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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. Box 33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 By: P.E. Noker and G.S. Gorman Southern Research Institute 2000 Ninth Avenue South 35205 P.O. Box 55305 Birmingham, Alabama 35255-5305 ABSTRACT The pharmacokinetics and urinary excretion o f perfluorohexanesulfonate were investigated in male and female cynomolgus monkeys. Three male and three female monkeys were administered a single iv bolus dose o f 10 mg/kg o f 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 o f intact perfluorohexanesulfonate. The lower limit o f 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 140,900 ng/mL in male monkeys and from 116,400 to 174,000 ng/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 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 o f perfluorohexanesulfonate 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 o f perfluorohexanesulfonate 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 o f 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 o f distribution o f 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 o f the compound were likely attributable to an overestimation o f the extrapolated AUC0.infinityvalues for two o f the female monkeys. Only very low levels (<0.01 to 0.11% o f the administered dose) o f perfluorohexanesulfonate were measured in urine during any given 24hour period o f sample collection between Day 1 and Day 70 after dosing. The results o f this study suggested that the pharmacokinetics o f perfluorohexanesulfonate 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 of time (>70 days) after iv administration. 1 TABLE OF CONTENTS Page SIGNATURE PAGE iii 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 5 5 5 5 5 4.0 DISCUSSION 7 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 Pharmacokinetic 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 Page 9 13 14 15 18 A-l B-l Ill Signature Page A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey --------------------------------------------------- V Patricia E. Noker, Ph.D., D.A.B.T. Study Director Supervisor, ADME & Pharmacokinetics / <3-/ q-^? Date Reviewed by: Charles D. Hbert, Ph.D., D.A.B.T. Director, Safety Assessment Date We, the undersigned, were responsible for the conduct o f the work and reporting o f the results in the listed sections. We concur with the views relative to our body o f work as expressed in the discussion and conclusions. 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 o f the study. There were no adverse circumstances that affected the quality or integrity o f the study. Patricia E. Noker, Ph.D., D.A.B.T. Study Director Date Study Dates: V Study Schedule and Personnel Study Initiation: Day of Dosing: Last Day o f 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. Hbert, 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. Hoskins, 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 1.0 Introduction The objectives o f this study were to determine the concentration of potassium perfluorohexanesulfonate in serum and to estimate urinary clearance at various times following administration o f a single intravenous dose to monkeys. A copy o f 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 BRF, Inc. (Houston, TX) and were an estimated 3-4 years o f 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 o f 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. 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 o f the daily ration was sufficient to meet nutritional requirements. In addition, the diet was supplemented with fresh ffuit/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 o f the study, the monkeys were housed in a room that was maintained at a temperature o f 67.7-72.8 F and a relative humidity of 29-74%. The humidity was within the required range (30-70%) over 90% o f the time during the study; excursions below the recommended humidity range were o f short duration and had no impact on animal health or the outcome o f the study. Room lights were controlled by an automatic timer set to provide 12 hours o f light (0600 to 1800 hours, CST) and 12 hours o f dark per day. Cage size and animal care conformed to the guidelines o f the Guidefo r the Care and Use o fLaboratory Animals, 7th edition(1) and the U.S. Department o f 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 o f Laboratory Animal Care International. With the exception o f animal 2053, all o f 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 o f 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 o f potassium 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 o f 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 o f the test article was the responsibility of the Sponsor. Vehicle: The vehicle used for the preparation o f the dose formulation o f 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 o f potassium perfluorohexanesulfonate prepared at 5 mg/mL, the required amount o f 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. 2.3 Experimental Design Group Assignment and Dose Procedure: As only one treatment group was used in this study, no formal randomization was required. On Day 0, each o f the three male and three female monkeys received a single intravenous (iv) dose o f 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 o f 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 o f toxicity. Additional clinical observations were performed on days o f 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 o f 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 T o r below) until analyzed. Bioanalytical Method Development and Sample Analysis: Serum and urine samples were 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 o f 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 o f the monkeys in this study survived to the end o f the study. 3.2 Clinical Observations No adverse drug-related clinical signs were noted for any monkey during the course o f 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 perfluorohexanesulfonate in three male and three female monkeys at various times through Day 171 after administration o f a single iv dose o f 10 mg/kg are presented in Table 2 and Figure 1. No sex-related differences were apparent in serum concentrations o f perfluorohexanesulfonate at any time o f sample collection. Peak serum concentrations o f 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 of perfluorohexanesulfonate (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 o f the samples had occurred. Subsequent to the time o f peak levels, serum concentrations o f perfluorohexanesulfonate 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 the three male monkeys and from 27,115 to 54,300 ng/mL in the three female monkeys. Serum 6 concentrations o f perfluorohexanesulfonate decreased at a slow rate between 24 hours and the end o f sample collection (171 days). During this period, fluctuations were 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 o f the large dilution o f 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 o f the data. No distinct differences were apparent between male and female monkeys in the estimated parameters; however, for two o f the three female monkeys, the serum half-life and clearance o f perfluorohexanesulfonate were shorter and faster, respectively, than observed for the three male monkeys and the other female monkey. AUC0.infinity values ranged from 6456 to 8745 g^day/mL in male monkeys and from 3849 to 8501 ,ugday/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 o f distribution o f perfluorohexanesulfonate ranged from 223 to 391 mL/kg in male monkeys and from 160 to 255 mL/kg in female monkeys. The amount o f 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 o f 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 o f the compound were present in urine on Day 70 (last day of urine collection) after dosing. 4.0 Discussion The results o f this study indicated that perfluorohexanesulfonate was slowly eliminated by male and female monkeys given a single iv dose o f 10 mg/kg. Throughout the period o f sample collection, serum concentrations o f perfluorohexanesulfonate were similar at each sample time in both male and female monkeys. In addition, the rate o f urinary excretion o f perfluorohexanesulfonate 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 o f the three female monkeys on the current study, the estimated serum half-life and total body clearance o f 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.infinitityvalues; these latter values were obtained from extrapolation o f the serum concentration time curve and may have contained considerable error. That the AUC0_infinity values for two o f the three female monkeys may have been overestimated is supported by the observation that estimated AUC0_last 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 o f 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 o f time (>70 days) after iv administration. 8 6.0 Record Archives Data, specimens, and a copy o f 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 o f the Sponsor's Monitor, the data and any samples/specimens will 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. 7.0 References 1. Institute o f Laboratory Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington D.C.; 1996. Table 1 A Pharmacokinetic Study o f 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 Page 1 of 4 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 63 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 Page 2 of 4 Table 1 (Continued) A Pharmacokinetic Study o f Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Body Weights: Females Group Sex IF 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 Page 3 o f 4 Table 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate 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 13 Table 2 A Pharmacokinetic Study o f Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentrations of Perfluorohexanesulfonate Timepoint 2053 Serum Concentration (ng/mL) Males Females 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) a Data represent an average of two samples. Page 1 o f 1 Table 3 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Pharmacokinetic Parameters Calculated from Serum Concentrations of Potassium Perfluorohexanesulfonate Male Parameter 2053 2054 221 l a Average SD C0O g /m L )b 214 159 169 181 29 AUCo-iast Og*day/mL)c 4044 4864 3309 4072 778 AUCo-infinity Ogday/mL)d 6456 8745 7186 7462 1169 ti/2 (day)6 100 124 200 141 52 Cl (mL/day/kg)1 1.5 1.1 1.4 1.3 0.2 Vdss (mL/kg)g 248 223 391 287 91 "L a m b d a z ranges were specified for the estimation o f the terminal ha lf-life bSerum concentration at time 0 cArea under the serum concentration time curve from 0 to the last time point dArea under the serum concentration time curve from 0 to infinity eHalf-life o f the terminal elimination phase f Total body clearance gVolume o f distribution at steady state 2058 178 3802 5031 72 2.0 225 2059 215 3619 3849 49 2.6 160 Female 206 l a 146 4417 8501 140 1.2 255 Average 180 3946 5794 87 1.9 213 SD 35 418 2418 47 0.7 49 Page 1 o f 1 15 Table 4 A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Urinary Excretion of Perfluorohexanesulfonate Animal ID 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 Urine Concentration Sex (ng/mL) M BQL M BQL M BQL F BQL F BQL F BQL M 48 M 193 M 79 F 22 F 60 F 484 M 65 M 17 M 10 F 60 F 193 F 709 M 28 M 16 M 182 F 64 F 15 F 121 Urine Volume (mL) Total Og) Baseline 740 - 1000 - 630 - 290 - 150 250 - Day 1 1000 48 360 69 640 51 320 7 150 9 90 44 Day 2 610 40 330 6 340 3 170 10 60 12 50 35 Day 7 490 14 210 3 210 38 290 19 180 3 70 8 Dose O g) 60,000 62,000 54,000 36,000 37,000 41,000 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 51,000 34,000 35,000 41,000 Percent of Dose in Urine (%) -- -- -- - -- -- 0.08 0.11 0.09 0.02 0.02 0.11 0.07 0.01 0.01 0.03 0.03 0.09 0.02 0.01 0.07 0.05 0.01 0.02 BQL = Below the quantitation limit (<1 ng/mL) Page 1 of 3 16 Table 4 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Animal ID 2053a 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 Urinary Excretion o f Perfluorohexanesulfonate Urine Concentration Sex (ng/mL) M5 M8 M7 F 167 F 72 F 129 M 10 M 27 M4 F 49 F 237 F 40 M5 M8 M 10 F 16 F 25 F 35 M 34 M 55 M 24 F 74 F 28 F 14 Urine Volume (mL) Total Og) Day 14 280 1 740 6 660 5 170 28 210 15 110 14 Day 21 440 4 320 9 530 2 100 5 110 26 90 4 Day 28 660 3 830 7 520 5 270 4 200 5 150 5 Day 42 450 15 440 24 820 20 220 16 160 4 180 3 Dose G^g) 60,000 62,000 54,000 36,000 37,000 41,000 60,000 62,000 54,000 36,000 37,000 41,000 60,000 62,000 54,000 36,000 37,000 41,000 60,000 62,000 54,000 36,000 37,000 41,000 Percent of Dose in Urine (%) <0.01 0.01 0.01 0.08 0.04 0.03 0.01 0.01 <0.01 0.01 0.07 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.03 0.04 0.04 0.05 0.01 0.01 BQL = Below the quantitation limit (<1 ng/mL) Page 2 of 3 17 Table 4 (Continued) A Pharmacokinetic Study o f Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Urinary Excretion of Perfluorohexanesulfonate Animal ID 2053" 2054 2211 2058 2059 2061 2053 2054 2211 2058 2059 2061 Urine Concentration Sex (ng/mL) M 15 M 48 M7 F8 F 11 F 70 M6 M 38 M3 F 20 F 28 F 177 Urine Volume (mL) Total (M) Day 56 510 8 450 22 510 4 330 3 160 2 200 14 Day 70 510 3 430 16 640 2 220 4 150 4 170 30 Dose (M) 60,000 62,000 54,000 36,000 37,000 41,000 60,000 62,000 54,000 36,000 37,000 41,000 Percent of Dose in Urine (%) 0.01 0.03 0.01 0.01 <0.01 0.03 0.01 0.03 <0.01 0.01 0.01 0.07 BQL = Below the quantitation limit (<1 ng/mL) Page 3 of 3 18 M2053 -e - Observed -- Predicted Serum Concentration (ug/mL) Serum Concentration (ug/mL) M2054 -- Observe -- Predicted Figure 1 A Pharmacokinetic Study o f Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate Serum Concentration (ug/mL) 1000 > 100 \ 1___________ e>--O-----O---- 3-- 0------- o 'o 10 19 M2211 u- ------ o -e- Observe -- Predicted 20 40 60 80 100 120 140 160 180 Time (Days) F2058 -- Observe -- Predicted Seum Concentration (ug/mL) Figure 1 (Continued) A Pharmacokinetic Study of Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate 20 F2061 Observe -- Predicted Serum Concentration (ug/mL) Serum Concentration (ug/mL) F2059 -- Observe -- Predicted Time (Days) Figure 1 (Continued) A Pharmacokinetic Study o f Potassium Perfluorohexanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorohexanesulfonate Appendix A Study Protocol 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 STUDY NO.: 9921.5 A -2 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 7,2001 P ase 2 o f 13 Test Article: Ship Unused Test Article to: John L. Butenhoff Date Perfluorohexanesulfonate, potassium salt D. Hakes Building B236 3M Center P.O. Box 33327 55133-3327 St. Paul, Minnesota 55144-1000 A-3 STUDY NO.: 9921.5 2.0 TITLE: February 7, 2001 Page 3 o f 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 f perfluorohexanesulfonate 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 Day of Treatment Urine and Feces Collections Serum Drug Levels Draft Report Due Final Report 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 o f the in-life phase 15 days after final Sponsor comments received Date(s) - Year 2001 2/9/01 2/9/01 2/10/01 2/11/01 2/16/01 2/23/01 3/2/01 3/9/01 3/23/01 4/6/01 4/20/01 5/11/01 2/9-10/01 2/11/01 2/13/01 2/16/01 2/23/01 3/2/01 3/9/01 ! 3/23/01 4/6/01 4/20/01 5/11/01 7/10/01 -- A-4 STUDY NO.: 9921.5 6.0 STUDY PERSONNEL: February 7, 2001 Page 4 o f 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 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 o f stability, as well as methods o f synthesis, fabrication, or derivation. Upon completion of the study, residual bulk test article will be returned to the Sponsor. 7.1 Identity of the Test Article: Name: Identification: Supplier: Lot Number(s): Special Handling: Perfluorohexanesulfonate, potassium salt T-7504 3M To be documented in the study data. None Characterization: Documentation o f the characterization o f the test article, including identity, purity, strength, and composition, as well as methods o f synthesis, fabrication, or derivation, is the responsibility o f the Sponsor. Copies o f characterization data have been provided to the testing laboratory. Stability & Storage: The bulk test article will be stored at room temperature. Stability o f the bulk test article is the responsibility of the Sponsor. A -5 STUDY NO.: 9921.5 7.2 Identity of the Vehicle: Febniary 7,2001 Page 5 o f 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 o f 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) o f 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 o f 5 mg/mL for intravenous administration; briefly, the required amount o f 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 o f 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 (M acaca fa sc ic u la ris) Charles River BRF, Inc. (Houston, TX) 3-4 years of age (estimated) 3-7 kg Males -3 Females -3 Animals were previously dosed with potassium perfluorobutanesulfonate in study 9921.1, potassium perfluorobutanoate in study 9921.2, potassium perfluorohexanoate in study 9921.3, and potassium perfluorooctanoate in study 9921.4. A-6 STUDY NO.: 9921.5 8.1 Justification: February 7,2001 Page 6 o f 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 minimum o f 10 air exchanges per hour. Controls will be set to maintain the animal room at a temperature o f 64-84 F and a relative humidity o f 30-70%. A 12-hour light/12-hour dark cycle will be routinely maintained. Animals will be acclimated in the same room used for study. 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 used for excrement absorption. Analyses o f the bedding, supplied by the vendor, will be reviewed by the Department o f Veterinary Medicine and Bioresources (DVMB) o f Southern Research to assure that no known contaminants are present that could interfere with or affect the outcome o f 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 fresh fruit offered daily and treats offered several times each week. The quantity o f the daily ration will be sufficient to meet nutritional requirements. Analyses o f the feed, supplied by the vendor, will be reviewed by the DVMB o f Southern Research to assure that no known contaminants are present that could affect the health o f 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 o f water from 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 o f the animals. A-7 STUDY NO.: 9921.5 8.6 Quarantine: February?, 2001 ___Page7 o f 13 All primates were selected from stock animals that were quarantined for a minimum o f 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 o f 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 Psychological Well-Being and Socialization: Nonhuman primates will be provided a psychological well-being program for social enrichment as directed by a veterinarian and approved by the LACUC and in accordance with the appropriate SOP. Nonhuman primates will be provided cage and feeding regimen modifications daily for their psychological well-being. The modifications include, but are not limited to: swings, perches, Kong toys, clean 2liter soft drink bottles, puzzle feeders, nutritionally sound primate treats, unshelled peanuts, and raw fruit. Where possible, primates will be housed proximate to one another for visual and vocal contact. 8.8 Animal Identification: 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 9.0 EX PERIM EN TA L DESIGN: February 7,2001 Page 8 o f 13 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 pre determined intervals. A synopsis o f the study design is presented in the following table. Study Procedures Day o f Stucly - l a 0 1 2 4 7 14 21 28 35 42 49 56 63 70 77 84 91 Health Check X Dose Preparation X D oing * MS ' i Clinical Observations XXXXXXXX X X X . X Body Weights X XXXXXXXXXXX X X X Urine & Feces X XX XXXX X X X X Serum Drug Levels X X X X X X X X X X X X Denotes week 9.1 Randomization & Group Assignment: As only one treatment group will be used in this study, no formal randomization will be required. 9.2 Dose Procedure: Each primate (three males, three females) will receive a single intravenous (TV) dose o f 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 o f dosing will be Day 0 o f the study. 9.3 Clinical Observations: Daily O bservations: 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 STUDY NO.: 9921.5 February 7, 2001 Page 9 o f 13 study for signs o f mortality/moribundity and overt toxicity. Animals found in extremis will be humanely sacrificed by an overdose o f barbiturate followed by exsanguination with appropriate approval. Detailed Observations: Each primate will be examined shortly after dose administration for detailed clinical signs o f toxicity. All findings will be recorded. Additional clinical observations will be performed and recorded on days o f blood 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: Urine and feces will be collected for approximate 24 horn intervals prior to dosing and on Days 1 (0-24 hours postdose), 2 (24-48 hours post dose), 7 ,1 4 ,2 1 ,2 8 ,4 2 , 56, 70, and 91. The volume o f each urine sample will be measured upon collection. All samples collected will be stored frozen at -2 0 C or below prior to analysis (urine) or until further notice by the Sponsor (feces). 9.6 Serum Drug Levels : Blood samples (approximately 3 mL) will be collected from each primate at approximately 0 (predose) minutes; 0.5,2, 4, 8, and 24 hours; and 2 ,4 , 7 ,1 4 ,2 1 ,2 8 , 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 frozen at -2 0 C or below until analyzed. 9.7 Bioanalytical Method Development: 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 and/or other tissues require analyses, these analyses will be negotiated with the Sponsor. 9.8 Bioanalytical Sample Analysis: The serum and urine samples from all monkeys will be analyzed for concentrations o f perfluorohexanesulfonate using the previously validated method. The data will be A-10 STUDY NO.: 9921.5 February 7, 2001 Page 10 o f 13 expressed as equivalents o f potassium perfluorohexanesulfonate. Feces will be analyzed only if requested by the Sponsor. 9.9 Animal Disposition: A t the end o f the study, monkeys will be maintained in the stock colony. In the event untoward reactions or other conditions warrant the euthanasia o f a monkey at any time during the sample collection period, a serum sample (5-10 mL) will be obtained from 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: Pharmacokinetic parameters (e.g., AUC, half-life, clearance) will be estimated from serum concentrations o f unchanged perfluorohexanesulfonate, as appropriate and feasible, using a standard pharmacokinetic program. The total amount o f perfluorohexanesulfonate in urine will be calculated and expressed in terms o f percent of dose. Mean values and standard deviations will be calculated for each time point and sample type, as appropriate. No other statistical analyses o f the data will be performed. 11.0 RECORDS: All raw data pertaining to the conduct o f 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 o f the final report by the Sponsor. After 1 year and with the permission o f the Sponsor's Monitor, the data and any samples/specimens will 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 o f the final report will be retained in the central archives at Southern Research. 12.0 FINAL R EPO R T: 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 o f the in-life aspects o f the study. The final report (electronic and hard copies) will be issued within 15 working days after receipt o f 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 -ll STUDY NO.: 9921.5 February 7, 2001 Page 1 1 o f 13 Dose formulation preparation Clinical observations Body weight data Serum drug level data Pharmacokinetic parameters Urine excretion data 13.0 REGULATORY R EFEREN CES: This study will be conducted in accordance with the protocol and the Standard Operating Procedures (SOPs) of Southern Research, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 protocol Amendments and deviations: Amendments: All changes in or revisions o f the approved protocol and the reasons thereof will be documented, signed, and dated by the Study Director, and the Sponsor's Monitor. Amendments will be maintained with the protocol. Written approval (a fax signature or electronic communication, such as email) for changes in the protocol may be granted by the Sponsor's Monitor, but a written amendment will follow. Deviations: All operations pertaining to this study, unless specifically defined in this protocol, will be performed according to the Standard Operating Procedures (SOPs) o f Southern Research and/or the protocol, and any deviations from protocol or SOP will be documented. 13.2 Regulatory Compliance: Good L aboratory 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 from this study may be submitted to the FDA in support o f an IND/NDA 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 Facilities Management and Animal Husbandry: February 7, 2001 Pape 1 2 o f 13 Animal care will be in compliance with the SOPs of Southern Research, the Guidelines fo r the Care and Use o f Laboratory Animals, 7thEdition (Institute o f 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 (AAALAC). 13.4 Animal Welfare Act Compliance: By signing this protocol, the Sponsor signifies that there are no generally accepted alternatives to the use o f 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 o f these agents has been justified in writing by the Study Director and/or Sponsor and approved by the IACUC. The number o f animals selected for use in this study is considered to be the minimum number necessary to meet scientific and regulatory guidelines for this type o f 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. A-13 STUDY NO.: 9921.5 14.0 PR O TO C O L APPROVALS: This protocol has been reviewed and approved. February 7,2001 Page 13 o f 13 Study Director: Patricia E. Noker, Ph.D., D.A.B.T. Study Director j-Q l__ Date Sponsor's Monitor: _________________________________________________________ INITIALS ONLY (See page 2) Date Appendix B Analytical M ethod for Determination of Perfluorohexanesulfonate in M onkey Serum and Urine B-l Page 1 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 1.0 2.0 2.1 2.1.1 2.1.2 PRINCIPLE Serum or urine samples are obtained from 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 % 5mM ammonium acetate, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS). The range o f reliable results extends from about 5 to 20,000 ng/mL o f PFHS in serum and from 10 to 500 ng/mL in urine. Samples containing PFHS at concentrations greater than 20,000 ng/mL may be diluted with control blank matrix so that the concentration o f PFHS will be within the range of reliable results prior to analysis. The mass spectrometry o f PFHS and PFOC is accomplished in the negative ion mode. The ion spray source voltage is set at - 2000 volts which is low enough to greatly reduce the formation o f 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. REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. Neat Reagents Water, deionized and organic free (from in-house purification system; e.g., Ingalls 21 ON) Methanol, HPLC grade B-2 Page 2 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.3 Perfluorohexanesulfonate (analyte), as provided by the client 2.1.4 Perfluorooctanecarboxalate (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 o f the analyst 2.2.1 5 mM Ammonium acetate in organic free water 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. 2.2.2 TBA Ion-Pairing Solution (0.5 M tetrabutylammonium hydroxide) B-3 Page 3 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.2.2.1 For example, to prepare 25 mL, dissolve approximately 4.24 g o f tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. Note: a more dilute solution of NaOH in water may be used to effect smaller pH adjustments. 2.2.3 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M) 2.2.3.1 For example, to prepare 100 mL, dissolve approximately 2.65 g o f sodium carbonate and approximately 2.10 g o f sodium bicarbonate in 100 mL o f deionized water. Mix well to ensure complete dissolution. 2.2.4 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) 2.2.4.1 For example, to prepare 100 mL, dissolve approximately 10.6 g o f sodium carbonate and approximately 8.4 g o f sodium bicarbonate in 100 mL o f deionized water. Mix well to ensure complete dissolution. 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 UCA-Vibrax platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with source o f nitrogen) 3.5 HPLC mobile phase filtration apparatus 3.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 pm) B-4 Page 4 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 3.16 3.17 4.0 4.1 4.1.1 Analytical balance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipettes Micropipettor(s) with tips Culture tubes with teflon-lined caps Centrifuge Assorted glassware and syringes Culture tubes (vials) for use with solvent-concentration apparatus 1 mL Plastic syringes with 0.2 pm PVDF syringe filters Variable speed horizontal platform shaker pH meter PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations o f the solutions may be made at the discretion o f the analyst. Actual dilutions will be documented on the preparation sheets. Main Stock Solution of PFHS ~I000 pg/mL Prepare an -1000 pg/mL solution o f 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 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 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 o f 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 ~200pg/mL solution o f 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-free water. 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), ~ 50pg/mL Prepare an ~ 50 pg/mL solution o f 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 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Working Stock Level (WSL) Approximate Concentration (ng/mL) Volume of PFHS solution 500,000 5 mL o f 1000 pg/mL 250,000 5 mL of 500,000 ng/mL 62,500 2.5 mL o f 250,000 ng/mL 31,250 5 mL o f 62,500 ng/mL 15,625 5 mL of 31,250 ng/ mL 5,000 50 pL o f 1000 pg/mL stock 2,500 5 mL of 5000 ng/mL stock 1,000 4 mL o f 2500 ng/mL stock 500 5 mL o f 1000 ng/mL stock 250 5 mL of 500 ng/mL stock Final Volume in deionized organic free water (mL) 10 10 10 10 10 10 10 10 10 10 B-7 Page 7 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.4.2 Summary o f concentrations of serum standards: Standard Level Volume and Spike Cone. A 20 pL of 500,000 ng/mL B 10 pL o f 500,000 ng/mL C 10 pL o f 250,000 ng/mL D 16 pL o f 62,500 ng/mL Approximate Concentration of PFHS in serum (ng/mL) 20,000 10,000 5,000 2,000 E 16 pL o f 31,250 ng/mL F 16 pL o f 15,625 ng/mL G 20 pL of 5,000 ng/mL H 10 pL o f 5,000 ng/mL I 10 pL o f 2,500 ng/mL J 10 pL o f 1,000 ng/mL K 10 pL o f 500 ng/mL L 10 pL o f 250 ng/mL 1,000 500 200 100 50 20 10 5 B-8 Page 8 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion o f the analyst. 5.1 Multiple (e.g., about three) sets o f matrix standards and a matrix blank (blank + IS) are analyzed with each set o f 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 o f organic-free water instead o f the working stock solution. Add the 10 pL o f internal standard stock (~50 pg/mL) to each tube except the blank-IS (pipet 10 pL of organic-free water instead) and vortex for ~5 seconds. 5.3 For serum samples add the following to each tube: 500 pL o f the TBA ion-pairing solution, 1 mL of 0.25M/0.25M carbonate/bicarbonate 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 o f TBA ion-pairing solution, 1 mL o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds. 5.4 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 5.5 Remove the tube from 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 ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). B-9 Page 9 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 5.7 Reconstitute the residue in 500 pL o f 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 o f each sample (e.g., 0.5 mL) into individual ~20-mL culture tubes. If necessary, dilute an aliquot o f any sample with blank matrix so that the expected concentration o f the test article being analyzed will fall within the concentration range o f 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 o f the TBA ion-pairing solution, 1 mL o f 0.25M/0.25M carbonate/bicarbonate 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 o f TBA ion-pairing solution, 1 mL o f 1.0M/1.0M carbonate/bicarbonate buffer and 1 mL o f deionized water. Vortex each tube for about 5 seconds. 6.3 Add 2.5 mL o f ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 6.4 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 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 ) with a gentle stream o f nitrogen and moderate heat (e.g., 50 C). 6.6 Reconstitute the residue in 500 pL o f 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 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 7.0 7.1 7.1.1 7.1.2 ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY (HPLC/MS/MS) Conditions are to be optimized if necessary. HPLC Conditions Analytical Column: Keystone Scientific Aquasil C l8,150 m m x 2 m m 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: 5mM 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 10%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 -ll Page 11 o f 14 ANALYTICAL METHOD Method No. : BACG-3606 Title: Determination o f Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter NC TEM OR RNG QO IQ1 ST ROl 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 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) Parameter IPE Value 0 MS/MS Acquisition Conditions Scan type: MRM Polarity: Negative Acquisition mode: Profile Pause time: 5 milliseconds Masses requested: PFHS: 0 1 M ass (amu) 398.9 0 3 M ass (am ui 398.9 D w e ll T im e Citisi 200 PFO C (IS) 0 1 M ass (amu) 412.9 Param eter R02 ST3 R03 QO IQ1 ST ROl IQ 2 0 3 M ass (amu) 368.9 S tart 35 45 37 18 19 23 19 20 Stop 35 45 37 18 19 23 19 20 D w ell Tim e (m st 200 B-13 Page 13 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 8.0 CALCULATIONS 8.1 At the end o f the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination o f 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 o f PFHS divided by the peak area response o f the IS (PFOC) from all standards versus the concentration of the test article in the standards. Alternatively, the peak heights may be used instead o f peak areas. Obtain the best curve fit o f the data (e.g., quadratic fit weighted with 1/concentration o f the test article or a quadratic fit). Note: The best curve fit may be dependent on the range o f 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, b, c Peak height response of PFHS divided by peak height response of the IS (PFOC) in standards, Concentration of the PFHS in standards, = Constants derived from the regression analysis. 8.3 Using the standard curve, calculate the level o f PFHS in each unknown sample. Correct the results o f samples for any dilutions. NOTE: Due to unresolvable interferences with the test article and/or internal standard from the matrix, external standard quantitation may be used at the discretion of the supervising mass spectrometrist. B-14 Page 14 o f 14 ANALYTICAL METHOD Method No.: BACG-3606 Title: Determination of Perfluorohexanesulfonate in Monkey Serum and Urine: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass 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% o f theoretical. 10.0 REPORTING 10.1 Results o f all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Author(s): Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group 7 /g ~S Date AUApproved by: Greg Gorman, Ph.D. Manager Bioanalytical Chemistry Group Date