Document e7J3w1oM3xgZKav6vjOMjGJq4

36/ft R A S I 6 - 1 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Southern Research Institute Study ID: 9921.5 April 22,2003 001599 Final Report on A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey To: SPt..OPa.3uBMlo,3xMCMe3inn3Ctn3eo2err,s7po2ot2raa455t5-iI5o1N1n34-304-43-1302070 By: P.E. Noker and G.S. Gorman B2i0rm0S0inoNguihtnhaPtemhr.OnA, .ARvBeleaosnbexuaaem5rcS5ah3o0Iu3n5t5hs2ti5tu53t-5e5230055 001600 ABSTRACT The pharmacokinetics and urinary excretion ofperfluorohexanesulfonate were investigated in male and female cynomolgus monkeys. Three male and three female monkeys were administered a single iv bolus dose of 10 mg/kg ofperfluorohexanesulfonate, 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 ofperfluorohexanesulfonate were similar in male and female monkeys and ranged from 104,600 to 140,900 ng/mL in male monkeys and from 116,400 to 174,000 ng/mL in female monkeys. Serum concentrations ofperfluorohexanesulfonate in each monkey decreased relatively rapidly during the first 8-24 hours after dosing. At 24 hours, serum concentrations of perfluorohexanesulfonate ranged from 24,870 to 40,405 ng/mL in male monkeys and from 27,115 to 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 ofperfluorohexanesulfonate ranged from 13,415 to 21,725 ng/mL in male monkeys and from 3,249 to 20,220 ng/mL in female monkeys. The serum concentration versus time data were subjected to non-compartmental pharmacokinetic analysis. The serum elimination half-life ofperfluorohexanesulfonate ranged from 100 to 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 perfluorohexanesulfonate was 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 and from 160 to 255 mL/kg in male and female monkeys, respectively. Apparent differences among individual monkeys in the estimated serum half-life and clearance of the compound were likely attributable to an overestimation ofthe extrapolated AUC^ ^ values for two ofthe female monkeys. Only very low levels (<0.01 to 0.11% of the administered dose) of perfluorohexanesulfonate were measured in urine during any given 24hour period of sample collection between Day 1 and Day 70 after dosing. The results ofthis study suggested that the pharmacokinetics ofperfluorohexanesulfonate were similar in male and female monkeys. Perfluorohexanesulfonate was eliminated in urine by both male and female monkeys at low levels for a prolonged period oftime (>70 days) after iv administration. 001601 1 TABLE OF CONTENTS ag SIGNATURE PAGE iii GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL v 1.0 INTRODUCTION 1 2.0 MATERIALS AND METHODS 1 22..12 TTeesstt ASyrstitcelme and Vehicle 21 TDVeoeshsteiAcFlerotircmleulation Preparation 233 2.3 DCEGxloriopnsueeicprFiamAol rOesmnsbitugaslenlarmDtvioaeentsniiotgAnannnsadlyDsoesse Procedure 3333 BUoridnye WanedigFhetcses Collection 43 BDSeiaortuaamnAaLlnyaetlivycesalelssMofePtheorfdluDoerovheleoxpamneesnutlafonndaSteample Analysis 444 3.0 RESULTS 333...132 MBColoidnryitcaWaliltyeOigbhsetsrvations 3.4 PSeerruflmuoarondheUxarinneesuClofonncaetnetrations of 5555 5 4.0 DISCUSSION 7 5.0 CONCLUSIONS 7 6.0 RECORD ARCHIVES 8 7.0 REFERENCES 8 001602 U 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 Pohf ParomtaascsoiukminePtiecrfPluaoraromheetxearsneCsaullcfuolnaateted from Serum Concentrations Urinary Excretion of Perfluorohexanesulfonate LIST OF FIGURES Serum Concentration Profile ofPerfluorohexanesulfonate LIST OF APPENDICES Study Protocol AinnMaloyntikceayl MSeertuhmodafnodr UDreitneermination ofPerfluorohexanesulfonate Page 9 13 14 15 18 A-l B-l 001603 m Signature Page A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey PStautrdiyciDa Eir.ecNtoorker, Ph.D., D.A.B.T. Supervisor, ADME & Pharmacokinetics Reviewed by: Date DChiraerclteosrD, S. aHfetbyeArt,sPsehs.sDm.,eDnt.A.B.T. `t- L Z .- td Date We, the undersigned, were responsible for the conduct of the work and reporting ofthe results in the alinstdedcosnecctliuosnios.nsW. e concur with the views relative to ourbody ofwork as expressed in the discussion MGraengaogreyr,SB. diooarnmaalynt,icPahl.CDh. emistry Group 9 /lj 3 Date 001604 IV Good Laboratory Practices Disclaimer This study described in this final reportwas 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 ofthe study. There were no adverse circumstances that affected the quality or integrity ofthe study. f. PSatutrdiyciDa Eir.ecNtoorker, Ph.D., D.A.B.T. Date 001605 Study Dates: 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. Charles D. Hbert, Ph.D., D.A.B.T. Norman D. Jefferson, B.A. Gregory S. Gorman, Ph.D. Darrell E. Hoskins, D.V.M., Ph.D., A.C.L.A.M. LaJuana A. Durbin, B.S. D, Wayne May, LATG Carolyn R. Oliver, B.S. Study Director Director, Safety Assessment Associate Director, Safety Assessment Manager, Bioanalytical Chemistry Group Veterinarian Supervisor, Large Animal Laboratory Supervisor, Animal Care Supervisor, Study Coordination 001606 1 1.0 Introduction The objectives of this study were to determine the concentration of potassium perfluorohexanesulfonate in serum and to estimate urinary clearance at various times following administration of a single intravenous dose to monkeys. A copy ofthe 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 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 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 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. 001607 2 Certified, commercial, dry monkey chow #5048 (PMI Feeds, file., 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 fruit/treats 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 o f67.7-72.8 F 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 ofthe 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 o fLaboratory Animals, 7th edition(1)and the U.S. Department ofAgriculture 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 this study were previously given a single iv bolus dose of perfluorobutanesulfonate (10 mg/kg) on 4/10/00 (Southern Research Study No. 9921.1); a single iv bolus dose of potassium perfluorobutanoate (10 mg/kg) on 6/13/00 (Southern Research Study No. 9921.2); a single iv bolus dose of potassium perfluorohexanoate (10 mg/kg) on 7/31/00 (Southern Research Study No. 9921.3); and a single iv bolus dose ofpotassium perfluorooctanoate (10 mg/kg) on 10/9/00 (Southern Research Study No. 9921.4). Monkey 2053 was naive. 2.2 Test Article and Vehicle Test Article: One bottle containing 5 grams ofpotassium perfluorohexanesulfonate (T-7504; expiration date not supplied; SRI E06/L-1) was supplied by 3M (St. Paul, MN) and received 001608 3 on May 15,2000. The test article was stored at room temperature until used. Stability ofthe test article was the responsibility of the Sponsor. Vehicle: The vehicle used for the preparation of the dose formulation of potassium perfluorohexanesulfonate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 0081050, expiration date August 2003). The vehicle was stored at room 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 refrigerated 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. 23 Experim ental Design Group Assignment and Dose Procedure: As only one treatment group was used in this 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 mg/kg 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 oftoxicity. Additional clinical observations were performed on days ofblood collection. Body Weights: Each primate was weighed on Days -1, 4, 7,14, 21,28, 35,42,49,56,63, 70, 77, 84, 91,98, 105,112, and 119. 001609 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 171 postdose. Samples were collected into tubes without anticoagulant and were allowed to clot at room temperature. The blood samples were then centrifuged, and the serum separated and stored frozen (approximately -20 C or below) until analyzed. Bioanalytical Method Development and Sample Analysis: Serum and urine samples w oe analyzed for perfluorohexanesulfonate using a previously validated HPLC/MS/MS method (Appendix B). Data Analyses: The serum concentration data for unchanged perfluorohexanesulfonate were subjected to non-compartmental pharmacokinetic analysis using WinNonlin (Standard Edition; Version 1.1; Scientific Consulting Inc.; Cary, NC). Mean values and standard deviations for each parameter were calculated using Microsoft Excel Software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorohexanesulfonate at each collection interval was calculated and expressed as a percent ofthe administered dose. No other statistical analyses of the data were performed. Q Q L & IQ 5 3.0Results 3.1 M ortality 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 ofthis study. 3.3 Body W eights 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 ofperfluorohexanesulfonate in 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 concentrations of 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 this time ranged from 116,400 to 140,900 ng/mL among these four monkeys. For the other 2 male monkeys (2053 and 2211), peak serum concentrations ofperfluorohexanesulfonate (104,600 and 128,500 ng/mL) were not observed until 2 hours after dosing. The possibility was investigated that the 0.5- and 2-hour serum samples collected from these two monkeys were switched during collection and/or analysis; however, it could not be 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 ofperfluorohexanesulfonate ranged from 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 001611 6 concentrations ofperfluorohexanesulfonate decreased at a slow rate between 24 hours and the end of sample collection (171 days). During this period, fluctuations were observed in the serum concentrations ofperfluorohexanesulfonate among individual monkeys. In that the fluctuations appeared to be random among die 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 perfluorohexanesulfonate in individual monkeys are presented in Table 3. The values were derived from non-compartmental analysis ofthe data. No distinct differences were apparent between male and female monkeys in the estimated parameters; however, for two ofdie three female monkeys, the serum half-life and clearance ofperfluorohexanesulfonate w oe shorter and faster, respectively, than observed for the three male monkeys and the other female monkey. AUC0.infiaityvalues ranged from 6456 to 8745 /g*day/mL in male monkeys and from 3849 to 8501 gday/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 from 49 to 140 days (mean: 87 days) in the three female monkeys. The total body clearance of perfluorohexanesulfonate was 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) ofperfluorohexanesulfonate 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 001612 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 perfluorohexanesulfonate was slowly eliminated by male and female monkeys given a single iv dose of 10 mg/kg. Throughout the period of sample collection, serum concentrations ofperfluorohexanesulfonate were similar at each sample time in both male and female monkeys. In addition, the rate ofurinary excretion ofperfluorohexanesulfonate appeared to be similar for male and female monkeys. Thus, there was no clear indication from 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 third 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 AUC0.infinidtyvalues; these latter values were obtained from extrapolation of the serum concentration time curve and may have contained considerable error. That the AUCnj..^ values for two of the three female monkeys may have been overestimated is supported by the observation that estimated A U C ^, values [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 perfluorohexanesulfonate was 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 (>70 days) after iv administration. 001613 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 ofthe final report by the Sponsor. After 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 die 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 ofthe final report will be retained in the central archives at Southern Research. 7.0 References 1. ICnsotuitnuctiel;oNfaLtiaobnoarlaAtocryadAemniymParleRsse;sWouarcshesin, gCtoonmDm.iCss.;io1n99o6n. Life Sciences, National Research 001614 Table 1 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Males Group Sex 1M Animal Number 2053 2054 2211 Mean S.D. N -1 6.0 6.2 5.4 5.87 0.42 3 4 6.1 6.4 5.5 6.00 0.46 3 Day numbers relative to Start Date 7 6.0 6.4 5.6 6.00 0.40 3 14 6.4 6.6 5.8 6.27 0.42 3 21 6.1 6.5 5.6 6.07 0.45 3 28 6.1 6.6 5.7 6.13 0.45 3 35 6.2 6.6 5.7 6.17 0.45 3 42 6.3 6.6 5.9 6.27 0.35 3 49 6.0 6.7 5.9 6.20 0.44 3 56 6.1 6.7 6.0 6.27 0.38 3 001615 Page 1of4 Table 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Males Group Sex 1M Animal Number 2053 2054 2211 Mean S.D. N S3 6.3 6.8 6.2 6.43 0.32 3 70 6.4 7.0 6.3 6.57 0.38 3 Day numbers relative to Start Date 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 6.7 6.9 6.5 6.70 0.20 3 98 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 Table 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Females Group Sex IP Animal Number 2058 2059 2061 Mean S.D. N -1 3.6 3.7 4.1 3.80 0.26 3 4 3.7 3.6 4.1 3.80 0.26 3 Day numbers relative to Start Date 7 3.7 3.6 4.0 3.77 0.21 3 14 3.8 3.8 4.3 3.97 0.29 3 21 3.5 3.6 4.1 3.73 0.32 3 28 3.6 3.5 4.1 3.73 0.32 3 35 3.6 3.5 4.1 3.73 0.32 3 42 3.7 3.7 4.2 3.87 0.29 3 49 3.7 3.6 4.3 3.87 0.38 3 56 3.7 3.6 4.2 3.83 0.32 3 001617 Page 3 of4 Table 1 (Continued) A Pharmacokinetic Study of Potassium Periluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Females Group Sex IF 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 3.6 3.6 4 .4 3.87 0.46 3 119 3.6 3.5 4.3 3.80 0.44 3 N> 001618 Page 4 of4 13 Table 2 A Pharmacokinetic Study ofPotassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentrations of Perfluorohexanesulfonate Serum Concentration (ng/mL) Males Females Timepoint 2053 2054 2211 2058 2059 2061 0 5.8 BQL BQL BQL BQL BQL 0.5 hrs 113,000 140,900 70,820 153,600 174,000 116,400 2 hrs 128,500 99,070 104,600 98,100 92,300 58,660 4 hrs 76,530 46,960 64,480 65,570 66,940 48,510 8 hrs 50,770 53,090 18,660 55,910 52,600 54,470 24 hrs 40,405 40,340 24,870 54,300 37,100 27,115 48 hrs 46,740 43,885 23,930 50,050 41,045 35,555 Day 4 37,930 54,850 16,640 44,850 37,250 48,200 Day 7 35,880 48,840 28,160 41,880 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 31,110 37,675 28,285 32,890 31,300 34,270 Day 56 16,960 24,990 19,960 16,010 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) aData represent an average of two samples. Page 1 of 1 001619 Table 3 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Pharmacokinetic Parameters Calculated from Serum Concentrations of Potassium Perfluorohexanesulfonate Parameter 2053 2054 22M11a*le Average SD 2058 2059 F2e0m6a1l*e Average SD VC*btAACiS/LdUlUo2ejCaC(Os(rmmodoui-(a-gimmbiLny/df)ms/Ii*andct7_LioaOktyz)yngg0c/(r)*k/aBeidggnna*/gtrydea/asmtyiow/Lmne)crLaet)dstipmeceif046i221e104410.d54584064for 4185694 3136099 8745 7186 21122.134 32190.410 the estimation of the 4108712 7462 21184.371 terminal ha 72798 1169 095.122 f-life 3187082 5031 2272.205 215 3619 3849 1246.960 4144167 8501 21154.250 180 3946 5794 2181.793 35 418 2418 044.977 cdcAHArareelafa-uluinfneddoeerrfttthhheee ssteeerrrmuumminaccloonenlcciemennittnrraaatttiiiooonnn ttpiimhmaeeseccuurrvvee ffrroomm 00 ttoo tihnefinlaitsyt time point fTotal body clearance gVolume of distribution at steady state Page l of 1 oC CD ct o 15 Table 4 A Pharmacokinetic Study ofPotassium Perfhiorohexanesulfonate in the Cynomolgus Monkey Urinary Excretion of Perfluorohexanesulfonate Urine Urine AnIiDmal Sex Co(nncge/nmtrLat)ion V(omluLm)e Total (ms) Baseline 22005534 MM 2211 M BBQQLL BQL 1704000 630 -- -- -- 2058 F BQL 290 -- 22006519 FF BBQQLL 215500 --- 2053 M 48 1000 Day 1 48 22201514 MM 17993 360 640 69 51 2058 F 22 320 7 22006519 FF 46804 19500 494 Day 2 2053 M 2054 M 65 17 610 330 40 6 2211 M 10 340 3 2058 F 22006519 FF 60 710939 170 6500 311502 2053 M 2054 M 2211 M 28 11862 490 Day 7 14 210 3 210 38 2058 F 64 290 19 22006519 FF 11251 17800 38 Percent ofDose Dose inUrine (ms) (%) 60,000 _ 6524,,000000 36,000 -- ---- 4317,,000000 ---- 60,000 62,000 00..1018 5346,,000000 00..0029 4317,,000000 00..1012 6620,,000000 0.07 0.01 54,000 0.01 433176,,,000000000 000...000339 66,000 0.02 64,000 0.01 51,000 0.07 34,000 4315,,000000 000...000152 BQL = Below the quantitation limit (<1 ng/mL) Page 1 of3 001621 16 Table 4 (Continued) A Pharmacokinetic Study ofPotassium Perfluorohexanesulfonate in the Cynomolgus Monkey Urinary Excretion of Perfluorohexanesulfonate Urine Urine AnDimDal Sex Co(nncge/nmtrLat)ion V(omluLm)e Total (ms) 2053* M 2054 M 5 8 Day 14 280 740 61 2211 M 7 660 5 2058 F 167 170 28 22006519 FF 17229 211100 1154 2053 M 2054 M 2211 222000655189 MF FF 10 247 49 24307 440 Day 21 4 352300 92 100 5 19100 246 22005534 M M 5 8 Day 28 660 3 830 7 2211 2058 MF 22006519 FF 1106 2355 257200 210500 45 55 Day 42 22005534 M M 34 55 450 440 15 24 2211 M 24 820 20 222000655198 FFF 74 2184 211268000 1436 Percent ofDose D(Mogse) inUrine (%) 665024,,,000000000 <000..00.0111 36,000 4317,,000000 00..0084 0.03 60,000 0.01 6524,,000000 <00.0.011 36,000 0.01 4317,,000000 00..0017 60,000 0.01 6524,,000000 00..0011 36,000 0.01 4317,,000000 00..0011 60,000 0.03 62,000 0.04 54,000 0.04 36,000 4317,,000000 000...000115 BQL = Below the quantitation limit (<1 ng/mL) Page 2 of3 001622 17 Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Urinary Excretion ofPerfluorohexanesulfonate I Animal 1 ID 1 222020515314* 2058 11 22006519 1 22005534 2211 222000655189 Urine Sex Co(nncge/nmtrLat)ion MMM F 15 48 78 FF 7110 MM M 6 338 FFF 1227087 Urine V(omluLm)e Total Og) Day 56 510 450 282 510 4 231036000 3 124 510 Day 70 3 463400 126 211275000 3440 Dose PercinenUt orifnDe ose Og) (%) 60,000 0.01 62,000 0.03 54,000 0.01 36,000 0.01 4317,,000000 <00.0.031 60,000 0.01 62,000 0.03 3564,,000000 <00.0.011 4317,,000000 00..0017 BQL = Below the quantitation limit (<1 ng/mL) Page 3 of3 001623 18 M2053 -e- observed -- Predicted Serum Concentration (ug/mL) Serum Concentration (ug/mL) M2054 - - Observe -- Predicted Figure 1 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate 001624 19 M2211 Serum Concentration (ug/mL) Time (Days) F2058 - e - O bserve -- Predicted Seum Concentration (ug/mL) -e - Observe -- Predicted 0 20 40 60 80 100 120 140 160 180 Time (Days) Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate 001625 Serum Concentration (ug/mL) 20 F2061 1000 100 r e ---------------- y- 10 CL _________ Q _ O _____ - O -------- -o - O bserve -- Predicted 1 0 20 40 80 80 100 120 140 160 180 Tim e (D ays) F2059 - - Observe -- Predicted Serum Concentration (ug/mL) Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate 0 lS 2 6 Appendix A Study Protocol 001627 A-l Study Protocol: A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Southern Research Study ID: 9921.5 February 7,2001 Southern Research INSTITUTE 001628 A-2 STUDY NO.: 9921.5 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: February 7,2001 Paie 2 of 13 Sponsor: P3SM.tO. P.CaBeuonl,xteM3r,3i2n22n20e0s-2oEta-0525133-3220 Sponso&r'sSRtuedpyreMseonntaittoivr:e John L. Butenhoff, Ph.D., D.A.B.T. B3S(6Mtu5. iP1lCd)aieu7nnl3g,t3eM2-r21i09n-6n22eE;so-F0tAa2 X55: 1(4645-13)272303-1773 Protocol Approval: (Initial last page also) Test Article: John L. Butenhoff Z Date Perfluorohexanesulfonate, potassium salt ot Ship Unused Test Article to: PDB3SM.tu.O. iHPl.CdaaBiueknonleg,xsteMB3r 23in33n62e7so t5a55153134-43-3120700 001629 A-3 STUDY NO.: 9921.5 2.0 TITLE: February 7,2001 Pace 3 of 13 A Pharmacokinetic Study o f Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey 3.0 OBJECTIVE: The objectives o f this study are to determine the concentration o fperfluorohexanesulfonate in serum and urine at various times following administration o f 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 5.0 KEY STUDY DATES: Event UDraiyneofanTdreFaetcmesenCtollections Serum Drug Levels Draft Report Due Final Report Due Sequence (Day) D atefs)- Year 2001 404pB7279272247022559611111:1112h88206065044aas0dCseael(ayiplnsereenad(dfpotaersreredd)f,aiony0saes.l)5aS,f2pte,o4rn,cso8ormacnpodlme2tmi4oenhnootusfrrstehpceoeisivntde-oldisfee 423472222344522223352233////////////////////////2621999122621111221291190//0/01136331-//633//10/000000001///////////////100111000000000001110010111111111111111/01 -- 001630 A-4 STUDY NO.: 9921.5 6.0 STUDY PERSONNEL: February 7,2001 Page 4of 13 The following are the primary contributors and supervisory personnel participating in this 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 P hJ),D A B .T . M.S., M .BA. D.V.M., M.S., D.A.C.VJ>. BA. M .S..M .B .A . A.A.S. D .V A t, A.C.LA.M . (D ipl) 7.0 TEST & CONTROL ARTICLES: tTohfhesetsatteubsditlyiat,yrrt,eiacssliedwwueailllllbabuselmksuetepthsptolaidersdtiocbflyesytwhneitlhlSebpseiosnr,esftoaurbr,rnwiechdatotioownti,hlolebrSedperorenisvspaootrin.osni.blUe pfoorndcoocmumpleentitoantioonf 7.1 Id e n t it y o f t h e T e s t A r t ic l e : INdaemntief:ication: LSuopt pNliuemr: bers): Special Handling: TP-e7r5fl0u4orohexanesulfonate, potassium salt 3M TNoonbee documented in the study data. C h aracterizatio n : rihDanesasocvplmceuoudebnmtieshneieogbnnditilapdsittreyiooonovfntiiftsdyotyeh,fndeptthutShoerpeisttohiycsne,,hssotafetrrars.aebtcCnirntigeocgtrpahitliz,iaeoaabstnnoioo,drfnaocctorohormadfyre.patrhcoitveseiartttiieiozosanntt,i,oaansirstwidctalehetlea,l Stability & Storage: The bulk test article will be stored at room temperature. SSptaobnisliotyr. of the bulk test article is the responsibility of the 001631 STUDY NO.: 9921.5 7.2 Identity of the Vehicle: February 7,2001 Page 5 of 13 Name: LSuopt pNluiexrn: ber(s): Special Handling: SCtoemrimleeSrcailainl esupplier NToonbee documented in the study data. C h aracterizatio n : Datotaciunmedenbtaytiorencoofrdthinegchaalrlacpteerrtiiznaetniotnionffothrme avteiohniclefrmomaytbhee cthoenrteaoinf,erinlabtheels,soturdbyy dreattaai.ninTghtehevechoinctlaeiniesralabceolms,morecrcoipailelys available product. Stability & Storage: eaSxptepprriirolaeptiroisaantleinlpyer.oiTvsihdceeobdnuslibkdyevreethdhiecsletamwbalienllubtfhearcostutuogrreherdthiwnehaedcncaoters(dtsao)nrecodef with the manufacturer's instructions. 7.3 Formulation: PoarfrteiScpleamrwga/timliloLbne:fmTo rhixeientdtersawtviaethrntoitchulese rwaedqilmul ibirneeidsftoarrmamtioouunlna; ttebodrfieisnftelysrti,eletrhisleealrsineaqeli,unaierneaddttahamecomonuicxnettnuotrrefawttieoislnlt breexefprsietgciertrereaddtetodunbutneiltsitlthauebsleteedfsfotorarwrdtieoceslkeinsigsw; vhfoiesrnimbsluoylasittnioorsneosdl.uotfiothne. teFsotramrtuicllaetiionnsstewriillel bsaelisnteoraerde DdoosseefForomrmuluatliaotniocnonCcoenntcreantiotrnataiondn haonmdoHgeonmeiotygewniellitbyeAconnadlyuscetse:d.No analysis of 8.0 TEST SYSTEM: ASSpugpeecpoileinesrD:&ayS t1r:a in : NWuemigbhetraot nraSntduodmy:ization: CChyanrolemsoRlgivuesrmBoRnFk,eIynsc.((MHaocuasctaonfa,sTcXic)ularis) MF33--e74amlykeaegslae-rs3s-o3f age (estimated) pA9o9nt2iam1s.s3aiul,smawndpeerperofpltuaresosvrioiuobmuustpalynerodfaolutseoerdinowosctiutthadnpyoo9att9aes2si1ni.u2sm,tuppdoeytarf9sls9ui2ou1rmo.4bp.uetrafnlueosurolfhoenxaatneoinatestiundsytu9d9y21.1, A-6 STUDY NO.: 9921.5 8.1 J ustification: FebPruaagrey67,o20f0113 Primates are commonly used in preclinical pharmacological and toxicological meviaglhutabtieonesxpoofsceodm. pounds used or intended for use in humans, or to which humans 8.2 Housing: Dstauirninlegssqsuteaeral,nstliante-/baoctctolimmactaigoens.anAdllstaundimy,aalsnwimilallbsewhiollusbeediinndaivriodoumalltyhahtopurosvedideins a minimum of 10 air exchanges per hour. Controls will be set to maintain the animal rilnoigothhmte/1as2ta-mahoteeurmrodpoaemrrkautcusyeredcloefofwr6si4lt-lu8bd4ey.roFuatinndelayrmelaaitnivtaeinheudm. idAitnyimoafl3s0w-7i0ll%be. aAcc1li2m-haoteudr 8.3 Bedding: Nexocnreemreeqnut iarbedsofroprticoangpinangse, qcuoimppmeedrcwiailthheflaut-sthreaabtleedphaanrsd.wFooord ccahgipesbeeqdudiipnpgewd iwll ibthe buseerdevfoierwexecdrebmy ethnet aDbesopraprttmione.ntAonfaVlyesteesrionaftrhyeMbeedddicininge, saunpdpBliieodrebsyotuhrecevsen(DdoVr,MwBil)l oinfteSrofeurtehewrnithReosreaafrfcehcttothaesosuurtceotmhaet onof tkhneoswtundyc.ontaminants are present that could 8.4 Diet: MDiOet)w. ilTl bhee cpormimmaetercsiawl CillerbtiefieodffPerriemdafteeeCdhtowwic#e50d4a8ily(P,MwIitFheeadpsp,rIonxci.m; SatteLlyouthise, wrecilolmbemseunpdpeldemdeaniltyedrawtioitnh afrveasihlafbrlueitaotfefaecrehdfedeadiliynganindtterrevaatls. oIfnfearedddisteiovne,rathl etimdieest rDeceaoVqcuuhMlidrwBeamefofeeekfnc.SttsotT.huheAthehneeqranaulylaRtshneetsositeofyatfrhotchefheatthnofeeimeaddsasa,luisslru.yeprtphaltaiietodnnobwykintlhloewbvenescnuodfnofitrac,miweniinltlatbnoetsmreaevreeietpwnreuesdterinbtityotnhthaaelt 8.5 W ater: rWqfaerruoveaamitrpeeawrrnethets(iBdeenneibartymnathintmihandetgasclhDtouafVudmalycMdipplBaiuetfybrfoielofwiccdStisoltwlhuveabtihtaeheerearpnanseluRtrahipuoeptosdolefyimcat)haracwletlhiyciatlnolwainbmaaaestalseylsurszuir.neepgdpth,lsiayaetnsdntdeoamtdkh.neloiSbawaintmnuamlcpyolsendestsuaormwifniwignllaatnthbeteesr 001633 A-7 STUDY NO. 9921.5 February 7,2001 _Page 7 of 13 8.6 Q u a r a n tin e : Aofll3p5rimdaaytessuwpeorne rseecleecitpetdaftroSmoustthoecrknaRniemseaalsrcthha. t wNeorepqrouparhaynltaincteidc foorr athmerianpimeuutmic tcroenatdmucetnetds dwuerrinegatdhmeiqnuisatrearnedtindeuprienrgiotdhwe eqrueaarasnftoinlleowpes:riod. Standard procedures a) A complete physical examination including a fecal examination for internal pteamrapseitreast,urecowmapsletpeerbfolormodedc;obu)ntth(rCeBe Ctu),bebrocduylinwteeisgtsht,atan2d-wbeoedkyin(treercvtaalls) prmSl(aeaeablderleoafmmaosrsoaurienmntroiefeserrmltoydleamerfoenondaqtrnuaeodiananfrcataSBhrlnyahtppsiivinrgasiielem;rp.luleaaas;bnt)ecrdt.a)iwtleetlPh)yrar;ea,silmbdluol)psaobirtontaeiamdgsifnstaeeaeatcesmdlattseelpwardlsnneeanadrmdteeergpaseauwlxeetbiyanvmmefefoloiiitrtdnroteseCcddauBfllaolttCtuorthlrweereaaeaanse(cstshtcoaienrlnsestdcteoeseesnuptpwi)senreneigwodedrkeeffooltnryoerrt bclyinaicvaelteorbinsearrviaantiaonnds oabnsdemrvoerdta(cliatyg/em-soidrieboubnsdeirtvya. tions) twice daily for abnormal uTnhdroeurgchoonudtittihoenssusbimseiqlauretnot thhoolsdeinfgoraqnudasrtaundtyinpee.riIondasd, mditoionkne, yqsuawrtiellrblyeemvaaliunatatiionneds ttdooetaebretmubpineerarcftiouorlnimn, eatednsdtobwnoidellyabctehememuptoehrnaakntueizyreedwmiiemlalsmiunercedlmiuadetenelty.t.uPbreimrcautleins thteasttrinesgp,obnoddpyoswiteivigehlyt 8.7 P s y c h o l o g ic a l W e l l -B e in g a n d S o c ia l iz a t io n : NeacnocrniochrhdumamneacnnetwparisitmhdtahitreeescatwpepdilrlobbpyeripaatreovvSeiOtdePerdi.nNaarpoiasnynhcuhamonladongapipcrpiamlrowavteeelsdl-wbbieylilnbtghepeprroIoAgvriCadmUedCfcoaragsneodcainiandl mpaflieneteoeaoddrntihiusnfeotigscrf,atfrtaodeinorrgidnvnimsikrsaiuebwnnaoclltfutmarlundeoidsetd,,.vipbfoWuiuccztaahzatlelireocreeonfnnespoetoaddtscelastir.iimsbly,liente,fuodptrrrtiiotmti:hoasentwiaerlsilnpywgsssyiol,cluphbneoedrlcohphgoreiiuscms,aeKladteowpnterrgolelxat-obitmsye,isanu,tgnec.sltehoaenTlolneh2dee- 8.8 A n im a l I d e n t if ic a t io n : Tcporhimoerbptionriambtilaootnoe.ds sPwaomislliptilbvineegi,idndedonisvteiifdiacudaamtliloiynnisiwdtreialntlitobifneie,rdaenqbduyiorebcdsheearsvfttaetrtiaoetntvo.eoryncuamgebecrhaonrgeleattnedr 001634 A-8 STUDY NO.: 9921.5 9.0 EXPERIMENTAL DESIGN: February 7,2001 Paw 8 of 13 As only one treatment group will be used in this study, no formal randomization will be required. Dthoesteesswt ailrltibcelea. dmEainchistperriemd abtye i(nthtrraeveenmoaulsesi,njtehcretieonfetmo adleetse)rmwiinllertehceepivhearamsaicnogkleindeotisces ooff potassium perfluorohexanesulfonate by injection into a superficial arm or leg vein. Bdseelloteeocrdmtesdinametidmpilenetsepfrovoiarnlstsse.rduumridnrgugthleevsetul ddyet.ermUirninaetioannsdwfielclebse wcoillllecatlesod fbroemcoelalecchtepdrimatatpereat A synopsis ofthe study design is presented in the following table. Study Procecures HDoeaselthPrCehpeacraktion -l1 0 1 2 4 7 14 21 Day 28 ofStuc 35 y42 49 56 63 70 77 84 X X (Clinical Observations BUoridnyeW& eFigechetss Serum Drug Levels X X X X X X X XXX XX XX XXX X X X X X X X X X XX X X X X X X X XX XXX XX XX X X * Denotes week 9.1 Randomization & Group Assignment: Abesroenqluyiroende. treatment group will be used in this study, no formal randomization will 9.2 Dose Procedure: EooD0rfaoopclsfehoetgtshpaewvrsisemsiiiltunluam.bdteyepD(.aethodrfrsmleeuesionmwriosaithellelerxesb,adetnhabertsaeauselevffdooenlmuuapmateoleen(s1o)t0hfwe2milmmgl /rLkoegsc/kte)girbve.yecTeiannhjsteeicindntgaidolyeinvoiinidfnturdtoaaovlaseibnsnougodpuwyesriw(flTilceVbiiae)gldhDaotrsasm.ye 9.3 Clinical Observations: 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 001635 91 X X X X A-9 STUDY NO. 9921.5 February 7 ,2001 Page 9 of 13 study for signs of mortality/moribundity and overt toxicity. Animals found in eexxtsraenmgiusinwaitliol nbewhituhmapanperolyprsiaacteriafipcperdovbayl.an overdose of barbiturate followed by Detailed Observations: Each primate will be examined shortly after dose Aaddmdiitnioisntraalticolinnficoarldoebtasielervdactiloinniscawlislilgbnespoefrtfooxrmicietdy.anAdllreficnodridnegds ownildlabyesreocfobrldoeodd. collection. 9.4 Body Weights: Each primate will be weighed on Days -1 , 4, 7, and weekly intervals thereafter through Day 91. 9.5 Urine and Feces Collections: Ua7n0rd,inaoenndaDn9da1y.fseTc1eh(se0wv-2oi4llluhbmoeuecrosoflpleeoacscttdheodusrfeion)r,e2asp(a2pm4rop-4xle8imwhaoitlrelnb2se4pmohsoetaudsrouisrneet)de,ru7vp,a1ols4n,pc2ro1ilol,e2rct8toi,o4dn2o.,s5iAn6gl,l osarmunptlielsfcuortlhleecrtendotwiciellbbyetshteorSepdofnrosozren(feacte-2s)0. C or below prior to analysis (urine) 9.6 Serum Drug Levels: B4ap2lop, or5od6x,ism7a0ma,teaplnlyeds09((1paprdepadryoossxeai)mftmeartiendluoytsei3sn;g0m..L5S),a2mw,4pi,ll8les,bawenidlclo2bl4leehccotoeuldlresc;ftareondmdi2net,o4ac,t7hu,b1pe4rsi,mw21aitth,e2o8ua,tt anticoagulant and will be allowed to clot at room temperature. The blood samples worilbletlhoewn buentcielnatnriafluygzeedd,. and the serum will be separated and stored frozen at-20 C 9.7 Bioanalytical Method Development: Bpvaneailrodifda/lounarotaelrodyotthhfioceearxrlaatcniscesmusurueealtscfhoyorneadaqn(tusde)iiridenewaasnliellayrlulwymsbielelsab,netdhdseeeusvnreiesnilatoeinvpameeldytaostretihsfcoeewrs1.ilpltpThbmeheeonrmedlgeeeotsetsthrimaoletdevi(ndesal).wtiwoIiftnihflel tcbhoeeesf Sponsor. 9.8 Bioanalytical Sample analysis: The serum and urine samples from all monkeys will be analyzed for concentrations of perfluorohexanesulfonate using the previously validated method. The data will be 001636 A-10 STUDY NO.: 9921.5 February 7,2001 Pace 10 of 13 eaxnaplryezseseddonalsyeiqf ureivqauleesnttesd obfy pthoetaSsspiounmsorp.erfluorohexanesulfonate. Feces will be 9.9 A n im a l D is p o s it io n : At the end ofthe 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 tfairnmodme tdthhueerinalingvitemhrealasnpadmriopbrlietloecoe(ulalteshcatminoaunscipahe.raiAosdftp,eaorsesseuirbtuhlmaen) sawasmiail,pltlhebee(5a-rn1eim0mmoalvLwe)diwllaiblnledbneesoctorborteapdisnieeaddt approximately -70 C. 10.0 DATA ANALYSIS: Pcohnacrmenatcraotkioinnestiocfpuanrcahmaentgeerds (pee.rgf.l,uAorUohCe,xhaanlfe-sluiflefo, nclaetaer,aanscaep)pwroilplrbiaeteesatnimd afeteadsifbrloem, usseinrugma standard pharmacokinetic program. The total amount o fperfluorohexanesulfonate in urine will be calculated and expressed in terms ofpercent of dose. Maseaapnpvroaplureiastea.ndNsotaontdhaerrdsdtaetvisiatitcioanlsanwaillylsbeescoaflcthuleatdeadtafowriellabche tpimerfeoprmoinedt .and sample type, 11.0 RECORDS: All raw data pertaining to the conduct of this study, and all samples/specimens collected in tahcicsespttuadnyc,ewoifllthbee sfitnoareldreipnothrtebAyrtchheivSepsoantsSoor.utAhefrtnerR1eyseeaarrcahnIdnswtiittuhtethfeorpueprmtois1siyoenaor affttheer btSoepytohonensdoSrpt'shoniMssoodrna'sitteod,re,ssutihgcehnadctaeotdnataianrncudheiadvnasyltofsaraacmgilepitlywe.si/lIslfpbemceaimtfeoerrinaaslswsapirelelcbtiofeicbsehfeirpeeptadeiednteetordmtihninetehSdepwaornictshhoirvtheoesr RSpeosenasrocrh.. A copy of the final report will be retained in the central archives at Southern 12.0 FINAL REPORT: Ainfborrimefalteiottnerisreapvoaritlasbulme.mAaridzrinafgt tfhineaslerreupmordtrwugilllebveelisrseuseudltswwitihlilnbe60iscsualeedndasarsodoanysasafttheer cwoonimltlhpbeleedtirisoasfnuteordefpwtohiretth.iinnT-lh1ief5efwiansoaprlekrcientpsgoodrftatfyhoseratshfttueedrpyrre.ecsTeenihptetsfotiunfdathylerweSpilpolorinnt sc(eolulre'dscetf,riobnnualitcrneaovntidenwehcaecrsodsmacromiplyeienbstes) limited to the following: 001637 A -ll STUDY NO.: 9921.5 FePbarguaer1y17,o20f0113 DBColoidsneyicfwaolreomigbuhsletardtviaoatntaiopnresparation Serum drug level data UPhrainrme eaxccorkeitnioenticdpaatarameters 13.0 REGULATORY REFERENCES: rPTerhqoiucsiersdetuumdreeysnwt(sS,ilOalsPbase)dcdoorfneSdssoueucdtthebdeerlnionwRae.csceoarrdcahn,caendwiinthatchceoprdraontocceowl iatnhddtiheeapSptalincdaabrlde rOegpuelraattoinrgy 13.1 protocol Amendments and deviations: AtShpmeorenenosodfrmw'seiMlnltosb:neiAtdolorl.ccuhAmamnegneentesddimn, esonirgtrsneewvdii,sliloabnneds omdfaatthienedtaaibpnyperdothvweeidtShptrutohdteyocpDorolirtaeonccdtootlrh.,eaWrneadrsitotthennes atfhopelploprowrov.taolc(oalfmaxasyigbneagturarnetoedr eblyectthreonSipcocnosmorm'suMniocnatiitoonr,,bsuutcha wasrietmtenaial)mtoenr dchmaenngtews iilnl DpworfeiolSvltooibacuetotihodl,enowrcsn:uilRmlAbeelselneotpaeperdcer.fhroaratimnodne/sdopraetchrcteaoirpndriionntggotctoootlth,hiaesnsSdttuaadnnyyd,adurednvlOeiaspstieosrpnaesticnfirgfoicPmarlolpycreoddteoufcirneoesldo(SirnOStOPhsiPs) 13.2 Regulatory Compliance: Ai5Ganp8dop)tmh.oliedcDinasLaitpsitaoitarrbniafto.rtiooroamfn,to'btshru(yitFswPDstriAulaldc)nytGiocmtoeroasedy:qLubTiearhbesiosusrbntarmotiocnitrtcytcleioPdnmritcaopaclttlihiacleanebcF(oeGDrwaLAtiPotihr)ny,restshugtuepudplUayot.riSwot.onilFsfloa(b2one1dIcNCaonDnFddR/NuDcPDrtaueArdgt QwauiudthaitlFeitdDybAAy'sstshuGerLaQnPucraeelgRituyelvaAiteisowsnu:sr,aAnnescietthhUiesnrsitthtuaedtyinSw-oliiulfltehnaeocrtntbivReietciseoesnandrcuohcr.ttehdeinfinsatrlicretpcoomrt pwliiallnbcee 001638 A-12 STUDY NO.: 9921.5 13.3 Facilities Management and Animal Husbandry: FePbarueear1y27,2o0f0113 AGnuiimdeallinceasrfeo rwtihlle bCearien acnodmUplsieanocfeLwabitohratthoerySAOnPims aolfs,S7o*uEthdeirtnionRe(sIneastricthu,tethoef NAantiimonaal lRAecsoaduercmesy, PCreosms;mWissaisohninogntonL,ifDe CS;ci1e9n9c6e)s,, aNnadtitohnealUR.Se.seDaercphartCmoeunntcoil;f ARegsreicaurclthurIenstthitruoteugishhtihlley AacncirmedailteWd beylftahree AAmcter(iPcaunblAicssLoacwiati9o9n-f1o9r8A).ccrSeoduittahtieornn of Laboratory Animal Care (AAALAC). 13.4 Animal Welfare Act Compliance: By signing this protocol, the Sponsor signifies that there are no generally accepted nalotetrunnantievceesstsoartihlye duuseploicfaatneipmraelvsi,oaunsdlythcaotnddiuecstteuddoyrdreespcorirbteeddebxyptehriismpernottso.col does Ptoromceindiumreizseuoserdavinoitdhicsapursointogcpoalianr,eddisetsriegsnse, dortodicsocnofmorfmorttoinatchceepatneidmparlas.ctiIcnetshaonsde cmothirroecamuSnmeetusnsdtthtayaernyDticcoiesrrseuscinlntilogerwhsasthnptihdcaeh/ionwrroeSiqrthpudhoiirosnetlsrddoeisrnssatg,untodhdyfeathppaneprosirmeocevaadeglsuderwnbetysisllhtahrareeescIbelAiiekveCeenlUyajupCtspo.triofcipearudiasietnemwanorairtleignetghsibacnys 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. 001639 A-13 STUDY NO.! 9921.5 14.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. Febniary7,2001 Page 13 of 13 Study Director: PSatutrdiyciDa Eir.eNctoorker, PhD ., D.A.B.T. `X j l ! eD( ate Sponsor's Monitor: INITIALS ONLY (See page 2) Date oi/nfaManagement Approval: WardR. Director, ity AssessW nt DepartAm.Cen.Vt,JSo. uthern Research/InstitDutaete 001640 Appendix B Analytical M ethod ifnorMDoentkeermy Sineartaiomn aonfdPeUrfrliunoerohexanesnlfonate 001641 B-l Page 1 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: PD(HreePtpeLarmCra/itMnioaSnti/oMnanSod)f PAenrfalluyosrioshebxyanHesPuLlfConaMteaisns MSopnekcteryamSeertruym/Maansds USripneec:trSoammeptrlye 1.0 PRINCIPLE Serum or urine samples are obtained from cynomolgus monkeys treated with Piraseseacxecmetrfroftoaalpnurctltseoetitif,rsioiotefunahidtlreweteexwdiratteihhnitdnheee,sn9tuaha5nlmnyf%odliinnxatmcaetreetadeertnant(wshtaPefalieF.tnrhHsorTtealSahdnc)ne.doteoianTotrhtdhnaaye-iulnp(tsIaioaSencisr)rgeau,itmnmaP1gtpe.eo5rlrlerfe%alruauygrvoeifenirroanoeirlstoms,(,crebeitaca.mugnnfa.ode,fcevc0iaraed.5ndr,aba,mn5loeydLxv%zaa)welpcda5aootmtebrneaytrM(ta,PeiHdnfFoaiPOtnmloLlgComdCw()rP.oyeMFnndHTieuabshSmssyes), rSwfSerpaolitimemhacbptcrallooeebmnsortecuresootturln5ylbtt/astMloainpna2irknsi0sogm,r0SPt0apoFt0erHicanxtnSrgao/slammoytsLetcihtsoroa.yntfc(tPHehnFePtHcrLaoSCtnio/icMnnesnSsteg/rMraruetmiSaot)ne.arTontdhhf ePafnrFroaH2mn0Sg,e01w00oi0fltlornebg5lei/0amw0bLlinethgmri/enmastuyLhlbetisenreaduxnirtlgeiuennteedo.dsf TtThheheefmoioramnssasptsiropaneycostrofouomrtcheeetrrvypooolttfaegPneFtiHiaslSlsyeatinnadtte-Prf2Fe0Or0inC0gvisioolantcsscweoxhmtircpahlcisitsehdelodfwrionemtnhotehuengehmgtaoattrigivxree.aitolyn rmedoudcee. pCiunrnAcoilcvuUeeddTriunsIrOagelbsNplto:rooeSbdciean, pucueltsaieopsdmnriswma.haanetRendsehsfmeearrnautydmolci,nSaagrOrreuyPntaopnnrbueumesmecbrobevneresrdSidoRpefrIrziem2od-oa5ants-eo5bstiiefsoossh,ruaaeazl.ladrudensspcarrneipsdetihrovanneddolfteidsssawufeeittshy, 2.0 REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 2.1 Neat Reagents 2.1.1 Water, deionized and organic free (from in-house purification system; e.g., Ingalls 21ON) 2.1.2 Methanol, HPLC grade 001642 B-2 Page 2 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: DP(HreePtpeLarmCra/itMnioaSnti/oMannSod)f PAenrfalulyosrioshebxyanHesPuLlfConaMteaisns MSopneckteryomSeertruym/Maansds USripneec:trSoammeptrlye 2.1.3 Perfluorohexanesulfonate (analyte), as provided by the client 2.1.4 Perfluorooctanecarboxalale (internal standard), 97% 2.1.5 Ammonium acetate, HPLC grade 2.1.6 Blank control monkey serum 2.1.7 Sodium Carbonate, Certified ACS Grade or equivalent 2.1.8 Sodium Bicarbonate, Certified ACS Grade or equivalent 2.1.9 Ethyl Acetate, HPLC grade 2.1.10 Tetrabutylammonium Hydrogen Sulfate, Aldrich 97% 2.1.11 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.1.12 Blank control monkey urine 2.1.13 Formic Acid, 88% 2.2 Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 2.2.1 5 mM Ammonium acetate in organic free water 2.2.1.1 Fifnioltroraertgxioaanmnicap-plfepr,eateroawtpurase.tpearre(e4.gl.i,te4rsL, m). eaMsuirxe wouetllamanmdofnilituemr tahcreotuagteh(eH.gP.L, 1C.5m42ogb)ileanpdhaadsde 2.2.2 TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) 001643 B-3 Page 3 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: DPreetpeanrnaitnioantionanodf PAenrialulyosrioshebxyanHesPuLlfConaMteaisns MSopneckteryomSeertruym/Maansds USripneec:trSoammeptrlye (HPLC/MS/MS) 2.2.2.1 FhNaydoodjrtureeos:xtgamaemnmepnsolutersl,ef.atodteiplirunetpedaesrioeoln2ui5tzieomdnLwo, adftiNesrsaoaOlnvHdeaaidnpjpuwrsotaxtthiemer apmtHealyyto4b.1e204uwsgeitodhft5toe0t%reafbfNeuctaytOlsaHmmasmolloleunrtiiupomnH. 2.2.3 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) 2.2.3.1 Faeopnrpseuroxreaxmicmopmaletep,ltlyoet2pe.r1de0ipsagsroeoluf1ts0ioo0dnmi.uLm, bdiicssaorblvoenaaptepirnox1i0m0amtelLyo2.f6d5eigonoifzseoddwiuamtecr.arMboinxawteeallntdo 2.2.4 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) 2.2.4.1 Faepnopsrureorxexaicmmopmalteep,llyteot8ep.4dreigspsaoorfleust1oi0od0niu.mmLb,idciasrsboolvneataepipnro1x0i0mmatLelyof1d0.e6iognoizfesdodwiuatmer.caMrbioxnwateelalntod 3.0 INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents may be used. 3.1 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and DCA-Vibrax platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with source ofnitrogen) 3.5 HPLC mobile phase filtration apparatus 3.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 pm) 001644 B-4 Page 4 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample P(HrePpLaCra/tMioSn/ManSd) Analysis by HPLC Mass Spectrometry/Mass Spectrometry 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-concentration apparatus 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 4.1 oMAfpatphinreoSpanrtioaacltyeksctS.hoaAlnugctteiuosanilnodtifhlPuetFciooHnnSscew~n1itrl0la0bti0oenpdsgoo/cmuftmLheenstoeldutoionntshme paryebpearmataiodne asthteheetsd.iscretion 4.1.1 Paocrrgecpuanararicete-flayrenwe-we1i0gath0e0ratbopogdu/itmss1Lo0lvmseog.luDPtiFioluHntSeotiofnttPohFeaHmS1a0r-ikmn. LdAevlitooelnrunimzaeteidvtreioclyrgf,laawnsikec)i-g.frheAtehdewdcadotemeiropn(oeiuz.gnedd., 001645 B-5 Page 5 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: PD(HreePtpeLarmCra/itMnioaSnti/oMnanSod)f PAenrfalluyosrioshebxyanHesPuLlfConaMteaisns MSopneckteryomSeertruym/Maansds USripneec:trSoammeptrlye iwnatotear.n Mapipxrowperlila.teTvreasnssefel r(eth.ge.,scoululttuiorenttuobae)claenadnavdedss1e0l imf dLeosifredde.ionized organic-free 4.2 Stock Solution of Internal Standard (PFOC), ~200 pg/mL 4.2.1 Pwttddohreeeeeidspioigiarcnsehrosiedozma.aelbvndpoeo~uoua2trnn0g1dd0a0dpnimiginlc/utg-motfeirLneatetosoaotwnhaluea5tatim0epor-pna.mrrookLMfpwPvriiFioxatOhltuewdCmeeviielenolt.srnsdiiceezTlieforld(aanensoi.zkgsrefg).,ed.arncoAiutrchgdl-etadfurnresdieeoce-liwtufourtanebitoeieeznr)we.dtAaaotnoeltdrraeg(raaencnd.lageitdc.ia,v-na5efcr0lveyceeu,mswrwsaLeetaelitgloeyihrff 4.3 Spiking solution of Internal Standard (PFOC), ~ 50pg/mL 4.3.1 P2MrmeixpLawroeeflalt.hne~25000ppgg//mmLLssoolluuttiioonnoinftPoFaOcCulitnurdeetiuobneizaenddoargdadn6icm- fLreoefwdaetieornbizyepdipweattteinr.g 4.4 W orking Stock Solutions of PFHS 4.4.1 tmTaobolpderi.feipPeadrreedpiwaluroetrioiknnin1gs0cs-htmeomcLkevsocoallunumtbioeentursis,cemfdlaaaksnekdsthdoeorpcorutomhpeeernratdpeidpluritonioptnrhisaetaesstguslhdaoyswsrwencaoirnred.thsI.effdoellsoirweidnga 001646 B-6 Page 6 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: DP(HreePtpeLarmCra/itMnioaSnti/oMnanSod)f PAenrfalluyosrioshebxyanHesPuLlfConaMteaisns MSopneckteryomSeertruym/Maansds USripneec:trSoammeptrlye WApoprkroinxgimS(atnotgec/kmCLoLne)vceeln(trWatSioLn) Volume ofPFHS solution 500,000 250,000 62,500 31,250 15,625 5,000 2,500 1,000 500 250 5 mL of 1000 pg/mL 5 mL of 500,000 ng/mL 2.5 mL o f250,000 ng/mL 5 mL of 62,500 ng/mL 5 mL of 31,250 ng/mL 50 pL of 1000 pg/mL stock 5 mL o f5000 ng/mL stock 4 mL of 2500 ng/mL stock 5 mL of 1000 ng/mL stock 5 mL of 500 ng/mL stock dFferiienoeanlwizVeaodteluormr(gmeaLnin)ic 10 10 10 10 10 10 10 10 10 10 001647 B-7 Page 7 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample P(HrePpLaCra/tMioSn/ManSd) Analysis by HPLC Mass Spectrometry/Mass Spectrometry 4.4.2 Summary of concentrations of serum standards: StLaenvdealrd A B C D Volume and Spike Cone. 20 pL of 500,000 ng/mL 10 pL of 500,000 ng/mL 10 pL of 250,000 ng/mL 16 pL of 62,500 ng/mL CPoAFnHpc(pSnergnoi/ntmxraismLteio)rauntmeo f 20,000 10,000 5,000 2,000 E 16 pL o f31,250 ng/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 UU1648 B-8 Page 8 of 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: DP(HreePtpeLarmCra/itMnioaSnti/oMnanSod)f PAenrfalulyosrioshebxyanHesPuLlfConaMteaisns MSopnekcteryomSeertruym/Maansds USripneec:trSoammeptrlye 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Aofpdpireopanriaaltyesct.hanges in the concentrations ofthe solutions may be made at the discretion 5.1 Mbaneuaallyntizapellydez(weedi.gtih.f,edaaebcsohiurestedtth.orefeu)nskentsowofnmsaamtripxlesst.aAndmaradtsriaxnddoaumbleatbrilxanbkla(nbkla(nbkl-aInSk) m+ aISy)aalsroe 5.2 Ipooanpriftpgpoieanrntotienip1crd0rn-iifaavprtlieLedseutvoawaonflalodu~traemg2rrda0ein-nsmaitscsotL-ecdfakrecdesu(e)c~lwart5uinb0ardeetpedvtgruoi/inmbrnteestLshtxe,)eafptdootiaprobe~ealfte5ctbhhaslebeatucownbovkoenermdekfsxoia.ncrtgreeipxasttco(thehc.eksgt.bas,nol0adln.u5aktri-domI.nSL.F(Ap)o.idrpPdteihtpthee1ebt0li1pan0nLtkphosLe,f 5.3 Fosfc5ooarosglrlrleubaoctosnwioeonincrinanudt,fgmesr1/.ebtemsiocawaLmerabapotceolfehrn.0sa.tt2aVue5dbobMderutt/efh10fxee.2mre5faaoLMnclhdlooctw1faurmbiTbneLogBnfAotoaorftediae/oebbaniciooc-hupnataritzbui5reobidsnneeg:awcto5easn0tobed0lurus.f.ptfiVLFeornoo,o,rratfmen1txdhimnee1eaLTcmshBaoLmtAufopb1ifleoe.d0nsfeoM-aiprod/aan1dibi.r0ziotnheMudget 5.4 Asedttdin2g..5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed 5.5 Rmeinmuotevse. the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 5.6 Rh(eee.agmt.,o(e~v.eg5.t0,h5me0tion*pCu)t.eesthiynltahceeTtauterbloay-Veraapnd )pwutitiht iantgoeantclelesatnretaumbeoafnnditervoagpeonraatnedtomdordyenreastes