Document qd0g5KZz9B8V676MQ7YgB6EpG

A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Southern Research Institute Study ID: 9921.5 April 22,2003 Final Report on A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey To: 3M Corporation P.O. BOX33327 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 ABSTRACT The pharmacokineticsand urinary excretion of perfluorohexanesulfonatewere investigated in male and female cynomolgus monkeys. Three male and three female monkeys were administered a single iv bolus dose of 10mgkg of perfluorohexanesulfonate,potassium salt (T-7504). At various times after dosing, serum and urine (24-hour collections) samples were obtained and analyzed by HPLC/MS/MS for levels of intact perfluorohexanesulfonate. The lower limit of detection of the analytical method was 5 ng/mL for serum samples and 1 ng/mL for urine samples. Peak serum concentrations of perfluorohexanesulfonate were similar in male and female monkeys and ranged from 104,600 to 140,900ng/mL in male monkeys and from 116,400 to 174,000ng/mL in female monkeys. Serum concentrations of perfluorohexanesulfonate in each monkey decreased relatively rapidly during the first 8-24 hours after dosing. At 24 hours, serum concentrations of perfluorohexanesulfonate ranged fkom 24,870 to 40,405 ng/mL in male monkeys and fkom 27,115 to 54,300 ng/mL in female monkeys. Subsequently, serum concentrations of perfluorohexanesulfonate decreased at a slower rate between 24 hours and the end of sample collection (171 days). On Day 171,serum concentrations of perfluorohexanesulfonateranged fiom 13,415to 21,725 ng/mL in male monkeys and from 3,249 to 20,220 ngmL in female monkeys. The serum concentration versus time data were subjected to non-compartmental phannacokinetic analysis. The serum elimination half-life of perfluorohexanesulfonateranged from 100to 200 days (mean: 141 days) in male monkeys and from 49 to 140 days (mean: 87 days) in female monkeys. The total body clearance of perfluorohexanesulfonatewas 1.1 to 1.5 mL/day/kg in male monkeys and 1.2 to 2.6 mL/day/kg in female monkeys. The volume of distribution of perfluorohexanesulfonate ranged from 223 to 391 mLkg and from 160 to 255 mLkg in male and female monkeys, respectively. Apparent differences among individual monkeys in the estimated serum half-life and clearance of the compound were likely attributable to an overestimation of the extrapolated AUC,M,, values for two of the female monkeys. Only very low levels (<0.01 to 0.11% of the administereddose) of perfluorohexanesulfonatewere measured in urine during any given 24hour period of sample collection between Day 1 and Day 70 after dosing. The results of this study suggested that the phannacokinetics of perfluorohexanesulfonatewere similar in male and female monkeys. Perfluorohexanesulfonatewas eliminated in urine by both male and female monkeys at low levels for a prolonged period of time (270 days) after iv administration. i TABLE OF CONTENTS ... SIGNATURE PAGE 111 GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL V 1.0 INTRODUCTION 1 2.0 MATERIALS AND METHODS 1 2.1 Test System 1 2.2 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 4 Serum Levels of Perfluorohexanesulfonate 4 Bioanalytical Method Development and Sample Analysis 4 Data Analyses 4 3.0 RESULTS 3.1 Mortality 3.2 Clinical Observations 3.3 Body Weights 3.4 Serum and Urine Concentrations of Perfluorohexanesulfonate 4.0 DISCUSSION 5.0 CONCLUSIONS 7 6.0 RECORD ARCHIVES 8 7.0 REFERENCES 8 11 TABLE OF CONTENTS (Continued) LIST OF TABLES Table 1: Table 2: Table 3: Table 4: Figure 1: Appendix A: Appendix B: Body Weights Serum Concentrations of Perfluorohexanesulfonate Pharmacolunetic Parameters Calculated from Serum Concentrations of Potassium Perfluorohexanesulfonate Urinary Excretion of Perfluorohexanesulfonate LIST OF FIGURES Serum Concentration Profile of Perfluorohexanesulfonate LIST OF APPENDICES Study Protocol Analytical Method for Determination of Perfluorohexanesulfonate in Monkey Serum and Urine Pape 9 13 14 15 18 A- 1 B- 1 ... 111 Signature Page A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey , &+7/ F ,%&J Patricia E. Noker, Ph.D., D.A.B.T. Study Director Supervisor,ADME & Pharmacokinetics Reviewed by: d / / 2 2 /*3 Date Charles D. Hkbert, Ph.D., D.A.B.T. Date Director, Safety Assessment We, the undersigned, were responsible for the conduct of the work and reporting of the results in the listed sections. We concur with the views relative to our body of work as expressed in the discussion and conclusions. lreg'%dg Gyman, Ph.D. Date Manager, Bioanalytical Chemistry Group iv Good Laboratory Practices Disclaimer This study described in this final report was not conducted in strict compliance with the 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 Southern Research Quality Assurance Unit. However, the study was conducted according to 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 standard of GLPs. The final report accurately reflects the raw data obtained during the performance of the study. There were no adverse circumstances that affected the quality or integrity of the study. Patricia E. Noker, Ph.D., D.A.B.T. Study Director 5g.z/os Date Study Dates: V Study Schedule and Personnel Study Initiation: Day of Dosing: Last Day of Sample Collection: Study Completion: February 7,2001 February 9,2001 July 29,2001 April 22,2003 Study Personnel: Patricia E. Noker, Ph.D., D.A.B.T. Study Director Charles D. Hkbert, Ph.D., D.A.B.T. Director, Safety Assessment Norman D. Jefferson, B.A. Associate Director, Safety Assessment Gregory S. Gorman, Ph.D. Manager, Bioanalytical Chemistry Group Darrell E. Hosluns, D.V.M., Ph.D., A.C.L.A.M. (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 1 1.O Introduction The objectives of this study were to determine the concentration of potassium perfluorohexanesulfonate in serum and to estimate urinary clearance at various times following administration of a single intravenous dose to monkeys. A copy of the protocol and any applicable amendments can be found in Appendix A. 2.0 Materials and Methods 2.1 Test System The three male and three female cynomolgus monkeys designated for use in this study were selected from an in-house colony of monkeys that were housed at Southern Research Institute (Southern Research) prior to use on this study. These monkeys were purchased from Charles River B W , 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 of drugs used or intended for use in humans. During the quarantine period, a complete physical examination including a fecal examination for internal 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 Shigellu) 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. 2 Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed to the monkeys 2-3 times each day. The quantity of the daily ration was sufficient to meet nutritional requirements. In addition, the diet was supplemented with fresh hithreats 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 0 to the end of the study, the monkeys were housed in a room that was maintained at a temperature of 67.7-72.8 OF and a relative humidity of 29-74%. The humidity was within the required range (30-70%) over 90% of the time during the study; excursions below the recommended humidity range were of short duration and had no impact on animal health or the outcome of the study. Room lights were controlled by an automatic timer set to provide 12 hours of light (0600 to 1800 hours, CST) and 12 hours of dark per day. Cage size and animal care conformed to the guidelines of the Guidefor the Care and Use of Laboratory Animals, 7th 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 Southern Research. The study design was approved by Southern Research's Institute Animal Care and Use Committee. Southern Research is fully accredited by the American Association for Accreditation of Laboratory Animal Care International. With the exception of animal 2053, all of the monkeys used on t h s study were previously given a single iv bolus dose of perfluorobutanesulfonate (10 mgkg) on 4/1O/OO (Southern Research Study No. 9921.1); a single iv bolus dose of potassium perfluorobutanoate (10 mgkg) on 6/13/00 (Southern Research Study No. 9921.2); a single iv bolus dose of potassium perfluorohexanoate(10 mgkg) on 7/31/00 (Southern Research Study No. 9921.3); and a single iv bolus dose of potassium perfluorooctanoate (10 mgkg) on 10/9/00(Southern Research Study No. 9921.4). Monkey 2053 was nayve. 2.2 Test Article and Vehicle Test Article: One bottle containing 5 grams of potassium perfluorohexanesulfonate (T-7504; expiration date not supplied; SRI E06/L-1) was supplied by 3M (St. Paul, MN) and received 3 on May 15,2000. The test article was stored at room temperature until used. Stability of the test article was the responsibility of the Sponsor. Vehicle: The vehicle used for the preparation of the dose formulation of potassium perfluorohexanesulfonatewas sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 0081050, expiration date August 2003). 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 perfluorohexanesulfonate prepared at 5 mg/mL, the required amount of test article was weighed out in a volumetric flask. Sterile saline was added and the formulation was stirred until in solution. The formulation was stored refigerated and used for dosing within 1 day 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: As only one treatment group was used in t h s study, no formal randomization was required. On Day 0, each of the three male and three female monkeys received a single intravenous (iv) dose of perfluorohexanesulfonate at 10 mgkg by injection into a superficial arm or leg vein. Doses were based upon the Day -1 individual body weights. Doses were administered at a volume of 2 mL/kg. Clinical Observations: All animals were observed twice daily for signs of mortality/moribundity. Each primate was examined shortly after dose administration for clinical signs of toxicity. Additional clinical observationswere performed on days of blood collection. Body Weights: Each primate was weighed on Days -1,4,7, 14,21,28,35,42,49,56,63, 70, 77,84, 91,98, 105, 112, and 119. 4 Urine and Feces Collection: Urine and feces were collected for 24-hour intervals on the following days: prior to dose administration (Day -1; baseline), on Day 1 (0-24 hours postdose), on Day 2 (24-48 hours postdose), and on Days 7,14,21,28,42,56, and 70. 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 Levels of Perfluorohexanesulfonate: Blood samples (approximately 3 mL) were collected from each primate at approximately 0 (predose) minutes; 0.5, 2, 4, 8, 24 and 48 hours; and on Days 4,7, 14,21,28,42, 56,70, and 171postdose. Samples were collected into tubes without anticoagulant and were allowed to clot at room temperature. The blood samples were then centrifuged, and the serum separated and stored fiozen (approximately -20 "C or below) until analyzed. BioanalyticalMethod Development and Sample Analysis: Serum and urine sampleswere analyzed for perfluorohexanesulfonateusing a previously validated HPLCMSMS method (Appendix B). Data Analyses: The serum concentrationdata for unchanged perfluorohexanesulfonatewere subjected to non-compartmental phannacokinetic analysis using WinNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc.; Cary, NC). Mean values and standard deviations for each parameter were calculated using Microsoft Excel@ Software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorohexanesulfonate at each collection interval was calculated and expressed as a percent of the administered dose. No other statistical analyses of the data were performed. 5 3.0 Results 3.1 Mortality All of the monkeys in this study survived to the end of the study. 3.2 Clinical Observations No adverse drug-related clinical signs were noted for any monkey during the course of this study. 3.3 Body Weights Body weights are presented in Table 1. Each monkey either gained weight or maintained essentially a constant weight between Day -1 and Day 119 (last day during the study that body weights were obtained). 3.4 Serum and Urine Concentrations of Perfluorohexanesulfonate Serum concentrations of perfluorohexanesulfonatein three male and three female monkeys at various times through Day 171 after administration of a single iv dose of 10 mg/kg are presented in Table 2 and Figure 1. No sex-related differences were apparent in serum concentrationsof perfluorohexanesulfonate at any time of sample collection. Peak serum concentrations of perfluorohexanesulfonate were observed in 1/3 male monkeys and 3/3 female monkeys at 0.5 hours (earliest time point) after dosing. Serum concentrations of perfluorohexanesulfonate at t h s time ranged from 116,400to 140,900 ng/mL among these four monkeys. For the other 2 male monkeys (2053 and 221l), peak serum concentrations of perfluorohexanesulfonate(104,600 and 128,500ng/mL) were not observed until 2 hours after dosing. The possibility was investigated that the 0.5- and 2-hour serum samples collected fkom these two monkeys were switched during collection andor analysis; however, it could not be determined if the switching of the samples had occurred. Subsequent to the time of peak levels, serum concentrations of perfluorohexanesulfonate in each monkey decreased relatively rapidly during the first 8-24 hours after dosing. At 24 hours, serum concentrations of perfluorohexanesulfonate ranged fkom 24,870 to 40,405 ng/mL in the three male monkeys and from 27,115 to 54,300 ng/mL in the three female monkeys. Serum 6 concentrations of perfluorohexanesulfonate decreased at a slow rate between 24 hours and the end of sample collection (171 days). During this period, fluctuationswere observed in the serum concentrations of perfluorohexanesulfonate among individual monkeys. In that the fluctuations appeared to be random among the monkeys, they may have been attributable to analytical error introduced as a consequence of the large dilution of each sample that was required prior to HPLC/MS/MS analysis. On Day 171, serum concentrations of perfluorohexanesulfonate ranged from 13,415 to 21,725 ng/mL in the male monkeys and from 3,249 to 20,220 ng/mL in the female monkeys. Pharmacokinetic parameters calculated from serum concentrations of perfluorohexanesulfonatein individual monkeys are presented in Table 3. The values were derived from non-compartmental analysis of the data. No distinct differenceswere apparent between male and female monkeys in the estimated parameters; however, for two of the three female monkeys, the serum half-life and clearance of perfluorohexanesulfonatewere shorter and faster, respectively, than observed for the three male monkeys and the other female monkey. AUCO-infvinailtuyes ranged from 6456 to 8745 pg.day/mL in male monkeys and from 3849 to 8501 ,ug*day/mL in female monkeys. The terminal half-life of perfluorohexanesulfonate in serum ranged from 100 to 200 days (mean: 141 days) in the three male monkeys and fiom 49 to 140 days (mean: 87 days) in the three female monkeys. The total body clearance of perfluorohexanesulfonatewas 1.1 to 1.5 mL/day/kg in male monkeys and 1.2 to 2.6 mL/day/kg in female monkeys. The volume of distribution of perfluorohexanesulfonate ranged from 223 to 391 mL/kg in male monkeys and from 160 to 255 mL/kg in female monkeys. The amount of perfluorohexanesulfonate eliminated in urine by individual monkeys at various times after dosing is presented in Table 4. Only very low levels (<0.01 to 0.11% of the administered dose) of perfluorohexanesulfonate were measured in urine during any given 24-hour period of sample collection between Day 1 and Day 70 after dosing. There was no clear indication of a sex-related difference in the urinary excretion of perfluorohexanesulfonate. The urinary excretion of perfluorohexanesulfonate was 7 prolonged; detectable levels of the compound were present in urine on Day 70 (last day of urine collection) after dosing. 4.0 Discussion The results of this study indicated that perfluorohexanesulfonatewas slowly eliminated by male and female monkeys given a single iv dose of 10 mgkg. Throughout the period of sample collection, serum concentrations of perfluorohexanesulfonatewere similar at each sample time in both male and female monkeys. In addition, the rate of urinary excretion of perfluorohexanesulfonate appeared to be similar for male and female monkeys. Thus, there was no clear indication &om the data that there was a sex-related difference in the elimination of the compound. For two of the three female monkeys on the current study, the estimated serum half-life and total body clearance of perfluorohexanesulfonate appeared to be shorter and faster, respectively, than observed for the thrd female money and for the three male monkeys. These apparent differences may have been artifactual in that both parameters (half-life and clearance) were calculated from AUCo-&itityvalues; these latter values were obtained from extrapolation of the serum concentration time curve and may have contained considerable error. That the AUCO-infinviatlyues for two of the three female monkeys may have been overestimated is supported by the observation that estimated AUCo.lasvtalues [0 to the last time point (Day 171)] for all three male and all three female monkeys were similar. 5.0 Conclusions No sex differences were apparent in serum concentrations and the urinary excretion of perfluorohexanesulfonate among male and female monkeys given an iv dose. The mean terminal serum half-life of perfluorohexanesulfonatewas 141 days in male monkeys and 87 days in female monkeys. Perfluorohexanesulfonate was eliminated in urine by both male and female monkeys at low levels for a prolonged period of time (270 days) after iv administration. 8 6.0 Record Archives Data, specimens, and a copy of the final report from this study will be stored in the Archives at Southern Research for up to 1 year after acceptance of the final report by the Sponsor. After 1 year and with the permission of the Sponsor's Monitor, the data and any sampleshpecimens will be shipped to the Sponsor or to the Sponsor's designated archival facility. If materials are to be retained in the archives beyond t h s 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 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. 9 . . . . . I ~m I w d r o ~ r nI m I I w w w worn I I I * . . 0 1 I ul O P u l N c r I I w w m I worn I I I I N cr m. u. m. wwm I . . . . . I I c m I m N W P dcr I rn I I w w m worn I I I . . . . . r n m I m d W P dcr I N I w w m worn I I . . . . . I P m I d rlmw 0 1 I N I I w w m worn I I I I . . . . . r N I cr c r w m N c r I d I w w m worn I I I . . . . . 0 - w 00 I 0- I r I Q+ w w m w o r n II 0 . . . . . I o w I cr r l - m I ocr II w w m worn I I I . . . . . I P N I d O N * m- I I I I wwm morn I I I j m i r l I I mmrl I OON I NNN I EfflZ I a g$ eh f f l Table 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Males Group Sex 1M Animal Number 63 2053 2054 2211 Mean S.D. N ____-- 6.3 6.8 _ _ 6_ ._ 2_ _ 6.43 0.32 3 70 6.4 7.0 6.3 ___--_ 6.57 0.38 3 D a y numbers r e l a t i v e t o S t a r t D a t e 77 6.5 6.9 _ _ 6_ ._ 2_ _ 6.53 0.35 3 84 6.5 6.9 6.7 ______ 6.70 0.20 3 91 98 6.7 6.9 6.5 ____-_ 6.70 0.20 3 6.4 7.0 _ _ 6_ ._ 5_ _ 6.63 0.32 3 105 6.5 6.7 _ _ 6_ ._ 4_ _ 6.53 0.15 3 112 6.6 6.5 6.5 ---___ 6.53 0.06 3 119 6.6 6.5 _ _ 6_ ._ 7_ _ 6.60 0.10 3 Page 2 of 4 11 m . . . , I ~ N I rU)Nlam I I.. mmq Imom I I I I I , r w *. irm I m ~ a mI .I.. mm-a I m o m I I I I I . . .irm I rrNlari I I.. I mm-a I m o m I I I I I . . .I ~ N I wmrllrm I I.. I mm-a I m o m I I I I w m r l Il r m~ N I I ...I.. 1 mm-a I m o m I I I I I . . . , I ~ N I mwrlirm I I.. mm-a I m o m I I I I I . . . . . , , ~ r mI a a m ~ m NI mm-a I m o m I I I I I . . .irrl I PWOI~N I I.. I mmq Imom I I I I I . . . IOU) I ~ w r l i mI ~ I.. I mm-a I m o m I I I I I . . . IOU) I U ) P , + I ~ NI I.. 1 mm-a I m o m I I I I I a rl Table 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Females Group Sex 1F Animal Number 2058 2059 2061 ---___ Mean S.D. N 63 3.8 3.6 _4_.2_ _ - 3.87 0.31 3 70 3.9 3.7 4.3 -___- 3.97 0.31 3 Day numbers relative to Start Date 77 3.7 3.6 4.4 ____- 3.90 0.44 3 84 3.7 3.7 4.4 --__3.93 0.40 3 91 3.8 3.6 4.3 ___-- 3.90 0.36 3 98 3.9 3.6 4.3 ______ 3.93 0.35 3 ______ 105 3.7 3.6 4.4 ______ 3.90 0.44 3 ______ 112 119 3.6 3.6 4.4 ___--- 3.87 0.46 3 ____-- 3.6 3.5 4.3 ___--- 3.80 0.44 3 ___--- Page 4 of 4 13 Table 2 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey cSerum Concentrations of Perfluorohexanesulfonate Timepoin Serum Concentration (ng/mL) Males I Females I 2053 I 2054 I 2211 I 2058 I 2059 0 0.5 hrs 2 hrs 5.8 BQL BQL BQL BQL BQL . I I 113,000I 140,900I 70,820 I 153,600I 174,000 116,400 ~~~~ 128,5001 99,070 104,600[ 98,100 92,300 58,660 4 hrs 76,530 46,960 64,480 65,570 66,940 48,5 10 8 hrs 24 hrs 50,770 53,090 18,660 55,910 52,600 54,470 40.405 I 40.340 I 24.870 I 54.300 I 37.100 27,115 48 hrs 46,740 43,885 23,930 50,050 41,045 35,555 Day 4 Day 7 37,930 54,850 16,640 44,850 37,250 48,200 35.880 I 48.840 I 28.160 I 41.880 I 41.500 41,250 Day 14 45,585 48,475 29,400 41,045 33,300 37,405 Day 21 42,295 37,210 22,285 39,015 40,930 42,200 Day 28 42,845 43,820 23,745 42,460 38,105 34,800 Day 42 Day 56 31,110 37,675 28,285 32,890 31,300 34,270 16,960 I 24,990 I 19,960 I 16,010 I 27,220 22,050 Day 70 18,020 22,710 18,680 17,850 22,580 21,610 Day 171a 16,680 21,725 13,415 11,805 3,249 20,220 BQL = Below the quantitation limit (<5 ng/mL) a Data represent an average of two samples. Page 1 of 1 Table 3 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonatein the Cynomolgus Monkey Pharmacokinetic Parameters Calculated from Serum Concentrations of Potassium Perfluorohexanesulfonate bSerum concentration at time o `Area under the serum concentration time curve fi-om 0 to the last time point dAreaunder the serum concentrationtime curve from 0 to infinity eHalf-lifeof the terminal eliminationphase Total body clearance gVolumeof distribution at steady state c. P Page 1of 1 15 Table 4 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Animal ID Urine Concentration Sex (ng/mL) Urine Volume (d) Total (Pd Dose (Pd Percent of Dose in Urine (%) 2053 M 48 1000 48 2054 M 193 3 60 69 2211 M 79 640 51 2058 F 22 320 7 2059 F 60 150 9 2061 F 484 90 44 2053 M 65 610 40 2054 M 17 330 6 2211 M 10 340 3 2058 F 60 170 10 2059 F 193 60 12 2061 F 709 50 35 2053 M 28 490 14 2054 M 16 210 3 2211 M 182 210 38 2058 F 64 290 19 2059 F 15 180 3 206 1 F 121 70 8 BQL = Below the quantitation limit (4ng/mL) 60,000 62,000 54,000 36,000 37,000 41,000 60,000 62,000 54,000 36,000 37,000 41,000 66,000 64,000 5 1,000 34,000 35,000 41,000 0.08 0.11 0.09 0.02 0.02 0.1 1 0.07 0.01 0.01 0.03 0.03 0.09 0.02 0.01 0.07 0.05 0.01 0.02 Page 1o f 3 16 Table 4 (Continued) A PharmacokineticStudy of Potassium Perfluorohexanesulfonatein the Cynomolgus Monkey Urinary Excretion of Perfluorohexanesulfonate BQL = Below the quantitation limit (4ng/mL) Page 2 of 3 17 Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonatein the Cynornolgus Monkey Animal ID Urine Concentration Sex (ng/mL) Urine Volume (mL) 2053" M 15 2054 M 48 2211 M 7 2058 F 8 2059 F 11 2061 F 70 2053 I M I 6 2054 M 38 I 2211 M 3 2058 F I 20 2059 F 28 2061 F 177 510 450 510 330 160 200 510 I 430 640 220 150 170 Total (iug) 8 22 4 3 2 14 3 16 2 4 4 30 Dose (iud Percent of Dose in Urine (%I 60,000 62,000 54,000 36,000 37,000 4 1,000 0.01 0.03 0.01 0.01 <0.01 0.03 I 60,000 I 62,000 54,000 36,000 37,000 4 1,000 0.01 0.03 <0.01 0.01 0.01 0.07 BQL = Below the quantitation limit ( 4 ng/mL) Page 3 o f 3 I000 . 3 E 0) 3 v .-C 0 II!I I C 1 a, 0 C 0 0 E 2 Q) (I) 18 M2053 -8 Observed - Predicted 10 0 20 40 60 80 100 120 140 160 180 Time (Days) 1000 M2054 h -I E . m 3 v C .0- - 2 I c 100 a, 0 C s E 2 a, (I) +j- Observe - Predicted 10 0 20 40 60 80 100 120 140 160 180 Time (Days) Figure 1 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate 19 M2211 1000 , I I I I I I I I I I I I I I I I I I I 1 I I I . h 1 E 20) 100 I I I I I I I I s K .0_ c - c C a0 , c a"" n u C F---~ -8 Observe 0 - Predicted 1 0 20 40 60 80 100 120 140 160 180 Time (Days) 1000 F2058 . h 1 E u) 3 100 v 5 K .0- c 2 c C a, 0 K 0 0 10 ma, 8- Observe - Predicted 1 0 20 40 60 80 100 120 140 160 180 Time (Days) Figure 1 (Continued) A Pharmacolunetic Study of Potassium Perfluorohexanesulfonatein the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate 1000 . 3 E 01 3 v 100 C .0- CI 2 - C a, 0 C sE 10 2 al (I) 20 F2061 8 Observe - Predicted 1 0 20 40 60 80 100 120 140 160 180 Time (Days) Iuuu . h 1 E 01 3 v 100 !i C .0- CI 2 c C 0 0 E 10 2 a, (I) F2059 -e- Observe - Predicted 1 0 20 40 60 80 100 120 140 160 180 Time (Days) Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonatein the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate Appendix A Study Protocol A- 1 Study Protocol: A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Southern Research Study ID: 9921.5 February 7,2001 SOUTHERNRESEARCH INSTITUTE A-2 STUDY NO.: 9921.5 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: Sponsor: 3M Center, 220-2E-02 P.O. Box 33220 St. Paul, Minnesota 55133-3220 Sponsor's Representative & Study Monitor: John L. Butenhoff, Ph.D., D.A.B.T. 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-3220 (651) 733-1962; FAX: (651) 733-1773 Protocol Approval: (Initial last page also) February 7,200 1 Page 2 of 13 Test Article: Ship Unused Test Article to: John L. Butenhoff -- Date Perfluorohexanesulfonate,potassium salt D. Hakes Building B236 3M Center P.O. BOX33327 55133-3327 St. Paul, Minnesota 55144-1000 A-3 STUDY NO.: 9921.5 2.0 TITLE: February 7,2001 page 3 of 13 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey 3.0 OBJECTIVE: The objectives of this study are to determine the concentration of perfluorohexanesulfonate in serum and urine at various times following administration of a single intravenous dose of potassium perfluorohexanesulfonate to monkeys. 4.0 TESTING LABORATORY: Southern Research Institute 2000 Ninth Avenue South 35205 P.O. Box 55305 Birmingham, AL 35255-5305 (205) 581-2335; FAX: (205) 581-2044 Event Day of Treatment Urine and Feces Collections Serum Drug Levels Draft Report Due Final ReDort Due Sequence (Day) 0 Baseline (predose) 1 2 7 14 21 28 42 56 70 91 0: 0 (predose), 0.5,2,4, 8 and 24 hours postdose 2 4 7 14 21 28 42 56 70 91 60 Calendar days after completion of the in-life phase 15 davs after final SDonsor comments received Date(s) - Year 2001 2/9/01 21910 1 2/10/01 211 1/01 211 610 1 2/23/01 31210 1 31910 1 3/23/01 41610 1 4/20/0 1 511 1/01 219- 1010 1 211 1/01 211 310 1 2116/01 2/23/01 3/2/01 , 31910 1 3/23/01 41610 1 4/20/0 1 511 1/01 7/10/01 A-4 STUDY NO.: 9921.5 February 7,2001 6.0 STUDY PERSONNEL: The following are the primary contributors and supervisory personnel participating in t h s study. Study Director: Alternate Study Director: Director, Safety Assessment: Associate Director: Manager, Bioanalytical Chemistry: Supervisor, In-Life Laboratories: Veterinarian: Patricia E. Noker James D. Johnson Ward R. Richter Norman D. Jefferson James D. Johnson LaJuana A. Durbin Darrell E. Hoskins Ph.D., D.A.B.T. M.S., M.B.A. D.V.M., M.S., D.A.C.V.P. B.A. M.S., M.B.A. A.A.S. D.V.M.,A.C.L.A.M.(Dipl.) 7.0 TEST & CONTROL ARTICLES: The test article will be supplied by the Sponsor, who will be responsible for documentation of stability, as well as methods of synthesis, fabrication, or derivation. Upon completion of the study, residual bulk test article will be returned to the Sponsor. 7.1 IDENTITY OF THE TESTARTICLE: Name: Identification: Supplier: Lot Number(s): Special Handling: Perfluorohexanesulfonate, potassium salt T-7504 3M To be documented in the study data. None Characterization: Documentation of the characterization of the test article, including identity, purity, strength, and composition, as well as methods of synthesis, fabrication, or derivation, is the responsibility of the Sponsor. Copies of characterization data have been provided to the testing laboratory. Stability & Storage: The bulk test article will be stored at room temperature. Stability of the bulk test article is the responsibility of the Sponsor. A-5 STUDY NO.: 9921.5 7.2 IDENTITY OF THE VEHICLE: February 7,2001 Page 5 of 13 Name: Supplier: Lot Number(s): Special Handling: Sterile Saline Commercial supplier To be documented in the study data. None Characterization: Documentation of the characterization of the 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. 7.3 FORMULATION: Preparation:The test article will be formulated in sterile saline at a concentration of 5 mg/rnL, for intravenous administration; briefly, the required amount of test article will be mixed with the required amount of sterile saline, and the mixture will be stirred until the test article is visibly in solution. Formulations will be stored refrigerated until used for dosing; formulations of the 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. 8.0 TEST SYSTEM: Species & Strain: Supplier: Age on Day 1: Weight at randomization: Number on Study: Cynomolgus monkeys (Macacafascicularis) Charles River BRF, Inc. (Houston, TX) 3-4years of age (estimated) 3-7 kg Males -3 Females -3 Animals were previously dosed with potassium perfluorobutanesulfonate in study 9921.1, potassium perfluorobutanoatein study 9921.2, potassium perfluorohexanoate in study 9921.3, and potassium perfluorooctanoate in study 9921.4. A-6 STUDY NO.: 9921.5 I 8.1 JUSTIFICATION: February 7,2001 Page 6 of 13 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. 8.2 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 a minimumof 10air exchanges per hour. Controls will be set to maintain the animal room at a temperature of 64-84 OF and a relative humidity of 30-70%. A 12-hour lightIl2-hour dark cycle will be routinely maintained. Animals will be acclimated in the same room used for study. 8.3 BEDDING: None required for caging equipped with flushable pans. For cages equipped with excrement absorption pans, commercial heat-treated hardwood chip bedding will be i used for excrement absorption. Analyses of the bedding, supplied by the vendor, will be reviewed by the Department of Veterinary Medicine and Bioresources (DVMB) of Southern Research to assure that no known contaminants are present that could interfere with or affect the outcome of the study. 8.4 DIET: 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 fi-esh fruit offered daily and treats offered several times each week. The quantity of the daily ration will be sufficient to meet nutritional requirements. Analyses of the feed, supplied by the vendor, will be reviewed by the DVMB of Southern Research to assure that no known contaminants are present that could affect the health of the animals. 8.5 WATER: Water (Birmingham public water supply) will be supplied ad libitum during the quarantine and study periods via an automatic watering system. Samples of water fi-om the animal facility will be periodically analyzed, and the analyses will be reviewed by the DVMB of Southern Research to assure that no known contaminants are present that could affect the health of the animals. A- 7 i STUDY NO.: 9921.5 I 8.6 QUARANTINE: February 7,2001 Paee 7of 13 All primates were selected from stock animals that were quarantined for a minimum of 35 days upon receipt at Southern Research. No prophylactic or therapeutic treatments were administered during the quarantine period. Standard procedures conducted during the quarantine period were as follows: a) A complete physical examination including a fecal examination for internal parasites, complete blood count (CBC), body weight, and body (rectal) temperature was performed; b) three tuberculin tests at 2-week intervals (administered intrapalpebrally, using alternate eyelids for each test) were performed on each primate. Primates tested negative to all three tests prior to release from quarantine. ;c) the blood sample drawn for CBC was also used for measurement of B virus titer; d) a fecal sample for culture (screening for Salmonella and Shigella) was obtained and submitted to an independent laboratory for analysis; and e) all primates were examined at least once weekly by a veterinarian and observed (cage-side observations) twice daily for abnormal clinical observations and mortality/moribundity. Throughout the subsequent holding and study periods, monkeys will be maintained under conditions similar to those for quarantine. In addition, quarterly evaluations to be performed on each monkey will include tuberculin testing, body weight determination, and body temperature measurement. Primates that respond positively to a tuberculin test will be euthanized immediately. 8.7 PSYCHOLOGICALWELL-BEINAGND 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 h i t . Where possible, primates will be housed proximate to one another for visual and vocal contact. 8.8 ANIMAL IDENTIFICATION: 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-8 STUDY NO.: 9921.5 February 7,2001 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 perfluorohexanesulfonate 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 synopsis of the study design is presented in the following table. a Denotes week 9.1 RANDOMIZATION& GROUPASSIGNMENT: As only one treatment group will be used in this study, no formal randomization will be required. 9.2 DOSEPROCEDURE: Each primate (three males, three females) will receive a single intravenous (n? dose of potassium perfluorohexanesulfonate(10 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 a volume of 2 mL/kg. The day of dosing will be Day 0 of the study. 9.3 CLINICAOLBSERVATIONS: Daily Observations: All monkeys will be observed once daily during the holding period and twice daily, morning and afternoon, at least 4 hours apart, during the A-9 r STUDY NO.: 9921.5 February 7,200 1 study for signs of mortality/moribundity and overt toxicity. Animals found in extrernis will 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 signs of toxicity. All findings will be recorded. Additional clinical observations will be performed and recorded on days of blood collection. 9.4 BODYWEIGHTS: Each primate will be weighed on Days -1, 4, 7, and weekly intervals thereafter through Day 91. 9.5 URINE AND FECESCOLLECTIONS: Urine and feces will be collected for approximate 24 hour intervals prior to dosing and on Days 1 (0-24 hours postdose), 2 (24-48 hours post dose), 7,14,21,28,42,56, 70, and 91. The volume of each urine sample will be measured upon collection. All samples collected will be stored fiozen at -20 "Cor below prior to analysis (urine) or until further notice by the Sponsor (feces). 9.6 SERUMDRUGLEVELS: Blood samples (approximately 3 mL) will be collected fiom each primate at approximately 0 (predose)minutes; 0.5,2,4,8, and 24 hours; and 2,4,7, 14,21,28, 42, 56, 70, and 91 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 fiozen at -20 "C or below until analyzed. 9.7 BIOANALYTICMALETHODEVELOPMENT: Bioanalytical method(s) will be developed for the determination of perfluorohexanesulfonate in serum and urine matrices. The method(s) will be validated for accuracy and ideally will be sensitive to the 1 ppm or less level. If feces andor other tissues require analyses, these analyses will be negotiated with the Sponsor. 9.8 BIOANALYTICSAALMPLEANALYSIS: The serum and urine samples fi-om all monkeys will be analyzed for concentrations of perfluorohexanesulfonateusing the previously validated method. The data will be A-10 I STUDY NO.: 9921.5 February 7,2001 Page 10 of 13 expressed as equivalents of potassium perfluorohexanesulfonate. Feces will be analyzed only if requested by the Sponsor. 9.9 ANIMALDISPOSITION: At the end of the study, monkeys will be maintained in the stock colony. In the event untoward reactions or other conditions warrant the euthanasia of a monkey at any time during the sample collection period, a serum sample (5-10 mL) will be obtained fiom the animal prior to euthanasia. After euthanasia, the animal will be necropsied and the liver and bile (as much as possible) will be removed and stored at approximately -70 "C. 10.0 DATA ANALYSIS: Pharmacokineticparameters (e.g., AUC, half-life, clearance) will be estimated fiom serum concentrations of unchanged perfluorohexanesulfonate,as appropriate and feasible, using a standard pharmacokinetic program. The total amount of perfluorohexanesulfonatein urine will be calculated and expressed in terms of percent of dose. Mean values and standard deviations will be calculated for each time point and sample type, as appropriate. No other statistical analyses of the data will be performed. 11.0 RECORDS: All raw data pertaining to the conduct of this 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 acceptance of the final report by the Sponsor. After 1 year and with the permission of the Sponsor's Monitor, the data and any samples/specimenswill be shipped to the Sponsor or to the Sponsor's designated archival facility. If materials 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 Southern Research. 12.0 FINAL REPORT: A brief letter 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 completion of the in-life aspects of the study. The final report (electronic and hard copies) will be issued within 15 working days after receipt of the Sponsor's final review comments on the draft report. The final report for the present study will include, but not necessarily be limited to the following: A-1 1 STUDY NO.: 9921.5 February 7,2001 Page 11 of 13 Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data 13.0 REGULATORY REFERENCES: This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs) of Southem Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 PROTOCOL AMENDMENTS AND DEVIATIONS: Amendments: All changes in or revisions of the approved protocol and the reasons thereof will be documented, signed, and dated by the Study Director, and the Sponsorls 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 specificallyd e h e d in this protocol, will be performed according to the Standard Operating Procedures (SOPs) of Southern Research andor the protocol, and any deviations fkom protocol or SOP will be documented. 13.2 REGULATORCYOMPLIANCE: Good Laboratory Practices: This nonclinical laboratory study will be conducted in the spirit of, but will not require strict compliance with, the U.S. Food and Drug Administration's (FDA) Good Laboratory Practice (GLP) regulations (21 CFR Part 58). Data &omthis study may be submitted to the FDA in support of an INDNDA application. Quality Assurance Review: As this study will not be conducted in strict compliance with FDA's GLP regulations, neither the in-life activities nor the final report will be audited by the Quality Assurance Unit at Southern Research. A-12 STUDY NO.: 9921.5 13.3 FACILITIEMSANAGEMENATND ANIMAL HUSBANDRY: February 7,2001 Page 12 of 13 Animal care will be in compliance with the SOPS of Southern Research, the Guidelinesfor the Care and Use of Laboratory Animals, 7thEdition (Institute 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 Research Institute is fully accredited by the American Association for Accreditation of Laboratory Animal Care ( M A C ) . 13.4 ANIMAL WELFARE ACTCOMPLIANCE: By signing this protocol, the Sponsor signifies that there are no generally accepted alternatives to the use of animals, and that the study described by this protocol does not unnecessarily duplicate previously conducted or reported experiments. Procedures used in this protocol are designed to 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 withholding of these agents has been justified in writing by the Study Director andor Sponsor and approved by the IACUC. The number of animals selected for use in this study is considered to be the minimum number necessary to meet scientific and regulatory guidelines for this type of study. 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. . 1 ' I J A-13 STUDY NO.: 9921.5 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. Study Director: Y& -;r,,,UJ Patricia E. Noker, Ph.D., D.A.B.T. Study Director Sponsor's Monitor: vzG INITIALS ONLY (See page 2) n I Management Approval: February 7,2001 Page 13 of 13 2/7/D/ I Date Date Appendix B Analytical Method for Determination of Perfluorohexanesulfonate in Monkey Serum and Urine B- 1 Page 1 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry OIpLCMSMS) 1.0 PRINCIPLE Serum or urine samples are obtained fiom cynomolgus monkeys treated with Perfluorohexanesulfonate(PFHS). The serum or urine (e.g., 0.5 mL) containing (PFHS) is fortified with an internal standard (IS), Perfluorooctanecarboxalate (PFOC). The samples are then mixed with an ion-pairing reagent, buffer and water, followed by extraction with ethyl acetate. The ethyl acetate layer is removed, evaporated to dryness, reconstituted in 95% methanol containing 1.5 % formic acid, 5 % 5 m M ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass SpectrometryMass Spectrometry@WLCMSNS). The range of reliable results extends from about 5 to 20,000 ng/mL of PFHS in serum and fiom 10 to 500 ng/mL in urine. Samples containingPFHS at concentrations greater than 20,000 ng/mL may be diluted with control blank matrix so that the concentration of PFHS will be within the range of reliable results prior to analysis. The mass spectrometryof PFHS and PFOC is accomplishedin the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the formation of other potentially interfering ions extracted from the matrix. CAUTION: Since primates may carry a number of zoonoses, all unpreserved tissues, including blood, plasma and serum, are to be considered as biohazards and handled 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. 2.0 REAGENTS AND SOLUTIONS The listed reagents or their equivalentsmay be used. 2.1 Neat Reagents 2.1.1 Water, deionized and organic fiee (fiom in-house purification system; e.g., Ingalls 210N) 2.1.2 Methanol, HPLC grade B-2 Page 2 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLC/MS/MS) 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.1.9 2.1.10 2.1.11 2.1.12 2.1.13 2.2 2.2.1 2.2.1.1 2.2.2 Perfluorohexanesulfonate(analyte), as provided by the client Perfluorooctanecarboxalate (internal standard), 97% Ammonium acetate, HPLC grade Blank control monkey serum Sodium Carbonate, Certified ACS Grade or equivalent Sodium Bicarbonate, Certified ACS Grade or equivalent Ethyl Acetate, HPLC grade TetrabutylammoniumHydrogen Sulfate, Aldrich 97% Sodium Hydroxide 50% solution, Certified grade or equivalent Blank control monkey urine Formic Acid, 88% Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 5 mM Ammonium acetate in organic free water For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-fkee water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) B-3 Page 3 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryNass Spectrometry (HPLC/MS/MS) 2.2.2.1 2.2.3 2.2.3.1 2.2.4 2.2.4.1 3.0 3.1 3.2 3.3 3.4 3.5 3.6 For example, to prepare 25 mL, dissolve approximately 4.24 g of tetrabutylasnmonium 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. CarbonateBicarbonate Buffer Solution for Serum (0.25W0.25M) For example, to prepare 100 mL, dissolve approximately 2.65 g of sodium carbonate and approximately2.10 g of sodium bicarbonate in 100mL of deionized water. Mix well to ensure complete dissolution. CarbonateBicarbonate Buffer Solution for Urine (1.OW1.OM) For example, to prepare 100 mL, dissolve approximately 10.6 g of sodium carbonate and approximately 8.4 g of sodium bicarbonate in 100 mL of deionized 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 8 platform mixer) Solvent-concentrationapparatus (e.g., Zymark Turbo-Vap 8 with source of nitrogen) HPLC mobile phase filtration apparatus Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 pm) B-4 Page 4 of 14 A N L Y T I C L METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometgdMass Spectrometry (HPLC/MS/MS) 3.7 Analytical balance 3.8 Volumetric flasks (e.g., 10 and 25 mL) 3.9 Disposable Pasteur pipettes 3.10 Micropipettor(s)with tips 3.11 Culture tubes with teflon-lined caps 3.12 Centrifuge 3.13 Assorted glassware and syringes 3.14 Culture tubes (vials) for use with solvent-concentrationapparatus 3.15 1 mL Plastic syringes with 0.2 pm PVDF syringe filters 3.16 Variable speed horizontal platform shaker 3.17 pH meter 4.0 PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrationsof the solutions may be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. 4.1 Main Stock Solution of PFHS -1000 pg/mL 4.1.1 Prepare an -1000 pg/mL solution of PFHS in deionized organic-free water (e.g., accurately weigh about 10 mg PFHS into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternatively, weigh the compound B-5 Page 5 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometryhlass Spectrometry (HPLCMSMS) 4.2 4.2.1 4.3 4.3.1 4.4 4.4.1 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. Stock Solution of Internal Standard (PFOC), -200 pg/mL Prepare an -2OOpg/mL solution of PFOC 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-fieewater. Alternatively,weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. Spiking solution of Internal Standard (PFOC), - 50pglmL Prepare an - 50 pg/mL solution of PFOC 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. Working Stock Solutions of PFHS 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. If desired a modified dilution scheme can be used and documented in the study records. B-6 Page 6 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryNass Spectrometry (HPLCIMSNS) Working Stock Level (WSL) Approximate Concentration (ng/mL) Volume of PFHS solution Final Volume in deionized organic fiee water (mL) I ~ ~~ 500,000 5 mL of 1000 p g / d 250,000 I 5 mL of 500,000 ng/mL 1 ~~ 2.5 mL of 250,000 ng/mL 10 10 ~ ~~ 10 31,250 5 mL of 62,500 n g / d 10 15,625 5 mL of 31,250 ng/ mL 10 5,000 50 pL of 1000 p g / d stock 10 2,500 5 mL of 5000 ng/mL stock 10 1,000 4 tnL of 2500 n g / d stock 10 500 5 mL of 1000 ng/mL stock 10 250 5 mL of 500 ng/mL stock 10 B-7 Page 7 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryDvlass Spectrometry (PLC/MS/MS) 4.4.2 Summary of concentrations of serum standards: Standard Level Volume and Spike Conc. Approximate Concentration of PFHS in serum (ng/mL) A 20 pL of 500,000 ng/mL 20,000 I B I 10 pLof500,000ng/mL I 10,000 I C 10 pL of 250,000 ng/mL, 5,000 D 16 pL of 62,500 ng/mL 2,000 E 16 pLof31,250ng/mL F 16 pL of 15,625 ng/mL, G 20 pL of 5,000 ng/mL H 10 pL of 5,000 ng/mL I 10 pL of 2,500 ng/mL J 10 pL of 1,000 ng/mL K 10 pL of 500 ng/mL L 10 pL of 250 ng/mL 1,000 500 200 100 ~ ~~~ 50 20 10 5 B-8 Page 8 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometryktass Spectrometry (HPLC/MS/MS) 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutionsmay 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 set of unknown samples. A matrix double blank (blank-IS) may also be analyzed if desired. 5.2 Into individual -20-mL culture tubes, pipet blank matrix (e.g., 0.5 mL ). Pipet in the appropriate volume as described in the table above for each standard. For the blanks, pipet 10 pL of organic-free water instead of the working stock solution. Add the 10 pL of internal standard stock (-50 &nL) to each tube except the blank-IS (pipet 10 pL of organic-fkee water instead) and vortex for -5 seconds. 5.3 For serum samples add the following to each tube: 500 yL of the TBA ion-pairing solution, 1 mL of 0.25W0.25M carbonatehicarbonate buffer, and 1 mL of deionized organic free water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of l.OM/l.OM carbonatehicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. 5.4 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube fkom the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 - Remove the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., 50 minutes in the Turbo-Vap 0)with a gentle stream of nitrogen and moderate heat (e.g., 50 'C). B-9 Page 9 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS) 5.7 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm 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. Pipet an aliquot of each sample (e.g., 0.5 mL) into individual-20-mL culture tubes. If necessary, dilute an aliquot of any sample with blank matrix so that the expected concentration of the test articlebeing analyzed will fall w i t h the concentration range of the standard curve. Add 10 pL of internal standard stock (- 50 pg/mL) to each tube and vortex for a couple of seconds. 6.2 For serum samples add the following to each tube: 500 pL of the TBA ion-pairing solution, 1 mL of 0.25W0.25M carbonatehicarbonate buffer, and 1 mL of deionized organic flee water. Vortex each tube for about 5 seconds. For urine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of 1.OWl.OM carbonatehicarbonate buffer and 1 mL of deionized water. Vortex each tube for about 5 seconds. 6.3 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 fiom the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. - 6.5 Remove the top ethyl acetate layer and put it into a clean tube and evaporate to dryness (e.g., 50 minutes in the Turbo-Vap 0 )with a gentle stream of nitrogen and moderate heat (e.g., 50 'C). 6.6 Reconstitute the residue in 500 pL of 5% 5 mM ammonium acetate: 95% methanol containing 1.5% formic acid and vortex briefly to mix. Filter the samples through 0.2 pm PVDF or Nylon syringe filters into autosampler vials. Cap vials for analysis. B-10 Page 10 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSrnS) 7.0 7.1 7.1.1 7.1.2 ANALYSIS BY HTGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLCMSMS) Conditions are to be optimized if necessary. HPLC Conditions 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 C 18 10 mm x 2 mm 600 pL/min. 5 PL A: 5 m M ammonium acetate buffer B: 1.5% formic acid in methanol Gradient Profile: Temperature: 0 - 3 min. 3 - 5min. 5 - 8 min. 8-10 min. Ambient 50%A : 50%B 20% A : 80% B linear gradient lO%A : 90% B step gradient 50%A : 50%B step gradient PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions Software: PE Sciex TurboQuan Turboion 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 IS Value -2000 B-11 Page 11 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLC/MSMS) Parameter NC TEM OR RNG QO IQ 1 ST R01 IQ2 R02 ST3 R03 DF CEM NEB CUR CAD QPE POL VCM Value 0 450 -20 -120 10 11 15 11 20 50 60 52 250 1800 15 6 5 0 1 0 B-12 Page 12 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLC/MS/MS) Parameter IPE Value 0 MSMS Acquisition Conditions Scantype: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFHS: 0 1 Mass (amu) 398.9 0 3 Mass (amu) 398.9 Dwell Time (ms) 200 PFOC (IS) 0 1 Mass (amu) 412.9 Parameter R02 ST3 R03 QO IQ1 ST RO 1 IQ2 0 3 Mass (amu) 368.9 stor, 35 35 45 45 37 37 18 18 19 19 23 23 19 19 20 20 Dwell Time (ms) 200 B-13 Page 13 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonatein Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryiMass Spectrometry (HPLCIMSNS) 8.0 CALCULATIONS 8.1 At the end of the analyticalrun,review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: Analyte PFHS PFOC Ion Profile 398.9 to 398.9 412.9 to 368.9 8.2 Plot the peak area response of PFHS divided by the peak area response of the IS (PFOC) from all standards versus the concentration of the test article in the standards. Alternatively, the peak heights may be used instead of peak areas. Obtain the best curve fit of the data (e.g., quadratic fit weighted with l/concentration of the test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: y = ax2+bx + c where y = x = a, byc Peak height response of PFHS divided by peak height response of the IS (PFOC) in standards. Concentration of the PFHS in standards, = Constants derived fiom the regression analysis. 8.3 Using the standard curve, calculate the level of PFHS in each unknown sample. Correct the results of samples for any dilutions. NOTE: Due to unresolvable interferences with the test article and/or internal standard fiom the matrix, external standard quantitation may be used at the discretion of the supervising mass spectrometrist. B-14 Page 14 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass SpectrometryiMass Spectrometry (HPLC/MS/MS) 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. Author(s): cf%L. ' Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group 4/7b 3 Date Approved by: Gieg &man, Ph.D. Manager Bioanalytical Chemistry Group