Document NGqEVozbyR2BwdDQgKqL9jgxw

11) e, Pharmacokinetic study in the Cynomolgus Monkey, T-7485.1 , 77 y s s SANITIZED DEC 0 9 2003 Final Report on the A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey To: 3M Corporation P.O. BOX33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 By: P.E. Noker Southern Research Institute 2000 Ninth Avenue South 35205 P.O. Box 55305 Birmingham, Alabama 35255-5305 ABSTRACT The pharmacokinetics and urinary excretion of perfluorobutanesulfonate 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 of perfluorobutanesulfonate, potassium salt. At various times through 3 1 days after dosing, serum and urine (24-hour collections) samples were obtained and analyzed by HPLC/MS/MS for levels of intact perfluorobutanesulfonate. The lower limit of detection of the analytical method was 0.5 ng/mL. No sex-related differences in serum concentrations of perfluorobutanesulfonate were apparent among the six dosed monkeys. In individual animals, the concentration of perfluorobutanesulfonate in serum ranged from 19,628 to 61,740 ng/mL at 2 hours after dosing (earliest time point). By 48 hours after dosing, the serum concentration of perfluorobutanesulfonate ranged from 463 to 8,172 ng/mL in individual monkeys. Perfluorobutanesulfonate was not detectable in serum collected from any monkey on Day 31 post dose. The serum concentration versus time data were best fit to a three-compartment model. Mean serum half-life values for perfluorobutanesulfonate were 0.04, 0.55, and 4.0 days in male monkeys, and 0.06, 0.47, and 3.5 days in female monkeys. The mean AUC value calculated from serum concentrations of perfluorobutanesulfonate was 24,258 or 35,401 ng*day/mL in male or female monkeys, respectively. The total body clearance of perfluorobutanesulfonate was 511 mL/day/kg in male monkeys and 368 rnL/day/kg in female monkeys. The volume of distribution at steady state (Vd,,) was 254 or 255 mL/kg in male or female monkeys, respectively. For 5 of 6 monkeys, from 3 3 . 8 to 85.8% of the dose was recovered in urine within 24 hours after administration of perfluorobutanesulfonate. On Day 14, individual monkeys excreted less than 0.01% of the dose within a 24-hour interval. The results of this study indicated that the pharmacokinetics of perfluorobutanesulfonate were similar in male and female monkeys. Urinary excretion was a major route of elimination of the compound by monkeys. 1 SIGNATURE PAGE TABLE OF CONTENTS Plus ... 111 GOOD LABORATORY PRACTICES DISCLAIMER iv STUDY SCHEDULE AND PERSONNEL V 1.o INTRODUCTION 1 2.0 MATERIALS AND METHODS 1 2.1 Test System 1 2.2 Test Article and Vehicle Test Article - 2 7 Vehicle 2 Dose Formulation Preparation 2 Dose Formulation Analyses 2 2.3 Experimental Design 2 Group Assignment and Dose Procedure 2 Clinical Observations 3 Body Weights 3 Urine and Feces Collection 3 Serum Levels of Perfluorobutanesulfonate 3 Bioanalytical Method Development and Sample Analysis 3 Data Analyses 3 3.0 RESULTS 3.1 Mortality 3.2 Clinical Observations 3.3 Body Weights 3.4 Serum and Urine Concentrations of Perfhorobutanesul fonate 4.0 DISCUSSION 5 5.0 CONCLUSIONS 5 6.0 RECORD ARCHIVES 6 7.0 REFERENCES 6 ii TABLE OF CONTENTS (Continued) LIST OF TABLES Table 1: Body Weights Table 2: Serum Concentrations of Perflurobutanesulfonate Table 3 Pharmacokinetic Parameters Calculated from Serum Concentrations of Perfluorobutanesulfonate Table 4 Urinary Excretion of Perflurobutanesulfonate LIST OF FIGURES Figure 1: Serum Concentration Profile of Perfluorobutanesulfonate in 1M2052 Figure 2 : Serum Concentration Profile of Perfluorobutanesulfonate in 1M2054 Figure 1: Serum Concentration Profile of Perfluorobutanesulfonate in 1M2211 Figure 1: Serum Concentration Profile of Perfluorobutanesulfonate in 1F2058 Figure 1: Serum Concentration Profile of Perfluorobutanesulfonate in 1F2059 Figure 1: Serum Concentration Profile of Perfluorobutanesulfonate in 1F206 1 LIST OF APPENDICES Appendix A: Study Protocol and Amendments Appendix B : Method Validation Report: Validation of Analytical Methods for Determination of Perfluorobutanesulfonate in Monkey Serum Using HPLC/MS/MS Appendix C: Analytical Method for the Analysis of Perfluorobutanesulfonate in Serum 7 8 9 10 11 12 13 .-.14 1s 16 A- 1 B-1 c-1 ... 1U Signature Page A Pharmacokinetic Study of Perfluorobutanesulfonatein the Cynomolgus Monkey LZ//&f/.<=Z,t,& Patricia E. Noker, Ph.D., D.A.B.T. Study Director Supervisor, ADME and Pharmacokinetics Reviewed by: 3JL / o l Date Ward R. kchter, D.V.M., M.S., D.A.C.V.P. Director, Safety Assessment ' Date We, the undersigned, were responsible for the conduct of the work and reporting of the results in the listed sections. We concur with the views relative to our body of work as expressed in the discussion and conclusions. Research Chemist d -- - -- -\ \ Tsu-HanLin, Ph.D. Staff Pharmacologist 3:..4h4 ,/ Date Date iv Good Laboratory Practices Disclaimer This study described in this final report was not conducted instrict compliancewith the U.S. Food and Drug Administration's (FDA) Good Laboratory Practice (GLP) regulations (21 CFR Part 58). Neither this report nor the study data were reviewed by the Southern Research Quality Assurance Unit (QAU). However, the study was conducted according to the protocol and the applicable Standard Operating Procedures (SOPS) of Southern Research. All study procedures, data recording, and reporting were performed in a manner consistent with the standard of GLPs. . I %& Patricia E. Noker, Ph.D., D.A.B.T. Study Director 3 6/0/ Date Study Dates: V Study Schedule and Personnel Study Initiation: Day of Dosing: Last Day of Sample Collection: Study Completion: March 17,2000 April 10,2000 May 11,2000 TBD Study Personnel: Patricia E. Noker, Ph.D., D.A.B.T. Study Director James D. Johnson, M.S., M.B.A. Manager, Bioanalytical Chemistry Group Gregory S. Gorman, Ph.D. Chemist Tsu-Han Lin, Ph.D. Pharmacokineticist Darrell E. Hoskins, D.V.M., Ph.D., A.C.L.A.M. (Dipl.) Veterinarian Carolyn R. Oliver, B.S. Preclinical Research Associate LaJuana A. Durbin, B.S. Supervisor, Large Animal Laboratory Joan B. Belzer Manager, Animal Facilities and Operations Deborah S. Bailey, B.S. Senior Supervisor, Animal Laboratory Donna B. Hogan, B.S. Senior Supervisor,Scientific Coordination 1 1.0 Introduction The objectives of this study were to determine the concentration of perfluorobutanesulfonate in serum and to estimate urinary clearance at various times following administration of a single intravenous dose to monkeys. A copy of the protocol and any applicable amendments can be found in Appendix A. 2.0 Materials and Methods 2.1 Test System The 3 male and 3 female monkeys designated for use in this study were selected from an inhouse colony of monkeys that were housed at Southern Research Institute (Southern Research) prior to use on this study. These monkeys were purchased from Charles Rwers BRF, Inc. (Houston, TX). 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.i-nc-luding 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. Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed to the monkeys 2-3 times each day. The quantity of the daily ration was sufficient to meet nutritional requirements. In addition, the diet was supplemented with fresh hithreats several times each week. Tap water (Birmingham public water supply) was available to the monkeys ad libitum during the quarantine and study periods. The monkeys were housed individually in stainless steel cages during the quarantine and the study periods. From Day 2 0 to the end of the study, the monkeys were housed in a room that was maintained at a temperature of 68.0-70.3 "F and a relative humidity of 22.2-65.7%. The humidity was within the required range (30-70%) over 90% of the time during the study; excursions below ' the recommended humidity range were of short duration. 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 Cuidefor the Care and Use of Laboratory Animals, 7th edition"' and the U.S.Department of Agriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPS)of Southern Research. 2.2 Test Article and Vehicle Test Article: One bottle containing 9.58 grams of perfluorobutanesulfonate,potassium salt, was received fkom 3M Corporation (St. Paul, MN; T7485; expiration date not supplied; SRI Lot E04L-1) on March 17,2000. The test article was stored at room temperature until used. Stability of the test article was the responsibility of the Sponsor. Vehicle: The vehicle used for preparation of the dose formulation of perfluorobutanesulfonate was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 8101069; expiration date October 2001). The vehicle was stored at room temperature and was considered to be stable when stored under these conditions. ._._ Dose Formulation Preparation: For the single dose formulation of perfluorobutanesulfonate prepared to contain 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 the test article was in solution. The formulation was stored refiigerated and used for dosing within 3 days after preparation; it was considered to be 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 of the three male and three female monkeys received a single intravenous (IV)dose of perfluorobutanesulfonate at 10 mgkg by injection into a superficial arm or leg vein. Doses were based upon the Day 0 individual body weights. Doses were administered at a volume of 2 mL/kg. Clinical Observations: All animals were checked twice daily for signs of mortality/moribundity. Each primate was examined shortly after dose administration for clinical signs of toxicity. Additional clinical observations (cage-side) were performed daily. Body Weights: Each primate was weighed on Days 0,4, 7, and 14. 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 0 (0-24 hours postdose), and on Days 7 and 14. The volume of each urine sample was measured upon collection. Samples were stored frozen at -20 "C or below. Fecal samples will not be analyzed unless specifically requested by the Sponsor. ._._ Serum Levels of Perfluorobutanesulfonate: Blood samples (approximately 2 mL) were collected from each primate at approximately 0 (predose) minutes; 2, 4, 8,24, and 48 hours; and on Days 4, 7, 11, 14, and 31 postdose. Samples were collected into tubes without anticoagulant and were allowed.to clot at room temperature. The blood samples were then centrihged, and the serum separated and stored frozen (-20 "C or below) until analyzed. Bioanalytical Method Development and Sample Analysis: A bioanalytical method was developed for the analysis of perfluorobutanesulfonate in serum and urine matrices. The method was validated for accuracy. This method was used for the analysis of serum and urine samples collected during the study (see Appendices B and C). Data Analyses: Pharmacokinetic parameters were estimated from serum concentrations of unchanged perfluorobutanesulfonate using WinNonlin (Version 1.1;Scientific Consulting, h c . ; Apex, NC) The data were fit to a three compartment model. Mean values and standard deviations for each parameter were calculated using Microsoft@ Excel software (Microsoft Corporation; Irvine, CA). The urinary excretion of perfluorobutanesulfonate at each collection interval was calculated and expressed as a percent of the administered dose. No , other statistical analyses of the data were performed. 4 3.0 Results 3.1 Mortality All of the monkeys in h s study survived to the end of the study. 3.2 Clinical Observations No adverse clinical signs were noted for any monkey during the course of this study. 3.3 Body Weights Body weights are presented in Table 1. The body weight of each monkey remained essentially constant between Days 0 and 14. - 1 3.4 Serum and Urine Concentrations ofLPerfluorobutanesulfo*~t~~ Pnor to the initiation of the in-life aspects of the study, an HpLC/MS/MS method was developed and validated for the analysis of@rfluorobutanesulfonatt$n monkey serum. Details of the method validation procedure and specifications of the method are provided in Appendix B. Details of the ana-lyhcal method are provided in Appendix C. This method was also applied to the analysis ofberfluorobutanesulfonate$n monkey urine. c Serum concentrations of/pefluorobutanesulfonat] in three male and three female monkeys at various times through Day 3 1 after administration of a single IV dose of the compound are presented in Table 2. No sex-related differences in serum concentrations of berfluorobutanesu1fonate)vere apparent. Among the six dosed monkeys, the concentration -_ of6duorobutanesulfonate)in serum ranged Erom 19,628 to 61,740 n g h L at 2 hours after -dosing (earliest time point). By 48 hours after dosing, the serum concentration of lperfluorobutanesulfonatl ranged from 463 to 8,172 n g / d in individual monkeys. lPerfluorobutanesulfonatdwasnot detectable in serum collected from any monkey on Day 31. For the analyses conducted on the Day 31 serum samples, the standard curve was extended to include $erfluorobutanesulfonat$oncentration of 0.5 ng/mL; thus, the serum concentration 1 !I of perfluorobutanesulfonat was below this level in all monkeys on Day 31. 1 r Pharmacokinetic data calculated from serum concentrations oflperfluorobutanesulfonate in individual monkeys are presented in Table 3 and are plotted in Figures 1-6. The serum concentration versus time data were fitted to a three-compment model. Mean serum half- life values for perfhorobutanesulfonate in the male monkeys were 0.04,0.55, and 4.0 days for the a-,p-, and y-phases, respectively; the corresponding values in the female monkeys were 0.06,0.47, and 3.5 days. The mean AUC value calculated f?om serum concentrations of perfluorobutanesulfonatewas 24,258 or 35,401 ng.day/mJ., in male or female monkeys, respectively. The total body clearance of perfluorobutanesulfonatewas 511 mL/day/kg in male monkeys and 368 mL/day/kg in female monkeys. The volume of distribution at steady state (Vd,,) was 254 or 255 mLkg in male or female monkeys, respectively. The amount .of perfluorobutanesulfonate eliminated in urine by individual monkeys at various times after dosing is presented in Table 4. For 5/6 monkeys, from 33.8% to 86.8% of the dose was recovered in urine within 24 hours after administration of perfluorobutanesulfonate. For the remaining monkey, less than 1% of the dose was recovered in urine during t h s same time interval. In that the serum concentrations of perfluorobutanesulfonate indicated this animal had been properly dosed, loss or spillage of urine excreted by this animal most likely occurred during the collection interval. With time after dosing, the rate of urinary excretion of perfluorobutanesulfonate, expressed as percent of dose per day, decreased. On Day 14, individual monkeys excreted less than 0.01%-of the dose within a 24-hour interval. 4.0 Discussion In male and female monkeys administered a single N dose of 10 mgkg of perfluorobutanesulfonate, no sex-related differences in serum concentrations of the compound were apparent. Serum concentrations of perfluorobutanesulfonate versus time were best fit to a three-compartment pharmacokinetic model. Mean half-life values for perfluorobutanesulfonate in serum were 0.04, 0.55, and 4.0 days in male monkeys and 0.06, 0.47, and 3.5 days in female monkeys. The small value calculated for the steady state of volume of distribution of perfluorobutanesulfonate in individual monkeys indicated the compound was not distributed to total body water. Urinary excretion was a major route of elimination of perfluorobutanesulfonate in that as much as 86.8% of the dose was eliminated as unchanged parent compound within the first 24 hours after dosing. 5.0 Conclusions No sex-related differences in serum concentrations and urinary excretion of perfluorobutanesulfonate were apparent among male and female monkeys administered a single IV dose of 10 m a g . The mean terminal serum half-life of perfluorobutanesulfonatein male and female monkeys was 3.5-4.0 6 days. In monkeys, urinary excretion was a major route of elimination of unchanged perfluorobutanesul fonate. 6.0 Record Archives All raw data pertaining to the conduct of this study, and all sampledspecimens collected in this study, will be stored in the Archives at Southern Research Institute for up to 1 year d e r acceptance of the final report by the Sponsor. After 1 year and with the permission of the Sponsor's Monitor, the data and any samples/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 of Animal Laboratory Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington D.C.;1996. 7 Table 1 A Pharmacokinetic Study of Perfluorobutanesulfonatein the Cynomolgus Monkey Body Weights Group Sex 1M 7F Day nLlTkrs r e l a t i v e t o S t a r t Date Animal Nunber 0 4 7 16 2052 5.8 5.9 5.9 6.0 2054 5.6 5.7 5.7 5.8 2211 4 . 4 4.4 4.4 4.5 2058 3 . 3 3.4 3.4 3.4 2059 3.4 3.4 3.3 3.4 2061 3 . 9 3.9 3.9 4.0 *.- Nominal Dose: Group 1 - 10 w / k g Page 1 o f 1 8 Table 2 A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey Serum Concentrations of Perfluorobutanesulfonate I/' Timepoint 2052 ~~ Serum Concentration (ng/mL) Males Females 2054 2211 2058 2059 2061 0 1.6 1.1 BQLa BQLa BQL' BQLa 2 hrs 19,628 39,400 37,560 61,420 61.740 41.920 1 4hrs I 29,2251 39,5401 29,060) 40,5801 30,1601 22,99011 1 :4Es 48 hrs 20,695 10,410 6,506 4,311 2,406 463 9,952 4,224 1,293 29,420 19,278 8,172 17,350 5,930 2,278 12,530 3,460 948 Day 4 1,999 15.3 52.4 2,683 326 58.7 Day 7 71.7 4.1 11.6 881.6 13.9 10.2 Day 1 1 6.8 2.7 2.5 43.8 1.9 BQL" Day 14 _._ 5.1 1.6 B Q L ~ 7 1.7 Day31b BQLb BQLb BQLb BQLb BQLb BQL a Below the quantitation limit (<1 ng/mL) Below the quantitation limit (COS ng/mL); Day 31 samples were analyzed during a separate analytical run. Page 1 of 1 Table 3 A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey Pharmacokinetic Parameters Calcu1ated from Seruni Conce11Irat iotis of I'c rfl u or0butancs111fon ;1t c Parameters fll2a (day)" tv2p t 1/27 (day)' AUC O-inf (ng.day/mL)d C1 (mL/day/kg)e Vdss ( m L k 9 ' 2052 0.01 0.79 3.8 41352 242 245 2054 0.04 0.42 6.0 13862 72 1 22 8 Male 221 1 0.05 0.44 2.1 17561 569 289 Mean 0.04 0.55 4.0 24258 51 1 254 SD 0.02 0.2 1 1.9 14918 245 31.5 2058 0.04 0.30 1.2 61338 163 234 2059 0.06 0.66 6.9 27548 363 243 Fen1aIe 206 1 0.06 0.45 2.3 17318 5 77 289 Mean 0.06 0.47 3.5 35401 368 255 SD 0.01 0.18 3.1 23037 207 29.5 bHalf-life of the P-phase 'Half-life of the y-phase dAreaunder the serum concentration versus time curve calculated from 0 to infinity 'Total body clearance , 'Volume of distribution at steady state Page 1 of 1 10 Table 4 A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey r Animal ID Urine Concentration Sex (ng/mL) Urine Volume Total (d) (P8) Day 0 Percent of Dose Dose in Urine (Pg) (%) I BQL = Below the quantitation limit (4ng/mL) a Sample vial was received broken but frozen. b Sample was contaminated. Diluted sample concentration was out of calibration range. Estimated concennation was based on the highest standard in the calibration curve x the dilution factor of the sample. The actual concentration was most likely greater than this amount. Page 1 of 1 100000 10000 1000 . `s ~ ........ I -I ---t-rI- --1- t- ~- I 0 2 4 6 8 10 12 14 16 Time (day) Figure 1 A Pharmacokinetic Study of Perfluorobutanesulfonatein the Cynomolgus Monkey Serum Concentration Profile of Perfluorobutanesulfonate in 1M2052 r;. E 10000 \ 0, C W C .+0- 1000 2 w C a, 0 C 100 0 0 E 2 a, 10 c h, c/) 1 I 0 2 4 6 8 10 12 14 16 Time (day) Figure 2 A PharmacokineticStudy of Perfluorobutanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorobutanesulfonate in 1M2054 100000 10000 1000 100 10 1 0 2 4 8.Observed - Predicted 6 8 10 12 14 16 Time (day) Figure 3 A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey Serum Concentralion Profilc of Perfluorobutancsulfonatein 1M2211 100000 10000 I -1 1000 100 10 I 0 2 4 - .-. . ... -4. Observed - Predicted I - - - I I---- - -. 1 . gT-: . . 6 a 10 12 14 16 Time (day) Figure 4 A Pharmacokinetic Study or Perfluorobutanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorobutanesulfonatein 1F2058 100000 E 10000 \ cn t v z.co 1000 w m, ?= C 100 0 u E 2 c0n IO ---I -.. .__- 1 0 2 4 6 a 10 12 14 16 Time (day) Figure 5 A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorobutanesulfonate in 1F2059 4 I n uunununun 10000 1000 100 10 8.Observed - Predicted 1 0 2 4 6 8 10 12 14 16 Time (day) Figure 6 A Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey Serum Concentration Profile of Perfluorobutanesulfonate in IF2061 ._ Appendix A Study Protocol and Amendments A- 1 11 Study Protocol: Pharmacokinetic Study of Perfluorobutanesulfonate in the Cynomolgus Monkey Serquest Study ID: 9921.1 March 17,2000 serquest m a division of Southern Research I n s t i t u t e A-2 STUDY NO.: 9921.1 1.0 SPONSOR REPRESENTATIVE AND CONTACTS: Sponsor: 3M Corporation P.O. BOX33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 March 17,2000 Pane 2 of u Sponsor's Representative & Study Monitor: Protocol Approval: (Initial last page also) Paul H. Lieder, Ph.D., D.A.B.T. P.O. BOX33327 55133-3327 St. Paul, Minnesota 55144-1000 (651) 737-2678; FAX: (651) 733-1773 Test Article: Ship Unused Test Article to: Paul H. Lieder Date Perfluorobutanesuifonate, potassium salt D. Hakes Building B236 3M Corporation P.O. BOX33327 55133-3327 3M Center, 224-IN-04 St. Paul, Minnesota 55144-1000 A-3 STUDY NO.: 9921.1 March 17,2000 2.0 TITLE: A Pharmacokinetic Study of Perfluorobutanesulfonatein the Cynomolgus Monkey 3.0 OBJECTIVE: The objectives of this study are to deternine the concentration of perfluorobutanesulfonate in serum and to estimate urinary clearance at various times following administration of a single intravenous dose to monkeys. 4.0 TESTING LABORATORY: Serquest (a division of Southern Research Institute) 2000 Ninth Avenue South 35205 P.O. Box 55537 Birmingham, AL,35255-5537 (205) 581-2335; FAX: (205) 581-2044 5.0 KJ2k' STUDY DATES: Event Day of Treatment Unne and Feces Collections Serum Drug Levels Draft Report Due Final Report Due Sequence (Day) -1 0 - 0-24 hours 7 14 Baseline 0 - 0 , 2 , 4 , 8 hours postdose 1 - 24 hours postdose 2 - 48 hours postdose 4 7 11 14 60 Calendar days after completion of the in-life phase 15 days after final Sponsor comments received > Datejs) - Year 2000 TBD TBD TBD TBD STUDY NO.: 9921.1 6.0 STUDY PERSONNEL: March 17,2000 4 o f 17 The following are the primary contributors and supervisory personnel participating in this study. Study Director: Clinical Pathologist & Manager, Clinical Pathology: Director, Safety Assessment: Associate Director: Manager, Bioanalytical Chemistry Supervisor, In-Life Laboratories Supervisor, Study Coordination Veterinarian: Patricia E. Noker Ronna Fulton Ward R. Richter Norman D. Jefferson James D. Johnson Deborah S. Bailey Donna B. Hogan Darrell E. Hoskins Ph.D., D.A.B.T. D.V.M., PbD., D.A.C.V.P. D.V.M., M.S.,D.A.C.V.P. B.A. M.S.,M.B.A. B.S. B.S. D.V.M., A.C.L.A.M.(Dipl.) 7.0 TEST & CONTROL ARTICLES: The test article will be supplied by the Sponsor, who will be responsible for documentation of stability, as well as methods of synthesis, fabrication, or derivation. Stability of the test article formulated in the vehicle will be assessed prior to treatment initiation. Upon completion of the study, residual bulk test article will be returned to the Sponsor. 7.1 IDENTITY OF THE TESTARTICLE: Name: Identification: Supplier: Lot Number(s): Special Handling: Characterization : Perfluorobutanesulfonate, potassium salt CAS NO. 29420-49-3 3M Corporation To be documented in the study data; the minimum number of lots necessary to complete the study will be used None Documentation of characterization, including identity, purity, strength, and composition, as well as methods of synthesis, fabrication, or derivation, is the responsibility of the Sponsor. [Copies of these data will be provided to the testing laboratory.] Stability & Storage: The bulk test article will be stored at room temperature. Stability of the bulk test article is the responsibility of the Sponsor. Copies of these data have been provided to the testing facility. A-5 STUDY NO.:9921.1 7.2 IDENTITY OF THE VEHICLE: March 17,2000 page 5 of 11 Name: Supplier: Lot Number(s): Special Handling: Sterile saline Commercial supplier To be documented in the study data; the minimumnumber of lots necessary will be used on the study. None Characterization: Documentation of characterization of the vehicle may be attained by recording all pertinent information from the container labels, or by retaining the container labels, or copies thereof, in the study data. The vehicle is a commercially available product. Stability & Storage: Sterile saline is considered stable through the date(s) of expiration provided by the manufacturer when stored appropriately. The bulk vehicle will be stored in accordance with the manufacturer's instructions. ._._ 7.3 FORMULATIONS: Preparation: The test article will be formulated in sterile saline at a concentration of 5 mg/mL for intravenous administration; briefly, the required amount of test article will be mixed with the required amount of sterile saline, and the mixture will be stirred until the test article is visibly in solution. Formulations will be stored refrigerated until used for dosing; formulations of the test article in sterile saline are expected to be stable for weeks when so stored. Dose Formulation Concentration and Homogeneity Analyses: No analysis of dose formulation concentration and homogeneity will be conducted. 8.0 TEST SYSTEM: Species & Strain: Supplier: Age on Day 0: Weight at randomization: Number on Study: Cynomolgus monkeys ('acaca farcicularis) Charles h v e r BRF, Inc. (Houston, TX) 2.5-3 years of age (estimated) 3-7 kg Males -3 Females -3 A-6 STUDY NO.: 9921.1 March 17,2000 8.1 JUSTIFICATION: Primates are commonly used in preclinical pharmacological and toxicological evaluations of compounds used or intended for use in humans, or to which humans might be exposed. 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 of 10 air exchanges per hour. Controls will be set to maintain the animal room at a temperature of 64-84 O F and a relative humidity of 30-70%. A 12-hour lightIl2-hour dark cycle will be routinely maintained. Animals will be acclimated in the same room used for study. 8.3 BEDDING: None required for caging equipped with flushable pans. For cages equipped with excrement absorption pans, commercial heat-treated hardwood chip bedding will be used for excrement absorption. Analyses of the bedding, supplied by the vendor, will be reviewed by the Department of Veterinary Medicine and Bioresources (DVMB) of Serquest to assure that no known contaminants are present that could interfere with or affect the outcome of the study. 8.4 DIET: Diet will be commercial Certified Primate Chow #5048 (PMI Feeds, Inc.; St. Louis, MO). The primates will be offered feed twice daily, with approximately the recommended daily ration available at each feeding interval. In addition, the diet will be supplemented with fresh fruit offered daily and treats offered several times each week. The quantity of the daily ration will be suficient to meet nutritional requirements. Analyses of the feed, supplied by the vendor, will be reviewed by the DVMB of Serquest to assure that no known contaminants are present that could interfere with and affect outcome of the study. 8.5 WATER: Water (Birmingham public water supply) will be supplied ad libitum during the quarantine and study periods via an automatic watering system. Samples of water from the animal facility will be periodically analyzed, and the analyses will be reviewed by the DVMB of Serquest to assure that no known contaminants are present that could interfere with or affect the outcome of the study. A-7 STUDY NO.: 9921.1 8.6 QUARANTINE: March 17,2000 Page7of17 All primates will be selected fiom stock animals that will have been quarantined for a minimum of 35 days prior to study start. No prophylactic or therapeutic treatments will be administered during the quarantine period. Standard procedures to be conducted during the quarantine period are as follows: a) A complete physical examination including a fecal examination for internal parasites, complete blood count (CBC), body weight, and body (rectal) temperature will be performed; b) three tuberculin tests at 2-week intervals (administered intrapalpebrally, using alternate eyelids for each test) will be performed on each primate (primates must test negative to all three tests prior to release from quarantine; primates that respond positively to a tuberculin test will be euthanized immediately, while non-responding primates housed in the same room will be held in quarantine for an additional 90 days); c) the blood sample drawn for CBC will also be used for measuring of B virus titer (primates found to have a positive B virus titer will be euthanized immediately); d) a fecal sample for culture (screening for Salmonella and Shigella) will be obtained and submitted to an independent laboratory for analysis- (if any primates are found to be positive for Salmonella, Shigella, or parasites, the Study Director, in conjunction with the staffveterinarian, will determine the course of action); and e) all primates will be examined at least once weekly by a veterinarian and observed (cage-side observations) twice daily for abnormal clinical observations and mortality/moribundity. b) A baseline serum sample will be collected fiom each monkey and stored at or below -20 "C. 8.7 PSYCHOLOGICAL WELL-BEINAGND SOCIALIZATION: Nonhuman primates will be provided a psychological well-being program for social enrichment as directed by a veterinarian and approved by the IACUC and in accordance with the appropriate SOP. Nonhuman primates will be provided cage and feeding regimen modifications daily for their psychological well-being. The modifications include, but are not limited to: swings, perches, Kong toys, clean 2liter soft drink bottles, puzzle feeders, nutritionally sound primate treats, unshelled peanuts, and raw h i t . Where possible, primates will be housed proximate to one another for visual and vocal contact. A- 8 STUDY NO.:9921.1 8.8 ANIMAL IDENTIFICATION: March 17,2000 Paee 8 of I 7 During quarantine, the primates will be individually identified by chest tattoo number or letter combination. Positive identification Will be required after every cage change and prior to blood sampling, dose administration, and observation. 9.0 EXPERIMENTAL DESIGN: As only one treatment group will be used in this study, no formal randomization will be required. Doses will be administered by intravenous injection to determine the pharmacokmetics of the test article. Each primate (3 males, 3 females) will receive a single dose of perfluorobutanesulfonate by injection into a superficial arm or leg vein. Blood samples for serum drug level determinations will be collected from each primate at seven time points during the study. A synopsis of the study design is presented in the following table, .*.- a Denotes Week 0-24 hours 0 minutes @redose);2,4, and 8 hours. 9.1 RANDOMIZAT&IOGNROUPASSIGNMENT: As only one treatment group Will be used in this study, no formal randomization will be required. 9.2 DOSEPROCEDURE: On Day 0, each primate (3 males, 3 females) will receive a single intravenous (N) dose of perfluorobutanesulfonate at 10 mgkg by injection into a superficial arm or A-9 STUDY NO.:9921.1 March 17,2000 Psgof13 leg vein. Doses will be based upon the most recent individual body weights. Doses will be administered at a volume of 2 m l k g . 9.3 CLINICAOLBSERVATIONS: Daily Observations: All monkeys will be observed once daily during quarantine and twice daily, morning and afternoon, at least 4 hours apart, during the study for signs of mortality/moribundity. Animals found in extremis will be humanely sacrificed by an overdose of barbiturate followed by exsanguination with appropriate approval. Detailed Observations: Each primate will be examined shortly after dose administration for clinical signs of toxicity. All findings will be recorded. Additional clinical observations (cage-side) will be performed daily; any adverse findings will be recorded. 9.4 BODYWEIGHTS: Each primate will be weighed on DajtS--l, 4, 7, and 14. 9.5 URINEAND FECESCOLLECTIONS: Urine and feces will be collected prior to dose administration (baseline), on Day 0 (0-24 hours postdose), and on Days 7 and 14. The volume of each urine sample will be measured upon collection. Samples will be stored frozen prior to analysis. Fecal samples will be collected and stored frozen, but samples will not be analyzed unless specifically requested by and at additional cost to the Sponsor. 9.6 SERUMDRUGLEVELS: Blood samples (approximately 2 mL) will be collected from each primate at approximately 0 (predose) minutes; 2 , 4 , 8,24, and 48 hours; and on Days 4, 7, 11, and 14 postdose. Samples will be collected into tubes without anticoagulant and will be allowed to clot at room temperature. The blood samples will then be centrifkged, and the serum will be separated and stored frozen until analyzed. 9.7 BIO-ANALYTICAMLETHODDEVELOPMENT: Bio-analytical method(s) are to be developed for the analysis of the compound and its likely metabolites in serum and urine matrices. The methods should be validated for accuracy and be sensitive to the 1 ppb or less level. The Sponsor will provide initial input on analytical methodology used for compound identification. If a A-10 STUDY NO.: 9921.1 March 17,2000 Paee 10 of 13 method for feces analysis needs to be developed, such a decision would result in additional cost. 9.8 BIO-ANALYTICASLAMPLEASNALYSIS: The serum and urine samples fiom all monkeys will be analyzed for the concentrations of perfluorobutanesulfonate using the previously validated method. Feces will be analyzed only if requested by the Sponsor. 9.9 ANIMADLISPOSITION: At the end of the study, monkeys will be maintained in the stock colony. 10.0 DATA ANALYSIS: Pharmacokinetic parameters (e.g., AUC, half-life, clearance) will be estimated from serum concentrations of unchanged perfluorobutanesulfonate and metabolites, as appropriate and feasible, using a standard pharmacokinetic program. .-.- The urinary clearance of perfluorobutanesulfonate (and metabolites) at each collection interval will be calculated. Mean values and standard deviations will be calculated for each time point and sample type, as appropriate. No other statistical analyses of the data will be performed. 11.0 RECORDS: All raw data pertaining to the conduct of this study, and all samples/specimens collected in this study, will be stored in the Archives at Southern Research Institute for up to 1 year after acceptance of the final report by the Sponsor. After 1 year and with the permission of the Sponsor's Monitor, the data and any samples/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. 12.0 FINAL REPORT: A draft final report will be issued within 60 calendar days after completion of the in-life aspects of the study. The final report (electronic and hard copies) will be issued within 15 working days after receipt of the Sponsor's final review comments on the draft report. The final report for the present study will include, but not necessarily be limited to the following: A-1 1 STUDY NO.:9921.1 Dose formulation preparation Clinical observations Body weight data Senun drug level data Pharmacokinetic parameters Urine excretion data March 17,2000 & P 13.0 REGULATORY REFERENCES: This study will be conducted in accordance with the protocol and the Standard Operating Procedures of Southern Research Institute, and in accordance with the applicable regulatory requirements, as addressed below. 13.1 PROTOCOL AMENDMENTS AND DEVIATIONS: Amendments: All changes in or revisions of the approved protocol and the reasons thereof will be documented, signed, and dated by the Study Director, and the Sponsor's Monitor. Amendments will be maintained with the protocol. Written approval (a fax signawe or electronic communication,such as email) for changes in the protocol may be granted by the Sponsor's Monitor, but a written amendment will follow. Deviations: All operations pertaining to this study, unless specifically defined in this protocol, will be performed according to the Standard Operating Procedures (SOPS) of SouthernResearch andor the protocol, and any deviations from protocol or SOP will be documented. 13.2 REGULATORCYOMPLIANCE: Good Laboratory Practices: This nonclinical laboratory study will be conducted in the spirit of, but will not require strict compliance with, the U.S. Food and Drug Administration's (FDA) Good Laboratory Practice (GLP) regulations (2 1 CFR Part 58). Data from this study may be submitted to the FDA in support of 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 Institute. 13.3 FACILITIEMSANAGEMEANNTD ANIMAHLUSBANDRY: Animal care will be in compliance with the Standard Operating Procedures (SOPs) of Southern Research Institute, the Guidelinesfor fhe Care and Use ofLaboratoly A-12 STUDY NO.: 9921.1 March 17,2000 ~~~ Animals, T" Edition (Institute of Animal Resources, Commission on Life Sciences, National Research Council; National Academy Press; Washington, DC; 1996), and the U.S.Department of Agriculturethrough the Animal Welfare Act (Public Law 99- 198). Southern Research Institute is fblly accredited by the American Association for Accreditation of Laboratory Animal Care ( M A C ) . 13.4 ANIMAWL ELFARAECTCOMPLIANCE: By signing this protocol, the Sponsor signifies that there are no generally accepted alternatives to the use of animals, and that the study described by this protocol does not unnecessarily duplicate previously conducted or reported experiments. Procedures used in this protocol are designed to conform to accepted practices and to minimize or avoid causing pain, distress, or discomfort in the animals. In those circumstances in which required study procedures are likely to cause more than momentary or slight pain or distress, the animals will receive appropriate analgesics or anesthetics unless the withholding of these agents has been justified in writing by the Study Director and/or Sponsor and approved by the IACUC. .-.- The number of animals selected for use in this study is considered to be the minimum number necessary to meet scientific and regulatory guidelines for this type of study. This study design was reviewed by the IACUC at Southern Research Institute and was approved on xx/xx/xxx; it was assigned IACUC tracking number xx-xx-xxx. A-13 STUDY NO.: 9921.1 i4.0 PROTOCOL APPROVALS: This protocol has been reviewed and approved. March 17,2000 Pae- Study Director: Patricia E. Noker, PbD., D.A.B.T. Study Director 3//3 / o 0 Date Sponsor's Monitor: INITIALS ONLY(See page 2) /////h Date Management Approval: Ward R. Richter, M.S., D.V.M., D.A.C.V.P. Director, Safety Assessment, Serquest 3//7//0 0 Date A-14 PROTOCOL AMENDMENT: 9921.1Ai Protocol 9921.1 is amended as follows: April 10,2000 Page 1 of2 1. Page 3 Section 5.0 (Key Study Dates): Key study dates were unavailable at the time of protocol finalization. This table has been amended as follows: I Event Day of Treatment Urine and Feces Collections Serum Drug Levels ' Draft Report Due Final Report Due 0 -1 0 - 0-24 hours 7 14 Baseline 0 - 0,2,4,8 hours postdose 1 - 24 hours postdose 2 - 48 hours postdose 4 7 11 14 60 calendar days after complenon of the in-life phase 15 days after final Sponsor comments received 4/10 419 4/10 - 411 1 4/17 4/24 4/10 4/10 411 1 4/12 4/14 411 7 412 1 4/24 6/23 TBD 2. Page9 3. Page 9 4. Page 12 Section 9.5 (Urine and Feces Collection): During protocol development, details on the freezing conditions for the urine and fecal samples collected were inadvertently omitted from the protocol. All samples collected will be stored frozen at -20 "C or below prior to analysis (urine) or until further notice by the Sponsor (fecal). Section 9.6 (Serum Drug Levels): During protocol development, details on the freezing conditions for the serum samples collected were inadvertently omitted fiom the protocol. All serum samples collected will be stored frozen at -20 "C or below prior to analysis. Section 13.4 (Animal Welfare Act Compliance): IACUC approval had not been received at the time of protocol finalization. Approval was received on March 24,2000 (L4CUC Tracking No. 00-03-019), and the last sentence of this section has been amended accordingly. Effective Date: April 7,2000 A-15 PROTOCOL AMENDMENT: 993-I . 1A[ Approval Signatures April. 10, 2000 Page 2 of 2 Study Director: %Z,,.` `C P/&J Patricia E. Noker, Ph.D., D.A.B.T. Study Director 5 4 0/cl v Date Sponsor 's klonitor: .-.- - Paul H. Lieder, Ph.D., D.A.B.T. Sponsor's Monitor ////3/0s Date hlan agemen t Approval : Ward R. Richttr, M.S., D.V.iM., D.A.C.V.P. ' Dat[ Director, Safety Assessment, Serques ._._ Appendix B Method Validation Report: Validation of Analytical Methods for Determination of Perfluorobutanesulfonate in Monkey Serum Using HPLCMSMS B-1 METHOD VALIDATION REPORT VALIDATION OF ANALYTICAL METHODS FOR DETERMINATION OF PERFLUOROBUTANESULFONATE IN MONKEY SERUM USING HPLC/MS/MS STUDY ID: 9921.1 Serquest 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 B-2 SUMMARY Serquest has successfully validated for 3M an analytical method (BACG 3523) entitled "Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLCMSMS)". Calibration standards were prepared by spikmg samples of blank monkey serum with known amounts of test article, perfluorobutanesulfonate (PFBS) and internal standard, perfluoropentanoic acid (PFPA). The concentration of test article covered a range of 0.5 to 500 ng/mL. Quantitation limitswere set at 1 ng/mL. Standard curves generated using internal standard quantitation produced correlation coefficients of 0.9919 or greater. A total of 39 standards in two composite calibration curves prepared on different days over a concentration range of 0.5 to 100 n g h L produced standard curves with a correlation coefficient of 0.9962 and 0.9979. A thxd composite curve containing a total of 33 standards and encompassing a range of 0.5 to 500 ng/mt produced a correlation coefficient of 0.99 19 thereby extending the validated calibration range. James D. Johnson, M.S. Manager Bioanalytical Chemistry Group Gregory S. Goman, Ph.D. Research Chemist 111 Bioanalytical Chemistry Group Lester Williams, B.S. Associate Chemist I1 Bioanalytical Chemistry Group B-3 KEY PERSONNEL B-4 1. OBJECTIVE The objective of this study was to provide a validated analytical method for the determination of perfluorobutanesulfonate in monkey serum. 2. SAFETY All necessary procedures to ensure safety of the analysts were based on information contained in the Material Safety and Data Sheets (MSDS), provided by 3M for the test article used in this study. 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. 3. EXPERIMENTAL 3.1 Analytical Procedures The sample preparation and analysis procedures as described in the analytical method entitled "Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS)" were employed for all analyses. For the preparation of the calibration standards, a known volume of a blank serum sample (e.g., 500 pL) was spiked with a known amount of test article (PFBS) and internal standard (PFPA). To the standard was added 500 pL of TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized water. This mixture was vortexed for approximately 5 seconds. Then 2.5 mL of ethyl acetate was added and`the mixture was placed on . a horizontal shake at a low speed for 1 hour. The mixture was then centrifuged at approximately 2500 rpm for 5 minutes. The ethyl acetate layer was transferred to a second tube and evaporated to dryness at approximately 55C under a stream of dry nitrogen. The residue was reconstituted in mobile phase (70 % 5 m M ammonium acetate in deionized water : 30% methanol) and filtered through a 0.2 pm syringe filter. 3.2 Method Validation Validation for BACG 3523 ``Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass SpectrometryMass Spectrometry (HPLC/MS/MS)" consisted of analyzing standard curves prepared in monkey serum over two different concentration ranges: 0.5 - 100 ng/mL and 0.5 - 500 ng/mL. B-5 0.5 - 100 w / m L Two composite calibration curves comprising a total of 5 analytical runs in the 0.5 - 100 ng/mL concentration range were generated from a total of 39 standards prepared on different days. The correlation coefficient for these composite curves are 0.9962 and 0.9979 respectively. One data point was dropped (0.5 ng/mL) from the first curve due to interference fiom matrix. An example total and extracted ion chromatogram for each component is shown in Figure 1. A statistical summary for each composite curve is shown below: STANDARDS COMPOSITE CURVE 1 (ndmL1 # of Standards 2 3 3 3 3 3 3 3 Mean % Accur. 93.6 99.8 I 92.2 113.1 108.6 88.1 102.6 99.7 Std. Deviation 0.014 0.085 1 0.192 0.523 0.717 0.474 3.44 6.28 STANDARDS COMPOSITE CURVE 2 (ndmL) I # of Standards 2 212i2 2 2 2 2 Mean % Accur. 81.8 101.2 j 113.1 1 112.4 94.6 95.3 I 102.1 99.7 Std. Deviation 0.131 I 0.146 0.182 I 0.308 0.211 1.01 1 3.14 3.91 0.5 - 500 n d m L A single composite curve consisting of 37 standards taken from an analytical run encompassing a concentration range of 0.5 - 500 ngYmL produced a correlation coefficient of 0.9919 (Figure 2). Of these data three points were dropped, 2 at the 0.5 ng/mL level and one at the 500 ng/mL level. This was due to matrix interference for one standard (0.5 ng/mL) and insufficient vial volume for the remaining two (0.5, 500 ng/mL). A statistical summary for the composite curve is given below: STANDARDS COMPOSITE CURVE (ndmL) # of Stds Mean%Accur. Std. Deviation 2 4 414 4 4 4 4 4 3 97.7 96.5 108 108 102 98.2 95.3 88.7 100 109 0.01 0.14 0.16 0.16 0.72 1.70 4.71 12.4 23.5 26.0 B-6 These results demonstrates the reproducibility and reliability of the sample preparation and analysis methodologies used to analyze serum samples. 3.3 Calculations Calculations were performed using TurboQuan (Version 1.0). The m o u n t of analyte in the serum extracts (ng/mL) was back calculated using a calibration curve generated from a set of calibration standards. The calibration curve was generated by a regression analysis to determine the best fit curve (e.g., linear, quadratic etc.) and amount of weighting. A quadratic fit with 1/X weighting was determined to be the best fit : y = & + bx + c where: y = Peak height response of PFBS divided by peak height response of the IS (PFPA) in standards. x = Concentration of the PFBS in standards. a, b, c = Constants derived from the regression analysis. 3.4 Compound Stability Blank serum samples spiked with PFBS at the 5 ng/mL level were subjected to two complete freeze thaw cycle then extracted and analyzed as described in BACG 3523. The results of the analysis are listed in the table below: 5 5.3 106 5 5.3 106 5 4.1 82 Average 4.9 98 4.0 Conclusion A quantitative method (BAGC 3523) has been developed and validated for the determination of perfluorobutanesulfonate in monkey serum utilizing LC/MS/MS. Quantitation for thk method is based on an internal standard (PFPA) whch produces standard curves with a correlation coefficients of 0.9919 or greater over a concentration range of 0.5 to 500 ng/mL. Analysis of B-7 spiked monkey serum after two complete freeze thaw cycles produced an average quantitative recovery of 98 % ._.- Figure 1 TIC from 50 nglrnL Sld 14 0.4 I? 0.. Exlracted ion lor PFPA (IS) I1 'I TucoOuanN. verson 1.O B-9 P n n t d : OYOWOO 09:07 Results Table: ...op Folder:May2000:da~Y6pfbs: pfbs 2nd CY08/00 08:58 Pace 1 of 1 Pnnted by: greg soman 4.0C 3.5c 3.0C 2.5C 2.00 1.so 1.oo 0.50 0.00 0.00 100.00 200.00 COmpwnd: Internal Standard: Fit: Fit Parameter: Weighting: Equation: Correlation Coefficient (r): Chi Squru: RBS 6 Ouadratic Area Ratio 1 +X'X y = - 6.7074e-6 * X * X bO.011OS 0 991897 0.00005 x + 0.00789 300.00 . 400.00 500.00 Appendix C Analytical Method for the Analysis of Perfluorobutanesulfonate in Serum c-1 Page 1 of 12 AN.ALYTI C A L METHOD Method No. : BACG-3523 Title: Determination of Perfluorobutanesulfonate in Monkey S e m : Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 1.0 , 2.0 2.1 2.1.1 PRINCIPLE Serum samples are obtained from cynomolgus monkeys treated with perfluorobutanesulfonate (PFBS). Serum (e.g., 0.5 mL) containing (PFBS) is fortified with an internal standard (IS), perfluoropentanoic acid (PFPA). The serum samples are then mixed with an ion-pairing reagent and buffer, followed by extraction with ethyl acetate. The ethyl acetate layer is removed, evaporated to dryness, reconstituted in mobile phase, filtered, and transferred to autosampler vials, and analyzed by HPLC Mass Spectrometry/Mass spectrometry (HPLC/MS/h.IS). The range of reliable results extends from about 0.5 to 500 ng/mL of PFBS in semm. Serum containing PFBS at concentrations greater than 500 ng/mL may be diluted with control blank serum so that the concentration of PFBS will be within the range of reliable results prior to analysis. - - The mass spectrometry of PFBS and PFPA 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 of other potentially interfering ions extracted from the serum. In addition, the collision energy is set rather high to reduce potential interferences from other matrix compounds. In order to maximize sensitivity, this method is based on mixed reaction monitoring of the negative parent ion (M-1) for both PFBS and PFPA. 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 A i 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 2 10N) c-2 ANALYTICAL, METHOD Page 2 of 12 Method No.: BACG-3523 Ti tie: Determination of Perfluorobumesulfonate in Monkey S e m : Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 2.1.2 Methanol, HPLC grade 2.1.3 Perfluorobutanesul fonate (analyte), as provided by the client 2.1.4 Perfluoropentanoic acid (internal standard), 975% 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 2.1.11 Sodium Hydroxide 50% solution, Certified grade or `equivalent 2.2 Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 2.2.1 5 mM Ammonium acetae 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) 2.2.2.1 For example, to prepare 25 mL, dissolve 4.24 g of tetrabutylammonium hydrogen sulfate in deionized water and adjust the pH to 10 with 50% NaOH solution. c-3 Page 3 of 12 ANALYTICAL METHOD Method No.: BACG-3523 Title: Determination of Perfluorobutanesulfonatein Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectromerry/bfass Spectrometry (HPLC/MS/MS) 2.2.3 2.2.3.1 Carbonate/Bicarbonate Buffer Solution For example, to prepare 100 mL, dissolve approximately 2.65 g of sodium carbonate and approximately 2.10 g of sodium bicarbonate in 100 mL of deionized water. Mix well to ensure complete dissolution. 3.0 INSTRUMENTS, MATERIALS, .A?JD APPAR4"ITS 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 IKA-Vibrax @ platform mixer) 3.4 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap @ with source of nitrogen) 3.5 HPLC mobile phase filtration apparatus i.6 Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 p m ) 3.7 Analytical balance 3.8 Volumetric flasks (e.:., 10 and 25 mL) 3.9 Disposable Pasteur pipets 3.10 Micropipettor(s) with tips 3.11 Culture tubes with teflon-lined caps 3.12 Centrifuge C-4 ANALYTICAL METHOD Page 4 of 12 Method No.: BACG-3523 Title: Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 3.13 Assorted glassware and syringes 3.14 Culture tubes (vials) for use with solvent-concentration apparatus 3.15 0.2 pm PVDF syringe filters 3.16 Variable speed horizontal platform shaker 4.0 PREPAFUTION OF STOCKS AiW WORKING STOCKS Appropriate changes in the concentrations of the solurions may be'-rmde at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. -- -- 4.1 4.1.1 , 4.2 4.2.1 lMain Stock Solution of PFBS - 1000 pg/mL Prepare an - 1000 p g / d solution of PFBS in deionized organic-free water (e.,.., accurately weigh about 10 mg PFBS into a 10-mL volumetric flask). Add deionized organic-free water to dissolve. Dilute to the mark. Alternarively, weigh the compound into an appropriate vessel (e.g., culture tube) and add IO mL of deionized organic-free . water. Mix well. Transfer the solution to a clean vessel if desired. Stock Solution of Internal Standard (PFPA), -200 pg/mL Prepare an -200 pg/mL solution of PFPA in deionized organic-free water (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organicfree 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. c-5 Page 5 of 12 ANALYTICAL iMETHOD Method No.: BACG-3523 Tide: Determination of Perfluorobutanesulfonatein Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 4.3 4.3.1 N'orking Stock Solutions of PFBS 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. I 1 Working Stock Level I 1 (WSU Volume of PFBS solution Final Volume in Approximate Concentration deionized organic (ng/d) free water (mL) 5000 2500 I 50 p L of 1000 p g / d stock 10 1 5 mL of 5000 ng/rnL stock 10 1000 4 mL of 2500 ng/mL stock 10 500 5 rnL of 1000 ng/rnL stock 10 250 5 rnL of 500 n g / d stock 10 I 100 4 mL of 250 ng/rnL stock 10 50 5 rnL of 100 ng/mL stock 10 25 *. 5 mL of 50 ng/mL stock 10 12.5 5 mL of 25 ng/mL stock 10 0 0 mL of 6.25 stock 10 C-6 Page 6 of 12 AWALYTICAL METHOD Method No.: BACG-3523 Title: Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrornetry/Mass Spectrometry (HPLC/MS/klS) ~~~ 4.3.2 Summary of concentrations of serum standards: Standard Level Appro xima re Concentration of PFBS in serum A2 500 ~ A1 A 200 - .- 100 B I 50 C 20 D 10 E 5 F 2 , I G I 1 I I H I 0.5 I I 0.25 I 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. 5.1 Multiple (e.g., about three) sets of serum standards and a serum blank (blank + IS) are analyzed with each set of unknown samples. A serum double blank (blank-IS) may also be analyzed if desired. c-7 A i a Y T I C A L , METHOD Pase 7 of 12 Method No. : BACG-3523 Title: Determination of Perfluorobutanesulfonatein Monkey Serum: Sample Preparation and Analysis by HPLC Mass SpectrornetrylMass Spectrometry (HPLC/MS/MS) 5.2 Into individual -20-mL culture rubes, pipet blank serum (e.g., 0.5 mL). Pipet in 50, 20, and 10 p L of the 5000 ng/mL working stock for b e 500, 200, and 100 ng/mL standard respectively. For the remaining standards pipet 10 pL of each subsequent working stock standard solution into separate tubes chat contain the blank serum. For the blanks, pipet 10 p L of organic-free water instead of the working stock solution. Add the 5 p L of internal standard stock (-200 pg/mL) to each tube except the blank- IS (pipet 10 pL of organic-free water instead) and vortex for - 5 seconds. 5.3 To each rube add 500 p L of the TBA ion-pziring solution, 1 mL of tcuabrbeofnoarraebhoicuatr5bosneactoenbdus.ffer, and 1 mL of deionized organic free water. Vortex each ._._ 5.4 Add 2.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed- ' Setling. 5.5 Remove the tube from the shaker and place in a centrifuge (e.g., 2500 rpm) for about 5 minutes. 5.6 Take off the dryness (e.g., -upper ethyl acetate layer and put it into a clean tube and 50 minutes in the Turbo-Vap @)with a gentle'stream of evaporate to nitrogen and 8 moderate heat (e.g., 50 'C). 5.7 Reconstitute the residue in 500 pl of mobile phase(70% 5 r;lM ammonium acetate: 30% methanol) and vortex briefly to mix: Filter the samples through 0.2 pm PVDF syringe filters into autosampler vials. 6.0 PREPARATION OF SAMPLES - 6.1 Allow each serum sample to thaw to room temperature. Vortex each sample briefly, but vigorously. Pipet an aliquot of each sample (e.g., 0.5 mL) into individual 20- mL culture tubes. If necessary, dilute an aliquot of the s e n m sample with blank serum so that the expected coilcentration of the test article being analyzed will fall within the concentration range of the standard curve. Add 5 p L of internal standard stock ( - 200 pg/mL) to each tube and vortex for a couple of seconds. C-8 Page 8 of 12 ANALYTICAL METHOD Method KO.: BACG-3523 Tirle: Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS) 6.2 To each tube add 500 p L of the TBA ion-pairing solution, 1 mL of carbonate/bicarbonate buffer, and 1 mL of deionized organic free water. Vortex each rube for about 5 seconds. 6.3 Add 3.5 mL of ethyl acetate and extract on horizontal mixer for 1 hour at a low speed setting. 6.4 Remove the tube from the shaker and place in a centrifuge (e.g., 2300 rpm) for about 5 minutes. - 6.5 Take off the upper ethyl acetate layif-and put it into a clean tube and evaporate to dryness (e.g., 50 minutes in the Turbo-Vap @)with a gentle stream of nitrogen and moderate heat (e.g., 50 'C). -- -- 6.6 Reconstitute the residue in 500 pL of mobile phase (70% 5 mif ammonium acetate: 30% methanol) and vortex briefly to mix. Filter the samples through 0.2 prn PVDF syringe filters into autosampler vials. Cap vials for analysis. 7.0 I 7.1 7.1.1 ANALYSIS BY HIGH PERFORMANCE LIQLTD CHROMATOGRAPHY MASS SPECTROh.IETRY/MASSSPECTROMETRY cHpLC/MS/h.IS) Conditions are to be optimized if necessary. J.CC . . w Analytical Column: Keystone Scientific Fluofix 120E, 5 micron, 150 mm x 2 mm ID, or equivalent GuardColumn: , Elution Flow rate: Injection volume: Mobile phase: Fluofix 120E or Fluophase RP 10 mm x 2 mm 200 pL/min. 50 pL A: S d v f ammonium acetate buffer B: methanol c-9 ANALYTICAL iMETHOD Page 9 of 12 Method No.: BACG-3523 Tide: Determination of Perfluorobutanesulfonate in hionkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Specrrometry (HPLC/MS/MS) Gradient Profile: Temperature: 0 - 2 min. 70% A : 30% B 2 - 6 min. 30% A : 70% B linear gradient 6-11 min. 30%A : 70 % B 11-15 min. 70%A : 30% B step gradient Ambient 7.1.2 , PE Sciex API 3000 Triple Quadrupole Mass Spectrometer Conditions S o h a r e : PE Sciex TurboQuan ._.- Note: Values listed under "MS/MS Acquisition Conditions" override parameters irr = this table. Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter Value Is -2000 - NC 0 , EM 450 OR -20 RNG -1zo QO 18 !Q 1 19 ST 23 RO 1 s 19 IQ2 20 R02 65 c-10 AiiALYTICAL METHOD Page 10 of 12 Method No. : BACG-3523 Title: Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrornecry (HPLC/MS/MS) Parameter R03 DF CE.U NEB CUR CAD QPE POL VCM IPE Value 65 250 2600 15 6 5 ._.0_ 0 0 0 Scan type: Polarity: Acquisition mode: -Pause time: Masses requested: PFBS: 298.9 exanluu R02 R03 sr3 MRM Positive Profile 5 milliseconds 298.9 SLxUdu 65 75 65 Dw-ll 200 su&ulc 65 75 65 200 sKu3lUc 45 55 45 c-11 Page 11 of 12 ANALYTICAL METHOD Method No. : BACG-3523 Tide: Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/hIS/MS) 8.0 CALCULATIOXS 8.1 At the end of the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: Analvte PFBS PFPA hn&Q?zk 298.9 to 298.9 218.9 to 218.9`-'- __ - 8.2 Plot the peak are3 response of PFBS divided by the peak area response of the IS (PFPA) from all standards versus the concentration of the test arricle in the standards. Alternatively, the peak heights may be used instead of peak areas. Obtain the best curve fit of the data (e.:., quadratic fit weighted with lkoncennation of the test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various . concentration ranges using the following: y = ax2+ bx + c where y = x = a, b, c Peak height response of PFBS divided by peak height response of the IS (PFPA) in standards. Concentration of the PFBS in standards. = Constants derived from the regression analysis. 8.3 Using the standard curve, calculate the level of PFBS in each unknown sample. Conect the results of samples for any dilutions. c-12 Page 12 of 12 ANALYTICAL METHOD Method No.: BACG-3523 Title: Determination of Perfluorobutanesulfonate in Monkey Serum: Sample Preparation and Analysis by HPLC Mass Spectrornetry/Mass Spectrometry (HPLC/MS/blS) NOTE: Due to unresolvable interferences with the test article and/or internal standard from che matrix, external standard quaatitation may be used at the discretion of the supervising mass spectromeuist. 9.0 ACCEPTANCE AND REJECTION CRITERIA 9.1 Refer to SOP SRI 9 1-3 for acceptance/rejection criteria except acceptable accuracy for standards is 80-12096 of theoretical. 10.0 REPORTING 10.1 Results of all analyses are tabulated, and the raw data, original chromatograms, and= = reports are to be filed in the appropriate study file. Authors: 0 Date