpreserved primate tissue. 20 REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 21 Neat Reagents 001698 c2 ANALYTICAL METHOD Page20f 13, Method No.: BACG-3548 Title: DePtreepramriantaitoinonaonfdPAnealrysfislbuy oHPrLoC oMacsistnaMSopnencketeryocmSeaetrrruym/bMaanosdsUxrSiapneecl:tSraaommteptlerey (HPLC/IMS/MS) _--_-- 211 a, deionized and organic free (from in-house purification system; e.g., Ingalls 212 Methanol, HPLC grade 213 Perfluorooctanccarboxalate (analyte), as provided bytheclient 214 Perfluorohexanesulfonate (internal standard), 97% 215 Ammonium acetate, HPLC grade 216 Blank control monkey serum 217 Sodium Carbonate, Certified ACS Grade or equivalent 218 Sodium Bicarbonate, Certified ACS Grade or equivalent 219 Ethyl Acetate, HPLC grade 2110 Tetrabutylammonium Hydrogen Sulfate 2.1.11 Sodium Hydroxide SO% solution, Certified grade or equivalent 2112 Blank control monkey urine 2113 Formic Acid 22 Prepared Solutions Appropriate changes in the solutions may be made at the discretion ofthe analyst 221 5mM Ammonium acetate in organic free water 001699 cs . Page3of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: DePtreepramriantaitoinonaonfdPAenalrysfislbuy oHPrLoCoMcasitsnaMSopnenckcterycomSeaetrrruym/bMaaonsdsxUrSaipneelc:traSoamtmepterlye (HPLC/MS/MS) _--mmm----eeeee 22.1.1 2F0odrienxoamrplge,atonprwieaptcaerer-(4cf.lgtr.er4se,Lme)e.aMsiurxewoeultlaamndmofinliteurtmhrsoceufgahteH(Pe.LgC.,m1o.b5i4l2egp)haansde filtration apparatus. 222 TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydroxide) 222.1 Fsuolrfaetexianmdpleei,ontiozepdrewpaatreer2a5ndmaLd,judsitstshoelvpeH4.t2o140gwoifthte5t0ra%buNtaylOaHmmsoonluituimonh.ydrogen Note: 2more adjustments. dilute solution ofNaOHin watermaybe used to effect smaller pH 223 Carbonate/Bicarbonate Buffer Solution 2.2.3.1 `Faonrd aepxparmopxliem,attoelpyre2p.a1r0ego10f0 smoLd,iudmibssioclavreboanpaptreoxiinma1t0e0lmyL2.o6f5dgeoifonsiozdeiduwmatcearr.bonMaitxe well to 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 source of nitrogen) 35 HPLC mobile phase filtration apparatus 36 Filters for HPLC mobile phase filtration apparatus (c.g., Nylon-66, 0.20 um) 001700 . ct Paged of 13 ANALYTICAL METHOD MethodNo.: BACG-3548 Title: DeterminationofPerfluorooctainnMeonckaeyrSberoumxaandlUaritnee: Sample PreparationandAnalysis by HPLCMassSpectromerSype/ctMroamestrsy (HPLC/MS/MS) _--_----m 37 Analytical balance 38 Volumetricflasks (e.g, 10an 25mdL) 39 Disposable Pasteur pipets 3.10 Micropipettor(s) with tips 341 Culture tbes with teflon-lined caps 312 Ceauifuge 3.13 Assorted glassware and syringes 314 Culture tubes (vials) for use with solventconcentration apparatus 315 1m Plastic syringes with 0.2 um PVDF syringe filters 3.16 Variable speed horizontal platform shaker 3.07 pH meter 40 PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets 41 Main Stock Solution of PFOC ~1000 jig/mL s 4.11 Prepare an ~1000 pg/mL solution of PFOC in deionized organic-free water (c.g., accurately weigh about 10 mg PFOC into a 10-mL volumetric flask). Add deionized `organic-free water to dissolve. Dilute to the mark. Alternatively, wetheicog mpoh und 001701 cs Page Sof 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: DeterminationofPerfiuoroocitnaMonnkeeycSaerrumbanodUxrianel:Saamtplee Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) intoanappropriate vessel(e.g., culturetube)andadd 10mLofdeionizedorganic-free water. Mix well. Transfer the solution to aclean vesselifdesired. 42 Stock Solution of Internal Standard (PFHS), ~200 pg/mL 42.1 Prepare an ~200 pg/mL solution of PFHS in deionized organic-free water (c.g., `accurately weigh about 10 mginto a 50-mLvolumetflraiskc). Adddeionizedorganic- fAlrteeernwaattiveerlyt,owediisgshotlhveecanodmdpiloutueinnttodo t2haenmaapprrkopwriitahtedeviesosneilze(d.go.r,gcaunlitcu-rferteuebew)ataenr.d "d 50 mLofdeionized organic-free water. Mixwell. Tratnhessolfutieonrto aclean `vessel if desired. 43 Spiking solution of Internal Standard (PFHS), ~ 50xg/mL 43.1 Preparean ~50ug/mLsolution of PFHSin deionized organic-froewaterbypipetting 2mLofthe 200 pg/mLsolution into aculturetubeandadd 6mLof deionizedwater. Mix well. 44 Working Stock Solutions of PROC 44.1 To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mL volumetric flasks or other appropriate glassware. Ifdesired a modified dilution scheme can be used and documented in the study records. `Working Stock Level (WSL) (g/mL) Approximate Concentration 250.000 Volume of PFOC solution | Final Volume in dferieoeniwzaetderor(gmaLn)ic smiotgml [0| SmLofs0000ngml | 10 | 001702 o ANALYTICAL METHOD Page 6 of 13 Method No.: BACG-3548 Title: DeteorfPmerfiluonroocatantccairbooxalnate in Monkey Searnd uUrimne: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) om |sTmowmeos||6 o0 wa hoem Tm emw ammo|ewe0nma o]]] oiwm T[esmmosommwwmm0 0mmss] ]]| Tm0w TTsoomtooommmmmmmesa]|0 0] ] 442 Summary of concentrationsofserum standards: Volume and Spike Conc.| Approximate Standard `Concentration of Level PFOC in serum 1 g/ml) EIT [C0eT Tsmo ots w||owso mno0mr|] 001703 or Page 7 of 13 ANALYTICAL METHOD Method No.: BACG-3548 `Title: DPreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbtyaneHcaPrLboCxalMaatsesin SMpoenckteryoSmeetrruym/aMansds USrpience:tSraommeptlrey -_ (HPLC/MS/MS) [r Tiosorsooo0nymr |0 [wmormsooomm| sow| LL] ror EE [+ [hsuorsiasompme|10| [0 [euorsesopn|so| [x [o0morsoompm|om| 0pof sg|100| [ [ouorzsomym | 50 | porto [ow| [0[ ouorswmm| 10 | Lr| wmorosomgm |5| 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS GAptprreopnroinatoef hcheanagnesysin the concentrations of the solutions may be made at the 51 Multiple (c.g. about three) sets of matrix standards and amatrix blank (blank + IS) `are analyzed with also be analyzed iefadceshisreetd.of unknown samples. Amatrixdouble blank (blank-IS) may 52 Into individual ~20-mL culture tubes, pipet blank matrix (e.g., 0.5mL). Pipet in the 001704 appropriate pipet 10 iL volume as described of organic-free water in the table above for each standard. insteadofthe working stock solution .FAodrdt he blan the 10 ks, uL. cs . Page 8 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: DeterminationofPerfluoroocitnaMonnkeeycSaerrumbanod xUrainel:Saamtplee Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) eee ------ ofinternalstandardstock (~50 g/mL)toeachtubeexcepttheblank-IS(pipet 10 uL oforganic-free water instead) and vortex for ~5 seconds. 53 To each tube add 500 uL oftheTBA ion-pairing solution, 1 mLof `carbonate/bicarbonate buffer, and 1mLof deionized organic free water. Vortex each tube for about 5 seconds. 54 Add 2.5mL ofethylacetateand extractonhorizontalmixerfor 1hour ata lowspeed setting. 55 Remove thetubefromthe shakerandplacein acentrifuge (e.g. 2500rpm) forabout 5 minutes. 56 Taoffk thee top ethylacetatelayer andput itinto aclean tubeandevaporateto dryness (e.g., ~ 50minutesintheTurbo-Vap ) with agentlestream ofnitrogenand `moderate heat (.g., 50 C). | 57 Reconstitute the residue in 500 uL of 5% 5 mM ammonium acetate: 95% methanol `containing 1.5% formic acid andvortex brieflyto mix. Fitlhetsameplresthrough0.2 um PVDF or Nylon syringe filters into autosampler vials. 60 PREPARATION OF SAMPLES 61 Allow each serum sample to thaw to room temperature. _ Vortex each sample briefly, but vigorously. Pipet an aliquotofeach sample (.g., 0.5 mL) into individual ~20mL culture tubes. If necessary, dilute an aliquot of any sample with blank matrix so that the expected concentration of the test article being analyzed will fall within the concentration range of the standard curve. Add 10 uLofinternal standard stock (~ 50 ug/mL) to each tube and vortex for a couple of seconds. 62 To each tbe add S00 pL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds 01.05 co Page 9 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Tite: DPreetpearrmaitniaotnionaonfdPeArnfalluyosriosoctbayneHcaPrLboCxalMaatsesinSMpoenckteroymSeterryu/mMaasnsd USrpience:trSoammeptrlye (HPLC/MS/MS) _--eeeee 63 Add2.5mLofethyl setting acetateandextract onhorizontalmixerfor 1 hourat a lowspeed 64 `Remove the minutes. tubefromtheshakerandplacein a centrifuge(e.g., 2500rpm)for about 65 dTraykneesosff(ct.h.e,t~op 50emthiynlutaecestiantethleaTyuerrbaon-dVapput9)itwiitnho aagecnltelaensttruebeamaonfdneivtarpoogreantaentdo `moderate heat (e.g., 50C). 66 Rcoenctoanisntiintgut1e.t5h%e freosrimdiuceaciind5a0n0duvLorotfex5b%rie5flmy tMo maimx.moFinlituermthaceetsaatme:pl9es5t%hrmoeutghha0n.o2l um PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. 7.0 MANAASLSYSSPIESCBTYROHMIEGTHRYP/EMRAFSOSRSMPAENCCTEROMLEITQURIYD(HCPHLRCO/MMSA/TMOS)GRAPHY 71 Conditions are to be optimized if necessary. 7.1.1 HPLCConditions Analytical Column: ~~ Keystone Scientific Aquasil C18, 150 mm x 2 mm ID, or equivalent Guard Column: Elution Flow rate: Injection volume: Mobile phase: Gradient Profile Keystone Aquasil C18 10 mm x 2 mm 400 L/min 3ul A: SmM ammonium acetate buffer B: 1.5% formic acid in methanol 0-0.5min. 50%A:S0%B 0.5-7.5 min. 10% A: 90% B lineargradient 7.58 min. 10%A : 90% B step gradient 001706 cto Method No.: BACG-3548 ANALYTICAL METHOD Page 100f 13 Title: DeterminationofPerfluoroocitnMaonnkeeycSearrumbanodxUrianel:Saamtplee: Preparation and (HPLC/MS/MS) Analysis by HPLC Mass Spectrometry/Mass Spectrometry _-- Temperature: Sllmin Ambient S0%A:S0%B 7.2 PE Sciex API 3000 Triple Quadrupole MassSpectrometerConditions . Software: PE Sciex TurboQuan `Turhoion SpraySource Note: Values this table. listed under "MS/MS Acquisition Conditions override parameters in Auxiliary Gas: Air (e.g.,Grade0.1) at85popuersnquadreisnch `Parameter Value is m0 xe 0 Ee or re 0 s PY . st sor . i 0 so 0 - = o sor 2 oF = a wn 001.07 cn ANALYTICALMETHOD Page 110f 13 Method No.: BACG-3548 Title: DeterofmPerfiluozrooactantecairbooxalante in Monkey SearndUurinme: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) _--m Parameter Value a is an . an 5 ae o ou ' vou o ve o MS/MSAcquisitionConditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: Sohiycon g5u5m'som DEtie 0 SpQ reis ai s) QHes Bwoorcce = s5a o&mm o 28 kD g Tm0 0o1, 08 ci Page 120f 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: PDreetpearrmaitniaotnionanodf PAenraflluyosriosocbtyaneHcaPrLboCxalMaatsesinSMpoenckteroymSeterryu/mMaasnsd USrpienec:trSoammeptlrey (HPLC/MS/MS) _--mm 80 CALCULATIONS 81 Atitmteh,epeeankdosfhatphee,anaanldypteiackalhreuing,htreavnidepweeaakcahrecahdreotmeartmoignraatmiotnoofentshuerteetshtaerrteitcelnetainodn tthheeIiSoanrperaofcicleepstaatblteh.eTfholeldoawtianmg amaysbse-tsom-ochoatrhgeedraastiaopsp:ropriate. Forquantitatiounse, APnFaOlCy PFHS I4o1n2Pr9o1f0i3le689 3989103989 82 (PlPoFtHSth)efpreoamkaalrsetaanrdeasrpdosnsveersoufsPtFheOcCodnicviednedtrbayotftihteohnepetaesktaarrteiaclreiesnptohnesestoafndtahredsI.S Afilttoefmtahtievedlayt,a (th.e.p,eaqkuahderiagthitcsfmitawyebieghutseedd wiinsttheaLdo/fcpoenackeanrtears.aotfOitbothaenintetshtearbteisctlecuorrvae c`uqruavderaatnicdfiitt).maNoyteb:eTnheecbeessstarcyutrvoefhiatvmeamyobreedtehpaenndoennetostnatnhdearradncguerovfetfhoerstvaanrdiaoruds `concentration ranges using the following: y=ad+brtc where y= Peak height responseofPOC divided bypeakheightresponseof the IS (PFHS) in standards. x = Concentration of the PFOC in standards. a,b,c = Constants derived from the regression analysis. 83 Using the standard curve, calculate Correct the results of samples for any the level of dilutions. PFHC in each unknown sample. 001,09 on . Page 13 of 13 ANALYTICAL METHOD Method No.: BACG-3548 `Title: DeterminationofPerfluoroocitnaMonnkeeycSearr umbanod Uxrianel: Saamtplee Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) NOTE: Due to unresolvable interferences with the test article and/or internal standardfromthematrix, external standardquantitation maybeusedatthe discretion of the supervising mass spectrometrist. 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 Results of all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Authors Ltdfone> Greg6ty .Gorman, Ph.D., Staff Chemist Bioanalytical Chemistry Group Ln)oo Date Approved by: les D. J 7ioanalytical (S, MBA, Manager mistry Group [2-27-00 Date woul, io