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ARR EG -- 1828 Final Report on A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey ~~T-757:! 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 Souther Research Institute 2000 Ninth Avenue South 35205 P.O. Box 55305 Birmingham, Alabama 35255-5305 2Ss 33388 z= EE a 000002 ABSTRACT `The pharmacokinetics and urinary excretion of perfluorooctanoate were investigated in male and female cynomolgus monkeys. Three male and three female monkeys were administered asingle iv bolus doseof 10 mg/kgof perfluorooctanoate, potassium salt. At various times after dosing, serum and urine (24-hour collections) samples were obtained and analyzed by HPLC/MS/MS for levels of intact perfluorooctanoate. The lower limitofquantitationofthe analytical method was 20 ng/mL forserum samples and 10 ng/mL for urine samples. At 0.5 hours after dosing, serum concentrations ofperfluorocetanoate were similar in male and female monkeys and ranged from 91,130 to 96,660 ng/mL in male monkeys and from 88,940 to 96,400 ng/mL in female monkeys. Serum concentrations of perfluorooctanoate subsequently declined but, beyond the first few days after dosing, the rateofdecrease appeared to be faster in male monkeys than in female monkeys. On Day 28, the concentrationsofperfluorooctanoate in serum ranged from 1863 t0 27,140 ng/mL in the male: `monkeys and from 7,145 to 33,680 ng/mL in the female monkeys. Dueto the relatively high serum concentrations of the 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 limitofquantitation (20 ng/mL) in male monkeysand between 885 and 4701 ng/mL in female monkeys. The serum concentration versus time data were subjected to non-compartmental `pharmacokinetic analysis. The terminal half-lifeofperfluorooctanoate in serum was 13.6, 13.7, and 35.3 days in the three malemonkeysand 26.8, 29.3,and 41.7 days inthethree female monkeys. The total body clearance was 4.0, 15.8, and 17.5 mL/day/kg for male monkeys and 3.1, 3.9, and 9.1 ml/day/kg 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 000003 monkeys. The volume of distribution of the compound at steady state (Vd) was similar for both sexes and ranged from 168 to 192 mL/kg for male monkeys and 133 to 270 mL/kg for female monkeys. Perfluorooctanoate was slowly excreted in urine by both male and female monkeys; levels ofperfluorooctanoate, representing as much as 1%ofthe administered dose, were present in urine 28 days afler dosing. The results of this study suggested that the pharmacokinetics of perfluorooctanoate may be different in male and female monkeys. Cen 000004 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 2.3 Experimental Design 3 Group Assignment and Dose Procedure 3 Clinical Observations 3 Body Weights 3 Urine and Feces Collection 3 Serum Levels of Perfluorooctanoate 4 Bioanalytical Method Development and Sample Analysis 4 Data Analyses 4 30 RESULTS 4 3.1 Mortality 4 3.2 Clinical Observations 5 33 Body Weights 5 3.4 Serum and Urine Concentrations of Perfluorooctanoate 5 40 DISCUSSION 7 50 CONCLUSIONS 8 60 RECORD ARCHIVES 8 7.0 REFERENCES 8 000005 i Table 1: Table 2: Table 3: Table 4: Figure 1: Appendix A: Appendix B: Appendix C: TABLE OF CONTENTS (Continued) LIST OF TABLES Page Individual Body Weights 9 Serum ConcentrationsofPerfluorooctanoate 10 Pharmacokinetic Parameters of Perfluorooctanoate Calculated from Serum Concentrations n Urinary ExcretionofPerfluorooctanoate 12 LIST OF FIGURES Serum Concentration ProfilesofPerfluorooctanoate in Monkeys 1 LIST OF APPENDICES Study Protocol and Amendments Al Method Validation Report: Validation of Analytical Methods for Determination of Perfluorooctancate in Monkey Serum and Urine Using B-1 HPLC/MS/MS Analytical Method for Determination of Perfluoroocianoate in Monkey Serum and Urine cl Sesh 000006 iii. Signature Page A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Fleer E 20AD Patricia E. Noker, Ph.D., D.A.B.T. Study Director Supervisor, ADME & Pharmacokinetics Reviewed by: 1)rus Date odo an Charles D. Hbert, Ph.D., D.A.B.T. Director, Safety Assessment fon Date We, the undersigned, were responsible for the conductofthe work and reportingofthe results in the listed sections. We concur with the views relative to our bodyof work as expressed in the discussion and conclusions. Ls < `Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Date . 000007 iv Good Laboratory Practices Disclaimer This study describedinthis final report was not conducted in strict compliancewiththe U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations (21 CFR Part 58), and neither this report nor the raw data were reviewed by the Souther Research Quality Assurance Unit, However, the study was conducted according 10 the protocol and amendments and the applicable standard operating procedures, and all study procedures, data recording, and reporting were performed in a manner consistent with the standardof GLPs. The final report accurately reflects the raw data obtained during the performanceofthe study. There were no adverse circumstances that affected the quality or integrityofthe study. Lz vad) Patricia E. Noker, Ph.D, D.AB.T. Study Director 1/rgfes Date Tent 000008 v Study Dates: Study Schedule and Personnel Study Initiation: DayofDosing: Last Dayof Sample Collection: Study Completion: October 4, 2000 October 9, 2000 February 9, 2001 January 14, 2003 Study Personnel: Patricia E. Noker, Ph.D, DAB.T. Study Director Charles D. Hbert, PhD, DAB.T. Director, Safety Assessment Norman D. Jefferson, B.S. Associate Director, Safety Assessment Gregory S. Gorman, Ph.D. Manager, Bioanalytical Chemistry Group `Tsu-Han Lin, Ph.D. Pharmacokineticist Darrell E. Hoskins, D.V.M, Ph.D., A.C.L AM. (Dipl) Veterinarian Laluana A. Durbin, B.S. Supervisor, Large Animal Laboratory D. Wayne May, LATG Supervisor, Animal Care Carolyn R. Oliver, B.S. Supervisor, Study Coordination . 000009 \ 1.0 Introduction `The objectivesofthis study were to determine the concentration ofpotassium perfluorooctanoate in serum and to estimate urinary clearance at various times following administration of 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 3 male and 3 female monkeys designatedforuse in this study were selected from an inhouse colonyof monkeys that were housed at Southem Research Institute (Southern Research) prior to use on this study. These monkeys were purchased from Charles River BRF, Inc. (Houston, TX) and were an estimated 3-4 years of age when placed on study. Individual animal identification was by chest tattoo. The cynomolgus monkey is an accepted species to support clinical studies ofdrugs used or intended for use in humans. `During the quarantine period, a complete physical examination including a fecal examination for intemal parasites, complete blood count (CBC), body weight, and rectal temperature was performed on each of the monkeys. The following procedures were performed on the `monkeys during quarantine: (1) Three tuberculin tests were administered to each animal 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 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 clinical 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 tothe monkeys 2-3 times each day. The quantityofthe daily ration was sufficietnto meet RE 000010 2 nutritional requirements. In addition, the diet was supplemented with fresh fruitireats several times each week. Tap water (Birmingham public water supply) was available to the monkeys ad libitum during the quarantine and study periods. The monkeys were housed individually in stainless steel cages during the quarantine and the study periods. From Day 010 the end of the study, the monkeys were housed in a room that was maintained at a temperature of 68.0-70.3 F and a relative humidity of 22.2-65.7%. The humidity was `within the required range (30-70%) over 90%ofthetimeduring the study; excursions below the recommended humidity range wereofshort duration. Room lights were controlled by `an automatic timer set to provide 12 hoursoflight (0600 to 1800 hours, CST) and 12 hours ofdark per day. Cage size and animal care conformed to the guidelinesofthe Guidefor the Care and UseofLaboratory Animals, Tth edition" and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPs) of Souther Research. The study design was approved by Southem 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 given a single IV bolus dose of perfluorobutanesulfonate (10 mg/kg) on April 10, 2000 (Souther Research Study No. 9921.1); a single IV bolus dose of potassium perfluorobutanoate (10 mg/kg) on June 13, 2000 (Souther Research Study No. 9921.2); and a single IV bolus dose of potassium perfluorohexanoate (10mg/kg) on July 31, 2000 (Southern Research Study No. 9921.3). 2.2 Test Article and Vehicle `Test Article: One bottle containing 5.2 gramsofpotassium perfluorooctanoate (T-7507; expiration date not supplied; Southern 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 room temperature. until used. Stabilityofthe test article was the responsibilityof the Sponsor. i. 000011 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 perfluorooctanoate prepared at mg/mL, the required amountoftest article was weighed out in a volumetric flask. Sterile saline was added and the formulation was stirred until in solution. The formulation was stored refrigerated and used for dosing within 4 days afier `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: As only one treatment group was used in this study, no formal randomization was required. On Day 0, eachofthe three male and three female monkeys received a single intravenous (IV) doseof perfluorooctanoate at 10 mg/kg by injection into a superficial arm or leg vein. Doses were based upon theDay -1 individual body weights. Doses were administered at a volume of2mL/kg. Clinical Observations: All animals were observed twice daily for signs of : `mortality/moribundity. Each primate was examined shorly after dose administration for clinical signsoftoxicity. Additional clinical observations were performed on daysofblood collection. Body Weights: Each primate was weighed on Days -1, 4,7, 14,21, and 28. Urine and Feces Collection: Urine and feces were collected for 24-hour intervals on the following days: prior to dose administration (Day -3; baseline), on Day 1 (0-24 hours pean 000012 4 postdose), on Day 2 (24-48 hours postdose), and on Days 7, 14,21, and 28. The volume of each urine sample was measured upon collection. Urine and feces samples were stored frozen (approximately -20 C or below). Fecal samples will not be analyzed unless specifically requested by the Sponsor. Serum Levelsof Perfluorooctanoate: Blood samples (spproximately 3 mL) were collected from each primate at approximately 0 (predose) minutes; 0.5, 2, 4, 8, and 24 hours; and on Days2,4,7, 11, 14,21, 28, 57, 79, 87, and 123 postdose. Samples were collected into tubes `without anticoagulant and were allowed 10 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 Development and Sample Analysis: A bioanalytical method was developedforthe analysisofperfluorooctanoate in serum and urine matrices. The method `was validated for accuracy. This method was used for the analysis of serum and urine samples collected during the study (see Appendices B and C). Data Analyses: The serum concentration data for unchanged perfluorooctanoate were subjected to non-compartment pharmacokinetic analysis using WinNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc.; Cary, NC). Mean values and standard deviations for ach parameter were calculated using Microsoft Excel software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorooctanoate at each collection interval was calculated and expressed as apercentofthe 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 euthanized on Day 79 becauseofrepeated episodesofself-mutilation; there was no indication that the self-mutilation was related to administrationofperfluorooctanoate. -. 000013 5 3.2 Clinical Observations No adverse drug-related clinical signs of toxicity were observed for any monkey. Male `monkey 2052 was observed on Day 57ofthe study to have several wounds on the right leg. The attending veterinarian diagnosed these wounds to be self-inflicted lacerations. The `wounds were cleaned and subsequently flushed with a weak betadine solution every 3-4 days for 4 coursesoftreatment. The social enrichment of the monkey was also augmented in order to provide the monkey additional stimulation and to change his focusof attention. On Day 79, the monkey was examinedand several severe lacerations were observed on the leg and footofthe animal. These lacerations were diagnosed as self-inflicted wounds. Due to the repeated instancesofself-mutilation and the likelihood of continued episodes of self`mutilation, the monkey was euthanized on Day 79 for humane reasons. 3.3 Body Weights Body weights for individual monkeys are presented in Table 1. The body weightofeach monkey remained the same between Days -1 and 28. 3.4 Serum and Urine Concentrations of Perfluorooctanoate During the study, an HPLC/MS/MS method was developed and validated for the analysis of perfluorooctanoate in monkey serum and urine. Details of the method validation procedure are provided in Appendix B. Details of the analytical method are provided in Appendix C. The lower limitofquantitationofthe method was 20 ng/mL for serum samples and 10 ng/mL for urine samples. en Serum concentrations of perfluorooctanoate in three male and three female monkeys at various times through Day 123 after administration of a single iv dose of 10 mg/kg are presented in Tabl2e and plotted in Figure 1. At0.5 hours after dosing, serum concentrations of perfluorooctanoate 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 concentrationsofperfluorooctancate subsequently declined ata slow rate; the rateofdecline appeared to be similar in male and female monkeys through at least the 000014 6 first two days after dosing. At 48 hours, serum concentrations ofperfluorooctanoate were between 48,530 and 65,810 ng/mL inthe male monkeys and 39,820 and 61,600 ng/mL in the female monkeys. Beyond this time, serum concentrationsof perfluorooctanoate in two ofthe three male monkeys decreased at a faster rate than that observed for the third male monkey and for the three female monkeys. On Day 28, the concentrations of perfluorooctanoate in serum ranged from 1863 to 27,140 ng/mL in the male monkeys and from 7,145 to 33,680 ng/mL in the female monkeys; due to these relatively high serum concentrations of the compound on this day, the lengthofserum collection was extended through Day 123. On Day 123, the serum concentrationofperfluorooctanoate was ator oly slightly above the limitof quantitation (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 concentrationsofperfluorooctancate in individual monkeys are presented in Table 3. The values were derived from non- compartmental pharmacokinetic analysisofthe data. AUCq.qga Values ranged from 571 to 2501 pgeday/mL (mean: 1235 ug.day/mL) for male monkeys and from 1094 to 3224 yigedaymL (mean: 2293 ugeday/mL) for female monkeys. The terminal halflife of perfluorooctanoate in serum was 13.6, 13.7, and 35.3 days in the three male monkeys and 26.8, 29.3, and 41.7 days in the three female monkeys. The total body clearance was 4.0, 15.8, and 17.5 mL/day/kg for male monkeys and 3.1, 3.9, and 9.1 mL/day/kg for female `monkeys. These estimated values for half-lifeand clearance indicated that twoofthe three `male monkeys eliminated perfluorooctanoate at a faster rate than did the female monkeys. `The volumeofdistributionofthe compound at steady state (Vd,,) was similar for both sexes and ranged from 168 to 192 mL/kg for the male monkeys and 133 to 270 mL/kg for the female monkeys. 000015 7 `The amount of perfluorooctanoate eliminated in urine by individual monkeys at various times after dosing is presented in Table 4. Sex differences in the urinary excretion of perfluorooctanoate were not readily apparent prior to Day 21; on Day 21 and Day 28, the female monkeys appeared to eliminate a higher percentageofthe dose in urine than did the `male monkeys. The urinary eliminationof perfluorooctanoate was prolonged for both the male and female monkeys; on Day 28 from 0.1 01%ofthe administered dose was recovered in urine collected from the individual monkeys. 4.0 Discussion Perfluorooctanoate was slowly eliminated by male and female monkeys given a single iv dose of 10 mg/kg. The resultsofthis study indicated that beginning on around Day 7 and continuing throughout the remaining periodofsample collection, serum concentrationsofperfluorooctanoate were lower in twoofthe three male monkeys than in the three female monkeys. On the final day of serum collection (Day 123), serum concentrationsof perfluorooctanoate were below or only slightly above: the quantitation limit (20 ng/mL) in the two surviving male monkeys, whereas, on the same day, serum concentrations of perfluorooctanoate ranged from 885 to 4701 ng/mL in the three female monkeys. These differences in the serum concentrationsof perfluorooctanoete were reflected in a shorter serum half-lifeof perfluorooctancate in these two male monkeys (13.6 or 13.7 days) than observed for the three female monkeys (range: 268 to 41.7 days). The clearance of perfluorooctanoate also appeared to be higher in twoofthe male monkeysthanin the three female monkeys. Although the number ofmonkeys was limited, these results suggested that 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 female monkeys than that observed for the other two male monkeys. It is possible tht the study procedures (c.g., handling, short-term restraint in a chair) or other unknown factors may have produced stress in this monkey resulting in 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 Cnet 000016 5 disposition of perfluorooctanoate in this male monkey. Iti also possible that the self-mutilation activitiesofthis monkey, which lead to the eventual euthanasiaofthe animal, were a response to stress. Although the serum levels of perfluorooctanate indicated that at least two of the three male `monkeys may have eliminated perfluorooctanoate at a faster rate than did female monkeys, it was difficult to discern a difference in the urinary excretion of the compound by male and female `monkeys. This may have been related to the fact that the rate ofurinary excretionofthe compound by all the monkeys on study was slow. Less than 20%ofthe administered dose was excreted in urine by either male or female monkeys within the first 48 hours afi dosing. Subsequently, on Day 28, perfluorooctanoate was present in urine collected from male or female monkeys at levels that represented as much as 1.0%ofthe dose. 5.0 Conclusions `The mean terminal serum half-lifeofperfluorooctanoate 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 copy of the final report from this study will be stored in the Archives at Souther Research for up to 1yearafter acceptanceofth final report by the Sponsor. Afer 1 year `and with the permission of the Sponsor's Monitor, the data and any samples/specimens will be shipped to the Sponsor or to the Sponsor's designated archival facility. Ifmaterials are to be retained in the archives beyond this date, such continued storage wil be for a specific fee determined with the Sponsor. A copyofthe final report will be retained in the central archives at Southern Research. 7.0 References 1. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research `Council; National Academy Press; Washington D.C. 1996. she 000017 s z =| [3 ie =5 o 2 by S2 ii. i 03 oe FH i: 1EE: Efr | Jaa 38 ) 32 i oF a 3 a oH 22 i3] 1s8lo2g [3 zl |E|E|E E|E { &l else] |=lle & = 8 Lok E2 S18 ! 000018 10 Tabl2e A Pharmacokinetic Studyof Potassium Perfluorooctanoate in the Cynomolgus Monkey Serum ConcentrationsofPerfluorooctanoate [ Co[ncSeenmwation(ngmi) | | [Ma ~~ TlFe emaless| (Timepoint] 2052 [ 2054 [22|120518T 2059 [ooT"msTeor 20 [57 [12533| [Lo|s91e,13s0| 96,660| 91,370| 96,400| 88.940 |2hrs [67,900 [91,630| 90,680| 74,280 71,600 81,620| 69,380 71,200| 60,860| 73,650| 79,570|61,520| 64,170 50.210| 50,930|64,160| 62,890| 60,760|41,940| 5540 7510 | 0 s920 65,810| 48,530| 48,930| 61,600 39.82|0 43,370| 42,940|30,490| 70,520| 41,640| 44,370 [our] 39,450| 19,320| 23,640| 38,980| 53,980| 37,500 [Da14y| 37,360| 11,220| 13,430| 40450| 45,790| 21.910 Day21| 33,600 | 3,985| 5,939| 38,750| 37,850 Day28 |27|, 1,81 63|4 3,20 41| 33,680| 24300| 7,145 | Days7 [154670 [71 80[0 31.6]00] 16870 ([Dowyeer|oos[o--s[ [ii ~ [wm| --~ 1wm| [Dey] ~[mou[ 35[36st]anor]sss | * Sample taken prior to humane sacrifice of animal. BQL = Below the quantitation limit (<20 ng/mL) Page loft . 000012 u FRRaEH 3 fSi ERAZR& i 38 | LR. I RERR S 3 | Lhlik i5:| blalillele| 22%s 2ST 5Ed5 | |Bellh EE fs cs E | shRER 33 3 B32 gt :E g: :2 cL aH 3 Fela 1 | EEiE,E B2l E [[iHEEll eEssEaia isaess2 s2s) lSeEpEES aiiiE:S 000020 2 Table 4 A Pharmacokinetic Studyof Potassium Perfluorooctanoate in the Cynomolgus Monkey Urinary Exeretionof Perfluorooctanoate: rrAnimal Urine | Unne | -- Percent of Dos|e Concentration| Volume Total Dose in Urine LE |Sex| (ng/ml) (mL) ug) | Go [| Cr wRTw Tam [20s TM BOL | #0| 1 eo | | zr w vo wo Tso (2s For ws TT --- sao| | [20% TFT Bor[70 |"3so00| -- | | wor [FJ Bor[0| J amew |] Dwi [202TMT 610[200 T 1250 T 66000[19 | [[20seTMT 9285 |a0| 5&9 |ea0| 61 | [2 TM 27050 | 550 | 9503 | S100| 186 | [2088 [F557[10| 1050 |34000| 1 | 209 [FT ior | se | des | mow sz[MwTwo| voTow 13 | [ 2056[M] Toa [T0 Te 2 o 0 | 00 5 | [20[Mare 1m0[ss stew| or (Zs [Fl sm 110| so sow| 17 | [[o2o0rT[FF{T s20w8 | 1e 660 ||""o15m7% ||a350w00 | | 05s9 || EE [20s [M1] Toso |0 |" 6005 | eas | tal | [2"[M] Toro ow sa [sow |e | [2058[F] as47 | 200 | 7% | e000 | 26 | [2059 [F] ass1000 | a8 |35000 | 13 | BQL = Below the quantitation limit (<10 ng/mL) * Concentrationofperfluoroostanoate in the sample greatly exceeded the highest concentration in the standard curve; therefore, urine concentration value may containsignificanterror "Due to technician error, urine was discarded afterthe 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 tor2 s 000021 5 Table4 (Continued) A Pharmacokinetic Study ofPotassium Perfluorooctanoate in the Cynomolgus Monkey Urinary Excretionof Perfluorooctanoate TT Om | ome | Animal i) Concentration| Volume Total (ngml) __w --T FereentofBowe| Dose inUrine | ww | oo mesn[wve esTw oe [|e3s5 seown[ [1 05 6] ] Moss[Fen[0[aw | saw[os] [209TFT S80 1m0|7 |35600 | 21 | (oosn[wws m[ [w0o|e aw seoow [ | ooes ]| [oaoss [T7F 223m | 00| 550 1 mmoww[|o_s| ] [2067[F1 6052 TT T00 | 635 | 4100| 15 | C FE s TMM a e r Te | sm T w weoaw[0o1r]] CoesnTv F s es Taso[[ 9%9 sosawwn [[0o2s ]| [Cosm[FrFso me | wmes0 | |30|aswo 05] BQL = Below the queniitation limit (<10 ng/mL) *Concentrationofperfluorooctante in the sample greatly exceeded the highest concentration inthe standard curve; therefore, urine concentration valu may containsignificanteror "Due to technician error, urine was discarded after the volume was obtained. "Concentrationofperfluorooctanaae inthe sample was slightly above the highest concentration in the standard curve; urine concentration value shouldhaveno significant eor. Page 2012 -- 000022 " wanes ---- _ _ge P r e r t r J iesms ss PF Eee ------ : Hil w Beam 1 = 5--" ---- _-- W ; . t . - erm ere------ i - pa Time (day) ibonm EE [FEE asa I me EE ferE EE ---- i EeE--Ea ee = e eee Bn BE d e--rr T rden F len] poms REE eee EYE M-- ne 1] o . a e .Tie a) = - = rcaovemnms Figur1e A Pharmacokinetic Studyof Potassium Perfluorooctanoate in the Cynomolgus Monkey . wae Serum ConcentrationProfilesofPerfluorooctanoate in Monkeys 000023 1s wazrs pr ywy pe ---- Ifee r--_-- -- ---- 1 = _ -- --= VEeet] i EV --1 ~ I ee ~~ ir on . ---- = " --. +-- --- A EE [ -- _ et ------ == TP f wp [ r E --- --- --- -- I ---- E- E P : ofp-- ameE === . PA e -- ee = = BEe= ro = So w Tine (i) 5 pa Fi1g(Cu ontr inue ed) A Pharmacokinetic StudyofPotassium Perfluorooctanoate intheCynomolgus Monkey til SerumConcentrationProfilesofPerfluoroocitnaMnocnaketyes ~~000024  vase 1000 Ess-------- ITE= 5 " F V re - = -F --- + =-- 1 -- Fol _------=TT | a = ---------- E _ ee Re W .T = T u = t T - T pA Time (day) -- Predicted Favor nTwe -p-L E p --ee a L e m tte . e es r =] 3 ee ae rr 5 CO a ----i i iEEl EE| 2 ST ---- -- t oddt= L| olCE Te TT eTTTTTTTTe TT Te TTe Time(day) + oe Pod Figure | (Continued) `A Pharmacokinetic Studyof Potassium PerfluorooctanoateintheCynomolgus Monkey Serum Concentration Profilesof Perfluorooctanoate in Monkeys 000025 Appendix A Study Protocol and Amendments enn 000026 . Study Protocol: A Pharmacokinetic Study of Potassium Perfluorooctanoate in the Cynomolgus Monkey Southern Research Study ID: 9921.4 October4, 2000 | 3 nN SERPS SOUTHERN RESEARCH INSTITUTE 000027 a2 STUDY NO.: 9921.4 October 4, 2000 B ------------------------ 10 SPONSOR REPRESENTATIVE AND CONTACTS: Sponsor: 3M Center, 220-26-02 P.0. Box 33220 St. Paul, Minnesota 55133-3220 Sponsor's Representative & Study Monitor: iPnriottaolcloaslt Apapgperaolvsoa)l: John L. Butenhoff, Ph.D., D.AB.T. 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-3226 (070 (651) 733-1962; FAX: (651) 733-1773 Test Article: gi B 2. ly 12/5 zoos John L. Butenhoff Date Perfluorooctanoate, potassium salt Ship Unused Test Article to: D. Hakes Building B236 3M P.0. Box 33327 + 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 "Rat. 000023 a3 STUDY NO.: 9921.4 _-- 2.0 TITLE: Octo4b, 2e00r0 ean A Pharmacokinetic StudyofPotassium Perfluorooctanoate in the Cynomolgus Monkey 3.0 OBJECTIVE: `The objectives of this study are to determine the concentration of perfluorooctanoate in serum and urine at various times following administrationofa single intravenous dose of potassium perfluorooctanoate to monkeys. 4.0 TESTING LABORATORY: S2o0u0t0hNeirnnthReAsveearnceh SIonsuttithut3e5205 P.O. Box 55305 Birmingham, AL 35255-5305 (205) 581-2335; FAX: (205) 581-2044 5.0 KEY STUDY DATES: [DUaryionefTanrdeFaetcemseCaotllections SerumDrug Levels Draft Report Due Date) -- Year 2000 [03 ~~ -- "TioTw06o0o0 ____-- 7} 2i 1100/1110//0000 7 101600 | 23114 111010//326030/00000 0: o0ur(psrepdoossted)o,se0.5, 2, 4, 8 and 24| 10/--9 10/00 2s 1101130/000 7 10/1600 un 10/20/00 221184 111100//3620/3/0/00000 ws em [| 60 Calendar days after completion of| 1/5/01 the in-life phase received co 000029 Ad STUDY NO.: 9921.4 Octo4b, 2e00r0 ees DURAL 6.0 STUDY PERSONNEL: `The following are the primary contributors and supervisory personnel participating in this study. StudyDirector: ~~ PatriEc.iNaoker Altemate Study Director: James D. Johnson Director, Safety Assessment: Ward R. Richter Associate Director: NomnanD. Jefferson Manager, Bioanalytical Chemistry: James D. Johnson Supervisor, In-Life Laboratories: ~~ Laluana A. Durbin Veterinarian: Darrell E. Hoskins PhD, DAB. MS, MBA DVM, MS.DACVP. BA. MS, MBA. AAS. DVM, ACLAM. (ipl) 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 retumed to the Sponsor. 70 IDENTITY OF THE TEST ARTICLE: Name: Perfluorooctanoate, potassium salt Identification: T-7507 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, as well 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 responsibilityofthe Sponsor. J 000030 as STUDY NO.: 9921.4 --_-- 72 IDENTITY OF THE VEHICLE: October 4, 2000 bweSoti Name: Supplier Lot Number(s): Special Handling: Sterile Saline Commercial supplier To be documented in the study data. None Characterization: Documentationofthe characterizationofthe vehicle may be attained by recording all pertinent information from the container labels, or by retaining the container labels, or copies thereof, in the study data. The vehicle is a commercially available product. Stability & Storage: Sterile saline is considered stable through the date(s) of expiration provided by the manufacturer when stored appropriately. The bulk vehicle will be stored in accordance with the manufacturer's instructions. 73 FORMULATION: Preparation: The test article will be formulated in sterile saline at a concentration of 5 mg/mL for intravenous administration; briefly, the required amount of test article will be mixed with the required amountofsterile saline, and the mixture will be stirred until the test article is visibly in solution. Formulations will be stored refrigerated until used for dosing; formulationsofthe test article in sterile saline are. 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: + SAupgpeloienrD:ayl: Weight at randomization Number on Study: Cynomolgus monkeys (Macacafascicularis) C3-h4aryleeasrsRoifvaergeBR(Fe,stIinmca.te(dH)ouston, TX) 37kg Males -3 Females -3 Animals were previously dosed with potassium perfluorobutanesulfonate in study 9921.1, potassium perfluorobutanoate in study 9921.2, and potassium - perfluorohexanoate in study 9921.3, or 000031 As STUDY NO.: 9921.4 Ocober 4, 2000 _--en bby 81 JUSTIFICATION: Primates are commonly used in preclinical pharmacological and toxicological evaluations of compounds used or intended for use in humans, or to which humans `might be exposed. 82 HousinG: During quarantine/acclimation and study, animals will be individually housed in stainless steel, slat-bottom cages. All animals will be housed in a room that provides aminimumof10 air exchangesper hour. Controls will be set to maintain the animal 100m at a temperature of 64-84 F and a relative humidity of 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 excrement absorption pans, commercial heat-treated hardwood chip bedding will be: . used for excrement absorption. Analysesofthe bedding, supplied by the vendor, will be reviewed by the DepartmentofVeterinary Medicine and Bioresources (DVMB) ofSouthern Research to assure that no known contaminants are present that could interfere with or affect the outcomeofthe study. 84 DET: Diet will be commercial Certified Primate Chow #5048 (PMI Feeds, Inc. St. Louis, MO). The primates will be offered feed twice daily, with approximately the recommended daily ration available at each feeding interval. In addition, the diet will be supplemented with fresh fruit offered daily and treats offered several times each week. The quantity of the daily ration will be sufficient to meet nutritional requirements. Analysesofthe feed, supplied by the vendor, will be reviewed by the DVMB of Souther Research to assure that no known contaminants are present that could affect the healthofthe animals. 85 Water: Water (Birmingham public water supply) will be supplied ad libitum during the quarantine and study periods via an automatic watering system. Samplesofwater from the animal facility will be periodically analyzed, and the analyses will be reviewbeyd the DVMBofSouthemn Research to assure that no known contaminants are present that could affect the healthofthe animals feet, 000032 a7 STUDY NO.: 9921.4 Octo4b, 2e00r0 -- heed 8.6 QUARANTINE: All primates were selected from stock animals that will have been quarantined for a `minimumof35 daysprior 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: a) A complete physical examination including a fecal examination for internal 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 each primate (primates must test negative to all three tests prior to release from quarantine; primates that respond positively to a tuberculin test will `be euthanized immediately, while non-responding primates housed in the same room will be held in quarantine for an additional 90 days); c) the blood sample drawn for CBC will also be used for measuring ofB virus titer (primates found to have a positivBe virus titerwill be euthanized immediately); d) 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 course ofaction); 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. 8.7 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 will be provided cage and 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 proximate to one another for visual and vocal contact. 8.8 ANIMAL IDENTIFICATION: During quarantine, the primates will be individually identified by chest tattoo number or letter combination. Positive identification will be required after every cage change and prior to blood sampling, dose administration, and observation. A 000033 As STUDY NO.: 9921.4 `October 4, 2000 -r ------ N. 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 determine the pharmacokinetics 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 samples for serum drug level determinations will be collected from each primate at selected time points during the study. Urine and feces will also be collected at predetermined intervals. A synopsisofthe study design is presented in the following table. eTEe ee Study Procedures [TTT 0[1[2[3[4567[[89[w0[nliz[B[al2]2s] feamhCheck [x|[| [T TTTT TTTT7 DosePrepanation |x| | 1 [TTTTTT TT 1 1 [Dosing= on i| ex NEITll aT|e ee fe ae cl | [ClinicalObservations] [x[x[x| [x[11x [7T [xT [ TxTx[2} BodyWeighs [x| [Tw[ |= [TTTTxTx|x} [rnedFeces TxITxTx][TTTlTTTTTTT[x (SerumDog levelsTTxTxTx]Tl 1 TT TT Tx T TxTx]x} * Denotes week, 9.1 RANDOMIZATION & GROUP ASSIGNMENT: As only one treatment group will be used in this study, no formal randomization will be required. 92 DOSE PROCEDURE: Each primate (three males, three females) will receive a single intravenous (IV) dose: ofpotassium perfluorooctano(a1te0 mg/kg) by injection into a superficial arm or leg. vein. Doses will be based upon the most recent individual body weights. Doses will be administered at volume of2 mL/kg. The day of dosing will be Day 0 of the study. 9.3 CLINICAL OBSERVATIONS: py Daily Observations: All monkeys will be observed once daily during quarantine and twice daily, morning and aftemoon, at least 4 hours apart, during the study for signsofmortality/moribundity and overt toxicity. Animals found in extremis will 000034 Ao STUDY NO.: 9921.4 October, 2000 _-- hen be humanely sacrificed by an overdoseofbarbiturate 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 daysofblood collection. 94 Boby WeiGHTs: Each primate will be weighed on Days -1, 4, 7, 14,21, and 28. 9.5 URINE AND FECES COLLECTIONS: Urine and feces willbe collected on Days =3, 1 (0-24 hours postdose), 2 (24-48 hours post dose), 7, 14, 21, and 28. The volume of each urine sample will be 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 DRUG LEVELS: 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 will then be centrifuged, and the serum will be separated and stored frozen at ~20 C or below 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 accuracy and should be sensititvoe the 1 ppm or less level. Iffeces and other tissue `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 perflucrooctanoate using the previously validated method. The data will be expressed as equivalents of potassium perfluorooctanoate. 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 000035 Alo STUDY NO.: 9921.4 Octo4b,2e00r0 --_-- Pally decisions concerning possible blood samplingofanimals beyond Day 28 postdose. Feces will be analyzed onlyifrequested by the Sponsor. 9.9 ANIMAL DISPOSITION: Atthe endofthe study, monkeys will be maintained in the stock colony. 100 DATA ANALYSIS: Pharmacokinetic parameters (.g., AUC, half-life, clearance) wil be estimated from serum concentrationsofunchanged perfluorooctanoate, as appropriate and feasible, using a standard `pharmacokinetic program. `The total amountofperfluorooctanoate in urine will be calculated and expressed in terms of percentofdose. 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. 110 RECORDS: All raw data pertaining to the conductofthis study, and all samples/specimens collected in this study, will be stored in the Archives at Southern Research Institute for up to 1 year after acceptanceofthe final report by the Sponsor. After 1 year and with the permissionofthe Sponsor's Monitor, the data and any samples/specimens will be shipped to the Sponsor or 10 the Sponsor's 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 copy of the final report will be retained in the central archives at Southem Research. 120 FINAL REPORT: Abrief letter report summarizing theserumdrug 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 afer receiptofthe Sponsors final review comments on the draft report. The final report for the present study will include, but not necessarily be: limited to the following; Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data. Shae 000036 At STUDY NO.: 9921.4 --_-- 13.0 REGULATORY REFERENCES: October 4, 2000 well "This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs) of Souther Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 PROTOCOL AMENDMENTS AND DEVIATIONS: Amendments: All changes in or revisions ofthe 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 (a fax signature or electronic communication, such as email) for changes in the protocol may be granted by the Sponsor's Monitor, but a written amendment will follow. Deviations: All operations pertaining to this study, unless specifically defined in this protocol, will be performed according to the Standard Operating Procedures (SOPs) of Southem Research and/or the protocol, and any deviations from protocol or SOP wil be documented. 132 REGULATORY COMPLIANCE: Good Laboratory Practices: This nonclinical laboratory study will be conducted inthe spirit of, but wil not require strict compliance with, the U.S. Food and Drug Administration's (FDA) Good Laboratory Practice (GLP) regulations (21 CFR Part 58). Data from this study may be submitted to the FDA in support ofan IND/NDA application. QwiutahliFtDyAA'sssuGrLaPncreegRuelvaitieown:s,Anseitthhiesrstthuedyinw-iilfenaocttibveitceosndnuocrttehdeifnisntarlicrtepcoormtplwiialnbcee 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 Guidelinesfor the Care and Use of Laboratory Animals, 7* Editon (stitute of Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, DC; 1996), and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198). Southern oRfesLeaabrocrhatIonsrtyitAunteimisalfulClayraec(crAeAdiAtLedACb)y.the American Association for Accreditation 000037 a2 1 STUDY NO.: 9921.4 October, 2000 ----r ---- r ------------------ AL 134 ANIMAL WELFARE ACT COMPLIANCE: aBlytesmiagtniivnegs tthoisthperoutsoecoolf, the Sponsor animals, and signifies that that the study there are no generally described by this prot accepted ocol does not unnecessarily duplicate previously conducted or reported experiments. Procedures used in this protocol are designed to conform to accepted practices and to minimize or avoid causing pain, distress, or discomfort in the animals. 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 agents has been justified in writing by the Study Director and/or Sponsor and approved by the IACUC. `The number ofanimals selected for use in this study is considered to be the minimum `number necessary to meet scientific and regulatory guidelines for this typeofstudy. This study design was reviewed by the IACUC at Southern Research Institute and `was approved on 07/26/2000; it was assigned IACUC tracking number 00-07-034. chu 000038 as STUDY NO.: 9921.4 - 140 PROTOCOL APPROVALS: `This protocol has been reviewed and approved. Study Director: ~ Fei Spe Patricia E. Noker, Ph.D., D.AB.T. Study Director Onoberd 200 wlan plufee Date Sponsor's Monitor: 7z& INITIALS ONLY (See page 2) TE Date Management approvals __L/01 1OL4AE soln `Ward R. Richter, M.S., D.V.M,, D.A.CV.P. Date Director, Safety Assessment Department, Southern Research Institute 000039 Appendix B Method Validation Report: ValidationofAnalytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine Using HPLC/MS/MS Rov 000020 B1 METHOD VALIDATION REPORT PERVFALLUIDOARTOIOOCNTOAFNEACNAARNBAOLXYATLIACTAEL MINEMTOHONDKEFYORSEDREUTMERAMNIDNAUTRIINOEN UOSFING HPLC/MS/MS STUDY ID: 9921.42 Southern Research Institute 2000 Ninth Avenue South P.0.-Box 55537 Birmingham, AL 35255-5537 000041 B2 SUMMARY S35o4u8t)heenrtiRtelsedea"rDcehteIrnmstiintautteiohnaosfsuPcecrefslsufourloloyctvaalnicdcaatrebdoxfyorla3teMiannMaonnalkyetyicaSlemreutmhaondd(UBriAneC:G (SaHmPpLlCe/MPSr/epMaSr)a"ti.onCaalnidbrAantailoynsistsabnydaHrdPsLwCerMeapsrsepSapreecdtrboymseptirkyi/nMgassasmSppleecstorfocmietthreyr blank (mPoFnOkCe)y saenrduimntoerrnhaulmsatnanudrairnde, pweirtfhlkunoroowhnexaanmeosuunltfsonoafttees(tPaHrtSi)cl.e,Tpheerfclounocreonotcrtaatnieocnaorfbotxeasltate: farrtoicmle5 i1n0 s5e0r0umngc/omvLe.redQuaanctoimtbatiinoendlrimaintgsewefrreomse1t0atto2100n0,g0/0m0Lnfgo/rmsLerwuhmilaentdhe10urnigne/meLxtfeonrded udreitnee.rmiCnoemtphoesciotreresltaatnidoanrcdocefufrivceiesntgse.nerFaotredthuessienrguimn,tetmhraelestcaonmdpaorsdiqtueanctuirtvaetsiconomwpeorseeudseod ftao t1o0ta0l,o0f0044ngi/nmdLi,vi1du0a0l-ca1l0i,b0r0a0tinogn/smtaLndaanrdds10ex~te5n0d0inngg/ovmeLrpcroondceuncterdatcoirornerlaatnigoenscforeoffmic5i0e0n0t of 0co.n9c8e7n4t,r0a.ti9o7n50raanngde0o.f9597--75r0es0pencgti/vmeLly.andAcosinntgalienicnogmp2o0siintdeivciudruvaelpcraelipbarraetdiofnrsotmaunrdianredsover a produced a correlation coefficientof 0.9960. PRT 000042 James D. Johnson, M.S., MBA' Manager Bioanalytical Chemistry Group Gregory S. Gorman, Ph.D. Staff Chemist Bioanalytical Chemistry Group Lester Williams, B.S. Associate Chemist II Bioanalytical Chemistry Group George Dollar Associate Biologist I Bioanalytical Chemistry Group B3 KEY PERSONNEL 000043 B4 1. OBJECTIVE `The objectiveof this study perfluorooctanecarboxalate was to provide a validated analytical in monkey serum and urine. method for the determination of 2. SAFETY Atlhel nMeacteesrsiaarlySparfoecteyduarnedsDtaoteansSuhreeetssaf(etMySoDfSt)h,eparnoavliysdtesd wbeyre3bMasfeodr otnheintefsotrmaarttiicolne cuosnetdaiinnedthiins study. CAUTION: Since blood, plasma and primates serum, may carry are to be a number of zoonoses, all considered as biohazards unpreserved tissues, and handled with including universal precautions. Refer to SOP number SRI 2-5-5 `when handling unpreserved primate tissue. for a description of safety procedures to be used 3. EXPERIMENTAL 3.1 Analytical Procedures T"DheetesrammipnlaetiporneopfarPaetrifolnuoarnodocatnaanleycsairsbporxoacleadtuereins MaosndkeescyriSbeerdumin atnhde Uarnianley:ticSaalmmpeltehPordepeanrtaittiloend and Analysis employed for by HPLC all analyses. Mass For the pSrpeepcatrraotmioentroyf/MtahsescalSipberacttiroonmesttarnydar(dHs,PLaCk/nMoSw/nMSv)o"lumweeroef ibnltaenrknaslesrtuamndoarrudri(nPeFH(Sc).g..,T5o00eaUcLh)swtaansdasrpdikweadswiatdhdaedkn5o00wnuaLmoofuTntBofAteisotn-apratiircilneg(PsoFlOutCi)ona,nd1 mL for of carbonate/bicarbonate buffer, approximately S seconds. Then and 2.5 1 mL of deionized water. mL of ethyl acetate was This added mixture and the was vortexed mixture was placed on centrifuged a horizontal platform at approximately 2500 shaker at rpm for 5 a low speed minutes. The for | ethyl hour. acetate The layer mixture was then was transferred to `aTsheecroensdiduteubweaasnrdeceovnasptoirtautteedd i1n05dr%ynSemssMatamapmpornoixiummataecleyta5te0inCduenidoenrizaedstwraetaemrof: d95r%y mniettrhoagneno.l containing 1.5% formic acid and filtered through a 0.2 pm syringe filter. 3.2 Method Validation Validation for Urine: Sample BACG 3548 Preparation "Determinationof Perfluorooctanecarboxalate in and Analysis by HPLC Mass Spectrometry/Mass Monkey Serum Spectrometry and (HPLC/MS/MS)" consisted of analyzing standard curves prepared in monkey serum of human Cyr 000044 Bs urine over various concentration ranges: 5,000 ~ 100,000 ng/mL, 10--0 10,000 ng/mL and 10 500 ng/mL for serum and -- 500 ng/mL for urine. 51 .0000 - 0, ng/0 mL(0 Ser0 um) One composite calibration curve comprising 3 sets ofcalibration standards in a single analytical run in the 5,000 -- 100,000 ng/mL concentration range generated from a total of 22 calibration standards produced a correlation coefficient of 0.9874. A statistical summary for the composite curve is shown below: STANDARDCOMPOSITECURVE 1(ng/mL) FCoonfcS.id(sng/mL) 3 121273[5a]3] 5000 10,000 20,000 25,000 50,000 75,000 100,000 [Mean%Accur.| 93.5 |T02|9181.973| 1[309177||1200116[953| 102| 11 00-0.ng0 /mL0 (Ser0 um) `Two composite curves consisting of atotalof 12 calibration standards encompassing a concentration range of 100 -- 10,00 ng/mL produced correlation coefficient of 0.9750 The 5,000 ng/mL standards weredroppeddue to preparation error. Astatistical summary for the composite curve is given below: STANDARDCOMPOSITECURVE2(ng/ml) [fCoofncS.e(nsg/mL)[2 100 [2 200 15020 11000222000 210,]000 [(MSeda.nDe%viAactciuonr.|| 1943..55 ||3120.91|[110164||9134.87||380671|| 180522|] 10-500ng/mL(Serum) A single composite curve consisting of 11 standards taken from an analytical run encompassing is given below a concentration range of 10 -- 500 ng/mL producedacorrelation coefficientof 0.9977. No calibration standards were dropped from the run. Astatistical summary for the composite curve. | 000045 Be STANDARD COMPOSITECURVE 3(ng/mL) [[MfCeooanfcn.S%(inAsgc/cmLa)r[._| 926:160[|120200[] T25o0r 7|1102060|1290030% |21500]0] [Ste Deviation | 051 |097 | 104 |208 |-- | 381] 5-500ng/mL(Urine) A composite curve prepared in urine consisting of 20 standards over a range of5 -- 500 ng/mL. produced a correlation coefficient of 0.9960. Only one calibration standard, 5 ng/mL was dropped due to low intemal standard response. A statistical summary for the composite curve is given below: STANDARD COMPOSITE CURVE 4 (ng/mL) [MCFeoonafcnS.%i(snAgc/emaL[ r). |29457[|130170[|130270 [| 35207[| 1130007[|1230010 39590]06 | [SiDeviation | 007 |027[3.01 |420| &2 | 90|Tac] 3.3 Calculations Calculations were performed using TurboQuan (Version 1.0). The amount of analyte in the serum extracts (ng/mL) was back calculated using a calibration curve genrated from a set of calibration standards. The calibration curve was generated by a regression analysis to determine the best fit curve (e.g, linear, quadratic etc.) and amount of weighting. A quadratic fit with 1/X `weighting was determined to be the best fit : y=axi+ bx +c where: y= Peak height response of PFOC divided by peak height responseofthe 1S (PFHS) in standards. x= Concentration of the PFOC in standards. a,b. =Constants derived from the egresion analysis NOS 000046 B7 4.0 Conclusion A quantitative method (BAGC 3548) has been developed and validated for the determination of periluorooctanecarboxalate in monkey serum utilizing LC/MS/MS. Quantitation for this method is based on an intemal standard (PFHS) using a 1/x weighted quadratic equation. The total concentration range for a serum matrix is 20 ~ 100,000 ng/mL and 5 ~ 500 ng/mL for urine with limitsofquantitation of 20 and 10 ng/mL respectively. we 000047 Appendix C Analytical Method for Determination of Perfluorooctanoate in Monkey Serum and Urine pu 000048 cl Page 1 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: DPreetpearrmaitniaotnionanodf PAenrafllyuosriosocbtyancHcaPrLboCxalMaatsesin SMpoenckteroymSeetrryu/mMaansds USrpienec:trSoammeptrlye (HPLC/MS/MS) --_--_--mm--e 10 PRINCIPLE SPeerrfulmuorooroctuarnicncearsbaomxpalleaste a(rPeFOoCb)ta.inTedhefsreormumcyornoumroilnegus(e.mg.o,n0k.e5ysmLt)recaotnetdaiwniitnhg (`TPhFeOsCa)mpilsefsoratriefietdhewnitmhixaendinwtietrhnaalnstiaonn-dpaaridri(n1Sg),repaegrefnltu,orbuofbfeexranaensdulwfaotneart,e f(oPlFlHoSw)e.d dbryyneexstsr,actriecoonnwstiitthuteetdhyilna9ce5ta%te.metThhaenoelthcyolntaaceitnaitneg l1a.y5er%is froermmoivceda,cide,va5po%ratSedmtMo aHmPmLoCnMiausmsaScpeetactter,omfeilttreyre/dM,asasndSptercatnrsofmererterdy t(oHaPuLtCo/sMamSp/lMeSr).viaTlhs,e arnadngaenoaflyrzeelidabblye rnegs/ulmtLs eixnteunrdinse.froSmaambpoluets20contota1i0n0i,n0g00PFngO/CmLatocfoPncFeOntCraitnisonesrugmreaantderfrthoamn1100t0o,500000 ng/mL will be may be diluted with control within the range of reliable blank results matrix prior to so that the analysis. concentration of PFOC `The `The mass spectrometry ion spray source of PFOC voltage is and set PFHS is accomplished at - 2000 volts which in is the negative low enough ion to mode. greatly Irnedourcdeerthteo fmoarxmiamtiiozne osfenostihteivritpyo,tetnhtiisalmleytihnotderfiserbiansgeidoonsn emxitxraecdterdeafcrtoiomntmhoenmiattorrixi.ng of the negative fragment ion (M-COOH) for PFOC. CinAcUluTdIinOgNb:loSoidn,ceplpraismmaateasndmasyercuamr,ryaraentuombbeercoonfsziodoenroesdesas, balilohuanzparredsseravnedd htaisnsduleesd, `with universal precautions. safety procedures to be used Refer to SOP number SRI 2-5-5 for a description when handling unpreserved primate tissue. of 20 REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 21 Neat Reagents " 000049 c2 Page 2 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Tide: Determination of Perfluorooctanecarbosalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) --_-- 2.11 Water, deionized and organic free (from in-house purification system; e.g. , Ingalls 210N) 212 Methanol, HPLC grade 213 Perfluorooctanccarboxalate (analyte), as provided by the client 214 Perfluorohexanesulfonate (internal standard), 97% 215 Ammonium acetate, HPLC grade 21.6 Blank control monkey serum 21.7 Sodium Carbonate, Certified ACS Grade or equivalent 21.8 Sodium Bicarbonate, Certified ACS Grade or equivalent 219 Ethyl Acetate, HPLC grade 21.10 Tetrabutylammonium Hydrogen Sulfate 2.111 Sodium Hydroxide 50% solution, Certified grade or equivalent 2112 Blank control monkey urine 21.13 Formic Acid 22 Prepared Solutions Appropriate changes in the solutions may be made at the discretionofthe analyst 221 SmM Ammonium acetate in organic free water apy 000050 c3 Page 3 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 22.1.1 222 222.1 223 223.1 3.0 31 32 33 34 35 36 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g, 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydroxide) For example, to prepare 25 mL, dissolve 4.24 g of tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. Note: a more dilute solution of NaOH in water may be used to effect smaller pH adjustments. Carbonate/Bicarbonate Buffer Solution For example, to prepare 100 mL, dissolve approximately 2.65 g of sodium carbonate `and approximately 2.10 g of sodium bicarbonate in 100 mLofdeionized water. Mix well to ensure complete dissolution. INSTRUMENTS, MATERIALS, AND APPARATUS `The following or their equivalents may be used. HPLC pump(s), autosampler, and triple quadrupole mass spectrometer Autosampler vials with inserts Vortex mixers (e.g., touch mixer and IKA-Vibrax platform mixer) Solvent-concentration apparatus (e.g. Zymark Turbo-Vap with source of nitrogen) HPLC mobile phase filtration apparatus Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 um) Lo 000051 ca Page d of 13 ANALYTICAL METHOD Method No.: BACG-3548 Tide: DPreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbaynecHaPrbLoCxalMaatessin SMpoenckteryomSeetrryu/mMaansds USrpience:trSoammeptlrey HPLCMSIMS) 37 38 39 310 311 312 303 314 315 316 317 40 41 411 Zenbn Aualytical balance Volumetric flasks (c.g., 10 and 25 mL) Disposable Pasteur pipets Micropipettor(s) with tips Culture tubes with teflon-lined caps Ceaifuge Assorted glassware and syringes Culture tubes (vials) for use with solvent-concentration apparatus 1mL Plastic syringes with 0.2 um PVDF syringe fillers Variable speed horizontal platform shaker pHmeer PREPARATION OF STOCKS AND WORKING STOCKS Adipspcrroeptriioanteof changes in the analyst. the concentrations Actual dilutions of wil the be solutions may be made at the documented on the preparation sheets Main Stock Solution of PROC ~1000 pg/mL Pacrceupraarteelaynwe~ig1h00a0bogut/1m0LmsgolPutFiOonCoifntPoFaO1C0-inmLdevioolnuimzeetdriocrgfalnasikc)-.freAedwdatdeerio(nci.zge.d, organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound 000052 cs ANALYTICAL METHOD Page Sof 13 Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MSIMS) into an appropriate vessel (e.g., culture tube) and add 10 mL of deionized organic-free `water. Mix well. Transfer the solution to a clean vessel if desired. 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 (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organic- free water to dissolve and dilute to the mark with deionized organic-free water. Alternatively, weigh the compound into a an appropriate vessel (.g., culture tube) and : add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. 43 Spiking solutionof Internal Standard (PFHS), ~ S0xg/mL 43.1 Preparane ~ 50 ug/mL solution of PFHS in deionized organic- free water by pipetting 2 mL of the 200 pg/mL solution into a culture tube and add 6 mL of deionized water. Mix well. 4.4 `Working Stock Solutions of PFOC 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 documentedin the study records. Working Stock Level Approximat(eWSCLo)ncentration (ng/mL) 250,000 Volume of PFOC solution dFeiinoanliVzeodluormgeaniinc free water (mL) smbotioougm | 10 SmLofs00000ngml | 10 | 000053 cs Page 6 of 13 ANALYTICAL METHOD Method No. BACG-3548 Tite: DPreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbtyaneHcaPrLboCxalMaatsesin Monkey Serum and Spectrometry/Mass Urine: Sample Spectrometry (HPLCIMS/MS) J -- 62,500 25mLof25000ngmL| 10 | 31,250 5mLof62,500 ng/mL 15,625 smSemLraoif3ro12s5gg0nngs emL ||0 e10] ]| 0 iw TTeommuummomwamrmsann]|0 w] ] smiorsoongmisock [10| 442 Summary of concentrations of serum standards: Standard Volume and Spike Conc. |_ Approximate Concentration of Level PROC in serum (ng/mL) 0 TBiamaroTrmoanm|so] 10 Lo 1000 ym aah 000054 ANALYTICAL METHOD Page 7 of 13 Method No.: BACG-3548 `Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine:Sample mee. Preparation and (HPLC/MS/MS) Analysis by HPLC Mass Spectrometry/Mass Spectrometry poem ] [Gro aAm nem wstoarssovnmm||o 0w ]| Tn Toptooonmngmma|so|] To [warmer |0] 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst 5.1 Multiple (e.g., about three) sets of matrix standards and a matrix blank (blank + IS) are analyzed with each setof unknown samples. A matrix double blank (blank-1S) may also be analyzedif desired. 5.2 Into individual ~20-mL culture tubes, pipet blank matrix (e. ., 0.5 mL). Pipet in the `appropriate volume as described in the table above for each standard. For the blanks, pipet 10 uL. of organic-free water instead of the working stock solution. Add the 10 uL. 000055 cs Page 8 of 13 ANALYTICAL METHOD Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectromeiry/Mass Spectrometry (HPLC/MS/MS) --_--_---- ee of internal standard stock (~ 50 pg/mL) to each tube except the blank-IS (pipet 10 pL of organic-free water instead) and vortex for ~5 seconds. 53 To cach whe add 500 uL 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. 54 Add 2.5mLof ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 55 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes 56 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., ~ 50 minutes in the Turbo-Vap ) witha gentle stream of nitrogen and `moderate heat (e.g., 50C). | 57 Reconstitute the residue in 500 uL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 um PVDF or Nylon syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipe an aliquotofeach sample (e.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 L of internal standard stock (~ 50 g/mL) to each tube and vortex for a couple of seconds. 62 To each tube add S00 uL of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex cach tube for about 5 seconds as 00056 co Page 9 of 13 ANALYTICAL METHOD Method No. BACG-3548 Title: _-- Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 63 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting, 6.4 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 65 Take off the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g. ~ 50 minutes in the Turbo-Vap ) with a gentle stream of nitrogen and `moderate heat (e.g., 50C). 66 Reconstitute the residue in 500 uL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 wm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. 70 ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) 7.1 Conditions are to be optimizedifnecessary. 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 Keystone Aquasil C18 10 mm x 2 mm 400 L/min. 3ul A: SmM ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: 0-0.5min. SO%A: SO%B 0.5-7.5 min. 10% A : 90% B linear gradient 7.58min. 10%A : 90% B step gradient | 000057 co ANALYTICAL METHOD Page 10 of 13 Method No.: BACG-3548 `Title: PDreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbtyaneHcaPrLboCxalMaatsesin SMpoenckteryomSeetrryu/mMaasnsd USrpineec:trSoammeptlrey (HPLC/MS/MS) 7.12 pn `Temperature: 8-11 min. Ambient 50%A : S0%B PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan Turhoion Spray Source Note: Values listed under "MS/MS Acquisition Conditions" override parameters in this table. . Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch `Parameter Value BN amo xe o En a or 0 me m0 s EY . st 0 sot ` @ so 0 sm w ror 2 or 0 can TM 000058 cn Method No.: Tide: _-- Page 11 of 13 ANALYTICAL METHOD BACG-3548 Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and (HPLC/MS/MS) Analysis by HPLC Mass Spectrometry/Mass Spectrometry Pacameter Value xen is ar ` ` a s are o rou ' vou o ve o MS/MSAcquisition Conditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time 5 milliseconds Masses requested: QparsMoc: ay bQeuam pL55r8Ms9 a5)aay QEunesoo sbImoc s5a =@Sw wo 92 D0uel Tine (my DEegtTine me 000059 . cn ANALYTICAL METHOD Page 12 of 13 Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.0 CALCULATIONS 8.1 At the end of the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and tthhee IiSonarpreoafcicleepstaatblteh.e Tfohlelodwaitnagmmaayssb-etos-mcohoatrhgeedraatsioasp:propriate. For quantitation, use Analyte PFOC PFHS lonProfile 412.910 368.9 398.910398.9 82 Plot the peak area response of PFOC divided by the peak area response of the IS (PFHS) from all standards versus the concentration of the test article in the standards. Alternatively, the peak heights may be used insteadofpeak areas. Obtain the best curve fit of the data (e.g., quadratic fit weighted with 1/concentration ofthe test article or a quadratic fit). Note: The best curve fit may be dependent on the rangeofthe standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: y=ad+bx+c where y= x= a,b,c Peak height response of PFOC divided by peak height response of the IS (PFHS) in standards. Concentration of the PFOC in standards. = Constants derived from the regression analysis. 83 Using the standard curve, calculate the level of PFHC in each unknown sample. Correct the results of samples for any dilutions. ; 00006Y on ANALYTICAL METHOD Page 13 of 13 Method No.: BACG-3548 Title: Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine:Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry TY(HPLC/MS/MS) ae erer------e NOTE: Due to unresolvable interferences with the test article and/or internal standard from the matrix, external standard quantitation may be used at the discretionofthe supervising mass spectrometrist. 9.0 9.1 10.0 10.1 ACCEPTANCE AND REJECTION CRITERIA Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-120%of theoretical. REPORTING Results of all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Authors: Didfn> Greg6ry.Gorman, Ph.D., StaffChemist Bioanalytical Chemistry Group ALnjoo Date Approved by: {es D. Johnsdn MS, MBA, Manager Bioanalytical `mistry Group _fa-azes Date 180 ON NIVANGD 000061